Ijmrhs Vol 4 Issue 3

International Journal of Medical Research
&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
th
Received: 20 April 2014
Research article
Coden: IJMRHS
Copyright @2014
ISSN: 2319-5886
th
Revised: 10 Jan 2015
Accepted: 18th April 2015
A COMPARATIVE STUDY OF ORAL OLANZAPINE AND ORAL HALOPERIDOL ON GLUCOSE

TOLERANCE LEVELS IN PATIENTS WITH SCHIZOPHRENIA
*G N S Sangeetha Lakshmi
Asst Professor, Dept of Pharmacology, Osmania University, Hyderabad
*Corresponding author email: asangeethalakshmi@yahoo.co.in
ABSTRACT
Background: Schizophrenia is a mental disorder characterized by persistent defects in the perception, thinking or
the expression of reality. The term "schizophrenia" translates roughly as "shattered mind," and comes from the
Greek (schizo, "to split" or "to divide") and (phrn, "mind"). Material and Methods: The study was designed to
be a prospective control study. Schizophrenic patients taking Olanzapine and Haloperidol were selected and
follow up at three weeks and six weeks was done. Results: In this prospective control study, Olanzapine and
Haloperidol were associated with an increase in Blood Glucose Levels. The mean changes in Glucose remained
within clinically normal range in this six week study. Conclusion: Antipsychotic treatmemt leads to the
development of Diabetes mellitus in a significant 10.1% of patients within 6 weeks. Given the serious
implications for morbidity and mortality attributable to diabetes mellitus, clinicians need to be aware of these risk
factors when treating patients with chronic schizophrenia
Keywords: Schizophrenia, Olanzapine, Haloperidol, Blood Glucose levels
INTRODUCTION
Schizophrenia is often described in terms of
"positive" and "negative" symptoms. Positive
symptoms include delusions, auditory hallucinations
and thought disorder and are typically regarded as
manifestations of psychosis. Negative symptoms are
so named because they are considered to be the loss
or absence of normal traits or abilities, and include
features such as flat, blunted or constricted affect and
emotion, poverty of speech and lack of motivation.
Some models of schizophrenia include formal
thought disorder and planning difficulties in a third
group, a "disorganization syndrome."[1]
The most commonly used criteria for diagnosing
schizophrenia are from the American Psychiatric
Association's Diagnostic and Statistical Manual of
Mental Disorders (DSM) and the World Health
Organization's International Statistical Classification
of Diseases and Related Health Problems (ICD). The
most recent versions are ICD-10. [2]
Cause: Genetic: Some researchers estimate

schizophrenia to be highly heritable (some estimates
are as high as70%). Environmental [3] - There is also
considerable evidence indicating that stress may
trigger episodes of schizophrenia psychosis.
Neurobiological influences: Role of dopamine: In
adult life, particular importance has been placed upon
the function (or malfunction) of dopamine [4] in the
mesolimbic pathway in the brain. This theory, known
as the dopamine hypothesis of schizophrenia, largely
resulted from the accidental finding that a drug group
which blocks dopamine function, known as the
phenothiazines, reduced psychotic symptoms. These
drugs have now been developed further and
antipsychotic medication is commonly used as a first
line treatment. Role of glutamate and the NMDA
receptor: Interest has also focused on the

neurotransmitter glutamate and the reduced
477
Lakshmi
Int J Med Res Health Sci. 2015;4(3):477-482
function of the NMDA glutamate receptor in the

development of schizophrenia.
Treatment: Medication and hospitalization
The first line treatment for schizophrenia is usually
the use of antipsychotic medication. Therefore,
antipsychotic drugs are only thought to provide
symptomatic relief from the positive symptoms of
psychosis. The newer atypical antipsychotic
medications [5] (such as clozapine, risperidone,
olanzapine, quetiapine, ziprasidone and aripiprazole)
are usually preferred over older typical antipsychotic
medications
(such
as
chlorpromazine
and
haloperidol) due to their favourable side-effect
profile.
While the atypical antipsychotics3 are
associated with less EPS and TD than the
conventional antipsychotics, some of the agents in
this class (especially olanzapine and clozapine)
appear to be associated with metabolic side effects [6]
such as weight gain, hyperglycemia and
hypertriglyceridemia that must be considered when
choosing appropriate pharmacotherapy.
Drugs: Haloperidol is a butyrophenone [7] with
general properties similar to those of the
phenothiazine, cholorpromazine.
It is an
antipsychotic with actions most closely resembling
those of phenothiazines with a piperazine side-chain.
Olanzapine is a thienobenzodiazepine atypical
antipsychotic.
It has affinity for serotonin,
muscarinic, histamine-H1 and adrenergic (a1)
receptors as well as various dopamine receptors.
Olanzapine is used for the management of
schizophrenia and for the treatment of moderate to
severe mania associated with bipolar disorder.
Olanzapine may share some of the adverse effects
seen with the classical antipsychotics, the incidence
and severity of such effects may vary. The most
frequent adverse effects with Olanzapine are
somnolence and weight gain; hyperprolactinaemia is
also common, but usually asymptomatic.
More
severe abnormalities of glucose homeostasis [8] occur
uncommonly;
severe
hyperglycaemia,
or
exacerbation of pre-existing diabetes, sometimes
leading to ketoacidosis or coma, has been reported.
Clinical monitoring for hyperglycaemia [9] has been
recommended, especially in patients with or at risk of
developing diabetes. Olanzapine has been associated
with a low incidence of extrapyramidal symptoms
including tar dive dyskinesia although extrapyramidal
symptoms may be more likely at high doses.
Neuroleptic malignant syndrome has been reported

rarely. [10]
Lambert BL [11] states that exposure to olanzapine is
associated with a 34-41% increase in the developing
of type 2 diabetes among recipients with
schizophrenia and compared with older generation
antipsychotics, exposure to olanzapine is associated
with an increased risk of hyperlipidemia among
people with schizophrenia. Still prospective,
randomized trials are needed to confirm these
retrospective, observational finding.
MATERIALS AND METHODS
A prospective study was conducted in the Psychiatry
department at Institute of Mental Health, Chennai on
patients admitted between October, 2005 to January,
2006. Patients who were newly started on Olanzapine
and Haloperidol and those who had been
antipsychotic free for the last six months formed the
sample of the study. The Olanzapine group had 38
patients and Haloperidol group had 25 patients.
Inclusion
Criteria:
Outpatient/Inpatient
in
Psychiatry department, Age 20 60 years, Patient
Sex-Male and Female Diagnosed by ICD-10 for
Schizophrenia, Patients taking oral Olanzapine 5-20
mg/day or Haloperidol 5-20 mg/day., Patients who
had given informed consent.
Exclusion Criteria:. Patients with other Mental
illness like bipolar mood disorders, Patients on
substance abuse or alcohol abuse, Pregnant or
lactating patients
Procedure: The study was a naturalistic study;
patients were diagnosed by the Consultant
Psychiatrist as suffering from Schizophrenia
according to ICD-10 [2].Informed consent was taken
from all the patients. Treatment was started and
monitered by a psychiatrist. The patients were
either started on Haloperidol or Olanzapine based
on
the
psychiatrists opinion. Patients could
continue taking their concomitant drugs like
Benzhexol, Benzodiapines, and Multivitamins and
anti hypertensives.
After taking informed consent, a semi structured
proforma was used to collect information about
relevant socio demographic data details about family
history and previous medical history.
The duration of the study was six weeks. During the
study cases took only one antipsychotic drug i.e. oral
478
Lakshmi
Olanzapine and controls took oral Haloperidol along

with their concomitant treatment.
After the patients fulfilled the inclusion criteria,
Random Plasma Glucose was taken before the start of
treatment patients who had above normal random
blood glucose (Normal RBS-120-140 mg/dl.) were
not taken into the study.
After the patient was started on an antipsychotic i.e.
in the first week, Blood was taken for Fasting Blood
Glucose and Post Prandial blood glucose. When the
patient came for review after three weeks and again
after six weeks fasting blood glucose and post
prandial blood glucose was taken. At each visit 2ml
Blood was taken under aseptic conditions. From the
blood sample sent to the laboratory, serum was
separated immediately after clotting.
Fasting blood glucose and post prandial blood
glucose was estimated by standardized enzymatic
procedure (applying glucose oxidase peroxidase
method).
Enzymatic method yields maximum
specificity for the procedure. From the blood sample
sent to the laboratory, serum was separated
immediately after clotting. Samples were used on the
same day. Haemolysed or grossly contaminated
samples were not used.
The following reference values were used in the
Laboratory.
Random Blood Sugar =
120 140 mg/dl.
Fasting Blood Sugar
=
60 90 mg/dl.
Post prandial blood sugar =
140 160 mg/dl.
RESULTS
Basic Description of Data: A total of seventy
patients who satisfied the inclusion criteria and who
signed the consent form were selected for the study.
Seven patients were lost in the follow up. The
remaining sixty three patients constituted the main
study group. The Olanzapine study group had 38
subjects (n=38) and the Haloperidol control group
had 25 subjects (n=25).
The mean age of study group was 34.5 9.9 years.
The mean age of Olanzapine group was 33.1 9.8
and the Haloperidol group was 35.6 + 10 years(Table
I). The Olanzapine group had Male 22 (57.9%) and
female 16 (42.1), Haloperidol group had male 11
(44%) and female 14 (56%). The Olanzapine group
had 5 subjects (13.2%) with family history of
Diabetes Mellitus and Haloperidol group had 5

subjects (20%) with family history of Diabetes
Mellitus. There was no statistical difference with
regard to family history of Diabetes Mellitus between
the two groups. When Chi square test was done, p
was 0.500 (P<0.05 it is significant). Most of the
subjects belonged to low socio-economic status
(83.3%) and the rest middle socio-economic status
(16.7%). The most common diagnosis was Paranoid
Schizophrenia;
in
the
Olanzapine
group
Undifferentiated Schizophrenia (18.4%), Paranoid
Schizophrenia (55.3%) and chronic schizophrenia
(26.3%). In the Haloperidol group undifferentiated
schizophrenia (2%), Paranoid Schizophrenia (68%)
and Chronic Schizophrenia (24%).
The mean dose of Olanzapine used was 13 4.7
mg/day and the mean dose of Haloperidol that was
used in the study was 14 + 3.2 mg/day.
Table 1: Comparison Between Olanzapine and
Haloperidol Groups (Baseline Characteristics)
Student Independentt Test
Olanzapine Haloperidol p
Mean SD Mean SD Value
(n=38)
(n=25)
Age (Yrs.)
33.19.8
35.610.0
0.33
Height (cms.)
164.3 8.9 165.2 7.7
0.678
Weight (kgs.)
63.809.6
65.8 8.4
0.389
BM I
23.5 1.7
24.1 2.1
0.234
Random Blood
Glucose
99.5 15.1 101.1 23.0 0.74
(mg/ml)
P<0.05 Significant
There is no statistically significant difference between
the Olanzapine group and Haloperidol group in Age,
Weight or Random Blood Sugar levels.
Regarding the Change in Fasting Blood Glucose and
Post Prandial Blood Glucose between the Olanzapine
and Haloperidol groups there was no statistically
significant increase. The change in the Fasting Blood
Glucose for the 1st and 3rd week was P=0.434, for
the 1st and 6th week was P=0.805, for the 3rd and 6th
week was P=0.68. The change in the Post Prandial
Blood Glucose for the 1st week and 3rd week was
P=0.429, 1st and 6th week was P=0.922, 3rd and 6the
week was P=0.236 (Table 2)
479
Lakshmi
Table2: Comparison between Olanzapine and Haloperidol groups Student Independentt test
Olanzapine
Haloperidol
Blood Glucose
Time Point
P Valve
Mean SD
Mean SD
1st week
78.1 17.
84.1 19.2
0.211 (NS)
Fasting Blood
3rd week
84.8 17.0
88.8 17.2
0.372 (NS)
Glucose
6th week
88.3 16.7
95.0 17.9
0.132 (NS)
1st week & 3rd week
6.7 7.7
4.7 12.8
0.434 (NS)
Increase in Fasting
1st week & 6th week
10.2 7.6
10.9 14.0
0.805 (NS)
Blood Glucose
rd
3 week & 6th week
3.5 5.7
6.2 6.1
0.068 (NS)
1st week
108.7 22.0
113.7 31.9
0.465 (NS)
Post Prandial
3rd week
115.2 23.3
117.3 23.7
0.728 (NS)
Blood Glucose
6th week
121.323.6
126.8 29.9
0.420 (NS)
6.5 9.7
3.6 18.9
0.429 (NS)
Increase in Post Prandial 1st week & 3rd week
Blood Glucose
1st week & 6th week
12.6 7.4
13.1 24.5
0.922 (NS)
3rdt week & 6th week
6.1 9.8
9.512.5
0.236 (NS)
glucose.
For
fasting
blood
glucose
there was no
P < 0.05 Significant; NS-Non Significant
increase for 1st and 3rd week was P=0.237, the
increase for 3rd and 6th week was P = 0.003, 3rd and
There is no statistically significant difference between
6th week P = <0.001. For Post Prandial Blood
Olanzapine and Haloperidol group for Fasting blood
glucose and post prandial blood glucose between 1st
and 3rd week, 1st and 6th week and 3rd and 6th week.
Significant increase in Blood glucose was seen in the
Olanzapine group. In Fasting Blood Glucose, the
significant increase for 1st and 3rd week was
P=0.001, for 1st & 6th week was P=0.001 and for 3rd
and 6th week was P=0.003.In Post Prandial Blood
Glucose the significant increase for 1st & 3rd week
was P=0.001, 1st & 6th week was P=0.001 and 3rd &
6th week was P=0.001
Table3: Comparison of Blood Glucose within
Olanzapine Group n=33 (between 1st, 3rd and 6th
week) Students Pairedt Test
Blood
Time Point
Change
P Valve
Glucose
6.7 7.7
Glucose
1st and 6th week 10.2 7.6
(mg/dl)
4.7 12.8
0.237
blood
1st and 6th week
10.914.0
0.003*
Glucose
3rd and 6th week
6.2 + 6.1
<0.001*
<0.001*
Post
1st and 3rd week
3.6 18.9
<0.001*
Prandial
1st and 6th week
13.124.5
0.039*
Blood
3rd and 6th week
9.5 12.5
0.003*
3rd and 6th week
3.5 5.7
0.003*
Post Prandial
1st and 3rd week
6.5 9.7
<0.001*
Blood
1st and 6th week 12.6 7.4
<0.001*
Glucose
3rd and 6th week
<0.001*
6.1 9.8
MeanSD
1st and 3rd week
(mg/dl)
1st and 3rd week
Glucose
Fasting
MeanSD
Fasting Blood
glucose there was no significant increase for the 1st

and 3rd week P=1.000, there was significant increase
for 1st and 6th week P=0.039, and 3rd and 6th week
P=0.003.
Table 4: Comparison of Blood Glucose within
Haloperidol, Group n =25 (between 1st, 3rd and
6th week) Student Pairedt Test
Blood
Time Point
Change
P Valve
* P<0.05 Significant
There is statistically significant difference for
Olanzapine group for fasting blood glucose and post
prandial blood glucose for 1st and 3rd week, 1st and 6th
week and 3rd and 6th week (Table3). For Haloperidol
group there was a significant increase in blood
Glucose
* P<0.05 Significant
There is statistically significant difference for
Halperiodol group for fasting blood glucose and post
prandial blood glucose between 1st and 6th week, and
3rd and 6th week. There is no statistically significant
difference between 1st and 3rd week for fasting blood
glucose and post prandial blood glucose within
Haloperidol patients group (Table 4)
480
Lakshmi
Fig1: Mean change in Fasting Blood Glucose

between different time interval for Olanzapine
and Haloperidol group.
Fig2: Mean change in Post Prandial Blood

Glucose between different time interval for
Olanzapine and Haloperidol group.
The relevant findings of the study are
Olanzapine produced significant increase in
fasting blood glucose and Post Prandial blood
glucose at 3rd week and 6th week
Haloperidol produced significant increase in
fasting blood glucose and Post Prandial blood
glucose at 6th week.
When Olanzapine and Haloperidol groups were
compared there was no statistically significant
difference between the increase in fasting and Post
Prandial blood glucose.
DISCUSSION
There is a re-emerging and controversial issue of
glycaemic control in schizophrenia and its possible
relationship to antipsychotic drug therapy. Obesity
and physical inactivity, which are common in patients
with schizophrenia, are known to increase the risk of
developing diabetes. It is reported a rate of diabetes
of 1.2% for persons age 18 to 44 years and 6.3% for
persons age 45 to 64 years. In patients with

schizophrenia, the prevalence of diabetes was 6 to 8%
in patients<45 years of age, 15 to 19% in patients 45
to 64 years of age, and 19 to 21% in patients>65
years of age. [12]
Case reports have also associated atypical
antipsychotic agents with exacerbation of pre-existing
diabetes,
new-onset
diabetes
and
diabetic
[13, 14]
ketoacidosis (DKA).
There are significantly
more reports associated with olanzapine.
In a pharmacoepidemiological study in >58,000
patients receiving a single antipsychotic, the overall
frequency of diabetes was about 3 times that found in
the reference general population. This result is very
similar to that found in studies to determine the rate
of diabetes in patients with schizophrenia done prior
to the widespread use of atypical agents.
In this report we are comparing the simultaneous
effect of two antipsychotic medications on a
important metabolic measure, indexing glucose in
patients with schizophrenia.
We found that
haloperidol was associated with significantly elevated
mean glucose levels after 6 week of treatment, that
olanzapine was associated wtih significantly elevated
glucose levels after 3 weeks of treatment. The mean
increases were modest and remained within clinically
normal ranges (one patient given Haloperidol
developed abnormally high glucose levels >125
mg/dl during the course of study treatment). The
Olanzapine-treated groups had significant elevations
in post prandial glucose levels when compared with
haloperidol-treated patients. Among antipschotics,
Olanzapine seems to have diabetogenic potential
when measured from baseline to endpoint.
Haloperidol fares better. [15, 16]
In our study, the typical antipsychotic haloperidol
was associated with an elevation of Blood glucose
levels within a clinically normal range. Haloperidol
has been reported to increase insulin resistance and to
be associated with higher fasting glucose levels in
obese women compared with control subjects.
Haloperidol has also been reported to be associated
with higher glucose levels in schizophrenia subjects.
Increased insulin resistance in peripheral tissues can
be caused by hyperprolactinemia and may be
involved in the mechanism underlying hyperglycemia
in patients treated with typical antipsychotics.
481
Lakshmi
Limitations:
Short follow up.
Confounding variables of co-medications.
Short duration requires 6th month or 1 year
follow up study.
More specific test can be used like Hydroxylated
Haemoglobin A (HbA) which will give the blood
glucose level for the previous 6 weeks.
CONCLUSION
In this prospective study, Olanzapine and Haloperidol
were associated with an increase in Blood Glucose
Levels. The mean changes in Glucose remained
within clinically normal range in this six week study.
Given
the
concerns
regarding
endocrine
dysregulation in the context of treatment of
schizophrenia patients with antipsychotic medication
the baseline and 6th week monitoring of fasting
blood glucose and post prandial blood glucose levels
be obtained in routine clinical practice with both
antipsychotics in order to monitor the risk for
development of hyperglycaemia.
Given the serious implications for morbidity and
mortality attributable to diabetes mellitus, clinicians
need to be aware of these risk factors when treating
patients with chronic schizophrenia.
6.
7.
8.
9.
10.
11.
12.
13.
Acknowledgement: I thank the Director of Institute

of Mental Health,Chennai,Dr.Murugappan and the
staff for helping me to do this study.
14.
Conflict of interest: Nil
15.
REFERENCES
1. Kraepelin, E. Text book of psychiatry (7th ed).
London : Macmillan, 1970; 525-20.
2. Turner, T. 'Schizophrenia'. A History of Clinical
Psychiatry, London,1999; 10th edition;110-50
3. Buse JB, Cavazzoni P, Hornbuckle K.
Antipsychotic induced type 2 diabetes: evidence
from a large health plan database. J Clin
Epidemiol; 2002;167-70
4. Uvnas-Moberg K, Ahlenius S, Alster P. Effects
of selective seratonin and dopamine agonists on
plasma levels of glucose, insulin and glucagon in
the rat. Neuroendocrine logy.1996;63:269-274
5. Leucht S, Wahlbeck K, Hamann J, Kissling W.
New generation antipsychotics versus low
potency conventional antipsychotics: a systematic
16.
review
and
meta-analysis.
Lancet,
2004;361(9369), 1581-9.
Weyer C,Hanson K,Bogardus C,Pratley RE.Long
term changes in insulin action and insulin
secretion associated with gain ,loss regain and
maintainance of body weight Diadetologia.2000;
36-46
Martin Dale Extra Pharmacopenia; 38th edition;
675-12.
Newcomer JW, Haupt DW, Fucetola,et
al.Abnormalities in glucose regulation during
antipsychotic treatment of Schizophrenia.Arch
Gen Psychiatry.2002;337-45.
Canadian Psychiatric Association. Canadian
clinical practice guidelines for the treatment of
schizophrenia, 1998; 43:255-05.
Lindenmayer JP, Patel R. Olanzapine-induced
ketoacidosis with Diabetes Mellitus Am J
Paychiatry.1999; 156:1471.
Pharmacoepidemiol Drug Saf. 2005 Mar 22. e J
Clin Psychopharmacol. 2005; 25(1):12-8.
Canadian Diabetes Association 2003 Clinical
Practise Guidelines for the Prevention and
Management of Diabetes in Canada,2003;27:5152
Lindenmayer JP, Patel R. Olanzapine-induced
ketoacidosis with diabetes mellitus Am J
Psychiatry. 1999;156:1471
Mukherjee S, Decina P, Bocola V, et al. Diabetes
mellitus in schizophrenic patients. Compr
Psychiatry. 1996; 68-73.
Saddicha, ManjunathaN, AmeenS, Akhtar.S.
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drug?Results from a randomised, double blind
controlled prospective study in first episode
Schizophrenia ;2000.
Ramaswamy K, Masand PS, Nasrath HA.Do
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482
Lakshmi
DOI: 10.5958/2319-5886.2015.00093.4

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
Received: 25th Oct 2014
Research article
Coden: IJMRHS
Copyright @2014
ISSN: 2319-5886
Revised: 10th Nov 2014
Accepted: 13th Nov 2014
SEROPREVALENCE OF HEPATITIS B IN A TERTIARY CARE CENTRE IN BIJAPUR,

KARNATAKA: A TWO YEARS PROSPECTIVE STUDY
*Preeti B. Mindolli1, Manjunath P. Salmani2
1
Associate Professor, Department of Microbiology, Al Ameen Medical College, H &RC, Athani Road, Bijapur,
Karnataka, India.
2
Associate Professor, Department of Microbiology, SBMP Medical College, H &RC, Solapur Road, Bijapur,
Karnataka, India.
*Corresponding author email: drmindolli@rediffmail.com
ABSTRACT
Background: Hepatitis B virus infection is endemic throughout the world especially in tropical and developing
countries. Clinical data collected in the hospital gives the estimation of burden of disease in the community as
patients with different background attend the hospital. With this background the present study was designed. It is
a prospective study estimating the prevalence of HBV infection in a tertiary care centre. Objective: Study was
conducted to know the prevalence of hepatitis B virus infection in a tertiary care centre in Bijapur, Karnataka.
Methodology: Patients attending Out-Patient Department (OPD) and In-Patient Department (IPD) with various
diagnosis who were advised for HbsAg testing were included in this study. Immunochromatographic method
(Hepacard) was used for qualitative detection of HbsAg to diagnose HBV infection. Results: A year wise
seropositivity showed there was slight increase in the HBV positive cases. In 2012 prevalence rate was 1.54% and
in 2013 it was 1.65%. Male preponderance compared to females was seen. More number of cases was seen in
active age group i.e. 31-40 years. Conclusion: The present study shows there is slight increase in number of cases
in 2013 compared to 2012. This study also highlights that hospital based studies can be an option for community
based studies.
Keywords: Hepatitis B; Immunochromatography; Seroprevalence
INTRODUCTION
Hepatitis B virus (HBV) is common human pathogen
and causes acute and chronic liver disease throughout
the world. Chronic illness develop in 5-10% 0f
infected adolescents or adults and up to 90% in
infected neonates. Chronic HBV infection is a major
cause of liver cirrhosis and primary cell carcinoma.[1]
Hepatitis B is endemic throughout the world,
especially in tropical and developing countries and
also in some regions of Europe. Its prevalence varies
from country to country and depends on behavioral
environment and host factor.[2]
Mindolli et al.,
More than two billion people worldwide have

evidence of past or current HBV infection and 350
million are chronic carriers of the virus, which is
harbored in liver and causes an estimated 6, 00, 000
deaths from cirrhosis of liver and hepatocellular
carcinoma.[3]
In Middle East and Indian subcontinent, an estimated
2-5% of general population is chronically infected
and falls in intermediate category according to World
Health Organization (WHO) classification.[2,3]
483
483
483
Several surveys for HbsAg screening have been

carried out at different places for blood donors,
pregnant women. Surveys for screening HBsAg have
been primary, simple and most useful mode for
determining HBV infection rate.[4]
Source of data: The study group comprises of
patients of all age groups and both sexes admitted
during January 2012 to December 2013 in IPD of all
departments of Shri B.M. Patil Medical College,
Hospital and Research Centre, Bijapur, Karnataka.
Type of study: Prospective analysis
Ethics committee approval: The study was
approved by the Institutional Ethics Committee
(IEC).
Methodology: Two ml of blood sample was collected
with aseptic conditions. The serum was separated
and it was used for the present study. Specimens
containing visible precipitates or cloudy specimens
are clarified prior to testing by high speed
centrifugation i.e. 10,000 revolutions per minute for
fifteen minutes before testing. The test was performed
within twenty four hours from the sample collection.
For qualitative detection of HbsAg, test was done by
Immunochromatographic method (Hepacard) to
diagnose HBV infection performed and test card was
labeled with identification number[5]. The test was
performed
and
interpreted
according
to
manufacturers instructions. The kit has sensitivity
and specificity of 100%.
Data collection: Patients personal details like age,
sex, address was noted down. The HBsAg test result
(positive or negative) was noted of individual person.
The collected data is represented in tabular form and
prevalence rate was calculated. The speed, sensitivity,
ease to perform and interpret the results makes it
more useful for both individual as well as large scale
studies.[5, 6]
RESULTS
The study was conducted from January 2012 to
December 2013. A total of 18, 372 samples were
screened for HbsAg during this period and year wise
prevalence rate was calculated.
From January-December 2012, 8,944 samples were
screened, out of which 138 were positive and
prevalence rate was 1.54%. From January-December
Mindolli et al.,
2013, 9428 samples were screened out of which 156

were positive and prevalence rate was 1.65%.
There is slight increase in prevalence rate in 2013
compared to 2012 (Table 1).
Male preponderance is seen compared to females
(Table 2).
Increased prevalence of HBV infection is seen in 3140 years age group followed by >50 years age group
(Table 3).
Table 1 Seropositivity of HbsAg among hospital
based population
Year Total No. Total No. of Total
of cases
HBsAg positive positive (In
%)
2012 8944
138
1.54%
2013 9428
156
1.65%
Table 2: Sex distribution of seropositivity of
HbsAg in hospital based population
Sex
No. of Total No. of Total positive
HbsAg positive (In %)
sera
tested
Male
9,268
159
1.71%
Female 9,104
135
1.48%
Table 3: Age distribution of hospital based general
population for HbsAg postivity
Age
Total No. Total No. Total
(Years) of
sera of HbsAg positive (In
%)
tested
positive
0-10
820
08
0.97%
11-20
2727
40
1.46%
21-30
4040
65
1.60%
31-40
4632
84
1.81%
41-50
3932
59
1.50%
>50
2221
38
1.71%
DISCUSSION
In our study of hospital based population the
prevalence rate of HbsAg in year 2012 was 1.54%
and in 2013 it was slightly increased to 1.65%. This
may be due to increased awareness about HBV
infection and number of samples to be tested has also
increased.
Similar studies on prevalence of hepatitis B are
conducted in India. A study conducted by Singh et al
among blood donors in Mangalore showed
prevalence as 0.62%.[7] Another study conducted by
Ronald Roche et al in Mangalore in 2012 showed
prevalence rate of HbsAg as 1.56%.[8] According to
484
484
484
WHO definition one could categorize Karnataka into

a HBV low endemic state.
Another review of hepatitis B prevalence in India by
Lodha et al has concluded that it is between 1-2%.[9]
Smita sood and Shirish malvankar have noted 0.87%
prevalence which is hospital based study similar to
us.[10] A study conducted by Bhatta CP et al in
Kathmandu Medical College teaching hospital in
2003 showed prevalence rate of HbsAg as 2.5%.[11]
Our study has reported higher prevalence among
males (1.71%) compared to females (1.48%).
Many studies shows male preponderance compared to
females. Dutta et al reported 35.3% in males and
19.3% in females.[12] Singh et al reported 0.65% in
males and 0.25% in females. Higher prevalence
among males is also noted in Smita Sood et al
study.[10] It is hypothesized that females clear HBV
more efficiently compared to males.
In our study higher prevalence rate was seen in the
age group of 31-40 years followed by > 50 years.
Similar findings were noted in Smita Sood et al
study10. This may be due to higher chances of
exposure to HBV infection due to sexual activity.
CONCLUSION
The present data is limited to patient population
served by our hospital and not applicable to other
centers. Hospital based studies can be alternate option
to community studies which are difficult to conduct.
The present study provides good reference to
formulate strategies to reduce the seroprevalence rate.
The patient attending our hospital represents cross
section of Bijapur district population with mix of rich
and poor and urban and rural population. Therefore
our study highlights HBV infection rate of this part of
state and shall provide reference for future studies on
epidemiology of HBV infection.
REFERENCES
1. Brian WJ. Hepatitis B. In: Topley and

Wilson's Microbiology and Microbial
Infections: 10th edn Vol 2 London: Arnold
Publishers; 2005 :1226
2. Park K. In: Parks textbook of Preventive and
Social Medicine.21st ed. Jabalpur, India: M/s
Banarasidas Bhanot Publishers; 2011: 231-32
3. WHO (2009), Weekly epidemiological
records, N 040, 2nd Oct 2009.
Mindolli et al.,
4. Sayed A. Quadri, H.J. Dadapeer, K.

Mohammed Arifulla and Nazia Khan.
Prevalence of Hepatitis B Surface Antigen in
hospital based population in Bijapur,
Karnataka. Al Ameen J Med Sci 2013; 6(2)
:180-82.
5. Kaur H, Dhanao J, Oberoi A. Evaluation of
rapid kits for detection of HIV, HbsAg and
HCV infections. Indian J Med Sci 2000; 54:
432-34.
6. Torlesse H, Wurie IM, Hodges M. The use of
immunochromatography test cards in the
diagnosis of hepatitis B surface antigen
among pregnant women in West Africa. Br J
Biomed Sci 1997; 54 (4): 256-59.
7. Sing K, Bhat S, Shastry S. Trend in
seroprevalence of Hepatitis B virus infection
among blood donors of coastal Karnataka.
Indian J Infect Dev Ctries 2009; 3 (5): 37679.
8. Ronald Roche, Shriyan Amrita, Leslie,
Ranjana Nayak. Prevalence of the Human
Immunodeficiency Virus, the Hepatitis B
Virus and the Hepatitis C Virus among the
Patients at a Tertiary Health Care Centre: A
Five Year Study. Journal of Clinical and
Diagnostic Research. 2012 May; 6(4): 62326.
9. Lodha R, Jain Y, Anand K, Kabra SK,
Pandava CS. Hepatitis B in India: A review
of disease epidemiology. Indian Pediatr
2001: 38: 1318-22.
10. Sood S and Malvankar S. Seroprevalence of
Hepatitis B surface Antigen, Antibodies to
Hepatitis
C
virus
and
Human
immunodeficiency virus ina Hospital based
population in Jaipur, Rajasthan. Indian J
Community Med 2010; 35 (1): 165-69.
11. Bhatta CP, Thapa B, Rana BB.
Seroprevalence of Hepatitis B in Kathmandu
Medical
College
teaching
hospital.
Kathmandu Univ Med J 2003; 1: 113-16.
12. Dutta S, Shivanand PG, Chatterjee A.
Prevalence of hepatitis B surface antigen and
antibody among hospital admitted patients in
Manipal. Indian J Public Health 1994; 38:
108-12.
485
485
485
DOI: 10.5958/2319-5886.2015.00094.6

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3 Coden: IJMRHS
Copyright @2014
th
th
Received: 25 Dec 2014
Revised: 20 Feb 2015
Research article
ISSN: 2319-5886
Accepted: 26th Apr 2015
HISTOPATHOLOGIC AND CYTOMORPHOLOGIC CORRELATION IN LEPROSY

*Sharayu A Sarode1, Anil T Deshmukh2
2
HOD, 1Department of Pathology, Panjabrao Deshmukh Medical College, Maharashtra, India
*Corresponding author email: sharayukalmegh@gmail.com

ABSTRACT
This study was conducted in Dr. Panjabrao Deshmukh Medical College we tried to evaluate and compare the
histological and cytological procedure for classifying leprosy lesion. Method: Total sample size was 60.Skin
punch biopsy was done and sample was evaluated for histology after H & E and Fite Faraco staining. In some
cases where histological diagnosis was confirmed we also took sample for cytology which were stained by MGG
and modified ZN technique. Results Our study group consists of total 60 leprosy patients, out of which 34
(56.66%) were males and 26 (43.44%) were female between 10 years to 68 years of age. Complete
cytohistological correlation was seen in 36 (60%) cases. Correlation was fairly strong in polar group of leprosy
like in TT i.e. (62.5%) and LL (60%). Conclusion In cases of polar leprosy cytological diagnosis parallels
histological diagnosis, within the constraint of cytological interpretation the cases in borderline unstable spectrum
of leprosy can be classified broadly. Histopathological correlation is required to determine appropriate position in
RJ spectrum. Similarly in cases where aspirate was inadequate histology is required to confirm or rule out type of
leprosy.
Keywords: Tuberculoid Leprosy, Borderline Tuberculoid, Borderline Lepromatous, Lepromatous Leprosy.
INTRODUCTION
Leprosy is one of the oldest disease known to
mankind. Currently The Ridley-Jopling (RJ)
classification currently in use for classifying leprosy
is based on widely acknowledged clinical,
bacteriological, immunological, and histological
parameters. [1] Application of the RJ scale in the
classification of leprosy helps in understanding the
immunology of the patient to predict prognosis and
possible complications. Histopathology is considered
to be a gold standard for diagnosing leprosy but it is
an invasive procedure and leads to a biopsy scar,
which may not be cosmetically acceptable. Slit skin
smear technique stained with Ziehl-Neelsen (ZN) is
considered as a simple field procedure for the
diagnosis of leprosy but many practical problems
affect the reliability of skin-smears.[2, 3, 4, 5,6,7]This
present study was undertaken to evaluate and
compare the histological and cytological procedure

for classifying leprosy lesion.
Sharayu et al.,
MATERIAL AND METHODS

Present study was undertaken in department of
pathology, Dr Panjabrao Deshmukh Memorial
Medical college after receiving clearance from
institutional ethical committee. Study done over a
period of 2.5 years from June 2011 to Oct. 2013. All
the new clinically suspected cases of leprosy
attending Dermatology OPD in Dr PDMM College
were enrolled. Informed consent was taken from
patients. Histologically confirmed cases of leprosy
were enrolled into the study.
Skin punch biopsy was performed by dermatologist.
Biopsy material was immediately fixed into 10%
formalin. After adequate fixation for 10 12 hours
486
sample was submitted for routine processing,

& FNA or Cytopuncture was done in case of nodular
following which paraffin embedded section of 5 m
lesion, slides were air dried and stain with May
thickness were stained with H and E for
Grunwald Giemsa stain & modified Ziehl-Neleson
histopathological analysis and fite faraco staining for
stain for acid fast bacilli. Cytological procedure were
identifying bacilli. After studying histopathological
consider adequate if the cellular yield of
feature & noting bacteriological index the diagnosis
inflammatory cells was heavy or when eccrine sweat
of leprosy was confirmed & classified as per Ridley
glands were seen in presence of low inflammatory
jopling classification.
cells. Cytological criteria for classifying the cases is
For cytology sampling procedure depend on type of
shown in table 1
lesion. Slit skin smear was done in cases of flat lesion
Table 1: Cytomorphological features in leprosy [5]
Types of leprosy
Morphological features
Cellularity
Granuloma
lymphocytes
AFB
Tuberculoid
Cellular smear
Cohesive granuloma
Numerous lymphocytes
AFB 0
leprosy (TT)
consists of epitheloid
not infiltrating granuloma
cell & lymphocytes
Borderline
Fairly cellular
Poorly cohesive
Few lymphocytes
AFB 1+,2+
tuberculoid(BT)
granuloma composed
mixture of epitheloid
cell and macrophages
Borderline
Moderate
Singly dispersed
Numerous lymphocytes
AFB 3+,4+
lepromatous (BL) cellularity
macrophages no
epitheloid cell
Lepromatous
Heavy
Numerous foamy
Few lymphocytes
AFB 5+,6+
leprosy (LL)
cellularity
macrophages
RESULTS
Our study group consists of total 60 leprosy patients,
out of which 34 (56.66%) were males and 26
(43.44%) were female between 10 years to 68 years
of age. majority of patients 18 (30%) were between
age group of 20 to 29 years.12 patients (20%) were
involved in farm related activity either they were
labour or farmer.
Majority of female patients 20 (33.33%) were
housewife. 6 patients (10%) in our study were
baggers they were detached from family because of
leprosy stigma.
Out of 60 cases of leprosy, on histology majority
25/60 (41.66%) were of borderline tuberculoid
leprosy followed by tuberculoid leprosy 16/60
(26.66%). Borderline lepromatous leprosy was seen
in 3 (5.0%) cases.
There were 10/60 (16.66%) cases of lepromatous
leprosy. We also found 1 case of indeterminate
leprosy, 2 cases of histoid leprosy and 3 cases of
ENL.
All cases (16/16) of histologically confirmed

tuberculoid leprosy were paucibacillary whereas
24/25 (96.0%) cases of borderline tuberculoid leprosy
were paucibacillary. All the cases of BL and LL were
multibacillary leprosy.
There were 2 cases of histoid leprosy all were
multibacillary. Out of 3 cases of ENL 2 were
paucibacillary and 1 case was multibacillary whose
bacillary index was more than 1+.
Complete cytohistological correlation was seen in 36
(60%) cases. Correlation was fairly strong in TT
leprosy i.e. (62.5%), borderline (50%), LL (60%). 2
cases of TT on cytology showed feature suggestive of
BT. 1 case of Histologically confirmed LL showed
feature suggestive of BL on cytology.
Two smears one each of TT and BT showed chronic
inflammatory cells on cytology. Remaining 24
smears for cytology were inadequate for
interpretation (table 2)
487
Sharayu et al.,
Table 2: cytohistological correlation along RJ spectrum

Histological
classification
classification
TT
BT
BL
LL
Histoid
ENL
Indeterminate
Total
Cytological classification
No. of
cases
TT
Borderline
(BT,BL)
LL
Histoid
ENL
Indeterminate
Chronic inflammatory
cells
16
25
3
10
2
3
1
60
10
01
-----11
2
14
-1
---17
---6
---6
-------0
-------0
-------0
1
1
-----2
DISCUSSION
In present series of 60 cases we found more no of
cases in age group of 20 29 years around 30%. We
could not found single case below 10 years of age
similarly in other studies also incidence of leprosy
below 10 years of age was very low [2,3,4,5,6]. Probable
cause for this finding may be long incubation period
of leprosy [7, 8]. Histology is considered to be a gold
standard for diagnosis of leprosy. In present series of
60 cases we did biopsy from lesion site from every
case.
The most commonly encountered type of leprosy was
BT 41.66% (25/60). Second common type was TT
26.66% (16/60), BL was seen in 5% of cases.
Borderline group constituted the major spectrum
46.66% 28 biopsies, which include BT, BB, BL. A
sizeable portion of leprosy patient will be in a
continuous changing immunological spectrum i.e.
BT, BB, BL so majority of cases belong to borderline
group[8]. According to many observer features of both
tuberculoid and lepromatous leprosy can occur in
same section or in serial sections or in different lesion
of same borderline cases immunological instability in
this borderline cases make them move in either
direction along the borderline spectrum. With
treatment they move toward tuberculoid pole or
without treatment they tend to move towards
lepromatous pole. If the disease is recognized at an
earlier stage and biopsy is taken, it will be in BT
stage or if disease is recognized at latter stage and
biopsy is taken, it may be in BL stage [9].
In our study overall cytohistological correlation was
seen in total 60% (36/60) cases, Cytohistological
correlation was more prominent in polar group of
leprosy. In TT leprosy 62.5% (10/16) cases were

diagnosed on cytology whereas 60% (6/10) cytology
showed
feature
suggestive
of
LL
type.
Cytohistological correlation was around 50% (14/28)
borderline group of leprosy.
Slit skin smear for AFB have conventionally been
used in assessing bacteriological index in leprosy.
Marine Ridley examined cellular exudates in slit skin
smear by ZN technique for AFB this generate more
information about leprosy lesion than only BI and MI
alone. She suggest that by studying nature of
exudates it was possible to place lesion in its
approximate position of RJ scale however she failed
to differentiate epithelioid cells from macrophages or
comment on cohesiveness of granuloma. This is a
limitation of ZN stain which does not provide
morphological detail comparable to that with MGG.
In present study cytological sub classification of
Histologically diagnosed leprosy was done on RJ
spectrum as per the criteria led down by ridley and
also the one adapted by N singh et al. Cytological TT
is characterized by cohesive epithelioid granuloma
with lymphocytes not infiltrating the granuloma as
the disease progress toward the lepromatous pole
cohesion between the cells of granuloma diminishes,
concurrent with increasing infiltration of lymphocytes
within them thus epitheloid granuloma of TT
transform to macrophage granuloma of LL with
heavy bacterial load. This is similar to feature
described in histology. The largest no of lymphocytes
are seen in BL leprosy where these predominate cell
type [7].
Histological criteria for diagnosis of leprosy is
applicable to cytological smear even though nerve
damage could not be detected on cytology the overall
cytodiagnostic accuracy of skin lesion has been 60%
488
Sharayu et al.,
in present study this is lower than 76.6% as reported

by Singh et al[10]. We observed uniform
cytohistological correlation in leprosy skin lesion.
However cytologic feature in cellular exudates may
be similar across Borderline tuberculoid (BT),
borderline borderline (BB), Borderline lepromatous
(BL) area.
Out of 10 cases of LL on MGG stain foamy
macrophages were seen in 6 cases i.e. 60%. 1 case of
LL was diagnosed as borderline on cytology and
remaining three smears were inadequate. Thus
complete cytohistological correlation was achieved in
60% cases of LL.
A 14 (50%) out of 28 (25 BT + 3 BL) cases were
diagnosed as borderline group mostly BT because it
shows inflammatory cell with few epithelioid cell.
Out of remaining 14 cases 7 showed nonspecific
inflammatory cells, 6 were inadequate and 1 case was
diagnosed as TT.
10 cases 62.5% out of 16 cases were diagnosed as TT
on cytology, 2 cases were diagnosed as BT, 1 showed
nonspecific inflammatory cells and remaining 3
inadequate smears.
It thus become evident that cytological examination
of cellular exudates from leprosy skin lesion provides
information similar to one obtained on histological
preparation of skin biopsy in cases of polar leprosy of
either type. In borderline cases however keeping in
view
the
recommendation
on
cytological
interpretation of a leprosy skin lesion made by
Marine Ridley [10] can be placed broadly in BT, BB,
BL area of spectrum. It seems, in such a cases,
histologic confirmation to place the cases in
appropriate borderline group is required.
CONCLUSION
In conclusion cytomorphology study of leprosy using
MGG and Z-N Stain for AFB can act as a useful
adjuvant to histopathology. In cases of polar leprosy
cytological diagnosis parallels histological diagnosis
within the constraint of cytological interpretation the
cases in borderline unstable spectrum of leprosy can
be classified broadly. Histopathologic correlation is
required to determine appropriate position in RJ
spectrum. Similarly in cases where aspirate was
inadequate histology is required to confirm or rule out
type of leprosy.
ACKNOWLEDGEMENT: None
Conflict of Interest: Nil
REFERENCES
1. Jopling WH, McDougall AC. Definition,
epidemiology and world distribution. In: Jopling
WH, McDougall AC, editors. Handbook of
Leprosy. 5 th ed. New Delhi: CBS Publishers;
1996; 1.
2. Sehal VN,SinghJ.Slit skin smear in leprosy.int J
Lepr 1990; 29:1
3. Thenvent, miyazaki, Rosche P,ShresthaI.
Cytological needle aspiration for diagnosis of
pure neural leprosy. Indian j lepr 1996; 6(5):1
4. Kaur S,Kumar B,Gupta SK,fine needle aspiration
of lymphnode in leprosy.a stdy of bacteriological
and morphological indices.Int j of lepr 1971; 45:4
5. TS Jaswal,VK Jain,Vandana Jain,Manmeet
Singh,Kamal Kishor,Sunita Singh. Evaluation of
leprosy lesion by skin smear cytology in
comparison to histology. Indian jor of
pathol.microlbiol 2001; 44:3
6. Georgiev, G. D. and McDougall, A. C. Skin
smears and the bacterial index (BI) in multiple
drug therapy leprosy control programs: an
unsatisfactory and potentially hazardous state of
affairs. Int. J. Lepr. 1988; 56 101-04.
7. WHO, World Health Organization Expert
Committee on Leprosy. Sixth report, 1988. 768
8. Thangasay RH, Yawalkar SJ. Historical
Background. In: Leprosy for Medical Practioners
and Paramedical Workers. Basle: 1986; 5: 14.
9. Pandya SS. Leprosy Control in India Historical
Aspects. In: Valia RG, VAlia AR, editors,
Textbook and Atlas of Dermatology. Bombay:
Bhalani Publishing 1994; 1422-1426.
10. Charles K. Job, Joseph Jayakumar, Michael
Kearney and Thomas P. Gillis Transmission of
Leprosy: A Study of Skin and Nasal Secretions of
Household Contacts of Leprosy Patients Using
PCR Am J Trop Med Hyg 2008 ;78(3): 518-21
489
Sharayu et al.,
DOI: 10.5958/2319-5886.2015.00095.8

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
Coden: IJMRHS
th
Received: 27 Jan 2015
Revised: 25th March 2015
Research article
Copyright @2015
ISSN: 2319-5886
Accepted: 9th May 2015
PREVALENCE AND AT EARLY AGE ONSET OF HYPO AND HYPERTHYROIDISM IN POSTIODIZATION ERA: A HOSPITAL BASED STUDY FROM SOUTH INDIA
Fathima Nusrath1, Baderuzzaman2, Anees Syyeda 2, N Parveen4, Siraj M3, N, *Ishaq M1
1
Department of Genetics, Osmania University, Hyderabad-500 007, Telangana, India

Department of Biochemistry,3Department of Medicine, Princess Esra Hospital, 4Salar-E-Millat Sultan Salahuddin
Owaisi Centre for Cellular and Molecular Medicine, PEH, Deccan College of Medical Sciences, Kanchanbagh,
Hyderabad, Telangana, India
2
*Corresponding author email: ishaq_50@rediffmail.com

ABSTRACT
Background: Thyroid dysfunction has been considered as one of the most common endocrine disorder in clinical
practice throughout the world. Its increasing prevalence had led to the screening of general population in different
parts of the world in order to investigate causes for rising incidence. A nationwide survey on epidemiology of
thyroid dysfunction in selected cities of India suggested the need for further studies in order to have a more
comprehensive analysis of epidemiological aspect for better awareness and control of this endocrine disorder.
Aim: The major objective of the present study was to identify the prevalence and early age at onset of hypo and
hyperthyroidism in post-iodization era based on a hospital based study. Materials and Methods: A total of 516
subjects visiting department of Medicine, Princess ESRA Hospital, Hyderabad, in age group of 10 to 75 years
were included in the study from June 2013 to January 2014. Serum TSH, T3, and T4 assays were assessed by
chemiluminescence method. Based on thyroid dysfunction test results, subjects were classified into
Hypothyroidism, Subclinical Hypothyroidism and Hyperthyroidism. Results: The prevalence of hypothyroidism
was highest in the females 33.52 % (n=173) as compared to males 2.32% (n=12) and hyperthyroidism in females
4.06% (n=21) and 0.19% (n=1) in males. Subclinical hypothyroidism in females was 7.55% (n=39). Conclusions:
An inordinately high increase in the prevalence rate in women was observed particularly in the age group 2130years. Monitoring of thyroid profile is necessary to prevent adverse outcome at clinical and subclinical levels
related to infertility, pregnancies and other complications.
Keywords: Hypothyroidism, Hyperthyroidism, Subclinical hypothyroidism, T3 (Triiodothyronine), T4
(Thyroxin), TSH (Thyroid stimulating hormone)
INTRODUCTION
Thyroid dysfunction (TD) has been considered as
one of the most common endocrine disorder in
clinical practice throughout the world. [1] Its
increasing prevalence had led to the screening of
general population in different parts of the world in
order to investigate causes for rising incidence. The
world wide prevalence of hypothyroidism is
Fathima et al.,
estimated to be around 5% [2, 3] and that of

subclinical hypothyroidism worldwide is 4-15%. [3, 4]
In this scenario there is a need for further
epidemiological studies in view of high prevalence
reported by some initial studies both hospitals based
as well as population surveys. Some recent reports
from Europe have claimed that the non-toxic goiter
490
Int J Med Res Heath Sci. 2015;4(3):490-495
in the post-iodization era appears to be of autoimmune type i.e. hypothyroidism. The important
signs and symptoms of hypothyroidism are fatigue,
weight gain, constipation and cold intolerance
whereas anxiety, palpitation, weight loss, increased
appetite and sweating are important signs of
hyperthyroidism. In a study by Doufas et al (1999)[5]
it has been reported that apart from genetic and other
environmental factors iodine excess in postiodization era is considered to play an important role
in the rising incidence of hypothyroidism of
autoimmune type. Due to less awareness as well as
less attention towards diagnostics of hypo and hyper
thyroidism in India a considerable percentage of
population, particularly women suffer from thyroid
dysfunction which is considered as a common organ
specific autoimmune disorder.[1]
A nation-wide study by Unnikrishnan et al. (2013)[ 6]
on epidemiology of thyroid dysfunction in selected
cities of India suggested the need for further studies in
order to have a more comprehensive analysis of
epidemiological aspects for better awareness and
control of this endocrine disorder. Important factors
that have been considered in such studies are age at
onset, and gender ratio, and the relative prevalence
rates of hypothyroidism and hyperthyroidism. In the
present study emphasis has also been laid on the
prevalence of subclinical hypothyroidism which may
have various consequences such as increasing risk of
cardiovascular disease, hyperlipidemia, somatic and
neuromuscular symptoms, reproductive and other
consequences as thyroid stimulating hormone (TSH)
levels above the normal range can cause such ailments.
In 1983 when India adopted the universal salt
iodization programme it is noted that in post iodization
period there was decline in goiter prevalence in several
parts of the country. But the prevalence is estimated
about that still 42 million people suffering from
thyroid dysfunction. [7] There is a need for further
studies on the prevalence of thyroid disorder in post
iodization period in order to investigate the new
emerging trends in the prevalence of TD.
The study was conducted after taking approval from
Institutional Review Board, Deccan college of Medical
Sciences, Hyderabad.
This was a unicentric, prospective based study. A
total of 516 subjects visiting Department of
Medicine, Princess ESRA Hospital, Hyderabad for

general health checkup in the age group of 10 to 75
years (those male cases who reported for thyroid
profile checkup were also included) were screened
after taking informed consent from them. Each
subject was given a reference number for further
reference. Only cases with primary thyroid disorders
were considered to be included under inclusion
criteria whereas cases suffering with secondary and
tertiary hypothyroidism and chronic illness were
excluded from the study.
Methodology: All the subjects underwent the
assessment of physical examination, medical history
and laboratory investigations. From each patient
aseptically 2ml of blood was collected and serum
was separated. The serum was used for the following
thyroid profile biochemical analysis within 24 hours
from collection. Serum TSH (Thyroid stimulating
hormone), T3 (Triiodothyronine), and T4 (Thyroxin)
assays were done by chemiluminescence method
(Helfand et al) [8] (Biomerieux, France, 3rd
Generation). Based on TD test results, subjects were
classified as shown in Table 1. Statistical analysis:
Statistical analysis was performed for all the subjects
enrolled in the study as per the protocol. All
statistical analysis was performed using Graph Pad
Prism software Version 5.0 (San Diego, CA, USA).
The prevalence of hypo, hyper and subclinical
hypothyroid was expressed as counts and
percentage. Distribution of various parameters such
as age and gender among hypo, hyper and
subclinical hypothyroidism was calculated using
Fishers exact test. P value 0.05 was considered
statistically significant for the all variables.
RESULTS
A total of five hundred and sixteen (516) subjects
were included in the study from June 2013 to
January 2014. Out of these four hundred and eighty
seven (487) subjects were females and the remaining
29 subjects were males falling in the age range of 10
to 75. However, the percentages of cases suffering
from hypothyroidism were 185(35.85%), sub
clinical
hypothyroidism
39(7.55%),
and
hyperthyroidism 22(4.26%) (Table 2 and Fig. 1).
The number of hypothyroid cases were significantly
higher than hyperthyroid as well as subclinical
hypothyroid cases (p<0.05).
491
Fathima et al.,
Subjects who were already diagnosed as suffering

from thyroid dysfunction and were on Levothyroxin/
Eltroxin/ Carbimazole etc constituted a total of
31.25% (n=145) with hypothyroidism and 1.93%
(n=9) with hyperthyroidism (Table 3). The number of
newly diagnosed hypo cases was 39 (7.55%) and that
of hyperthyroid cases 13 (2.52%). The total number of
already
diagnosed
hypothyroid
cases
were
significantly higher than that of newly diagnosed
hypothyroid cases (p<0.001). Gender-wise analysis
total cases revealed that the total prevalence newly
diagnosed and already known cases revealed the
highest prevalence of hypothyroidism (36.99%) and
hyperthyroidism (52.38%) was in the age group of 2130years in females and 31-40 (50%) in males.
The prevalence of hypothyroidism was highest in the

females 33.52 % (n=173) as compared to males
2.32% (n=12) and hyperthyroidism in females
4.06% (n=21) and 0.19% (n=1) in males.
Subclinical hypothyroidism in females was 7.55%
(n=39) and no male subclinical subjects were found.
Details of prevalence rates of hypo and
hyperthyroidism and subclinical thyroid condition
are shown in table 4.The percentage distribution
curves of hypothyroid as well as subclinical
hypothyroid cases are shown in figure 2. However
distribution for hyperthyroid cases is not shown here
due to small number of cases (n=22). The statistical
analysis revealed highest prevalence in age-group
21-30 years compared to other age groups
(p<0.001).
Table1: Normal and elevated levels of thyroid profile to segregate disease type
Serum TSH
Total T3
Total T4
Normal
Range
Hypo
thyroidism
0.3-5IU/ml
0.6-3.3 nmol/L
60-120nmol/L
7IU/ml
3.3 nmol/L
120nmol/L
Subclinical
Hypothyroidism
5-7IU/ml
Normal
Normal
Hyper
thyroidism
0.2IU/ml
0.6nmol/L
60nmol/L
Hypothyroidism versus hyperthyroidism and subclinical hypothyroidism cases were found statistically significant
(p<0.05)
Table 2: Percentages of patients suffering from hypothyroid, subclinical hypothyroidism and hyperthyroid
cases.
Number
Percentage
Total number
of subjects
Hypo
thyroid
Hyper
thyroid
Subclinical
hypothyroidism
516
100
185***
35.85%
22
4.26%
39
7.5%
***p<0.0003
Table 3: The number and percentage of newly and already diagnosed cases of hypo and hyperthyroidism
Hypothyroidism
Newly
Diagnosed
*Already Diagnosed
Total
Hyperthyroidism
Female
37 (7.17%)
Male
3 (0.38%)
Female
12 (2.32%)
Male
1(0.19%)
136 (26.35%)
173 (33.52%)
9 (1.93%)
12 (2.32%)
9(1.74%)
21(4.06%)
0
1(0.19%)
*Already diagnosed hypothyroid cases versus newly diagnosed hypothyroid cases p<0.001
Table 4: Prevalence of hypo and hyperthyroidism in males and females in different age groups
Subclinical
Hyperthyroidism
Age group Hypothyroidism
hypothyroidism
(Year)
F
M
F
M
F
M
10-20
27 (15.60%)
1(8.33 %)
2(5.12%)
0
3 (14.28%)
1
*21-30
64 (36.99%)
1(8.33%)
13(33.33%)
0
11 (52.38 %)
0
31-40
39 22.54%)
6(50.0%)
5(12.82%)
0
6 (28.57%)
0
41-50
20 11.56%)
2(16.6%)
9(23.07%)
0
0
0
51-60
10 (5.78%)
2(16.6%)
4(10.25%)
0
1 (4.76%)
0
61-70
12 (6.93%)
0
4(10.25%)
0
0
0
71-75
1 (0.57%)
0
2
0
0
0
492
Fathima et al.,
*Number of cases in the age group of 21-30 was

significantly higher than other age groups in females
with hypothyroidism (p<0.001)
Fig 1: Percentage of hypothyroid, subclinical

hypothyroid and hyperthyroid cases
Fig2: Percentage distribution of overt and subclinical hypothyroidism cases (females only)
DISCUSSION
Post-iodization studies on the occurrence of thyroid
disorders in different populations across the globe
have indicated trends of increase in the prevalence
rates of these disorders particularly in hypothyroidism
and hyperthyroidism. [6] Even the incidence of antithyroid peroxidase (TPO) antibody positive subjects
has been shown to be significantly increased. [9]
In the present study we report a prevalence rate of
35.84% of hypothyroidism (185/516) which is
similar to another hospital based study published by
Shekhar et al. (2012)[10] where the authors reported
have found 31.3% cases with overt hypothyroidism
in a cross sectional hospital based study from coastal
Andhra Pradesh, India. In our study hypothyroidism
cases (35.84%) included 7.55% (n=39) newly
diagnosed and 28.29% (n=146) already diagnosed
cases who were already on hormone replacement

therapy (Table 3). The prevalence of subclinical
hypothyroidism was 7.55% (n=39) and all these
cases were females. The prevalence of
Hypothyroidism and Hyperthyroidism was found to
be highest in females (37.58%) which are similar to
some previous studies. [10-12] The number of female
cases was inordinately high with a ratio of 14
females versus 1 male. However, population based
surveys have reported somewhat lesser rates of
prevalence which show approximately a ratio of 3
females versus 1 male. [6] Female preponderance
with variable female to male ratio has been reported
in almost all epidemiological studies. [13] However,
in post Iodization period this ratio appears to be
tilting more towards female cases due to increasing
number of females getting affected with this
disorder. Whereas endogenous environmental and
genetic factors are being attributed to high
vulnerability of females to these condition.
Age specific prevalence rate was found to be highest
in the age range 21-30 years 36.99% with overt
Hypothyroidism and 33.33% with subclinical
Hypothyroidism (Table 4). The reason for highest
incidence in the age range 21-30years in women
may be attributed to problems related to infertility
for which they report to the Gynecology and
Obstetrics Department of the hospitals. In general
our data represents that prevalence of thyroid
dysfunction (TD) is highest in adults of age group
21-30 years(Fig2) specifically females, even in the
subclinical group which constitutes all female cases.
The peek incidence was noted in the third decade of
life (21-30 years) (Table 4) both in overt and
subclinical hypo cases.
In order to analyze incidence of hypothyroidism in
younger and old age groups a cutoff age of 35 years
was also considered. The incidence of the disorder in
cases below the age of 35 years was 21.5% (n=111)
while in those above 35 years of age it was 12%
(n=62). These observations also clearly indicate the
peek incidence is in younger age group.
Considerable women report to hospital for infertility
problems with raised TSH levels which may be the
reason for higher female to male ratio. In a report on
prevalence of hypothyroidism in infertile women by
Verma et al (2012), 395 women who reported for the
first time; of these 25% were diagnosed as
493
Fathima et al.,
hypothyroid and responded to the treatment with

levothyroxin.[14]
Various endogenous factors are attributed to higher
predisposition of females particularly in younger age;
these include hormonal imbalance at puberty,
pregnancy, higher level of estrogen which cause rise in
TSH levels which in turn induces expression of major
histo compatibility (MHC) class II molecules on cells
including thyrocytes which may present self antigens
released due to infection of the thyroid there by setting
in motion the process of auto-immunity. [12]
The skewed X-chromosome inactivation (XCI)
hypothesis for high susceptibility to hypo is also
gaining more credence as it claims that the existence
of XCI in females results in self antigen express ion in
the thymus or in other peripheral sites which is
involved in tolerance induction. Because of this
reaction skewed XCI has been identified as a
predisposition factor for the development of AITD. [15,
16]
Multiple genes are implicated in the causation of

thyroid disorders which are mainly grouped into those
that are responsible for coding thyroglobulin, TSH
receptors etc and the other group comprises of those
genes that are responsible for immunoregulatory
molecules and cytokines. Single nucleotide
polymorphisms in some of these have been reported to
be associated with increased risk of thyroid
dysfunction. [17]
In the present study the protocol did not include
specific reasons for getting the thyroid profile tested.
However it appears that a systematic analysis of
thyroid profiles of patients visiting hospital appears to
be of significant importance in identifying emerging
trends in the prevalence of hypothyroidism.
These reports appear to represent emerging trends of
high prevalence of both overt and subclinical hypo in
women. These studies lend support to the results of
high prevalence of hypothyroidism reported in this
study, and indicate that the TSH levels are very crucial
as they may have serious consequences, they may also
affect
cardiovascular
and
neuromuscular
[18]
manifestations.
It is suggested that iodine intake of a population
should be kept within a relatively narrow range
interval that prevents iodine disorders, but not higher.
[12]
Limitations of our study are that it was performed

on a modest sample size. Secondly no test was
performed for screening for anti-TPO or antibodies.

Finally, with regard to the significant cause of TD
etiological factors may be one of the better
explanations along with iodine sufficient status. It
is suggested that further studies on similar lines
may be carried out both at the population base as
well as in the hospital with relatively larger sample
size in order to draw more definitive conclusion.
CONCLUSION
This appears to be an increasing trend in the
prevalence of thyroid disorders particularly
hypothyroidism in the post-iodization era in India.
An inordinately high increase in the prevalence rate
in women is observed particularly in the age group
21-30years (Fig. 2) and indicating the need to
explore possible molecular mechanisms underlying
this trend. Monitoring thyroid profile and adopt to
suitable measures to prevent adverse outcome as
clinical, subclinical levels have been related to
infertility, pregnancies and also cardiac problems
particularly heart failure.
ACKNOWLEDGEMENTS: None
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DOI: 10.5958/2319-5886.2015.00096.X

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
rd
Received: 3 Feb 2015
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
th
Revised: 28 Feb 2015
Accepted: 16th Mar 2015
UNMET NEED OF SEX EDUCATION AMONG ADOLESCENTS IN URBAN SLUM AREA: AN

INTERVENTIONAL STUDY
*Tamboli Kshitij S1, Avachat Subhada S2, Tamboli Suchit S3
1
Medical Intern, PDVVPFs Medical College, Ahmednagar, Maharashtra, India

Associate professor, Department of Ccommunity Medicine, PDVVPFs Medical College, Ahmednagar,
Maharashtra, India
3
Consultant, Chiranjiv clinic and child development centre, Ahmednagar, Maharashtra, India
2
* Corresponding author email: tambolikshitij@gmail.com

ABSTRACT
Context: Adolescents comprise one-fifth of Indias total population. There is widespread ignorance associated
with unprotected sex, contraceptives, among young people. As majority adolescents in slum areas have illiterate
and ignorant family backgrounds; they are misguided by the myths. Hence providing sex education for them is the
need of the hour. Aims: 1) To assess the knowledge and awareness of adolescents in an urban slum area
regarding some aspects of reproductive health. 2) To assess the need of sex education among them. 3) To study
the impact of sex education on their knowledge Material and Methods: An interventional study was done on
132 adolescents of urban slum area, selected by simple random sampling. Informed consent was obtained from
the participants. Data was collected with the help of structured questionnaire prepared by literature search.
Response of adolescents was recorded through questionnaires. A sensitization workshop was organized as
intervention. The same questionnaire was given to them and the effect of intervention was assessed. Statistical
analysis of data was done using percentage, proportion and appropriate tests of significance. Result and
Conclusions: Only 31.06% adolescents had discussed the topic of reproductive health with some or other person
and out of them friends were the major sources (39.2%) of information. Only 38.63% knew the hazards of teenage
pregnancy which significantly rose to 89.4% after intervention workshop. The study concludes that the slum
adolescents profoundly lack adequate knowledge of sexuality related matters. Even before intervention workshop,
unmet need of reproductive health education was 59.1% and 93.93% was the felt need in the post test.
Keywords: Adolescent health, Sex education, Urban slum area, Intervention
INTRODUCTION
The period between 10-19 years is referred to as
"adolescence" by the World Health Organization
(WHO). There are 225 million adolescents
comprising nearly one-fifth (22 %) of Indias total
population. Of the total adolescent population, nearly
10% are in the 15-19 years age group.
Adolescent sexuality is still a taboo in many societies;
there is widespread ignorance about the risks
associated with unprotected sex, contraceptives etc.
Kshitij et al.,
among the young people [1]. Teenagers are still

hungry for accurate, adequate information about sex
and sexuality and yearn to hear about it openly and
honestly [2]. There seems increase in the prevalence of
adolescent sexual problems due to lack of knowledge
in them. It is now thought that around 2.39 million
people in India are living with HIV. Highest HIV
prevalence among the age group 15-19 is 0.04%. It is
0.01%in males, 0.07% in females, according to
496
NFHS-3 2005-06, India: Volume- 1. Teenage

pregnancy is one of the main problems in the world,
its prevalence rate varies from 2 to 25% [3].
Sex education includes information regarding human
sexual behaviour, sexually transmitted diseases
(STDs), HIV/AIDS and other aspects of human
sexuality such as body image [4]. Adolescence is a
fascinating period of life that makes the transition
from being a dependent child to becoming an
independently functioning adult [5]. Providing
adolescents with proper sex education will minimize
their risks and hence the prevalence of sexually
transmitted diseases and will in turn help to control
the problems of HIV/AIDS, teenage pregnancy.
As majority adolescents in slum areas have illiterate,
ignorant family backgrounds, they may be misguided
by the information from peers & media. Right
guidance regarding reproductive health may not be
available for such adolescents and they may not
utilize the available health facilities despite having
queries on such a topic.
Educational intervention programs can help in
creating and promoting awareness among the youth
and women. A study by Dongre et al. (2006) showed
significant improvement in personal hygiene of
students and concluded that the school health
education programs with active involvement of
school teacher lead to improvement in personal
hygiene in school children and reduction in related
morbidities [6]. Twenty-two international studies were
done in various developing countries which showed
sufficiently strong evidence that these interventions
reduces risk behaviour [7].
Keeping above background in mind, an intervention
study in adolescents of slum area, including
awareness programs in the form of workshops and
lectures was deliberately planned, by which we could
identify their thrust areas and give proper emphasis
on them to provide solutions.
Study design: An intervention type of study.
Study area: Urban slum area in Ahmednagar,
Maharashtra, India.
Study participants: Adolescents in the age group of
15 19 years.
Study duration: April 2014 to September 2014.
Inclusion Criteria: 1) Adolescents living in the slum

area, belonging to the age group 15 -19 years. 2)
Subjects ready to participate in the study.
Exclusion Criteria:1) Adolescents who were not
willing to participate in the study. 2) Adolescents
from the age group 10-14 years.
Sampling technique: Simple random sampling
technique was used to select urban slum from
amongst 8 slums and the participants from the study
area. Sample size: 132 adolescents (44 boys and
88girls).
Ethical approval: The study was initiated after the
approval was taken from the institutional ethical
committee of Medical College.
Data collection: Informed consent was obtained from
the participants /parents (for minors) and
confidentiality regarding personal information of the
participant was maintained. Data collection was done
with the help of structured questionnaire prepared by
literature search [3],[8].NOTE: A female attendant
accompanied while collecting the data from the girls.
The questionnaire had questions testing the
knowledge of participants regarding puberty,
reproductive health, sexuality, physical and mental
changes and sexually transmitted diseases. Few
questions were open ended and a few close ended..
The questionnaire was translated in the local
language (Marathi). Response of adolescents was
recorded in writing format by giving 1 copy of
questionnaire to each subject. A sensitization
workshop was organized as a part of the intervention,
after collection of data that is after the pre-test. This
workshop was held separately for boys and girls. The
various methods used for health education were
informative pamphlets, lectures, group discussions,
CDs and posters regarding various aspects of
reproductive health, role plays were taken separately
for boys and girls. Need regarding various aspects of
sexuality was identified, thrust areas were noted and
accordingly the solutions were given in the workshop.
Immediately after this, same questionnaire was given
to the participants for post test and data was
statistically analyzed to see the effect of intervention
on the subjects.
Statistical analysis of the data: Data was compiled
and put up in an excel sheet. It was then analyzed
using percentage, proportion and appropriate tests of
significance were used.
497
Kshitij et al.,
RESULTS
Sociodemographic distribution: Age distribution of
data is shown in Fig 1. Out of the total (n=132), 67%
(88) were males and 33% (44) were females.
Knowledge of reproductive health: Only 41 (31.06%)
adolescents
out
of
132
had
discussed
sexuality/reproductive health some time with
someone earlier in their life. Those who wrote the
answer as yes, they discussed the subject with
friends (39.02%), parents (12.2%), doctor/health
counsellor (14.63%), elder brother / sister (19.51%).
Knowledge of adolescents in the study regarding
signs of puberty is depicted in table 1. Age of onset
of puberty was known to 10.6% of adolescents before
intervention while it improved to 77.27% after the
intervention. 39.4% of adolescents had knowledge
regarding Legal age of marriage in India which
improved significantly to 92.42% after the
intervention. Only 17.42% of adolescents knew the
safe minimum age of the pregnancy, which improved
significantly to 74.24% after the intervention.
Knowledge regarding contraception: 40.15% knew
about the places where contraception was available
before intervention which increased to 93.18% after
the intervention. Only 3 (2.72%) adolescents out of
the total sample were able to enlist the names of
various contraceptives in the pretest while in the post
test, 31 (23.48%) of the adolescents were able to do it
correctly. 70.5% of males thought that a same
condom can be used more than once before
intervention while after intervention 96% of males
answered that, a same condom cannot be used twice.
Knowledge regarding hazards of teenage pregnancy
is shown in table 2. Knowledge of adolescents
regarding hazards of unsafe sex is displayed in table
3. In post test67% of adolescents were able to write
all 4 modes of transmission of HIV/AIDS. Only
20.5% boys had information regarding masturbation
before intervention while 79.54% had knowledge
post intervention. Only (48.86%) females wrote that
someone (any source) earlier discussed with them
about menses before menarche while 45(51.14%)
wrote that nobody had discussed with them regarding
menses earlier. Those who had discussed the subject
wrote mother (81%) as a source mostly followed by
siblings (7%) and friends (7%) and others (5%).
Type of material used during menses by girls is
depicted in Fig 2.
Table 1: Knowledge of adolescents in the study

regarding signs of puberty :(n=132)
Correct
Incorrect
Total
Pre test
Post test
01(0.75%)
63(47.73%)
131(99.2%)
69(52.27%)
132
132
p < 0.001, Significant.
Total
64
200
Table 2: knowledge regarding hazards of teenage

pregnancy(n=132)
Pre-test
Post-test
Total
Correct(options1,2,3)
51(38.63%)
118(89.4%)
169
Dont know(option4)
81(61.36%)
14(10.6%)
95
Total
132
132
p < 0.001, Significant.
Table 3: Knowledge of adolescents regarding

Hazards of unsafe sex: (n=132)
Correct answers
Dont know
total
Pre-test
Post-test
44(33.33%)
118(89.4%)
88(66.66%)
14(10.6%)
132
132
P < 0.001, Significant
Total
162
102
Table 4: Response of adolescents to necessity of

sexual education: table showing unmet need of
sexual education: (n=132)
Pre-test
Post-test
total
78(59.1%) 124(93.93%) 202
YES
54(40.9%) 08(6.06%)
62
NO
total
132
132
p < 0.001 Significant
Fig 1: Age distribution of data (n=132):
Fig 2: Showing type of material used during

menses by girls
498
Kshitij et al.,
Unmet need of reproductive education and its value

in prevention: Response of adolescents to necessity of
reproductive education is mentioned in table
4.Providing reproductive health education will help in
preventing Sexually transmitted diseases, Unwanted
pregnancy, Physical & psychological problems in
puberty was the response of 130 (98.5%) of the total
adolescents in the study. The preferred sources of
Information for reproductive education were: school
teachers (48%), health workers and doctors (36%),
parents and siblings (7%), media (6%), magazines
and other sources (3%).
DISCUSSION
In our study, most of the adolescents 69.94% had no
access to information regarding sex education.
Majority of them wrote friends (39.02%) and
siblings (19.51%) as prominent sources through
which they got to know information about this
subject. In a similar intervention study done in rural
sector, 217 adolescents were involved,69.58% gave
friends as source of information[9]In a similar study
done in the slums of Mumbai, 5.66% of the students
had no access to information regarding sex education
[10]
.
In our study,67% of the adolescents were able to
enlist all the modes of transmission of HIV/AIDS
after the intervention. The NACO reported that about
30% of the boys (15-19 years old) know how the HIV
virus is transmitted, with slight variations between
urban and rural boys. An ICMR study stated that
about one-half of the adolescents were not aware of
condoms and were confused about the various modes
of HIV/AIDS transmission.
In our study, only 0.75% of adolescents gave correct
answers about signs of puberty in males and females
before intervention which significantly rose to
47.73%.This indicates clear lack of knowledge

regarding puberty. It should be noted that legal age of
marriage which is a basic important aspect was not
known to many adolescents before intervention. In a
Study by Singh.D [11], it is noted that the slum
adolescents profoundly lack appropriate and adequate
knowledge of sexuality related matters. 3.7% only
could correctly answer about puberty. Merely 12
percent of slum adolescents knew that Nightfall and
genital development are the initial features of
pubertal changes among males and 88 per cent had no
knowledge of these changes that taken place in their
body.
In our study,38.63% of the adolescents only correctly
knew the hazards of teenage pregnancy in present
study which significantly rose to 89.4% after
intervention. Thus, emphasizing on health education
of girls would be a successful strategy for delaying
marriage of girls and consequently preventing
adolescent pregnancy [12].
Our study shows that females also lacked adequate
knowledge regarding various aspects of reproductive
health but they were relatively more informed than
males. 48.86% of females had already discussed
about menses with some source before onset of
menses. Prominent source was mother for 81% of
them. An ICMR study concluded that most girls had
not been informed about menarche and other pubertal
changes prior to its onset.
In our study, the unmet need of reproductive
education is clearly noted as even before intervention
59% adolescents agreed to the necessity of obtaining
the knowledge. 94% adolescents felt the need of such
education during and after intervention. Preferred
sources of information as noted in the study are
school teachers (48%),health worker/doctor (36%). In
a study by Singh D, the findings suggest that these
slum adolescents need an intensive education
regarding 'how and what physical changes' take place
when an individual pass through adolescent period.
Similarly, poor baseline knowledge and increase in
knowledge after intervention has been observed in
other studies [13,14] . In a study by Shubhagna Sharma
et al , the intervention regarding reproductive and
child health showed a significant effect in the
knowledge levels of girls as seen by t-test value of
7.924 at 1 percent level of significance [15] .In a study
by Padhyegurjar Mansi et.al , need of sex education in
school curriculum was perceived by 94.72 % of the
499
Kshitij et al.,
students even before intervention. After the sessions

on sex education, this increased significantly to 97.36
% and preferred sources of knowledge of sex
education , a large majority of 83.02 % students
preferred doctors to impart sex education, followed
by teachers (40%) and television (30.19%).
CONCLUSIONS
The present study was done in the urban slum area.
The prominent sources of information were friends
for most of the adolescents. The study concludes that
the slum adolescents profoundly lack appropriate and
adequate knowledge of sexuality or sexuality related
matters. Regarding pubertal changes, the girls had
better knowledge as compared to boys. Knowledge of
adolescents regarding puberty and regarding of the
hazards of teenage pregnancy rose significantly after
intervention program. Before intervention workshop,
unmet need of reproductive health education was
noted among adolescents and felt need increased in
the post test. Most of the adolescents want school
teachers
as the source of reproductive health
education.
Advantages of this study were: we could bring forth
the unmet need of sex education in urban slums areas
that we selected. We could give them intervention
workshop which would help them in their past and
future unanswered questions regarding this topic.
Limitations: Due to time constraint we could not
include all the subtopics about sexual health while
providing intervention program.
Recommendations borne out of this study: Timely
assessment of adolescents health and development
needs by similar type of studies will help to find
aspects like unmet need of reproductive health
education in this age group. Sex education
incorporated in the school/college curriculum is the
need of the hour that can help to fill up the gaps by
updating their knowledge. Involvement of parents in
reproductive education: Educating the parents and
conducting education programs to increase awareness
of reproductive health can be another important task
which will help in turn in giving proper information
to adolescents. Involvement of NGOs to a much
greater extent: Informational and educational
activities through NGOs can be accelerated to
enhance knowledge of adolescents. PHCs can take
lead role to cover this neglected aspect by properly
utilizing medical and paramedical workers and

strengthening of the public health care system at all
levels, to deliver RCH services. Thus, empowering
adolescents to take care of their own health as well as
protect themselves from possible health problems like
unwanted pregnancies, risk of STDs in their future
life will be important and should be done by
implementing the recommendations.
ACKNOWLEDGMENTS
We are grateful to Mrs. Neha Tamboli, counselor for
conducting workshop amongst girls, Snehalaya
Ahmednagar and Mr. Hanif for their cooperation in
conducting workshop. We are grateful to Dr.Mrs
Zambre for guidance and Miss Shradha for technical
support.
This research paper was presented as oral
presentation at 11th WARSAW INTERNATIONAL
MEDICAL CONGRESS held at Poland in May,
2015, and has received best presentation award.
REFERENCES
1. Park K, Parks Textbook of Preventive and Social
Medicine. Jabalpur: Bhanot Publishers;2009;20th
edition:511- 2.
2. Nair MKC, Sexual reproductive health of young
people. Jaypee Publishers: 2006; First edition: 27.
3. Bhave S, Bhaves textbook of adolescent
medicine.
Jaypee
publishers:2006;first
edition:178-184.
4. Bhatlavande P, Gangakhedkar R On the
Horizon of Adulthood.New Delhi: UNICEF;
1999: 148-52.
5. Nair MKC, Bodhankar U. Adolescent health
.Teens journal.2002,;1-8.
6. Dongre AR, Deshmukh PR, Garg BS. The Impact
of School Health Education Programme on
Personal Hygience and Related Morbidities in
Tribal School Children of Wardha District. Indian
Journal of Community Medicine. 2006; 31(2):
81-82
7. Kirby D,Obasi A. The effectiveness of sex
education in schools in developing countries:
preventing HIV/AIDS in young people. World
Health Organ TECH REP SER 2006; 398:10342.
8. Nair MKC; Teens- journal of teenage care &
premarital counseling, 3(12) 2003:85-93.
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9. Avchat S, Phalke D , Phalke V .Impact of sex

education on knowledge and attitude of
adolescent school children. JIMA; November
2011 :33-35.
10. Padhyegurjar M , Padhyegurjar S , Adsul B
Assessment of felt needs and effect of health
education intervention on knowledge regarding
reproductive health of school students in a slum
in
Mumbai.
Retrieved
May
24,2014
http://njcmindia.org/uploads/3-2_221-226.pdf
11. Singh.D- Knowledge of slum adolescents about
common changes of Secondary sexual characters.
Health and Population- Perspectives and Issues
2000;23 (4): 198-204.
12. Thekkekkara T, Veenu J - Factors Associated
with Teenage Pregnancy: Indian Journal of
Community Medicine April - June, 2006-31 (2):
83-85.
13. Abraham L. Understanding Youth Sexuality: A
study of college students in Mumbai city. The
Indian Journal of Social Work 2001;62(2):233248.
14. Russel-Brown P, Rice JC, Hector O. The
effect of sex education on teenagers in St. Kitts
and Nevis. Bull Pan Am Health Organ.
1992;26(1):67-79.
15. Shubhangna Sharma , Shipra Nagar and Goldy
Chopra- Health Awareness of Rural Adolescent
Girls: An Intervention Study ;J. Soc. Sci,
2009;21(2): 99-104
501
Kshitij et al.,
DOI: 10.5958/2319-5886.2015.00097.1

&
Health Sciences
www.ijmrhs.com
Copyright @2015
th
th
Revised: 20 Mar 2015
Research article
ISSN: 2319-5886
HOW DO MEDICAL STUDENTS LEARN? A STUDY FROM TWO MEDICAL COLLEGES IN SOUTH
INDIA A CROSS SECTIONAL STUDY
*Christofer Thomas1, Praveen K Kodumuri2, Saranya P3
1
Department of Physiology, Sapthagiri Institute of Medical Science and Research Center, Bangalore, Karnataka
Department of Physiology, Mamata Medical College, Khammam, Telangana
2,3
* Corresponding author email: christytomchristy@gmail.com

ABSTRACT
Introduction: "Learning style" is defined as an individual's preferred method for approaching learning and
gaining knowledge. As a teacher, it is important to understand the different learning styles of the students in
acquiring the information, and hence one can make the necessary changes that best match the learning style of the
students. Assessment of learning styles can be done in various ways but Visual Auditory Reading Kinesthetic
(VARK) questionnaire is the most accepted one among them. The present study was undertaken to determine the
learning preferences of first year medical students in South India. The study was also aimed at determining
whether males and females have similar pattern of learning styles. Materials and Methods: This study was
jointly conducted in Mamata Medical College, Khammam, Telangana and Sapthagiri Institute of Medical Science
and Research Center, Bangalore, Karnataka. VARK-questionnaire Version 7.8 was used after obtaining
permission. The VARKquestionnaire along with information about age and sex was distributed among 200 first
year students of both colleges on the same day and response rate was 80%( Males49% and Females 51%)
Results: VARK questionnaire results revealed that none of our respondents preferred unimodal method. 3% were
bimodal, 32% were trimodal and 63% were quad modal. There was no statistical difference between the
individual scores between male and female respondents. Implication: The implication of this study is applicable
to teachers to understand his/her students pattern of learning. Although none of the students learn only by one
method, all the multimodal learners will have a predominance of a particular learning style either it be
visual/auditory/read/kinesthetic.
Keywords: Learning style, VARK questionnaire, Trimodal, Quadmodal.
INTRODUCTION
The term "learning style" is defined as an individual's
preferred method for approaching learning and
gaining knowledge [1]. As a teacher, it is important to
understand the different learning styles of the
students in acquiring the information, and hence one
can make the necessary changes that best match the
learning style of the students.
The major three learning theories or models are: 1.
Adult Learning Theory by Malcolm [2], 2. Visual,
Auditory, Read/Write and Kinesthetic (VARK)
Thomas et al.,
Learning Style Model by Neil Fleming[3] and 3.

Kolb's Experiential Learning Theory and Learning
Styles Model[4].
One of the commonly used
inventories is the VARK questionnaire developed by
Neil Fleming. The alphabets in VARK correspond to
visual, auditory, reading/writing and kinesthetic
respectively. Hence depending upon the preference of
learning one can be a visual learner who learns by
seeing or auditory learners who learns by hearing or
reading/writing learners who learns by interactions or
502
kinesthetic learners who learns by self-practice or

performing activity. One can adapt multiple ways of
learning and hence they can be called as unimodal,
bimodal, trimodal and quardmodel learners. The
present study was therefore undertaken to determine
the learning preferences of first year medical students
in two medical schools in South India using VARK
questionnaire. The study was also aimed at
determining whether males and females have similar
pattern of learning styles.
This cross-sectional study was jointly conducted in
Sapthagiri Institute of Medical Science and Research
Center, Bangalore Karnataka and Mamata Medical
College, Khammam, Telangana. After obtaining
informed-consent from the students, VARK
questionnaires in hard copy were administered to 200
first year MBBS students in both the colleges. Along
with the questionnaire, information regarding the age
and sex of the students were also taken. The VARK
questionnaire [3] has 16 multiple choice questions
which have four answer options. Students can choose
one or more than one answer depending on their
preference. Each option shows his or her preference
of learning either visual, auditory, read/write and
kinesthetic. Each student took an average of 15
minutes to mark their preferences. The questionnaires
were administered on the same day in both the
colleges. The study was approved by the Institutional
ethics committee of Sapthagiri Institute of Medical
Science and Research Center, Bangalore and of
Mamata Medical College, Khammam, Telangana.
Permission to use VARK questionnaire was obtained
from the developers and the copy of the questionnaire
along with the analysis guidelines was sent from the
developers via e mail. Statistical analysis: The
response from the students was entered in a Microsoft
Excel sheet. The distributions of the VARK
preferences were calculated in accordance with the
guidelines sent. Descriptive statistics were used for
each VARK component. To calculate the percentage
of students for each VARK component the number of
students who preferred each learning style modality
was divided by the total number of students.
students of two medical colleges in south India. The

VARK questionnaire was distributed to 200 students
of both colleges on the same day. A total of 160
students response were received making the response
rate of 80%. Out of 160 respondents 78 were males
and 82 were females with mean age of 18 years. The
results are shown in table 1.
Table 1: Shows the demographics of the
respondents with their mean age
Total
Mean age SD
Males
78 (48.75%)
180.69
Females
82 (51.25%)
180.75
VARK questionnaire results revealed that none of our
respondents preferred unimodal method. 3.125%
were bimodal, 31.875% were trimodal and 63.125%
were quadmodal.
Further, the results were reanalyzed by dividing the
respondents into males and females. The mean score
of individual V, A, R, K were calculated which is
shown in figure 1. There was no statistical difference
between the individual scores between male and
female respondents. Our results showed that 2.56%
were bimodal, 20.51% were trimodal, 74.36% were
quadmodal in males as shown in figure 2. 3.66%
were bimodal, 40.24% were trimodal, and 52.44%
were quadmodal learners in females as shown in
figure 3.
Fig 1: Shows the mean V, A, R, K scores

individually for both male and female
respondents. Results showed no significant
difference between the mean scores between boys
and girls. Data showed as Mean SD.
RESULTS
The study population comprised of first year medical
503
Thomas et al.,
Fig 2: Percentage of Males that preferred Bimodal

or Trimodal or Quadmodal preference.
Fig 3: Percentage of Females that preferred

Bimodal or Trimodal or Quadmodal preference.
DISCUSSION
This study was aimed in understanding different
learning styles of medical students and the influence
of sex on learning. Our study revealed that none of
the students preferred unimodal way of learning.
Maximum student preferred quadmodal (63%) then
trimodal (32%) and bimodal (3%). Another major
finding of the study is that both males and females
preferred quadmodal way of learning compared to
bimodal or trimodal way of learning. We also
observed that female respondents chose more
trimodal method compared to boys who preferred
quadmodal method compared to female respondents
(Figure 2 & 3). Our results showed that 2.56% were
bimodal, 20.51% were trimodal,74.36% were
quadmodal in males and 3.66% were bimodal,
40.24% were trimodal, 52.44% were quadmodal
learners in females. We also observed that both males
and females had less mean score for read modality
when compared to visual/auditory/kinesthetic
Thomas et al.,
modality and there was no significant difference

between the mean scores between male and female
students.
Literature review from both national and international
levels has shown different results in students studying
different disciplines using VARK inventory. Most of
the studies points out that student prefer multimodal
way of learning irrespective of the discipline they
study. In a study by Lujan and DiCarlo from a US
medical school observed that first year medical
students prefer multimodal way of learning and
kinesthetic was the most preferred modality [5].
Similar reports from medical school were also
reported from Malaysiya [6], Saudi Arabia [7], Turkey
[8]
, China [9]. Similar observation is also made in a
recent study from India by Shenoy et al.[10] which
showed that 70% of the respondents preferred
multimodal way of learning and they opted for
kinesthetic. It was also found that there existed no
correlation between the learning style and
performance in their previous exams. Our results are
also similar the existing findings except for the fact
that none of our respondents preferred unimodal way
of learning. There is also a study to show that
students preferred unimodal way of learning than
multimodal. A study by Rajaratnam et al on dental
students has shown that 55%preferred unimodal
learning and preferred were either aural (47%) or
kinesthetic (41%) [11]. The influence of sex on
learning styles is an area of active research. In our
study, most of the female undergraduate medical
students (68.3%) were multimodal. Among
multimodal learners, most of the female students
preferred trimodal way of learning compared to their
male counterparts. Apart from this difference there
was no difference found in the pattern of learning
between boys and girls.
CONCLUSION
Majority of the students from the two medical
colleges preferred multimodal way of learning and
there were no unimodal learners in our study group.
The found no statistical difference between the
individual scores between male and female students.
We also found that the percentage of trimodal
preference in female respondents were more when
compared with the male students. Implication: The
implication of this study is applicable to teachers to
understand his/her students pattern of learning.
504
Although none of the students learn only by one

method, all the multimodal learners will have a
predominance of a particular learning style either it
be visual/auditory/ read/ kinesthetic. A Visual learner
learns by looking at pictures, graphs, videos, and
graphics. An auditory learner receives learning by
listening method, by speaking or from music,
discussion, and explanation. Read modality student
prefer words and texts as an information obtaining
method. They like presentation style, by text or
writing and kinesthetic learners are more likely to
experience through physical movement aspect while
studying, such as, touch, feel, hold, perform and
move something. They prefer hands on work,
practical, project, and real experience. The
knowledge of the students dominant pattern of
learning allows a teacher to shift from his/her own
preferred mode of teaching towards the learning
preferences of students which may help to develop
their knowledge, skills and attitudes.
ACKNOWLEDGMENT
Authors are thankful to the Principal, Sapthagiri
Institute of Medical Science and Research Center,
Bangalore and Mamata Medical College, Khammam
and Ist year MBBS students of both the colleges.
7.
8.
9.
10.
11.
in a Malaysian medical college. Int J Med Public

Health. 2013; 3:60-3.
Nuzhat A, Salem RO, Quadri MS, Al-Hamdan N.
Learning style preferences of medical students: A
single-institute experience from Saudi Arabia. Int
J Med Edu. 2011; 2:70-3.
Baykan Z, Naar M. Learning styles of first-year
medical students attending Erciyes University in
Kayseri, Turkey. AdvPhysiolEduc. 2007; 31:15860.
Yuemin Ding, Jianxiang Liu, Hong Ruan, Xiong
Zhang. Learning Preferences to Physiology of
Undergraduate Students in a Chinese Medical
School, I.J. Education and Management
Engineering. 2012; 2: 81-85.
Shenoy N, Shenoy A, Ratnakar UP. The
Perceptual Preferences in Learning among Dental
Students in Clinical Subjects, Journal of Clinical
and Diagnostic Research. 2013; 7(8): 1683-85.
NavinRajaratnam, Suganthi V, Suzanne Maria
Dcruz. Learning preferences of students
studying Physiology in South India. IOSR
Journal of Dental and Medical Sciences (IOSRJDMS).2013;7(1):15-19

REFERENCES
1. Cuthbert, P. F. The student learning process:
Learning styles or learning approaches? Teaching
in Higher Education. 2005; 10(2):235-49
2. Knowles MS. The modern practice of adult
education: from pedagogy to andragogy: New
York 2nd ed.; Cambridge Books; 1980; 222-247
3. Fleming, N.D. & Mills, C. Not another Inventory,
Rather a Catalyst for Reflection. To Improve the
Academy, 1992: 11, 137-155.
4. Kolb, David. Experiential learning: Experience as
the source of learning and development.
Englewood Cliffs; NJ: Prentice-Hall Publishers; 1
1984;1: 21-25
5. Lujan HL, DiCarlo SE. First-year medical
students prefer multiple learning styles.
AdvPhysiolEduc Educ. 2006: 30:13-6
6. Sinha NK, Bhardwaj A, Singh S, Abas AL.
Learning preferences of clinical students: A study
505
Thomas et al.,
DOI: 10.5958/2319-5886.2015.00098.3

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 2
th
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
th
Accepted: 26th Mar 2015
EFFECT OF OCIMUM SANCTUM LINN. IN STRESS INDUCED GASTRIC ULCERS IN RATS

*Ayesha Vaseem, Ghulam Subhani, Khuteja Afshan, Mazher Ali, Md. Mohiuddin A Khan, Mujtaba T Rumana
Department of Pharmacology, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad, Telangana, India
*Corresponding author email: drayeshamazher@gmail.com
ABSTRACT
Aims & Objectives: Ocimum sanctum L. popularly known as Tulsi is a medicinal plant that has been used for
curing various diseases since ages. In the present study we used leaf extract of Ocimum sanctum for its anti-ulcer
property by inducing stress ulcers on rats. Materials and Method: Albino rats were randomly allocated to
different experimental groups and aqueous leaf extract of Ocimum sanctum was given for 7 days. Stress ulcers
were induced by restraint and ethanol administered methods and results were compared with standard drug
ranitidine. After that animal was sacrificed and stomach was dissected out and stomachs were observed for the
ulceration with the help of magnifying lens and studied its external, internal surface and ulcer index was evaluated
according to the severity of ulcers. The stomach were stored and fixed in 5% formalin and studied for
histopathological changes. Results: The ulcer index was high in control group. Animal pretreated with Ocimum
sanctum at the dose 100 & 200 mg/kg showed few signs of mucosal injury and the percentage of damage were
less compared to control group. Conclusion: In the present study pretreatment with Ocimum sanctum at dose
100-200 mg/kg caused a significant anti-ulcer effect in rats in comparison with control group and its effect is
comparable to the standard drug ranitidine, as evidenced by the reduction in the ulcer scores.
Key words: Ocimum sanctum, stress induced ulcers, peptic ulcer.
INTRODUCTION
Stress has become a very common problem in every
household and it leads to many diseases.One among
them is peptic ulcer [1, 2] Psychological stress not only
causes peptic ulcer but also can exacerbate it [3].Peptic
ulcer impairs the quality of life and is associated with
increased morbidity and mortality hence its treatment
is essential. Although there are many drugs available
for the treatment of peptic ulcer they are associated
with side effects hence there is always a need for a
better drug. Plants are one of the most important
sources of medicines and many drugs are derived
from it. Ocimum sanctum commonly called as Tulsi
grown easily in household is a medicinal plant used
since ages for various properties [4]. It has been used
as antiasthamatic, antifungal, antiulcerogenic[5],
antipyretic, antiviral, antibacterial, insecticidal and
antimalarial.
It
possess
antioxidant,
Antiinflammatory, Immunostimulant and antistress[6,
7]
properties [8]. It is considered to be an adaptogen,
balancing different processes in the body and helpful
for adapting to stress [9]. In present study we have
used leaves of Krishna tulsi for its antiulcer activity
against restraint induced and ethanol treated peptic
ulcer.
MATERIALS AND METHODS:
Study design: An experimental animal based study
Ethical Approval: The study was approved by the
animal ethics of our institute and the procedures are
followed the format given in form-B of

Committee for the Purpose of Control and
506
Ayesha et al.,
Supervision of Experimentation on Animals

(CPCSEA).
Animals: Albino rats of either sex weighing 180-200
g were procured and the animals were kept in wire
bottomed cages. They were randomly allocated

to different experimental groups and placed
individually in cages and cages were kept under
standard condition of illumination with a 12 - h
light-dark cycle at 25 1 C, 45-70% relative
humidity. They were provided with tap water ad
libitum and balanced diet.
Ocimum sanctum leaves extract (aqueous) obtained
from S. J. Herbals and Health Care. Bengaluru,
Karnataka, India. The extract was stored in cool and
dry place.
Extract of Ocimum sanctum leaves suspended in
distilled water and administered orally (intragastric)
Once a day (OD) for 7 days (Pretreated) to rats by
using feeding tube. (Group II to IV, VII to IX)
Ranitidine: Obtained from Saraca Laboratories
Limited and used as standard drug. It was suspended
in distilled water and administered orally
(intragastric) Once a day (OD) for 7 days
(Pretreated) to rats by using feeding tube. (Group V,
X)
Experimental design: To produce the ulcer we have
selected two types of stress models.
1. Restraint stress (Group I-V)
2. Ethanol induced Stress (Group VI-X)
Each stress model subdivided into 5 groups (n=6 in
each group) as follows (Table 1)
Table 1: Grouping and dose of drugs
Model of stress
Drug and doses
Restraint stress
Group I
Control(received D/W)
Group II
OSLE 50 mg/kg
Group III
OSLE 100 mg/kg
Group IV
OSLE 200 mg/kg
Group V
Ranitidine 10 mg/kg
Ethanol induced Stress
Group VI
Control (received D/W)
Group VII
OSLE 50
Group VIII
OSLE 100
Group IX
OSLE 200
Group X
Ranitidine 10
O.S.L.E = Ocimum sanctum leaf extract, D/W:
Distilled water
After 7 days pretreatment in both the stress

model the animals were kept fasting for 24 hours.
After this the animals were subjected to stress
Restraint stress [10]: Both upper and lower
extremities along with tail are tied together. The rats
are placed in galvanized steel window screen; the
screen is moulded around the animal and kept for 24
hours. Then animals are sacrificed by cervical
dislocation.
Ethanol induced oxidative stress [11, 12]: Absolute
ethanol was administered by orogastric tube at a dose
of 5 ml/kg. After 1 hour of ethanol administration the
animals are sacrificed.
The abdominal cavity was opened through a

midline abdominal incision to expose stomach.
The stomach dissected out, opened along the greater
curvature and washed in normal saline. Those
stomachs were fixed on wooden board with the help
of pins. The stomachs were observed for the
ulceration with the help of magnifying lens and
studied its external, internal surface and ulcer index
was evaluated according to the severity of ulcers.
The stomach were stored and fixed in 5% formalin
and studied for histopathological changes.
The percentage of ulcer inhibition calculated by
formula as described by Njar et al. (1995) [13]
Mean UI (control) - Mean UI (treated)
% of Ulcer inhibition=-------------------------------------------- x 100
Mean ulcer index of control
[UI= Ulcer index]

Evaluation of ulcers score [14]: 0 - No pathology, 1 A small ulcer(1-2mm), 2- Medium ulcer (3-4 mm), 4
Large ulcer (5-6 mm), 8 Large ulcer (> 6 mm)
Total severity of score
Ulcer index (UI) = ---------------------------------Number of animals
Statistical analysis: Data is expressed as mean

SEM. Data was by one-way ANOVA followed by
LSD and Scheffes multiple comparisons test. The
significance of difference was accepted at P <0.01.
507
Ayesha et al.,
RESULTS
Table 2: Effect of Ocimum sanctum leaf extract (OSLE) against Restraint and ethanol induced Gastric
Ulcers in rats (N = 30)
Treatment
Dose
Restraint
Ethanol treated
(mg/kg)
Ulcer index
% of Ulcer Protection
Ulcer index
% of Ulcer Protection
Distilled water 1ml/kg
7.33 2.07
5.88 1.04
O.S.L.E
50
6.67 1.63
09.0
3.94 1.42
20.95
O.S.L.E
100
1.83 0.75** 75.03
1.38 0.88**
84.65
O.S.L.E
200
2.50 0.98** 65.89
1.63 0.21**
79.86
Ranitidine
10
1.33 0.84** 81.85
1.01 0.55**
89.23
ns
Mean
SD,
Data presented as
* P < 0.05; ** P < 0.001; P > 0.05
with Ocimum sanctum at the dose 100 & 200 mg/kg
showed few signs of mucosal injury and the
percentage of damage were less compared to control
group. Correspondingly the ulcer index also was
reduced. These features were suggestive of anti ulcer
activity of Ocimum sanctum. Animals treated with
ranitidine maintained near normal pattern. The ulcer
index was markedly reduced.
DW - Distil water, OS Ocimum sanctum, Ran Ranitidine
Fig 1 : Bar diagram Comparing Ulcer Index in Restraint
Fig 3: Gross appearance of gastric mucosa in rats
method
Fig 2: Bar diagram Comparing Ulcer Index in ethanol

induced method
Gross appearance of gastric mucosa in rats:
On gross examination the gastric mucosa in restraint
and ethanol administered group showed dilated blood
vessels, haemorrhagic sites and large number of pin
point ulcers of varying sizes with central clots,
features of perforation in the stomach. The ulcer
index was high in control group. Animal pretreated
Fig 4: Microscopic observation of normal rat

stomach in 10x
508
Ayesha et al.,
Fig 5: Microscopic observation of restraint induced

ulcer rat stomach pre-treated with Ocimum sanctum
100mg/kg [10Xs]
Fig 6: Microscopic observation of restraint induced ulcer rat

stomach pre-treated with Ocimum sanctum 100mg/kg
[100Xs]
DISCUSSION
Psychological stress has been reported to be an
important contributing factor in the causation of
peptic ulcer. Mechanism by which stress causes
ulceration is by histamine release with increase in
acid secretion and reduction in mucous production.
Stress causes both sympathetic (causes direct
arteriolar vasoconstriction) and parasympathetic
(induces an increased motility and muscular
contraction) stimulation of stomach leading to local
hypoxia and near or actual ischemia. Formation of
excessive free radicals due to stressful conditions is a
major internal threat to cellular homeostasis of
aerobic organisms[15]. These free radicals are
extremely reactive and unstable and react with most
of the intracellular molecules[16]. They enhance the
process of lipid peroxidation[17]. The products of lipid
peroxidation are themselves reactive species and lead
to extensive membrane organelles and cellular
damage[18]. Lipid peroxidation causes loss of
membrane fluidity, impaired ion transport and

membrane integrity and finally loss of cellular
functions. Many studies have implicated oxygen free
radicals and lipid peroxidation, in ageing and various
diseases[19, 20] including peptic ulcer[21].The present
study is undertaken to produce gastric ulcers by
restraint and ethanol induced models of stress in
albino rats.
Most of the available drugs effective in treating
peptic ulcer were found to cause some potential side
effects. Efforts were made to find a suitable agent in
natural products of plant origin. Ocimum sanctum has
been used since ages and has been reported to possess
potent anti-ulcer and ulcer healing properties.
Ocimum sanctum contains a variety of chemical
constituents that have biological activity, including
saponins, flavonoids, triterpenoids, and tannins [22, 23]
which exhibit antioxidant and anti-inflammatory and
adaptogenic activities. Ocimum sanctum has effect on
neural pathways controlling acid secretion thereby
strengthening the animals physiological capabilities
to decrease stress and hence ulcers.
In the present study, we examined the effects of
Ocimum sanctum in restraint stress and ethanol
induced stress. Our data suggest that Ocimum
sanctum treatment has significant percentage of ulcer
protection in a dose dependent manner.
Antiulcerogenic activity of Ocimum sanctum was
seen at the dose 100mg/kg &200mg/kg and was
almost comparable to the standard drug ranitidine.
However, further study is required to know the exact
mechanism of action and to isolate the active
molecule responsible for the anti-ulcer activity.
CONCLUSION
Pretreatment of rats with Ocimum sanctum produced
a dose dependent protection in all models of stress
induced ulcer. Antiulcer effect of Ocimum sanctum is
statistically insignificant in comparison to ranitidine.
However, the protection index varies between
different models. Ocimum sanctum (Tulsi) 100, 200
mg/kg showed significant (p < 0.05) anti-ulcer effect
and its effect is comparable to the standard drug
ranitidine, as evidenced by the reduction in the ulcer
scores. However more experimentation and clinical
studies and detailed analysis are required for a
definitive conclusion.
509
Ayesha et al.,
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DOI: 10.5958/2319-5886.2015.00099.5

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
th
Received: 10 Mar 2015
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
th
Revised: 18 Apr 2015
Accepted: 5th Jun 2015
COMPARATIVE STUDY OF PULSE THERAPY WITH DAILY IMMUNOSUPPRESSIVE THERAPY

IN STEROID RESPONSIVE DERMATOSIS
*Naseem Begum 1, SwathiBurla2, Bhavika3
1
Department of Pharmacology, 2Department of Dermatology, Osmania Medical College, Hyderabad, Telangana
*Corresponding author email:naseembegum007@yahoo.com

ABSTRACT
Background: Pemphigus, scleroderma and SLE are diseases of unknown etiology for which no specific treatment
is effective. The introduction of corticosteroids and immunosuppressive drugs reduced the mortality rate.
Objectives: To correlate signs and symptoms and incidence of adverse effects in patients with steroid responsive
dermatosis before and during DCP therapy and daily immunosuppressive therapy. Material and Methods: 100
patients were enrolled in this study. They are divided into 2 groups. The treatment schedule in group 1 consists of
giving 100mg dexamethasone on 3 consecutive days and 500 mg cyclophosphamide on day two and repeating
these pulses (DCPS) every 4 weeks. In between the DCPS, the patient received only 50mg cyclophosphamide
orally daily and generally no corticosteroids. Group 2 patients received daily immunosuppressive therapy in the
form of tab prednisolone 1-2mg/kg body weight and tab cyclophosphamide 50 mg after food daily for 6months.
Results: At the end of 6 months of study period, based on clinical improvement, good response was seen in 82%
in group 1 and in 64% in group 2P<0.05 which is significant. Moderate response was seen in 10% in group 1 and
in 22% in groups 2.8% in group 1 and 14% in groups 2 recorded poor responses. Better response was seen with
DCP therapy. The incidence of adverse effects was less with DCP therapy when compared to daily
immunosuppressive therapy. P<0.0001 which is highly significant. Conclusion: DCP therapy is safe and effective
in the treatment of steroid responsive dermatosis. Incidence of adverse effects was less with DCP Therapy.
Keywords: Dexamethasone, Cyclophosphamide, Pulse therapy, Daily immunosuppressive therapy, Pemphigus,
systemic lupus erythematosus (SLE)
INTRODUCTION
Pulse therapy, the big shot [1] refers to administration
of large doses of drugs in an intermittent manner to
enhance the therapeutic effect and reduce the side
effects [2]
The auto-immune dermatologic disorders such as
pemphigus, systemic sclerosis [3], systemic lupus
erythematosus,
dermatomyositis,
pyoderma
[4]
gangrenosum, toxic epidermal necrolysis ,Stevens
Johnson's syndrome[5], lichen planus, alopecia aerate,
sarcoidosis and systemic vasculitis[6] are considered
to be serious diseases with high morbidity and
mortality. The outcome of these diseases before the

advent of Corticosteroids was death.
The introduction of corticosteroids in therapy reduced
the mortality rate. However the requirement of high
dose and long term steroid regimens resulted in
therapy related morbidity and mortality. Most of the
deaths occurred during the first few years of the
disease and if the patient survived for five years, the
prognosis was considered as excellent. The
introduction of immunosuppressive drugs as
adjuvants[7]further reduced the mortality rate but no
significant improvement was observed in the
511
Naseem et al.,
frequency
of
remissions.
Dexamethasone
cyclophosphamide pulse (DCP) therapy was then
suggested as an effective alternate therapy for the
treatment of these autoimmune dermatologic
conditions.
Pulse therapy consists of giving a very high dose of a
drug to bring about a quick result and then
withdrawing the drug completely till it is needed
again. Pulse therapy took its origin when
intravenously administered high dose steroids
(suprapharmacological doses of methyl prednisolone)
could successfully reverse the rejection of renal
transplantation without any undesirable effects
[8]
.Then it became the important mode of
management in all renal transplantation cases.
Later, the pulse therapy was extended to the
management of other disorders like lupus nephritis,
Polyarteritisnodosa (PAN), Rheumatoid arthritis
(RA), pyoderma gangrenosum [9]with the obvious
benefit of a quick recovery and without undesirable
sequelae. Quick recovery was attributed to the antiinflammatory and immunosuppressive effects of high
doses of corticosteroids.
The rapid elimination of intravenously administered
drug might be responsible for minimization of side
effects, prompt recovery of hypothalamo pituitary
adrenal (HPA) axis[10] function and minimal stigmata
of Hypercorticism. Life threatening complications
such as severe adverse cardiovascular effects reported
in some of the studies appeared to be restricted
mostly to the patients suffering from nondermatological disorders particularly with cardiac[11]
and renal disorders.
For the first time in India, Pasricha et al successfully
carried out pulse therapy with dexamethasone In a
patient with Reiters disease[12]. The promising results
encouraged them to try the same in potentially fatal
diseases like Pemphigus, systemic sclerosis,SLE, etc.
To achieve a prolonged remission, they arbitrarily
designed a regimen with dexamethasone and
cyclophosphamide termed the DCP therapy. The
treatment followed four phases (first three are
treatment phases, last phase being the post treatment
follow up). The choice for dexamethasone was based
on availability and economy. Dexamethasone being
the long acting steroid, treatment for three days was
found adequate reducing the hospital stay and
ensuring prompt recovery of HPA axis. The
negligible mineralocorticoid activity further reduced
Naseem et al.,
the incidence of life threatening complications. Of the

300 patients enrolled in their study for DCP therapy,
190 patients were reported to have completed the
treatment till date. The maximum duration of
remission with post treatment follow up was 9 years.
The major advantage was that almost every patient
could be induced into remission.Furthermore, the side
effects associated with conventional daily use of
corticosteroids such as diabetes, hypertension
osteoporosis, electrolyte imbalance, cushingoid
changes, cataract, weight gain, striae and acne were
either not seen or minimal. Dietary changeslike salt
or calorie intake restriction or supplements of calcium
or potassium were not required.
Encouraging results of these challenging trials made
the subject an interesting one from the view point of
therapeutic study. Hence the present study was
undertaken to compare the DCP therapy with daily
immunosuppressive therapy in steroid responsive
dermatosis.
Study design: Open label and parallel group,
prospective comparative clinical study between daily
immunosuppressive therapy and dexamethasone
cyclophosphamide pulse [DCP] therapy in patients
with steroid responsive dermatosis.
The study was conducted at department of
dermatology in OGH Hyderabad andthe duration of
study is for 6 months
Sample size: 100 patients with dermatosis (like
pemphigus vulgaris, SLE, scleroderma, etc.)
Ethical approval: Approval from the Ethics
Scientific Committee of Osmania Medical College,
Hyderabad was obtained (annexure). After selection
of the patients based on the above criteria, patient was
explained about the study in their own understandable
language & written informed consent was obtained.
Inclusion criteria: 1. Age group- Patients in the age
group 16-60yrs receiving oral immunosuppressive
therapy for steroid responsive dermatosis were
included in the study.2. Sex- Both male & female
patients were included in the study.
Exclusion criteria: Cardiac patients suffering with
hypertension, angina, myocardial infarction, cardiac
arrhythmias and stroke.Patients with uncontrolled
diabetes mellitus, psychiatric problems, renal and
liver diseases, on immunosuppressive drugs for other
conditions, any disorder where high dose
512
corticosteroids are contraindicated, evidence of major

systemic disease, and 8.Pregnant &lactating women.
The cases for this study were taken from
Dermatology department Osmania General Hospital,
Hyderabad. Patients showing signs and symptoms
suggestive of steroid responsive dermatosis like
[pemphigus, Systemic lupus erythematosis (SLE),
systematic sclerosis, etc.] were selected for this study.
Grouping:A total of 100 patients, divided into 2
groups [group1 and group2] of 50 patients each were
studied
Group 1: Received pulse therapy regimen called as
dexamethasone cyclophosphamide pulse [DCP]
therapy. It consists of giving 100mg dexamethasone
dissolved in 500ml of 5% dextrose as a slow
intravenous [IV] drip over 2 hrs repeated on 3
consecutive days every month and on 2nd day the
patient is given cyclophosphamide 500mg in the
infusion followed by + prednisolone 1mg/kg daily
and cyclophosphamide 50mg daily orally for 6
months.
Group2:Received daily immunosuppressive therapy.
Tab prednisolone 1-2mg/kg body wt after food along
with
an
antacid
before
breakfast.
Tab
cyclophosphamide 50mg after food daily orally for 6
months.
Methodology: For pulse therapy all the patients were
hospitalized (minimum 4 days for every pulse)daily
immunosuppressive therapy patients were treated in
the OPD.Recording of present complaints with
duration, personal and family history of autoimmune
dermatological conditions. General examination of
the patient.4. Dermatological examination of the
patient.Investigations:Before treatment following
investigations were done- haemogram, complete
urine analysis, blood sugar (fasting & post lunch)
blood urea, serum creatinine, serum electrolytes, liver
function tests, Electrocardiogram &stool examination
for occult blood was done X-ray chest and bones
(lumbar spine, proximal end of femur etc.) and
ophthalmic examination forcataract, glaucoma etc.
were done before starting the treatment and whenever
required and at the conclusion of study. Vital data and
weight were recorded at every visit. Serum cortisol
level estimation could not be done which helps to
know the HPA axis functional state, but
endocrinologists opinion was taken in this regard.
Treatment: Group1 patients received dexamethasone
cyclophosphamide pulse (DCP) therapy. It consists of
giving 100mg dexamethasone dissolved in 500ml of

5% dextrose given by a slow intravenous infusion,
over a period of not less than 2 hours and this was
repeated on 3 consecutive days. In addition, 500m g
of cyclophosphamide was added to dexamethasone in
the same drip on the second day. The dexamethasone
cyclophosphamide pulses [DCP] were repeated at
4weekely Intervals. In between the DCPs, the patients
were given 50mg cyclophosphamide orally every
day.
Group 2-Patients received Tab prednisolone 1-2mg
/kg body wt after food along with a proton pump
inhibitor before breakfast .In addition to this Tab
cyclophosphamide 50mg after food daily orally for 6
months.The treatment with DCP regimen has been
divided into four phases namely
Phase 1 It lasts till complete remission is
achieved.During this phase monthly DCP and daily
cyclophosphamide 50mg are given. Lesions heal very
quickly after pulse but reappear after a variable
period.
Recurrences become progressively less
severe with repeat pulses.
Phase 2- Once complete remission is achieved, the
patient is said to be in phase 2. Duration of phase 2 is
fixed and lasts for 6 months during which monthly
and daily cyclophosphamide are continued.
Phase 3-It lasts for a year during which time daily
cyclophosphamide is continued. The patient
continues to be disease free in this phase.
Phase 4-In this phase treatment is stopped and the
patient is followed up i.e., treatment free and disease
free followsup.
The clinical course of the disease, therapeutic benefits
and side effects of therapy were recorded in specific
proforma. The patients were advised to take regular
bath with soap and water even during the phase of
clinical activity. Topical medications like steroid and
steroid- antibiotic preparations were prescribed as per
requirement. Treatment of other concomitant diseases
was continued and drug induced complications such
as infections, diabetes, hypertension, acid peptic
disease etc. were simultaneously treated without
interrupting the specific treatment. Salt restriction and
potassium supplementation were not advised during
pulse therapy. However high protein diet and calcium
supplementation were advised to some patients who
received high dose steroids. The response rate based
on clinical improvement was graded as follows: [13]
1+ Poor response
513
Naseem et al.,
2+ Moderate response
3+ good response
The response rate was assessed on the basis of
clinical improvement.
Clinical improvement in
pemphigus was assessed by healing of old lesions,
decrease in new lesions and disappearance of oral
lesions. Clinical improvement in SLE was assessed
by improvement in rash, photosensitivty, arthralgia,
myalgia and decrease in proteinuria.
Response in scleroderma was assessed by softening
of skin, improvement in shortness of breath,
improvement in joint mobility, myalgias and
arthalgias. Follow up: This included recording of
any improvement in symptoms and signs, any adverse
effects of the treatment.End point: Primary end point
: Complete clinical improvement (assessed by healing
of old lesions, decreasing new lesions, decrease in
arthralgia and myalgia) Secondary end point: To
assess the incidence of any adverse effects.
Statistical analysis : All the values are expressed as
mean + SD. Improvement in clinical response and
incidence of adverse effects between the two groups
was done by using chi-square test and unpaired t test,
p value < 0.05 was considered as significant.
RESULTS
Table1: Age distribution of patients among two
groups
Age Group(years) Group I
Group II
N
%
N
%
16-25
6
12
9
16
26-35
13
26
11
22
36-45
23
46
13
26
46-60
8
16
18
36
Group I: DCP Therapy, Group II: Daily
immunosuppressive therapy
Table 2: Sex distribution of patients among two
groups
Group
Males
Females
n
%
n
%
Group I
12
24
38
76
Group II
17
34
33
66
Table3: Categories of patients
Group
Pemphigus
SLE
Scleroderma
Group I
Male
12
-
Female
36
1
1
Group II
Male
17
-
Fig 1:Duration of disease before treatment

Table 4:Side effects
Side effects
Group I
Group II
Cushingoid features
88
<0.0001
Pyoderma
16
74
<0.0001
Dermatophytosis
Striae
Diffuse hair loss
Acne
Candidiasis
Hyper acidity
18
18
14
24
24
18
66
66
62
58
56
54
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
Weight gain
11
46
<0.0001
Hyper pigmentation
Ecchymosis
Flushing
Hypertension
Loss of appetite
Diabetes mellitus
Cataract
Hirsutism
Psychosis
14
4
44
16
16
6
12
6
4
42
38
36
36
36
34
34
12
10
<0.0001
<0.0001
0.05
0.0001
0.0001
<0.0001
0.0001
> 0.05
> 0.05
Hiccup
Hemorrhagic Cystitis
16
2
8
6
> 0.05
> 0.05
6
6
4
< 0.01
>0.05
>0.05
HPA axis separation

0
Tuberculosis
2
Aseptic necrosis of
2
bone
Glaucoma
0
P value:< 0.0001, highly significant
P value
DISCUSSION
Female
28
3
2
Pemphigus, SLE and scleroderma are autoimmune

diseases associated with high morbidity and
mortality. Though several factors have been
514
Naseem et al.,
identified to have important role in determining the

prognosis, the course of the disease is variable and
unpredictable for a given patient. Hence early
diagnosis and effective therapy may greatly modify
the course of the disease. According to Lever and
Schaumberg Lever early intensive treatment can lead
to complete remission in pemphigus.[14]The use of
high dose steroids and immunosuppressive drugs for
prolonged periods could effectively control the
disease, but it was associated with significant
morbidity and mortality. Hence DCP therapy regimen
has been tried to achieve prolonged remission which
may amount to cure in pemphigus, SLE and
scleroderma.
The combined immunosuppressive [suppression of
the monoclonal antibody responses] and antiinflammatory effects of both, immunosuppressive
drugs
and
suprapharmacological
doses
of
corticosteroids have been utilized in pulse therapy to
induce immune tolerance. Adding to the benefits, the
rapid elimination of the intravenously administrated
drugs allows little time for the development of any
undesirable side effects.
The primary objective of our study was to compare
the safety and efficacy and the incidence of adverse
effects between the two treatment groups (DCP
therapy and daily immunosuppressive therapy) for 6
months. The first reported use of pulse steroid
therapy (PST) was by Kountz and Cohn in 1969 and
Bellet al in 1971 to prevent and treat rejection of
kidney transplantation. [15] The first use of DCP
therapy in dermatology was reported two decades
ago. At that time, it represented a radical change in
the approach to skin diseases as pulse therapy had
previously been used. Mainly to prevent transplant
rejection and in the treatment of lupus nephritis. [16]
In 1982, Pasricha et al introduced DCP therapy in
order to reduce the side effects of conventional
steroid therapy. The therapy has since been used to
treat a very large number of patients at several
centers[17-24] in India and elsewhere.[25-30] The reason
behind the effectiveness of pulse therapy is that the
treatment has evolved in response to observations of
the results of treatment in patients who were
receiving DCP therapy. Cyclophosphamide was
added to dexamethasone because relapses were
frequent with dexamethasone alone. Clinical studies
by Pasricha et al have also shown that DCP therapy
has produced quick control of the disease and reduced
hospital stay. Later it was noted to lead to long lasting

remissions (extending up to 9 yrs.) even after
stopping therapy, virtually amounting to cure[31]. The
efficacy of pulse steroid therapy reflects the complex
interplay of multiple physiologic and biochemical
events. Large doses of intravenous (IV) steroids
affect the numerous facets of the immunologic and
inflammatory
process
which
involve
immunoglobulins,
lymphocytes,
monocytes,
macrophages, polymorphonuclear leucocytes.
Studies carried by Pai et al[21]have enrolled 5 women
with scleroderma aged between 30-60yrs, all the
patients had symptomatic clinical improvement. The
vital capacity improved in three and post treatment
histopathologic regression was seen in two patients.
Whereas in our study there was only one patient of
scleroderma on DCP therapy and two patients on
daily immunosuppressive therapy. The patient on
DCP therapy showed good response whereas the two
patients on daily immunosuppressive therapy showed
moderate response. Several studies have reported the
effectiveness of DCP therapy in scleroderma.
Cathcart et al[32] treated SLE patients with DCP
therapy and observed that DCP therapy was effective
in treating these patients of bullous lupus
erythematosis. Our study enrolled one patient of SLE
on DCP therapy and 3 patients on daily
immunosuppressive therapy. A better response was
seen in patients on DCP therapy, the rash was
decreased glomerular filtration rate (GFR) improved.
Myalgiasand arthralgias improved.
At the end of six months of treatment in our study,
the incidence of adverse effects recorded in both the
groups showed that the adverse effects with DCP
therapy were less than with daily immunosuppressive
therapy. DCP therapy minimized the need for long
term, high dose oral steroid therapy (elimination of
the intravenously administered corticosteroids allows
little time for the development of any undesirable side
effects. The adverse effects of pulse therapy are those
of its constituent drugs, corticosteroids (infections,
diabetes mellitus, hypertension, hyperacidity and
osteoporosis) and cyclophosphamide (leucopenia,
hematuria, gonadal failure, hyperpigmentationand
hair loss). The major side effect reported with
immunosuppressive regimen in the literature is
increased susceptibility to infections like candidiasis,
dermatophytosis and pyoderma. Similarly in the
present study we observed that the infections were
515
Naseem et al.,
less frequent with DCP therapy than with daily

immunosuppressive therapy.
Pyoderma was found in 16% in group 1 and 74% in
group 2, dermatophytosis was found in 18% in group
1 and 66%. Candidiasis was seen in 24% in group 1
and in 56% in group 2 ((P<0.0001 which was highly
significant). Cushingoid features were seen in 4% in
group 1 and 88% in group 2. (p<0.0001 which was
highly significant) HPA axis suppression was seen in
6% of patients in group 2. Whereas none of the
patients in DCP therapy had HPA axis suppression
(p<0.0001, which was highly significant).
Ecchymosis was found in 4% in group 1 and in 38%
in group 2 (p<0.0001, which was highly significant),
Flushing was noted in 44% in group 1 and 36% in
group 2 (p<0.05, which was significant), 16% in
group 1 and 8% in group 2 had hiccups(p>0.05,
which was not significant)
Similarly, previous studies have reported that the side
effects peculiar to pulse therapy include hiccups,
facial flushing, diarrhea, weakness, generalized
swelling, muscle and joint pains[33]. These side effects
are usually observed with each pulse and last for a
few days afterwards. Most of the patients are able to
tolerate these symptoms and continue treatment.
White et al presented a review of adverse effects
associated with DCP therapy in various dermatologic
disorders. Of 188 patients reviewed in this category,
majority about 69% were treated for pemphigus
(especially in India) with a specific regimen called
dexamethasone cyclophosphamide pulse (DCP)
therapy. Most of the adverse effects reported were
mild and / or transient and included hyperglycemia,
metallic taste during infusion, facial flushing[34],
hiccups, malaise, mild hypertension and focal
infections. Serious adverse effects reported in 14
patients (7%) included death due to sepsis (four
patients), reactivation of pulmonary tuberculosis (3),
heart failure (2), perforated duodenal ulcer (one) and
euphoria followed by depression (one). However, all
the side effects could not be attributed to DCP
therapy alone, as the patients were also treated with
oral
corticosteroids
and
cyclophosphamide.
Furthermore there was no clear evidence to suggest a
correlation between DCP therapy and cardiovascular
side effects.
Kaur and Kanwar reported the
development of transient arrhythmias in two patients
with no changes noted in ECG. Barrett reported the
precipitation of left ventricular failure with DCP
therapy in a patient who was hemodynamically

unstable before receiving pulse therapy.
Severe adverse cardiovascular effects of pulse steroid
therapy were highlighted in some of the studies and
continuous cardiac monitoring wasrecommended
during steroid pulse administration. However the
complications were restricted to the elderly patients
and those with cardiac and renal diseases.
Subsequent cardiac monitoring of healthy volunteers
on DCP therapy detected no arrhythmias or other
significant changes. In the present study cardiac
monitoring was done for some patients during initial
courses of pulse therapy. Except transient increase in
the heart rate during pulse therapy. Cardiac
complications like ischemic heart disease, heart
failure or arrhythmias were not encountered. Cardiac
monitoring was not performed for all the patients but
Certain precautionary measures were taken to prevent
the complications. Elderly patients above 60 years
and those with compromised cardiac or renal
functions were not taken up for pulse therapy.
Careful monitoring of vital data was done during the
3 days of pulse therapy and ECG and serum
electrolytes were done before and after completion of
pulse therapy. In the present study, 16% in group 1
and 36% in group 2 recorded hypertension and they
were treated accordingly by anti-hypertensive drugs
(p<0.0001, which was highly significant).
Hemorrhagic cystitis a side effect seen with
cyclophosphamide was seen in 2 % in group 1 and
66% in group 2 (P>0.05, which was not significant),
Hemorrhagic cystitis was confirmed with complete
urine examination (CUE) and urologist opinion,
Tuberculosis was diagnosed in 2% in group 1 and 6%
in group 2 (p>0.05 which was not significant).
Aseptic necrosis of bone was seen in 2% patients in
group 1 and in 4% patients in group 2 (P > 0.05
which was not significant). Glaucoma was not seen in
either of the two groups. (Table 4)
The side effects observed during DCP therapy in 100
consecutive pemphigus patients reported in a study
are as follows.[35] Weight gain > 10% body weight
(11 patients), weight loss of >10% of body weight
(13), pyoderma (3), candidiasis (8), dermatophytosis
(7), Tuberculosis (1) diffuse hair loss (29),
generalized hyperpigmentation (3) cataract (5), loss
of appetite (3), diabetes mellitus (0), Hypertension
(3), hyperacidity (1), hemorrhagic cystitis (1), acne
(6), hirsutism (1), Striae (0), ecchymosis (3),
516
Naseem et al.,
glaucoma (1), hiccup (1), and aseptic necrosis of the

bones (0).
The other major complication of DCP regimen is
gonadal failureand all the patients treated with DCP
regimen had amenorrhea / oligomenorrhoea.
However the gonadal dysfunction was not a problem
in the present study as the patients who were married
and completed the family alone were chosen for this
treatment and possible complications were explained
to them before starting the treatment.
In our comparative study there was statistically
significant difference in safety and efficacy between
DCP therapy and daily immunosuppressive therapy.
Among the two groups we found that DCP therapy
was more effective in quick relief of signs and
symptoms and cure rate and decreased incidence of
adverse effects.
CONCLUSION
DCP therapy was found to have greater efficacy in
controlling the symptoms of steroid responsive
dermatosis than daily immunosuppressive therapy.
With pulse therapy remissions are long amounting to
cure as per the previous studies in the Dermatology
Department of Osmania General Hospital. With DCP
Therapy, quick healing of the lesions (within three
days of pulse) reduced the hospital stay to 4-5 days,
enabling the patients to resume their routine activities
earlier and to lead a normal life.The second major
advantage with pulse therapy is relative freedom from
the expected side effects of conventional dose of
corticosteroids and immunosuppressive drugs. These
side effects include disfiguring cushingoid changes,
infection, acid peptic disease, diabetes mellitus,
hypertension, myopathy, cataract and psychosis.
Thus, the treatment became more acceptable to the
patients on DCP therapy.
DCP therapy was avoidance of prolonged high dose
daily
immunosuppressive
therapy
and
its
consequences. Considering all the above factors DCP
therapy appears to be a better choice in the treatment
of steroid responsive dermatosis as compared with
daily immunosuppressive therapy. In conclusion
further dietary restrictions on salt or calorie intake
were not required.
Another main advantage with therapy with steroids
and immunosuppressive drugs is superior to daily
immunosuppressive therapy in terms of safety,
efficacy and longer remissions.
Naseem et al.,
Acknowledgement : It is my privilege and pleasure

to acknowledge the encouragement and guidance I
received from Department of Pharmacology,
Osmania Medical College.
I am infinitely obliged to express my feeling of pride
to my family members for their support. Lastly and
above all, I thank God almighty for giving me
strength and courage to complete this project.
Conflict of Interest: no conflict of interest
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Naseem et al.,
DOI: 10.5958/2319-5886.2015.00100.9

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
th
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
rd
Revised: 23 May 2015
A COMPARATIVE STUDY OF CAREGIVER BURDEN IN CANCER CERVIX AND CANCER BREAST

ILLNESSES
*Srinivasagopalan1, Nappinnai2, Solayappan3

1
Associate Professor, 2Clinical Psychologist, 3Formely Professor of Psychiatry, Department of Psychiatry,

Meenakshi Medical College & R.I., Enathur, Kanchipuram, Tamilnadu, India.
* Corresponding Author email: drsinivasagopalan1950@gmail.com
ABSTRACT
Background: Caregivers of individuals suffering from cancer illnesses are at risk of having subjected to mental
health consequences. There is a paucity of data comparing the caregiver burden of cancer breast and cancer cervix
patients. Aim: The aim of the present study is to compare the caregiver burden of cancer breast and cancer cervix
patients. To study the association of caregiver burden with demographic factors like age, gender, duration of
caregiving etc. Materials & Methods: This Cross sectional study is performed on the key relatives of patients of
31 cancer cervix and 31 cancer breast patients. Burden assessment schedule was used. Results: Our findings
suggest burden is more in male caregivers of breast cancer patients. It is not so in caregivers of cancer cervix
patients. Whenever the caregiver is closely related to the patients the burden is high in both groups. Whenever the
burden scores were high the depression scores were also high. Treatment modalities as a whole correlates with
burden scores in caregivers of breast cancer patients but not in cancer cervix patients. Conclusion: Caregivers
with breast and cervical cancer patients are vulnerable if the caregiver is male, from low socioeconomical
background, more closely related and when the patients received poor treatment modalities.
Keywords: Burden, caregiver, cancer breast, cancer cervix.
INTRODUCTION
Two important organs of women are uterus and
breast. They are associated with female image and are
also vital for reproduction and mothering. Both the
organs are prone to cancer and their loss is associated
with psychological and social consequences. A
diagnosis of cancer affects not only the patients but
also their significant others, especially when a lot of
care tasks are involved. Some care givers perceive the
care as a burden, while others consider it as a
challenge.[1] Care givers of individuals suffering
from cancer illnesses are at risk of being subjected to
mental health consequences. The care giver burden
can be quantified into objective and subjective
domains. There is a paucity of data comparing the
caregiver burden of cancer cervix and cancer breast

patients. [2] The term caregiver refers to anyone who
provides assistance to someone else who is in some
degree incapacitated and needs help.[3] An informal
caregiver is an individual such as a family member,
friend or neighbour who provides unpaid care. A
formal caregiver is a volunteer or a paid care provider
associated with a service system. [4] Objective burden
denotes the effects of caregiving on finances well
being, health activities of caregivers while subjective
burden denotes of depression, anxiety and somatic
symptoms associated with the caregiving role. In
India very few studies have been conducted in the
519
Srinivasagopalan et al.,
areas of family burden and the social support systems

of different kinds of cancer patients.
Most of the notable community based studies provide
that 18-47% of caregivers land in depression5.
A
widely accepted notion within developing country
societies of the family as endlessly supporting
caregivers may not be true.[6] Even where care is
exemplary, it is essential that the impact of providing
care on the family and on the wider community be
quantified.[7]
Aims and objectives
1. To compare the care giver burden in breast cancer
patients and cervix cancer patients.
2. To study the association of caregiver burden with
demographic factors like age, gender duration of
care giving and other variables.
METHODOLOGY
Study design: This cross sectional study is
performed on the key relatives of patients of cancer
cervix and cancer breast.
Grouping: Group I consist of caregivers of breast
cancer patients and groupII consists of care givers of
cancer cervix patients. The Patients were recruited
from Arignar Anna Cancer Institute, Karapettai,
Kanchipuram. It is a large regional centre for all
types cancer diseases. The study was conducted
during Dec. 2010 to May 2011.
Sample size: The study involved two groups of care
givers each of 31 members.
Ethics Approval: The institutional ethical committee
clearance has obtained. Informed oral consent from
all the caregivers and the patients were obtained.
A semi-structured interview proforma was used to
record the following details Demographic and
personal data of both patients and caregivers, clinical
diagnosis and details of the type, grading, duration of
illness, and duration of the care by caregivers etc.,
Inclusion criteria: 1) Age between 18 to 65 (both
inclusive for caregivers). 2) Confirmed primary
diagnosis of cancer breast and cancer cervix for the

patients.
Exclusion criteria: caregivers without severe
physical illnesses
The following scales were administered.
1) Hospital Anxity and Depression Scale (HADS)[8]
has been established as a much applied and
convenient self rating instrument for anxity and
depression in patients with both somatic and mental
problems and with equally good sensitivity and
specificity as other commonly used self rating
screening instruments.[9],[10]
2) Burden Assessment Schedule (BAS) [11] This is a
structured instrument with 40 items measuring both
subjective and objective burden. Each item is rated on
a 3 point scale. (Not at all, to some extent, very
much) Interrater reliability is good (Kappa 0.80)
regarding validity the reported co-efficient of
Correlation is 0.82. It assesses nine categories of
burden. (It is relevant for many chronic illnesses.)
3) Presumptive Stressful Life Event Scale by
Gurmeet Singh etal. (PSLES)[12]. The PSLES is
formulated by Singh etal to evaluate the life event
(that occurred within one year prior to illness). This is
a schedule which was standardized on Indian
population and has 51 items assessing various life
event experiences by the subject and the time frame
for assessing the occurrence of life events, in the year
proceeding the examination.
Care givers were included if they satisfied the
following criteria.
1) Healthy adults of 18 years and above.
2) Care givers who were continuously caring for the
last one year and spent a lot of time and emotions
caring the patients. Patients diagnosed for at least
one year and had received relevant treatments
(surgery/chemotherapy/radiotherapy etc.) and
were now attending the OPD or undergoing
treatment. They were in stable clinical condition
at the time of interview.
520
RESULTS
Table 1: Classification of caregivers based on the
demographic, economic and other patient related
variables
AGE GROUP
BREAST CA
CERVIX CA
NO.
%
NO.
%
20-30
13
41.9
9
29.0
30-40
5
16.1
11
35.5
40-50
3
9.7
3
9.7
50-0
6
19.4
3
9.7
60& ABOVE
4
12.9
5
16.1
SEX
MALE
17
54.8
13
41.9
FEMALE
14
45.2
18
58.1
MARITAL STATUS
MARRIED
24
77.4
23
74.2
SINGLE
7
22.6
8
25.8
EDUCATION
ILLETERATE
4
12.9
8
25.8
HIGH SCHOOL 19
61.3
16
51.6
HR.SEC
5
16.1
4
12.9
DIPLOMA
0
0.0
2
6.5
DEGREE
3
9.7
0
0.0
PG
0
0.0
1
3.2
OCCUPATION
Daily wages
10
32.3
14
45.2
Self employed
7
22.6
4
12.9
Private
4
12.9
4
12.9
Government
1
3.2
0
0.0
Housewife
7
22.6
8
25.8
Student
2
6.5
1
3.2
Income
BELOW 1000
0
0.0
3
9.7
1000-3000
9
29.0
13
41.9
3000-5000
10
32.3
10
32.3
5000-7000
8
25.8
4
12.9
7000-10000
3
9.7
1
3.2
ABOVE 10000 1
3.2
0
0.0
RELIGION
HINDU
27
87.1
26
83.9
CHRISTIAN
0
0.0
5
16.1
MUSLIM
4
12.9
0
0.0
OTHERS
0
0.0
0
0.0
FAMILY SYSTEM
NUCLEAR
20
64.5
23
74.2
JOINT
11
35.5
8
25.8
LIVING TYPE
Hut
0
0.0
2
6.5
Mud house
0
0.0
0
0.0
Thatched,
Sheet, Lightroof 18
58.1
19
61.3
Brick used
13
41.9
10
32.3
Table 2: Classification of caregivers based on the

Duration of illness, Hospitalization, treatment
DURATION OF
ILLNESS
BELOW 1 M
1-6 M
6-12 M
12-18 M
18-24 M
24-30 M
ABOVE 30 M
HOSPITALISATION
I TIME
2 TIMES
3 TIMES
4 TIMES
5 TIMES
6 TIMES
7 TIMES
8 TIMES
9 TIMES
Duration of treatment
BELOW 1 M
1-6 M
6-12 M
12-18 M
18-24 M
24-30 M
ABOVE 30 M
Mode of treatment
SURGERY
SURGERY
+
CHEMOTHERAPY
CHEMOTHERAPY
RADIOTHERAPY
CHEMOTHERAPY +
RT
SURGERY +RT
SURGERY + RT
+CHEMOTHERAPY
Time spent per week
< 8 HOURS
9-16 HOURS
17-32 HOURS
>32 HOURS
Relationship
PARENT
SPOUSE
DAUGHTER
SON
DAUGHTER IN LAW
SIBLING
OTHER RELATION
BREAST CA
No.
%
0
0.0
11
35.5
9
29.0
6
19.4
1
3.2
0
0.0
4
12.9
CERVIX CA
No.
%
0
0.0
15
48.4
10
32.3
4
12.9
0
0.0
0
0.0
2
6.5
4
11
5
1
2
5
1
1
1
12.9
35.5
16.1
3.2
6.5
16.1
3.2
3.2
3.2
10
10
4
5
2
0
0
0
0
32.3
32.3
12.9
16.1
6.5
0.0
0.0
0.0
0.0
0
12
10
4
1
0
4
0.0
38.7
32.3
12.9
3.2
0.0
12.9
1
23
4
2
0
0
1
3.2
74.2
12.9
6.5
0.0
0.0
3.2
0.0
0.0
10
7
0
32.3
22.6
0.0
10
7
0
32.3
22.6
0.0
1
2
3.2
6.5
1
2
3.2
6.5
11
35.5
11
35.5
0
1
3
27
0.0
3.2
9.7
87.1
1
1
3
26
3.2
3.2
9.7
83.9
1
10
5
5
5
2
3
3.2
32.3
16.1
16.1
16.1
6.5
9.7
3
8
11
3
2
2
2
9.7
25.8
35.5
9.7
6.5
6.5
6.5
521
Table : 3 Difference between the caregiver burden of

breast and cervix cancer patients
Breast
66.779.639
Cervix
70.7710.1
P value
0.116
The mean caregiver burden scores of breast cancer

and that of cervix cancer were compared using t
test.
Overall caregivers burden without breakdown for
cancer breast and cervix were worked out The burden
score for caregivers of cancer cervix is more by 4.
But the difference 4 is not statistically significant as
p(0.116) as p(0.116) > 0.05. Hence we conclude that
the difference 4 is due to chance.
Table 4: Burden between male and female
caregivers of breast & Cervix cancer patients:
Breast
Cervix
Male
70.69.079
72.9212.55
Female
62.799.04
69.227.93
P value
0.0341
0.3224
Among 31 care givers, 17 male & 14 female reported

for Breast cancer cases. As the difference of score
between Male and Female is significant i.e., P(0.034)
< 0.05, we conclude that male caregiver burden score
is significantly more when compared to female.
A test for comparison of burden score for cervix
cancer to find out if any gender difference was
correlated and the difference is insignificant as P =
0.3224, > 0.05. Hence we have no claim to record
that there is a difference in the burden score between
male & female caregivers.
Table 5: Education
burden score of cervix burden score of breast
cancer
cancer
r value
-0.167 r value
-0.158
P value
0.3687 P
value
0.3961
(two-tailed)
(two-tailed)
As the correlation between education and burden
score were not significant in both groups of care
givers i.e. p=0.3687 and p =0.3961 respectively for
cancer cervix group and cancer breast group, we
conclude that education does not influence the
burden. The r values are seen closed to 0.
Table 6: Occupation :
cancer
cancer
r value
-0.0516 r value
-0.27
P value
(two-tailed)
0.7826
P value
(two-tailed)
0.1418
The correlation co-efficient r = -0.05 and -0.27

leads to say that there is no relationship between
occupational and caregiver burden in both groups of
care givers. It is also seen that r value are not
significant as p = 0.7826 and P=0.1418 for cervix and
breast groups.
Table 7: Anxiety
cancer
cancer
r value
0.2982
r value
0.1465
P value
0.1032
P value
0.4315
(two-tailed)
(two-tailed)
The correlation co-efficient r = 0.2982 and 0.1465
leads to say that there is no relationship between
Anxiety and caregiver burden in both groups of care
givers. It is also seen that r value are not significant
as p = 0.1032 and P=0.4315 for cervix and breast
groups.
Table 8: Depression
burden score of cervix
cancer
r value
P value
(two-tailed)
burden score of breast cancer
0.4861 r value
0.0056 P
value
(two-tailed)
0.4526
0.0106
It is seen that there is a positive correlation between

depression and burden score in both groups of care
givers and the correlations are significant. We
conclude that whenever the burden score are more the
depression is also more.
Table 9: Relationship :
cancer
cancer
r value
-0.3963 r value
-0.6546
P value
0.0273 P
value P<0.0001
(two-tailed)
(two-tailed)
The correlation co-efficient r = 0.3963 is significant
as p=0.027 < 0.05 in cervix group and significantly
the r = -0.6546 and P < 0.0001 in breast group leads
to conclude that there is a significant correlation
between the relationship and burden score in both
groups. We may conclude that there is a big burden
when there is a close relationship.
Table 10: Family System :
cancer
cancer
r value
0.2434
r value
-0.2383
P value (two- 0.1871
P
value 0.1967
tailed)
(two-tailed)
522
The family system and burden score do not have

much relationship. The correlation coefficient
r=0.2434 and r = -0.2383 are not significant. We
conclude that the family system and the burden scores
are independent.
Table 11: Type of living :
cancer
cancer
r value
-0.32
r value
-0.02113
P value
0.0793 P
value 0.9102
(two-tailed)
(two-tailed)
Type of living and burden score do not have
significant relationship as r = -0.32 and r = -0.02
respectively in cervix group and breast group.
Table12: Treatment modality :
cancer
cancer
0.09942 r value
0.389
r value
0.5946 P
P value
value 0.0305
(two-tailed)
(two-tailed)
Treatment modality and burden score have good
correlation r = 0.39 in breast group and it is
significant, whereas it is not significant in cervix
group.
Table 13: Time spent
cancer
cancer
r value
0.3447
r value
0.2654
P value
(two-tailed)
0.0576
P
value 0.149
(two-tailed)
The time spent by caregivers is not significantly

correlated to burden score as p=0.0576 and p = 0.149
> 0.05. We conclude that apart from time spent there
are other factors to maximize the burden to the
individual caregiver.
Table 14: Duration of illness
cancer
cancer
r value
0.117
r value
0.3462
P value
0.5495 P
value
0.0564
(two-tailed)
(two-tailed)
The r = 0.117 and r = 0.34 are not significant as p =
0.549 ? 0.05 And p=0.059 > 0.05. We conclude that
duration of illness and burden score are not
correlated.
Table 15: Illness

cancer
cancer
r value
0.1734
r value
0.2441
P value
0.3509
P value
0.1857
(two-tailed)
(two-tailed)
The pearson r = 0.1734 shows that the relationship
between the illnesses and the burden score of
caregivers certify is positive. It can be concluded that
as the illness is more than the burden score is also
more but it is insignificant as p = 0.35 > 0.05.
Table 16: Income
cancer
cancer
r value
-0.2546
r value
-0.1776
P
value 0.1669
P value
0.3393
(two-tailed)
(two-tailed)
No relationship is established between the income
level and burdens of the caregivers of cancer cervix
as the p = 0.1669 > 0.05. But it is seen that as the
income becomes low the burden score becomes high
and not significant (r=0.2546)
DISCUSSION
Caregivers of breast cancer group were of 20-30 age
groups, but for cancer cervix it was 30-40 age group.
More no.of (54%) male caregivers belong to breast
cancer group than for cancer cervix group. Both
groups were represented by more number of married
people. Majority of caregivers in both groups (61.3%,
51.6%) completed school education. Daily wage
earners (32.3%, 45.2%) were more represented in
both groups. More no. of caregivers in both groups
were with income of below Rs.5000 per month.
Majority of them in both groups (87.1%, 83.9%)
belonged to Hindu religion. 64.5% of caregivers of
cancer breast and 74.2% of caregivers of cancer
cervix patients belonged to nuclear family. As far as
duration is concerned more no. of cases came under 6
months of caring. More number of patients underwent
(35.5%) all the three treatment modalities in both
groups. As far as time spent per week by the carers in
caregiving activities more than 32 hours (87% in
breast cancer and 83% in cancer cervix group) is the
largest group in both groups.
Sex: As far as breast cancer patients caregivers are
concerned the difference of score between male and
female is significant. i.e. p(0.034) < 0.05, we
523
conclude that male caregiver burden score is

significantly more when compared to female
caregivers. The reason being as explained in a
study[13] men were found to be deeply emotionally
engaged but they were hiding it, and were playing
protective, reassuring, minimizing role. And it has
been found that the coping strategies which husband
and wives used were largely independent of one
another[14] Mastectomized wives husbands most of
them reported good overall adjustment but a sub
group remained distressed and reported adverse
effects on their relationship with wives[15]. Another
study[16] says that breast cancer as an illness gave rise
to predominantly negative and dark association
among caregivers.
Income: As far as the income is concerned it is seen
that as the income becomes low the burden score
becomes high. It has been found caregivers with a
relatively low socioeconomic state are assumed to
report a higher burden and subsequently a poorer
health[17]. In another study[18] negative relationship
between income and caregiver outcomes has been
observed for only certain types of caregivers
(partners) whereas other types of caregivers as in our
study, report no relationships. The financial burden
was more problematic than the effect of caring on
family routines[19].
Relationship: Our results show that there is a
significant correlation between the relationship and
the burden score in both groups (cancer and cervix
patients caregivers). (p-0.027, p <0.0001). In the
case of family caregivers the burden of caring for
their relatives is associated with significant levels of
anxiety and depression.[20]
Duration of caregiving: As far as duration of
caregiving is concerned time spent by caregivers is
not significantly correlated to burden scores
(p=0.1497 > 0.05). In a study[21]
among cancer
patients and family members no significant effects of
the duration of care giving were found on outcome of
caregiving[22] [23].
Treatment modality: We have found that there is a
good correlation between treatment modality and
caregiver burden scores (r=0.389) in breast group and
it is significant whereas it is not significant in cervix
group caregivers.
In studies of spouses of
mastectomy patients it was found they were playing a
protective, reassuring role.[13]
Illness of caregivers : Our results show when the

physical illness were more in caregivers then the
burden scores were also more in both groups.
Caregivers physical wellbeing is at greater risk
because they have little time to rest, engage in fewer
self care behaviour or often fail to seek medical help
for themselves when sick.[24]
Depression: It is seen that there is a positive
correlation between depression and burden score in
both groups of care givers and the correlations are
significant. We conclude that whenever the burden
score are more the depression is also more.
Several authorities[25] [26] [27] [28][29] noted that
depression is the primary psychological symptom in
caregivers of cancer patients.
Anxiety: There is no relationship between anxiety
and burden scores in both groups of caregivers. In
some of the studies[30]. the authors demonstrated
positive experience of giving care rather than
negative experiences. They also argued that
caregivers who were most intensely involved in care
giving might have greater opportunity to derive
satisfaction from care giving. This may be the reason
why some of the caregivers were not anxious as in
our study.
Family System: There were no relationship between
the caregiver burden of both groups and the family
system.
PSLE score: Mean PSLE score for caregivers of
cancer cervix patients is significantly more than the
caregivers of breast cancer patients.
CONCLUSION
Male caregiver burden scores were significantly more
when compared to female caregivers of both groups.
Another finding is seen that as the income becomes
low the burden scores become high. And there is a
significant correlation between the relationship of the
caregiver and the burden score of both groups. There
is a good correlation between the type of treatment
modality and caregivers burden scores in breast
cancer patients only. Whenever the burden score were
high the depression scores were also high. Mean
PSLE scores for caregivers of cancer cervix patients
were significantly more than the caregivers of breast
cancer patients. Hence our findings suggest that the
families living with breast and cervical cancer
patients are vulnerable if the caregiver is male, from
524
low socioeconomical background, more closely

related and when the patients received poor treatment
modalities.
Limitations: Our sample is small. There is no rating
scale in vernacular language. We have translated it to
our local language. It may be a confounding factor.
Future directions: Patients and caregivers need to be
considered as a unit for attention in the clinical
setting and clinicians need to invest in the education
and support of family caregivers in order to enhance
their care giving roles. Patterns of caregiving changes
in relation to the course of the patients illnesses will
throw more light about the care giving process.
Source of support:- Nil.
Conflict of interest: None declared.
Acknowledgement: We would to express our
gratitude to the Director Arignar Anna Cancer
Institute, Karapettai, Kanchipuram for all the help.
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Anxiety and Depression Scale. Acta Psychiatrica
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Hospital Anxiety and Depression Scale, review of
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10. B Jelland Neckelmann.D, Tangen. Haug.T A
review of validation study of Hospital Anxiety
and Depression Scale. Journal of Psychiatric
Research 2002.
11. Thara.R, Padmavati.R, Suba Kumar & Latha
Srinivasan, Burden Assessment Schedule,
instrument to assess burden on caregivers; Indian
Journal of Psychiatry.1998.40(1), 21-29.
12. Gurmeet Singh, Dalbir Kaur , Harshevem Kour,
Hand book of Presumptive Stressful. Life Events,
Scale.National Psychological Corporation
2002. Agra ed;l. 8-9.
13. Sabo D.Brown.D.J and Smith.C The male group
and mastectomy : support groups and mens
adjustment. J.psycho Oncol 1986:4, 19-31.
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Ptacek, Social support in spouses of cancer
patients:what do they get and to what end?
Personal relationships 2005 4(4). 431-449.
15. Welliesh DK Jamison KR Pashall; Psychological
aspect of mastectomy the mens perspective AMJ
1978-135; 543-546.
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Gonyea.J.G
and
Hooyman.N.R caregiving and the experience of
subjective and objective burden. Fam. Relat.
1985:43:19-26.
18. Oberst M and Thomas SE; Cargiving demands
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psychosocial burden of caring for some Nigerian
women with breast cancer and cevical cancer.
Social Science and Medicine 1999:49;1541-1549.
20. Budger T Segrin ; Depression and Anxiety in
Women with Breast Cancer and their
Partners.Nurs. Res.2007;56;44-53.
21. Yang.C.T. and Kirshling.S.M Exploration of
factors related to direct care and outcomes of
caregiving Cancer Nurs. 1992:15; 173-181.
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Gerontologist 1991: 31;246-55.
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German.P.S., Preventive health behaviours
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Prev.Med.1997:26:162-169.
25. Schulz R, O Brien A.T., Bookwala.J Fleissner.K.
Psychiatric and physical morbidity effects of
dementia caregiving, prevalence, correlation and
causes. Gerontologist 1995:35; 771-791.
26. Kiccolt glaser.J.K et al Spousal caregivers of
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27. Holland.J.C. Anxiety and cancer. The patient and
the family. J.clinical psychiat 1989 20:20-25
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breast cancer Oncol Nurs. Forum 1989:16;511516.
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psychol.Ageing 1989; 4:402-408.
526
DOI: 10.5958/2319-5886.2015.00101.0

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Research article
ISSN: 2319-5886
Accepted: 2nd May 2015
A QUASI EXPERIMENTAL STUDY TO EVALUATE EFFECTIVENESS OF GLYCERIN

MAGNESIUM SULPHATE DRESSING ON PHLEBITIS AMONG PATIENTS UNDERGOING
PERIPHERAL INTRAVENOUS INFUSION IN SELECTED HOSPITAL,VADODARA
Ravindra HN1, *Patel Krupa D2
1
HOD of Medical-surgical Nursing, 2Department of Medical-Surgical Nursing student, Sumandeep Nursing

College, Vadodara, Gujarat, India
*Corresponding author email: krupa1491@gmail.com
ABSTRACT
Introduction: Intravenous therapy is indicated for many reasons. A significant number of patients admitted into
hospital receive some forms of intravenous therapy through peripheral venous cannula, which is a common
procedure carried out in hospital to allow rapid and accurate administration of medication. However, the
intravenous cannulation can have undesirable effects, the most of which is phlebitis, which is due to mechanical,
chemical or infectious cause. Method: In this study quasi-experimental research approach was used. Non
probability purposive sampling technique was used to select the sample from the selected hospital. The research
design adopted for the study was pre-test, post-test control group design. In the present study a sample of 60
hospitalised patients and who met the inclusion criteria was selected from the target population. In this study the
instruments used are baseline Performa, structured interview schedule to assess the subjective symptoms and
observation scale to observe the objective symptoms. Result: In experimental group post test mean score 1.10,
SD was 0.71 respectively. In control group post test mean score 2.53, SD was 0.78 respectively. The obtained
value 7.454 statistically was significant at 0.001 levels. So research hypothesis was accepted. So there was
significant difference between post intervention phlebitis among the experimental group and control group.
Discussion: In the research study findings revealed that Glycerin Magnesium sulphate dressing is highly effective
in decrease phlebitis level to the patients.
Keywords: Glycerin magnesium sulphate dressing, Phlebitis patients, Peripheral Intravenous infusion,
Effectiveness.
INTRODUCTION
Peripheral-catheter related phlebitis is caused by the
inflammation of tunica intima of a superficial vein
due to irritation of the tunica by mechanical, chemical
or bacterial sources. It is estimated that in U.K 2080% of patients with peripheral venous cannula
develop phlebitis.[1] A project was undertaken in Ball
memorial Hospital to determine the incidence of
peripheral intravenous therapy-related phlebitis in an
adult population, results showed that phlebitis rate
was 3.3%(10/305).[2] Chemical phlebitis is caused by
drug or fluid being infused through cannula. Factors

such as pH and osmolarity of substances have a
significant effect on the incidence of phlebitis. If left
untreated, it can lead to infection or thrombus
formation. Hence it is essential for the nurses to treat
the patients with phlebitis promptly with cost
effective thus preventing complications related to
phlebitis. [3] Phlebitis is defined as inflammation of a
vein, which can be categorized in as chemical,
mechanical, or bacterial. Chemical phlebitis can be
527
Krupa et al.,
caused by an irrigating medication or solution

(increased pH or high osmolarity of a solution), rapid
infusion rates, and medication in compatibities.
Mechanical phlebitis results from long periods of
cannulation, catheter in flexed areas, catheter gauges
larger than the vein lumen, and poorly secured
catheters. Bacterial phlebitis results from poor hand
hygiene, lack of aseptic technique, failure to check all
equipments before use and failure to recognize early
signs and symptoms of phlebitis. Other factor is poor
venipuncture technique phlebitis is characterized by
reddened warm area around the insertion site or along
the path of the vein, pain or tenderness at the site or
along the nein and swelling. Treatment consists of
discontinuing the IV line and restarting it in another
site and applying a warm, moist compress to the
affected site. Phlebitis also prevented by using aseptic
technique during insertion, using the appropriate-size
cannula or needle for the vein, considering the
composition of the fluids and medications when
selecting a site and observing the site hourly for
complications like phlebitis or any signs of
phlebitis.[4] Magnesium sulfate is a colorless, odorless
and a solid substance. It is slightly bitter in taste. It is
highly soluble in inorganic solvents like water. It is
partially soluble in organic solvents, like glycerin and
alcohol. Magnesium sulfate in its anhydrous form is
hygroscopic. It has a tendency to attract moisture. [5,6]
Aims & Objectives:
1. Assess the pre intervention phlebitis in
experimental group.
2. Assess the pre intervention phlebitis in control
group.
3. Assess the post intervention phlebitis in
experimental group.
4. Assess the post intervention phlebitis in control
group.
5. Determine the effectiveness of glycerin
magnesium sulphate dressing on phlebitis among
patient.
Type of the study: Experimental study
Ethical clearance was obtained from ethical
committee and informed consent was taken from the
participants. Duration of the study is 2 years.
Sampling technique: Non probability convenient
sampling technique was used in this study. In these
study 60 patients was selected 30 for experimental

group and 30 for control group.
Inclusion criteria:
1. Patients who were admitted to the Dhiraj hospital
during the period of study.
2. Patient above age of 14 years.
3. Patient those who can understand or speak, write
or read Gujarati, Hindi, English.
4. Patient who are on peripheral intravenous access
as treatment.
Exclusion criteria:
1. Patient who have condition like DM, HIV,
AIDS, Hepatitis, Renal Failure, skin diseases.
2. Patient who are having Central venous access &
Venisection.
Methodology: In this study quasi-experimental
research approach was used. The research design
adopted for the study was pre-test, post-test control
group design.
Grouping: there were two groups experimental and
control group.
In this study the instruments used are baseline
Performa, to assess the subjective symptoms and
observation scale to observe the objective symptoms.
Jacksons visual infusion phlebitis scale is use for
measure the phlebitis according to score.
In this 0 to 5 score in different stages. [9]
Score 0 is No signs of phlebitis.
Score 1 is possibly first signs of phlebitis.
Score 2 is Early stage of phlebitis.
Score 3 is Medium stage of phlebitis.
Score 4 is Advanced stage of phlebitis or start of
thrombophlebitis.
Score 5 is Advanced stage Thrombophlebitis.
Patients who are getting score 3, 4, 5 according to
scale those patients apply glycerin magnesium
sulphate dressing at affected site,
The study conducted in the following phases,
Phase 1: Pre test level of Phlebitis will be assessed
using Jacksons visual infusion phlebitis scale.
Phase 2: 20gram of magnesium sulphate diluted in
100 ml of glycerin and this combination applied on
site of phlebitis with help of roller bandage and the
limb will be elevated. This procedure will be repeated
two times in a day continuous for 2days
Phase 3: After second application of intervention the
post test level of Phlebitis assessed by using the
Jacksons visual infusion phlebitis scale.
528
Krupa et al.,
RESULTS
Table1: Analysis of observational score on effectiveness of glycerin
phlebitis among patient
Standard
GROUP
N Mean Deviation
Std. Error Mean
EXPERIMENTAL 30 1.10 0.71
0.130
POST CONTROL
30 2.53 0.78
0.142
Fig 1: Cone diagram showing Pre Test Phlebitis

Level among Experimental Group and Control
Group
Fig 2: Bar diagram showing Post Test Phlebitis

Level among Experimental Group and Control
Group
DISCUSSION
Joseph Jency.(2009), conducted a quasi-experimental
study was conducted in a selected hospital of
Mangalore to compare the effectiveness of selected
nursing interventions such as hot fomentation,
thrombophob and icthammol glycerine on patients
with phlebitis related to peripheral intravenous
infusion.[7] The sample consisted of 45 subjects who
had developed intravenous infusion related phlebitis,
Signs and symptoms of phlebitis was measured by
phlebitis measurement chart, erythema observation
magnesium sulphate dressing on

Mean
Difference
t value p value
-1.43
7.454
<0.001
check list and pain scale. Three treatments were

administered to 15 patients each for 3 days two times
a day. The data analyzed by using ANOVA andt
test. The findings of the study revealed that among
the three modalities of treatment of phlebitis, it was
found that warm icthammol glycerine dressing was
most effective in reducing in duration, swelling,
palpable venous cord, erythema and pain at p<0.001.
The pre-treatment pain score were 7.67 and it was
reduced to 1.47 on the 3rd post-treatment day. [8]The
pre test of experimental group that majority
20(66.7%) hospitalized patient had medium stage of
phlebitis, 9(30%) hospitalized patient had Advanced
stage of phlebitis or start of thrombophlebitis and
1(3.3%) hospitalized patient had Advanced stage
Thrombophlebitis.
The pre test of control group that majority 9(30%)
hospitalized patient had medium stage of phlebitis,
5(16.7%) hospitalized patient had possibly first signs
of phlebitis and 16(53.3%) hospitalized patient had
Early stage of phlebitis.
The post test of experimental group that majority
15(50%) hospitalized patient had possibly first signs
of phlebitis, 9(30%) hospitalized patient had Early
stage of phlebitis and 6(20%) hospitalized patient had
no sign of phlebitis.
The post test of control group that majority 15(50%)
hospitalized patient had medium stage of phlebitis,
10(33.3%) hospitalized patient had Early stage of
phlebitis had possibly first signs of phlebitis, 3(10%)
hospitalized patient had possibly first signs of
phlebitis and 2(6.7%) hospitalized patient had
Advanced stage of phlebitis or start of
thrombophlebitis. [9]
In experimental group post test mean score 1.10, SD
was 0.71 respectively. In control group post test mean
score 2.53, SD was 0.78 respectively. The obtained
value 7.454 statistically was significant at 0.001 level.
So research hypothesis was accepted.
529
Krupa et al.,
CONCLUSION
There was significant difference between post
intervention phlebitis among the experimental group
and control group. All the statistical evidence showed
in phlebitis scale, which is directly proportionate to
the effectiveness of the glycerin magnesium dressing
on phlebitis patient.
8. Anderson C, Bergbrant im, Frdin T. The

cochrane central register of controlled trials. The
cochrane
collaboration.
Available
on
http://onlinelibrary.wily.com/o/clcentral/article/0
42/cn-00408640/frame. html
9.Nursing times, Phlebitis: treatment, care and
prevention2011;107:36
ACKNOWLEDGEMENT
The researcher deeply in depted to the Almighty God,
for his omnipotent presence, bountiful blessings,
wisdom and inspiration through out the study. I
would like to express my heartfelt thanks and
gratitude to all faculties who guided me to complete
this study. It is my privilege to convey my sincere
gratitude and thanks to and the participants who has
participated in this study.
REFERENCES
1. Higginson R, Parry A. phlebitis: treatment, care
and prevention. Nursing times 2011 September
13;107(36):18-21
2. Sherril A RN, White J. Intravenous therapy
related phlebitis rates in an adult population.
Journal of IV nursing. 2001;24(1):19-24
3. Smeltzer SC, Bare BG, Hinkle JL, Cheever KH.
Brunner & Suddarths Text book of MedicalSurgical Nursing. 12th ed. New Delhi (India):
Wolters Kluwer; 2010;1: 308-09.
4. Kagel EM, Rayan GM. Intravenous catheter
complications in the hand and forearm. J Trauma
[serial online] 2004; 56(1):123-7.
5. Martin D, Joshy M, Jain N. Effectiveness of
Planned Teaching Programme Regarding
Peripheral Intravenous Infusion among Staff
Nurses. Ind J N Stud 2011; 2(1): 45-50.
6. Waitt C, Waitt P, Pirmohamed M. Intravenous
Therapy. [Online]. 2004 [cited 2011 Nov 13];
Available
from:
URL:http://pmj.bmj.com/content/80/939/1.full
7. Zheng GH, Yang L, Chu JF, Chen HY. Aloe
Vera for Prevention and Treatment of Infusion
Phlebitis. [Online]. [2011?] [cited 2011 Nov 20];
Available
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858.CD009162/pdf
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Krupa et al.,
DOI: 10.5958/2319-5886.2015.00102.2

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MORPHOMETRY OF THE ARTICULAR FACETS ON THE SUPERIOR, MEDIAL AND LATERAL

SURFACES OF THE BODY OF TALUS AND ITS CLINICAL RELEVANCE
*Goda Jatin B1, Patel Shailesh M2, Parmar Ajay M3, Agarwal GC4
1, 3
Assistant Professor, 4Professor & Head, Department of Anatomy, Pacific Medical College & Hospital, Udaipur
2
Professor & Head, Department of Anatomy, Government Medical College, Bhavnagar
*Corresponding author email: drjbgoda@gmail.com
ABSTRACT
Background: In the formation of Ankle joint, tibio-fibular mortice receives superior, medial and lateral articular
surfaces of body of Talus. Because of very limited availability of the data on the Morphometry of the articular
facets on the Body of the dry human tali, this study was undertaken. Aims: To prepare the database on the
articular facets on the superior, medial and lateral surfaces of body of talus, to find if there is statistically
significant difference between both the sides of measurements and to compare the results with the previous
studies. Methods and Material: 40 Dry Human Tali (20 Right and 20 Left) were measured with Digital vernier
caliper for the following Measurements: On the Trochlear surface: Medial length, Central length, Lateral length,
Anterior width, Central width, Posterior width. On the lateral triangular articular facet: Central height, Central
width. On the coma shaped medial articular facet: Central height, Central width. Results: Mean values of Medial,
Central and Lateral lengths were 31.02, 30.39 and 29.63mm on Right side and 31.79, 30.65 and 29.45mm on Left
side. Mean Anterior, Central and Posterior widths were 28.87, 28.16 and 21.59mm on right side and 29.08, 27.54
and 21.78mm on left side. On the medial articular surface, mean central height was 11.93mm on the right side and
11.29mm on the left side, Mean central width was 27.94mm on the right side and 28.29mm on the left side. On
the lateral articular surface, mean central height was 22.14mm on the right side and 22.63mm on the left side.
Mean central width was 18.93mm on the right side and 18.99mm on the left side. There is no significant
difference between right and left sides of measurements. Conclusion: The trochlear articular surface is wider in
front, measurements of opposite talus bone can be used as a control during talus bone replacement surgery, it may
help surgeons to plan pre-operatively the complex talar fracture surgeries, to design accurate talus bone prosthesis
and talus implants.
Keywords: Articular facets, Talus, Ankle joint, Trochlear surface, Talar implants.
INTRODUCTION
Talus receives the whole weight of the Body and
transmits it to the tarsal bones. Talus forms the
connecting link between the bones of the foot and the
leg. The superior surface and adjacent medial and
lateral surfaces of the Body of Talus are received by
the Tibio-fibular mortice and form the ankle joint [1].
The talar trochlear surfaces is convex parasagittally
and gently concave transeversely, being wider in
front. The talar articular surface for medial malleolus

is fairly flat, coma shaped and deeper anteriorly. The
larger lateral talar articular surface is triangular and
vertically concave [2]. In the studies on dead skeletal
material, Talus is convenient as it is preserved better.
A clear understanding of these articular surfaces has
applications in designing of ankle braces to ankle
implants and in total ankle replacements.
Jatin et al.,
531
Furthermore, it can be useful in explaining talar

morphology and ankle joint kinematics. Uptill now,
studies have been done on talar morphological
features like length, breadth, height, volume, Angles
of declination and inclination, Anatomical variations
of trochlear surface etc.[3,4,5]. This is one of very few
studies showing Morphometry of Articulating
surfaces on superior, medial and lateral surface of
Body of Talus.
In this study, 40 Dry HumanTali (20 Right and 20
Left) obtained from the Department of Anatomy,
Pacific Medical College and Hospital, Udaipur.
With the help of Digital vernier caliper the following
Measurements of Talus were taken irrespective of the
sex of bone:
On the Trochlear surface: Medial length, Central
length, Lateral length, anterior width, Central width,
Posterior width (Fig1)
On the lateral triangular articular facet: Central
height, Central width (Fig 2).
On the coma shaped medial articular facet: Central
height, Central width (Fig 3).
The Data was tabulated and Analysed statistically
using excel worksheet. Mean values and standard
deviation of each measurement were calculated.
Unpaired t-test was applied to find the signifance of
difference between Right and Left sides of
measurements.
Fig 2: Measurements on the triangular lateral

articular facet of Talus. Central height & Central
width.
Fig 3: Measurements on the coma shaped medial

articular facet of Talus. Central height & Central
width
RESULTS
The results found in this study are tabulated in Table
1. P values showed that there is no statistically
significant difference between the right and left sides
of parameters.
Table1: Measurements of the articular facets on
Superior, Medial and Lateral articular surfaces of
body of Talus
Superior Articular Surface
Paramerter
meanSD
Fig1: Measurements on the Trochlear surface of

Talus. First row shows Anterior, Central and
Posterior width from left to right and the second
row shows Lateral, Central and Medial length
from left to right.
meanSD
Right side(mm) Leftside (mm)

Medial Length
31.021.83
31.791.34
Central Length
30.391.63
30.650.91
Lateral Length
29.630.65
29.451.59
Anterior Width
28.871.73
29.082.73
Central Width
28.161.60
27.542.32
Posterior Width
21.591.42
21.781.47
Medial Articular Surface
Central Height
11.931.44
11.291.17
Central Width
27.942.90
28.291.79
Lateral Articular Surface
Central Height
22.141.71 22.633.24
Central Width
18.930.92 18.991.59
P
value
0.13
0.53
0.53
0.77
0.33
0.68
0.13
0.65
0.55
0.90
SD = Standard Deviation, *If p < 0.05, the difference

is significant between right and left sides.
532
Jatin et al.,
DISCUSSION
On the superior Articular surface, the mean values of
Medial, Central and Lateral length were 31.02, 30.39
and 29.63mm on Right side and 31.79, 30.65 and
29.45mm on Left side. Mean Anterior, Central and
Posterior widths were 28.87, 28.16 and 21.59mm on
right side and 29.08, 27.54 and 21.78mm on left side.
Mean central height on the medial articular surface
was 11.93mm on the right side and 11.29mm on the
left side, Mean central width on the medial articular
surface was 27.94mm on the right side and 28.29mm
on the left side. Mean central height on the lateral
articular surface was 22.14mm on the right side and
22.63mm on the left side. Mean central width on the
lateral articular surface was 18.93mm on the right
side and 18.99mm on the left side. This
measurements shows that trochlear articular surface is
respectively which were higher compared to present

study.
CONCLUSION
The trochlear articular surface is wider infront, there
is no significant difference between right and left
sides of measurements. The difference in the mean
values compared to other studies may be due to
inherent population variations which may be because
of genetic and environmental factors like climate,
nutrition etc. As there is no significant difference
between right and left sides of measurements,
measurements of opposite talus bone can be used as a
control during talus bone replacement surgery, it may
help surgeons to plan pre-operatively the complex
talar fracture surgeries, to design accurate talus bone
prosthesis and talus implants[9]. The limitation of the
study is that the mean values of the parameters may
differ in various ethnic groups suggesting the need for
different normograms in each group.
ACKNOWLEDGEMENT
We are thankful to Mr. Darshan Mavadiya for his
statistical assistance.
REFERENCES
wider in front.
Fig 4: Comparision of mean values of present
study with study of ShishirKumar.
Fig 4 shows the comparision of the measurements
taken on the superior articular surface of the body of
Talus between this study and the study done by
shishirKumar [6]. As shown in Graph, Mean values of
Medial, central and lateral lengths were higher on the
Both sides where as mean value of the posterior
width was higher on the left side in the study done by
Dr. ShishirKumar.
Gautham K[7] found in his study the mean maximum
transeverse width on the body of Talus was 37.94mm
on the right side and 36.80mm on the left side which
was higher compared to present study. Mean
Trochlear length was 30.62mm on right side and
30.44mm on the left side.
Ilhan Otag[8] found in his study that the mean values
of talar width, Trochlear length and Trochlear breadth
were 40.79, 33.45 and 31.69mm on right side and
43.39, 34.12 and 31.72mm on the left side
1. Datta AK. Essentials of Human Anatomy, Part 3,

3rd Edition, Current Books Internationals. 2004;
151-52.
2. Grays Anatomy. The Anatomical Basis of
clinical Practice. 40th Edition, Elsevier Churchil
Livingstone. 2008; 4279.
3. Javia MD, Patel MM, Kubavat DM, Dixit
Daksha, Singel TC. Morphometry of the Talus on
the Basis of Sexual Dimorphism. Indian Journal
of Research. June 2013, 3(5): 208-12.
4. Motagi MV, Kottapurath SR, Dharwadkar
Kavitarati. Morphometric analyses of human dry
tali of South Indian origin. International Journal
of Medical Science and Public Health 2015;4(2):
237-40.
5. Khadija I, Sundus A, Shirza N. Anatomical
Variations of Trochlear surface of Talus. JUMDC
2012; 3(1): 38-41.
6. Shishirkumar, Dr. Nambiar S, Dr. Arunachalam
Kumar, Dr. Patil GV. Morphometric Analysis of
Superior Articulating surface of Talus.
Jatin et al.,
533
International Journal of science and research,

2014; 3(6): 2387-91.
7. Gautham K, Clarista MQ, Sheela N,
Vidyashambhava P. Morphometric Analysis of
The Human Tali. CIBTech Journal of Surgery,
2013; 2(2): 64-68.
8. Otag I, Cimen M. Morphometric Measures of
Talus Bone in Skeleton Remains Belonging to
Anatolian Geography. Indian Journal of Applied
Research. 2003, 3(8), 530-31.
9. K. Islam, A. Dobbe, A. Komeili, K. Duke, M.
El-Rich, S. Dhillon, S. Adeeb, N. M. Jomha.
Symmetry analysis of talus bone. A Geometric
morphometric approach. Bone Joint Res. May
2014; 3(5): 13945.
534
Jatin et al.,
DOI: 10.5958/2319-5886.2015.00103.4

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Health Sciences
www.ijmrhs.com
Copyright @2015
th
th
Research article
ISSN: 2319-5886
REGENERATION OF ARTICULAR CARTILAGE UNDER THE IMPLANTATION OF BONE

MATRIX
Yuri M. Iryanov1, *Nikolay A. Kiryanov2, Olga V. Dyuriagina 3, Tatiana Yu. Karaseva4, Evgenii A. Karasev5
1
Head of the Laboratory of Morphology, Doctor of Biological Sciences, Professor. Russian Ilizarov Scientific
Center Restorative Traumatology and Orthopaedics (RISC RTO), Kurgan, Russia
2
Head of Department of Pathology of Izhevsk State Medical Academy, Doctor of Medical Science, Professor,
Russia
3
A Senior Researcher, Laboratory of Purulent Osteology and Limb Defect Filling, RISC RTO, Candidate of
Veterinary Sciences, Russia
4
Head, 5A Researcher, Department of Orthopaedics, RISC RTO, Candidate of Medical Sciences, Russia
*Corresponding author email: kirnik@list.ru
ABSTRACT
Background: The amage or loss of articular cartilage is costly medical problem. The purpose of this work morphological analysis of reparative chondrogenesis when implanted in the area of the knee joint cartilage of
granulated mineralized bone matrix. Material and Methods: The characteristic features of the knee cartilage
regeneration studied experimentally in pubertal Wistar rats after modeling a marginal perforated defect and
implantation of granulated mineralized bone matrix obtained according to original technology without heat and
demineralizing processing into the injury zone. Results: This biomaterial established to have pronounced
chondro- and osteoinductive properties, and to provide prolonged activation of reparative process, accelerated
organotypical remodeling and restoration of the articular cartilage injured. Conclusion: The data obtained
demonstrate the efficacy of in clinical practice for the treatment of diseases and injuries of the articular
cartilage.
Keywords: Articular cartilage, The mineral component of bone matrix, Implantation, Reparative chondrogenesis.
INTRODUCTION
The damage or loss of the articular cartilage due to
congenital anomalies, degenerative joint diseases or
injuries has a negative effect on the quality of life of
the injured in all age-related groups, and it is
expensive to treat [1]. Surgical methods are often
inadequate, and in many cases they do not give
positive results in terms of proper replacement of
articular hyaline cartilage [2, 3]. In this regard, the
development,
scientific
substantiation,
and
experimental morphologic evaluation of the
biomaterials which have chondrogenic activity are of
major importance in modern medicine. The most
widely used biomaterials (demineralized bone matrix,
matrices of polylactic and polyglycolic acids /PLA
and PGA/, collagen cryogels, analogs of bone

mineral, polysaccharides of natural origin) are
characterized by low plastic effectiveness, the
absence of chondrogenic activity, and limited
biocompatibility [4, 5, 6,7]. Our previous studies have
demonstrated that mineralized bone matrix (MBM)
obtained by using original technology without
thermal and demineralizing processing [8] has
osteoinductive properties, and it stimulates
osteogenesis effectively [9]. The research is aimed at
performing morphological analysis of reparative
chondrogenesis when this biomaterial is implanted
into the zone of the knee cartilage injury.
535
Kiryanov et al.,

All experiments in this study were performed in
accordance with guidelines for animal research and
were approved by our academy Ethics Committee.
Experimental groups. The experiments were
conducted on 25 pubertal Wistar rats male of 340
390 g weight (20 experimental and 5 intact ones). The
process of keeping, experimenting, and euthanizing
animals was regulated by the normative documents
[2]
. The operations on the animals were performed in
an operating room under general anesthesia (8 mg of
Rometar and 4 mg of Zoletil per 1000 g of body
weight intramuscularly). Non-through fenestrated
defects of 2.5-3 mm in diameter were made on the
patellar surface of the distal left and right femur with
the dental drill, the subchondral bone was penetrated.
Sterile granulated allogenic MBM (2-3-mg) obtained
from tubular bones of mongrel rats by original
technology [6] was placed into the right bone defect.
The defect in the left bone was left untreated under
blood clot and it was used as control. The knee
cartilage was studied in five intact normal rats for
comparison. The animals were taken out of the
experiment after 7, 15, 30, and 60 days after the
surgery (five animals were used for each time point).
Morphological evaluation. The pieces of femur bones
of the operated rats and those of intact ones were
fixed and embedded in paraffin (after decalcification)
and in araldite (without decalcification). Paraffin
sections were stained with hematoxylin-eosin, with
picrofuchsin according to Van Gieson, and with
Alcian blue (Alcian blue 8GS) at pH 2.5 and 1.0 to
reveal non-sulphated glycosaminoglycanes (NSGAG)
and sulphated ones (SGAG), respectively. The
content of glycosaminoglycanes was determined in
conventional units (c.u.) by defining Alcian blue
concentration in sections which was measured using
INCA-200
Energy
energy-dispersion
X-ray
spectrometer (X-ray electron probe microanalyzer)
(Oxford instruments, England) [4]. The structure of
MBM granules and regenerated bones in the injured
area was investigated using JSM-840 scanning
electron microscope and JEM-2010 transmission
electron microscope (Jeol, Japan). The results of
quantitative investigations were processed using the
methods of variation statistics. The significance of the
differences of the parameters compared was
calculated using Student's t-test. The differences were

considered significant at the level of <0.05.
RESULTS
The implanted MBM granules are of 50-200 m in
diameter and have a well-ordered highly porous
structure. The pores correspond to the places of
localizing bone lacunae and bone tubules from which
cells and other organic components have been
removed when obtaining the biomaterial.
Seven days after the surgery, the joint capsule in both
left and right limbs was hyperemic and edematic. The
cartilaginous coating was dull. The signs of
inflammatory reaction, and alternatively destructive
changes affecting all metaepiphyseal components
could be revealed around the injury. Foci of
hematoma were found. They were infiltrated by fibrin
clots, little-differentiated cell elements, neutrophilic
granulocytes, macrophages, mast cells, extravasal
erythrocytes, and lymphocytes. Leukocytic necrotic
masses containing lysed cells and fibrin strata could
be observed. Articular cartilage was scarified. The
defect zone was partially replenished by granular and
little-differentiated loose edematic connective tissue
and few vessels. Sporadic bone-osteoid foci and
isolated thin bone trabeculae were identified around
the injury of the right bone (experiment). Reparative
bone formation occurred by the type of
intramembranous osteogenesis. NSGAG and SGAG
content in the zone of articular cartilage injury was
reduced significantly comparing with the similar
values in intact animals (0.590.03, and 0.680.03
c.u., respectively), however, there were no significant
changes revealed in their content in control and
experiment at this stage (Table 1).
Table 1: Glycosaminoglycane content in the zone of the
knee cartilage injury in rats without defect plasty
(control), and when using allogenic MBM (experiment)
(Mm, c.u.)
The period
of the
experiment,
days
7
15
30
60
Control
Experiment
NSGAG
SGAG
NSGAG
SGAG
0.180.01
0.190.01
0.210.01
0.220.01
0.110.01
0.120.01
0.160.01
0.240.01
0.200.01
0.490.01*
0.560.02*
0.590.03*
0.120.01
0.260.01*
0.540.01*
0.700.01*
*Significant changes comparing with control values.

Student's t-test <0.05
536
Kiryanov et al.,
Fifteen days after the surgery, numerous leukocytes,

fibroblastic elements, bundles of collagen fibers,
granulation tissue, and a significant number of dilated
and filled with blood vessels could be identified in
the defect central zone of control bone. Cellular-andfibrillar elements of inflammation were not found in
the right bone (experiment), reparative bone
formation occurred not only by the type of
intramembranous osteogenesis, but by that of
enchondral osteogenesis as well. In the zone of
subchondral bone injury an extensive small-looped
network was formed consisting of thickened
trabeculae covered by rows of numerous large
osteoblasts which produced spongy bone closely
adhered with the surface of underlying bone.
Implanted MBM granules resembled cystic cavities
formed during decalcification of the sample and due
to biodestruction. Functionally active osteoclasts with
multiple nuclei and a brush-like border were located
within these cavities (Fig. 1). Numerous sinusoids
were surrounded by proliferating peri-vasculocytes.
Fig 1a
Fig 1b
ultrastructure of the osteoclast localized on the surface

of the implanted MBM granule, the arrow points to the
osteoclast brush-like border in the zone of contact,
ultrathin sections, transmission electron microscopy,
magnification 8000.
There were no hemorrhages and destruction foci in

these areas, as well as active proliferation of
fibroblasts, intense neoangiogenesis was observed.
The layers of osteo- and chondrogenic cells at
different differentiation stages were located on the
granule surface. The zone of articular cartilage defect
was partially filled with newly formed hyaline
cartilage. Its intercellular substance contained
NSGAG predominantly (Table 1). The defect edges
merged with the edges of maternal articular
cartilage where cell nests with isogenic groups were
identified. The presence of the latters was evidenced
by proliferative activity of some chondrocytes which
were one of the sources of the cartilaginous tissue
filling the defect. The cells located round MBM
granules were the second source, and the
chondrocytes in the zone of enchondral osteogenesis
in the sites of subchondral bone fracture healing
presented the third one. Glycosaminoglycane
concentration in the defect zone for experiment more
than twice exceeded control values (Table 1).
A significant part of bone defect was filled with loose
or dense connective tissue 30 and 60 days after
surgery in control (Fig. 2, , c). Newly formed
cartilaginous tissue was formed only in the area of
subchondral bone as a result of its injury, and it grew
from the defect edges in the form of strip-like
structures. In experiment the defect of articular
cartilage was almost completely filled with newly
formed hyaline cartilage having a smooth surface
(Figure 2, b) which grew not only from the
subchondral bone, but mainly from the defect edges
where the isogenic groups of cells were located
thereby, as evidenced by proliferative activity of
chondrocytes (Fig. 2d).
Fig 1: Zone of the defect of the rat knee cartilage 15

days after the surgery: a n osteoclast within
the implanted MBM granule which has the form of a
cystic cavity, paraffin sections, staining with
hematoxylin-eosin, magnification 400; b
537
Kiryanov et al.,
Fig2a
Fig 2b
Fig 2c
Fig 2d
Fig2: Zone of the defect of the rat knee cartilage 30

days after surgery: a, control, the defect is
filled with loose fibrillar connective tissue; b, d
experiment, the defect is filled with newly formed
hyaline cartilage.( a, b - paraffin sections, staining with
Alcian blue at 2.5, magnification 200; fibroblasts and collagen fibril, ultrathin sections,
transmission electron microscopy, magnification
5000; d - chondrocytes are arranged as isogenic groups,
scanning electron microscopy, magnification x 1300.)
The surface of newly formed hyaline cartilage

acquired the shine characteristics of the articular
cartilage of the intact animals. The cell population of
the regenerated cartilage was mainly represented by
proliferating chondrocytes; its structure did not
possess the characteristics of the articular cartilage.
Thirty days after surgery, the content of
glycosaminoglycane non-suplphated and sulphated
forms in the defect for experiment more than twice
exceeded control values, but they did not differ from
one another significantly. Sixty days after the
surgery, glycosaminoglycane sulphated forms
prevailed. It was evidenced by the higher maturity
degree of the newly formed cartilaginous tissue
(Table 1) which reached the maturity degree inherent
for the articular cartilage of intact animals.
DISCUSSION
Mature hyaline cartilage is known to have a slight
potential of restoration due to low density of cells and
little mitotic activity of chondrocytes [9, 14]. Large
defects of the articular cartilage are filled with
biomechanically improper fibrillar cartilage followed
by osteoarthritis development [10]. The advent of
tissue engineering techniques has provided alternative
possibilities to treat such patients by using cell
therapy combined with synthetic substitutes of
extracellular matrix and biologically active factors for
functional replacement of articular hyaline cartilage
[11, 12, 13]
. The studies have demonstrated that the
MBM granules when implanted into the defect zone
of articular cartilage are identified in all the periods
of further observation, they have marked
chondromodulating effect, demonstrate prolonged
activation of reparative chondro- and osteogenesis.
Regenerated cartilage is formed early in the zone of
articular cartilage injury, and it acquires the cell
specificity of hyaline cartilage tissue. Integrated
cartilage coating is formed, the disordered contours of
articular surface are aligned gradually, thereby
leading to complete or partial recovery of the joint
functional activity. The newly formed cartilage
dominated sulfated glycosaminoglycans, which
indicates a high degree of maturity [14, 15]. Growth
factors localized in the implanted MBM granules, as
well as bone morphogenetic proteins being released
during osteoclastic resoption provide chondro- and
osteoinductor properties for the granules [16, 17, 18].
538
Kiryanov et al.,
CONCLUSION
Thus, the studies have demonstrated that the use of
the implant of granulated MBM as chondro- and
osteogenesis stimulator and as a corrector of
destructive disorders in bone and cartilaginous tissue
in articular cartilage injuries seems theoretically
justified and promising.
10.
11.
ACKNOWLEDGEMENT
12.
We thank the staff of our institutions for their help in
carrying out experiments and supervision over
animals during all stages of work.
13.
14.
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Morfologiia. 2013;143(1):63-68.
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Morfologicheskie vedomosti. 2010;(3):77-81.
5. Lysenok LN. Biomaterials: contribution to the
progress of modern medical technologies.
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6. Patent RF. Biomaterial for filling bone defects
and a technique to obtain it. Ir'ianov IuM,
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7. Shishatskaia EI. Cell matrices of resorbable
polyhydroxyalkanoates. Kletochn Transplantol
Tkan Inzheneriia. 2007; 2(2):68-75.
8. Bessa PC, Casal M, Reis RL. Bone
morphogenetic proteins in tissue engineering: the
road from laboratory to clinic, part II (BMP
delivery). J Tissue Eng Regen Med. 2008; 2(23):81-96.
9. Brown TD, Johnston RC, Saltzman CL, Marsh
JL, Buckwalter JA. Posttraumatic osteoarthritis: a
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burden of disease. J Orthop Trauma. 2006;
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Buckwalter JA. Articular cartilage injuries. Clin
Orthop Relat Res. 2002; (402):21-37.
Danisovic L, Varga I, Zamborsky R, Bhmer D.
The tissue engineering of articular cartilage: cells,
scaffolds and stimulating factors. Exp Biol Med
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Duguy N, Petite H, Arnaud . Biomaterials and
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Eberli D, Atala . Tissue engineering using adult
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Strehl R, Tallheden T, Sjgren-Jansson E, Minuth
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Chung C, Burdick JA. Influence of threedimensional hyaluronic acid microenvironments
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Park KH, Na K. Effect of growth factors on
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DOI: 10.5958/2319-5886.2015.00104.6

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Health Sciences
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Volume 4 Issue 3
Coden: IJMRHS
th
Revised: 20th Apr 2015
Research article
Copyright @2015
ISSN: 2319-5886
EVALUATIONOF DIPYRIDAMOLE ON ACUTE AND SUBACUTE MODELS OF INFLAMMATION

IN MALE WISTAR RATS: AN EXPERIMENTAL STUDY
*Angadi Netravathi B1, Hiremath Shrishail V 2, Suranagi Vijayalakshmi V 3
1, 2
Department of Pharmacology, 3Department of Pathology, KLE Universitys J. N. Medical College, Nehru

Nagar, Belagavi,Karnataka, India.
*Corresponding author email: drnetra.angadi@gmail.com
ABSTRACT
Background: Atherosclerosis and its complications remains the major cause of death and premature disability.
Atherogenesis involves elements of inflammation, a process that now provides a unifying theme in the
pathogenesis of the disease. Anti-platelet drugs are currently used in the treatment of atherosclerosis and its
complications. Our study evaluated the influence of dipyridamole on acute and sub-acute models of inflammation
in male Wistar rats. Methods: Male Wistar rats (150-200g) were divided into three groups i.e. control, Aspirin
and dipyridamole (n=6 animals in each group). The effect of dipyridamole, administered orally, on inflammation
was studied using acute (carrageenan induced rat paw edema) and sub-acute (cotton pellet granuloma and
histopathological examination of grass piths) models. Experiment was conducted according to the Committee for
the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines. Analysis was done
using one way ANOVA followed by Post Hoc Test of Dunnets. P<0.05 was considered as statistically significant.
Results: Dipyridamole showed significant inhibition of rat paw edema in acute model (P<0.01) and granuloma
dry weight, in sub acute model of inflammation when compared to control (P<0.01). Histopathological
examination of grass pith revealed markedly reduced fibroblasts, granulation tissue, fibrous tissue and collagen in
dipyridamole group when compared to control. Conclusion: Dipyridamole exhibited a significant anti
inflammatory activity in acute and sub-acute models of inflammation.
Keywords: Dipyridamole, Aspirin, Carrageenan, Inflammation.
INTRODUCTION
Cardiovascular diseases remain the major cause of
death and premature disability in developed
societies.Current predictions estimate that by the year
2020 cardiovascular diseases, notably atherosclerosis
and hypertension will become leading global causes
of total disease burden. [1]
Atherogenesis involves elements of inflammation, a
process that now provides a unifying theme in the
pathogenesis of the disease. [2, 3, 4] Key inflammatory
factors in atherothrombosis include activated
endothelial cells, like inflammatory leucocytes,
smooth muscle cells and platelets. [4]
Platelet activation leads to surface expression of Pselectin, which promotes the formation of plateletleukocyte complexes, surfaceexpression of CD-40
Ligand and also platelet itself releases various
inflammatory mediators such as Platelet activating
factor (PAF), Platelet factor-4, RANTES (regulated
upon activation normal T-cell expressed and secreted)
and Tissue factor. Thus, drugs that simultaneously
block thrombotic occlusion and reduce inflammation
may have added benefits in the treatment of
cardiovascular disease. [4]
540
Netravathi et al.,
Dipyridamole is a vasodilator that inhibits platelet

function by inhibiting adenosine uptake and cGMP
phosphodiesterase activity. Certain in vitro studies
suggest that dipyridamole inhibits secretion of
monocyte chemo attractant Protein-1, matrix
metalloproteinase-9 (MMP-9) and tissue factor which
in turn leads to inhibition of platelet- monocyte
interaction. And also it acts indirectly through
adenosine and prostaglandin I-2, inhibiting
lymphocyte recruitment, activation and secretion of
pro inflammatory mediators suggesting its antiinflammatory activity. [5, 6]
Studies have shown that, inflammation drives all
phases of atherosclerosis, including initiation,
progression and thrombotic complications of the
lesion. [3]In view of paucity of anti-inflammatory
studies of dipyridamole, the present study was
planned to evaluate the effect of dipyridamole on
acute and sub acute models of inflammation in male
Wistar rats.
Study design: An experimental animal based study
The study was approved by Institutional Animal
Ethical Committee of KLE Universitys J. N. Medical
College, Belagavi. 18 male wistar rats (150- 200 g)
were used for present study. They were fed with
standard pellet diet and water ad libitum. All animals
were acclimatized 12:12 h light - dark cycle for one
week before the experiment session. All experiments
were done following the guidelines of CPCSEA
(Committee for the Purpose of Control and
Supervision of Experiments on Animals).
Grouping: Adult male healthy Wistar rats weighing
150- 200 g were obtained from the central animal
house, J. N. Medical College, Belagavi and were
acclimatized to 12:12 h light - dark cycle for 10 days
prior to the day of experimentation. Rats were
divided into three groups of six each, on which both
acute and subacute studies were carried out with a
gap of 2 weeks. Group I received 0.5ml of 1% gum
acacia suspension orally(control group), group II
received aspirin (Standard group) and group III
received dipyridamole (Test group).
Drugs administration: Aspirin was administered in
the dose of 200 mg/kg body weight of rat, equivalent
to 2222 mg of clinical dose orally in 1% gum acacia
suspension. [7, 8]
Dipyridamole was administered in the dose of 27

mg/kg body weight equivalent to 300 mg of clinical
dose orally.[7,8]
Carrageenan (Sigma Co. St. Louis) was administered
as a suspension in 1% warm normal saline given in
the volume of 0.05 ml per rat paw.
Acute inflammation was produced by injecting
carrageenan into right hind paws and sub-acute
inflammation by randomly implanting a foreign body
subcutaneously in axilla and groin as described
below.
1. Carrageenan induced rat paw edema [9]
Rats were divided into three groups of six each.
(Same animals were used for foreign body induced
inflammation)They were starved overnight with
water ad libitum prior to the day of experiment.
Group I (control) received 0.5ml of 1% gum acacia
suspension, orally; group II (standard) received
aspirin 200mg/kg orally in 1% gum acacia suspension
and group III received dipyridamole27 mg/kg orally
in 1% gum acacia suspension.[7,8] Aspirin was taken
as the standard anti-inflammatory drug.
Thirty minutes after aspirin and dipyridamole
administration, 0.05ml of 1% w/v carrageenan
suspension was injected into the sub-plantar region of
right hind paw. A mark was put on the hind limb at
the malleolus to facilitate uniform dipping at
subsequent readings. The paw edema volume in
milliliters was measured with the help of a
plethysmograph by mercury displacement [9] method
at zero hour i.e. immediately after injecting
carrageenan. The same procedure was repeated at 0.5,
1, 3, 4 and 5 hours. [9] The percentage inhibition of
edema in the various treated groups was then
calculated by using the formula,[9]
% of inhibition of edema =1-
Mean increase in treated group

Mean increase in control group
X100
2. Foreign Body Induced Granuloma Method[11,12]

Rats were divided into three groups of six each.
Under thiopentone anesthesia, each rat was implanted
subcutaneously with two sterile cotton pellets
weighing l0mg each and two sterile grass piths
(25x2mm) through a small incision in all rats.
Wounds were then sutured and animals were caged
individually after recovery from anaesthesia. Aseptic
precautions were taken throughout the experiment.
The treatment was started on the day of implantation
541
Netravathi et al.,
and was repeated every twenty-four hours, regularly,

for ten days. [10]
On the eleventh day, the rats were sacrificed to
remove the cotton pellets and grass piths. The grass
piths were preserved in 10% formalin for
histopathological studies. Sections were stained with
haematoxylin and eosin, and the grass pith with
granulation tissue in each group was studied
microscopically.
The pellets, free from extraneous tissue, were dried
overnight at 60C to note their dry weight. Net
granuloma formation was calculated by subtracting
initial weight of cotton pellet (10mg) from the
weights recorded. Mean granuloma dry weight for
various groups was calculated and expressed as
mg/100 gm body weight. [11, 12] The percentage
inhibition of granuloma dry weight was calculated
using the formula, [10]
Dry weight of granuloma in treated
% of inhibition of granuloma=1X100
Dry weight
Dry weight of granuloma in control
Statistical analysis: The data for all the groups was

expressed as Mean SEM and were analyzed by one
way ANOVA (Analysis of variance) followed by
Dunnets test using Graph pad prism software and P
< 0.05 was considered statistically significant.
RESULTS
In the present study, dipyridamole in therapeutic
equivalent dose was investigated for its possible anti
inflammatory effect, in acute and sub-acute models of
inflammation. Carrageenan induced acute
inflammation:
The edema volume in milliliters (ml), as measured by
mercury displacement using a plethysmograph, for
control group at h, 1h, 3h, 4h, and 5h, was 1.167
0.04, 0.85 0.01 , 0.82 0.02, 0.89 0.01 and 0.89
0.01 (Table- 1) respectively, while the
corresponding mean volumes in aspirin (200 mg/kg)
treated group was 1.033 0.06, 0.77 0.02, 0.34
0.01, 0.30 0.01 and 0.25 0.01 respectively (Table1, Fig 1), with percentage inhibition 12%, 10%,
58.53%, 66.29% and 71.9% respectively indicating
significant (P < 0.01) anti-inflammatory activity of
aspirin (Table - 1, Fig 2).
Dipyridamole in the dose of 27 mg/kg showed
significant inhibition (P < 0.01) of paw edema at h,
1h, 3h, 4h, and 5h, with mean edema volume of 1.008
0.03, 0.77 0.02, 0.43 0.02, 0.36 0.02 and 0.35

0.01 respectively (Table- 1, Fig-1) and percentage
inhibition 14%, 10%, 47.56%, 59.55%, and 60.67%
respectively (Table- 1, Graph- 2).The above results
clearly show the anti-inflammatory effect of
dipyridamole in acute model of inflammation when
compared to control.
Sub-acute inflammation (foreign body induced
granuloma method): The mean granuloma dry weight
of cotton pellet in control group was 22.83 1.138,
while in aspirin treated group, it was significantly
decreased (P<0.01) with the mean value of 150.81
and percentage inhibition of 34.29%.
Similarly, dipyridamole treated group exhibited
statistically significant decrease in granuloma weight
(P<0.01) with mean value of 13.50 0.50 (Table - 2),
with percentage inhibition of 40.86% when compared
to control (Table - 2).
The anti-inflammatory activity of dipyridamole as
observed in both, acute and sub acute studies was
further confirmed by histopathological studies.
The sections of grass pith when stained with
haematoxylin and eosin showed abundant fibrous
tissue in the control group, but revealed reduced
number of fibroblasts, decreased granulation tissue,
collagen content and fibrous tissue in aspirin and
dipyridamole treated groups. (Figures 3-5)
Fig1: Carrageenan induced paw edema when

compared with control group.
Post hoc analysis by Dunnets Test: * P < 0.05,

**P<0.01
542
Netravathi et al.,
Table 1: Effect of aspirin and dipyridamole treatment on carrageenan induced paw edema.
Time after
carrageena
n injection
Control Paw
edema in ml
(Mean SEM)
Aspirin
Dipyridamole
ANOVA
Result
Paw edema in ml
(Mean SEM)
inhibition
%
Paw edema in ml
(Mean SEM)
inhibition
%
P value
hr
1.1670.04
1.033 0.06
12
1.008 0.03*
14
>0.05
1 hr
0.85 0.01
0.77 0.02**
10
0.77 0.02*
10
<0.003
3 hr
0.82 0.02
0.34 0.01**
58.53
0.43 0.02**
47.56
<0.0001
4 hr
5 hr
0.89 0.01
0.89 0.01
0.30 0.01**
0.25 0.01**
66.29
71.9
0.36 0.02**
0.35 0.01**
59.55
60.67
<0.0001
<0.0001
Post hoc analysis by Dunnets Test: *P<0.05, **P<0.01

Table 2: Effect of aspirin and dipyridamole
treatments on granuloma dry weight
Drug treatment
Granuloma dry weight

mg/100 gm b/w
% inhibition
Control
Aspririn
Dipyridamole
22.801.13
150.81**
13.500.50**
-34.29
40.86
Fig 4: Aspirin group grass pith with granulation tissue

containing decreased number of fibroblasts, decreased
granulation tissue, collagen content and fibrous tissue.
(Hematoxylin& Eosin Stain X 10), G- Granulation
tissue, F- Fibrous tissue
Fig 2: Percentage inhibition of carrageenan induced

paw edema
Fig3: Control group grass pith with granulation tissue

containing abundant granulation tissue, fibroblasts, collagen
content and fibrous tissue. (Hematoxylin& Eosin Stain X
10) G- Granulation tissue, F- Fibrous tissue ( G and Fstands
for G- Granulation tissue, F- Fibrous tissue)
Fig 5: Dipyridamole group grass pith with granulation

tissue containing decreased number of fibroblasts,
decreased granulation tissue, collagen content and
fibrous tissue. (Hematoxylin& Eosin Stain X 10)GGranulation tissue, F- Fibrous tissue
Note: As compared to control group, Aspirin and

Dipyridamole group showed decreased number of
fibroblasts, decreased granulation tissue, collagen
content and fibrous tissue. (H& E Stain X 10)
543
Netravathi et al.,
DISCUSSION
Antiplatelet agents are the mainstay of preventive
care because they decrease the incidence of end-stage
vessel occlusion that is responsible for most
cardiovascular events. In addition to thrombosis,
however, it is now appreciated that inflammation
contributes to the development of atherosclerosis and
its complications. In some cases, inflammatory
pathways promote thrombosis, and conversely,
thrombotic events often exacerbate inflammatory
reactions. Thus, drugs that simultaneously block
thrombotic occlusion and reduce inflammation may
have added benefits in the treatment of cardiovascular
diseases. [13, 14, 15]
To prevent cardiovascular disease and its
complications, patients typically receive antiplatelet
therapy to suppress thrombotic events; however, the
inflammatory arm of treatment has not received as
much attention. In the European Stroke Prevention
Study-2 (ESPS-2), aspirin plus extended-release
dipyridamole showed a 23.1% reduction in the
relative risk of stroke events compared with aspirin
alone, indicating that the addition of dipyridamole
improves patient outcomes. [16]
Carrageenan induced rat hind paw edema method was
used to assess acute inflammatory activity. In our
study dipyridamole showed significant inhibition of
paw edema in carrageenan induced paw edema model
when compared to control group. In sub acute model
of inflammation; dipyridamole exhibited significant
decrease in the granuloma weight when compared to
control group in cotton pellet granuloma method. In
grass pith induced granuloma method, the sections of
grass pith when stained with haematoxylin and eosin
showed abundant fibrous tissue in the control group,
but revealed reduced number of fibroblasts, decreased
granulation tissue collagen content and fibrous tissue
in dipyridamole group.
In vitro studies have shown that anti-inflammatory
activity of dipyridamole may be attributed to its
potential to attenuate nuclear translocation of nuclear
factor kappa beta (NF- kB) [5] and property to block
the synthesis of monocyte Chemo attractant Protein1(MCP-1) at the transcriptional level. Dipyridamole
also blocks Interleukin8(IL-8) and matrix
metalloproteinase-9
(MMP-9)
generation
by
lipopolysaccharide-treated monocytes. [17, 18, 19] Since
Netravathi et al.,
inflammation drives all phases of atherosclerosis,

including initiation, progression and thrombotic
complications of the lesion. Drugs that
simultaneously block thrombotic occlusion and
reduce inflammation may have added benefit in the
treatment of cardiovascular disease by virtue of its
anti-inflammatory activity in addition to its antiplatelet activity.
CONCLUSION
Our study result shows that dipyridamole suppresses
the carrageenan induced paw edema and granuloma
formation, thereby acts as an anti- inflammatory
agent. This may be due inhibition of mediators of
inflammation. But these findings need to be verified
by further clinical studies.
ACKNOWLEDGEMENT
The authors would like to thank the staff of
Department of Pharmacology, KLE Universitys J. N.
Medical College, Belagavi for their support.
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Lindhoff-Last E, Harder S. Clopidogrel but not
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P, Lowenthal A. European Stroke Prevention
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the secondary prevention of stroke. Journal of the
Neurological Sciences 1996; 143: 1-13.
17. Weyrich AS, Denis MM, Kuhlmann-Eyre JR,
Spencer ED, Dixon DA, Marathe GK, et al.
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19. Chakrabarti S, Vitseva O, Iyu D, Varghese S,
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DOI: 10.5958/2319-5886.2015.00105.8

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
Received: 18th Mar 2015
Research article
Coden: IJMRHS
Revised: 10th May 2015
Copyright @2015
ISSN: 2319-5886
INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS DISSEMINATED

INTRAVASCULAR COAGULATION SCORING SYSTEM: IS IT A GOOD PROGNOSTIC
INDICATOR IN DIC
*Nisal Amit, Nimbargi Ravindra, Shelke Parineeta, Kelkar Anjali
Department of Pathology, Bharati Vidyapeeth Deemed University Medical College, Pune.
*Corresponding author email: dramitnisal@yahoo.com
ABSTRACT
Background: Disseminated intravascular coagulation is an acquired disorder characterized by intravascular
activation of coagulation due to variety of causes. There is excessive thrombin formation leading to fibrin
deposition in microcirculation and consequent ischemic organ damage. The diagnosis is essentially clinical
supported by laboratory parameters and a scoring system based on these. The mainstay of treatment is correction
of underlying cause and haemostatic support with replacement of coagulation factors. Aim: To evaluate the use
of ISTH DIC scoring system in patients of clinically suspected Disseminated Intravascular Coagulation.
Methods: 60 cases were studied over a period of one year. Patients were selected with a clinical suspicion of DIC
who are having an underlying predisposing condition. Clinical signs and symptoms were recorded. Routine
investigations and the tests necessary to calculate the ISTH score i.e. platelet count, Prothrombin time; D-dimer
and Fibrinogen were done. The scoring criteria of ISTH were applied in these cases. Chi square and Fishers exact
tests were used for analysis of the data. Results: The commonest cause of the underlying disorder in our study
was found to be Sepsis (66.7%) followed by trauma (10%), obstetric causes (8.3%) and solid malignancy (6.7%).
There was a significant association of ISTH score with outcome of the patients (P value <0.05). Raised APTT and
presence of schistocytes also had a significant association with a high ISTH score. Conclusion: ISTH DIC
scoring criteria more precisely define clinical and laboratory parameters of DIC. Its clinical utility will improve
the timely diagnosis, prediction of severity and will also aid in improving prognosis of DIC patients.
Keywords: Disseminated Intravascular Coagulation, International Society on Thrombosis and Haemostasis,
Disseminated Intravascular Coagulation scoring system, Schistocytes, Prothrombin time, Activated Partial
Thromboplastin Time
INTRODUCTION
Disseminated Intravascular Coagulopathy (DIC) is a
consumptive syndrome that is characterized by
simultaneous widespread microvascular thrombosis
and profuse bleeding from various sites. It is
described as the combination of thrombocytopenia,
decreased coagulation factors V and X causing
prolonged prothrombin time, together with decreased
fibrinogen and increased D-dimer levels. [1] The
normal haemostatic balance is disturbed in DIC.

There is excessive thrombin formation leading to
fibrin deposition in microcirculation and consequent
ischemic organ damage. The etiology is
multifactorial. A number of medical, surgical,
oncological and obstetrical conditions can cause DIC.
The diagnosis is essentially clinical supported by
laboratory parameters and a scoring system based on
546
Nisal et al.,
these. The mainstay of treatment is correction of

underlying cause and haemostatic support with
replacement of coagulation factors. [2] Although
hemorrhagic manifestations are more common in
DIC, yet it is the diffuse thrombosis that leads to endorgan damage and is associated for most of its
morbidity and mortality. DIC is thus a
thrombohemorrhagic disorder characterized by
procoagulant activation, fibrinolytic activation,
inhibitor consumption and biochemical evidence of
end organ damage or failure. [3] DIC is divided into
overt DIC and non-overt DIC. In overt DIC, the
haemostatic system is in decompensated state, and in
non-overt DIC, it is in compensated state. [4] There is
no single laboratory test that can establish or rule out
diagnosis of DIC. A diagnosis of DIC should be made
based on appropriate clinical suspicion supported by
relevant laboratory tests. Although the general
concept of DIC is known to most of the clinicians, a
uniform definition of the syndrome and
straightforward diagnostic criteria have never been
defined. A combination of tests when repeated in a
patient with clinically suspected DIC can be used to
diagnose the disorder with reasonable certainty in
most of the cases. This concept has been taken into
consideration by International Society of Thrombosis
and Haemostasis (ISTH). [5] The ISTH scoring criteria
takes into account Platelet count, Prothrombin time
(PT), D-dimer and Fibrinogen while calculating the
score. [6] The present study was aimed at evaluating
the usefulness of this scoring system for patients with
underlying disorder predisposing to DIC as a
prognostic indicator.
Study design: This was a prospective study carried
out in tertiary care hospital.
Inclusion criteria: Both paediatric and adult patients
were selected with an age range from new born to 80
years and included 35 male and 25 female patients
who had a clinical suspicion of DIC and having an
underlying condition associated with DIC.
Exclusion criteria: Patients with other hemolytic
anaemias such as Hemolytic Uremic Syndrome
(HUS) and Thrombotic Thrombocytopenic Purpura
(TTP) were excluded from this study.
Study period: 60 cases were studied over a period of
1 year. Ethics committee approval was done as per
the protocol and informed consent was taken from the
patients. In paediatric patients; consent from the

parents was taken.
Methodology: Clinical signs and symptoms of
predisposing condition were recorded which is
suspected to be leading to DIC i.e. fever, bleeding
and/or thrombosis, symptoms and signs of organ
failure etc. Along with the routine investigations the
tests necessary to calculate the ISTH score i.e.
platelet count, Prothrombin time (PT) [7], D-dimer[8]
and Fibrinogen[9] were done. The scoring criteria of
ISTH were applied in these cases. [6] Apart from these
tests, other investigations, such as Activated Partial
Thromboplastin Time (APTT) levels, Peripheral
Blood Smear for presence of schistocytes were also
assessed in these cases.
Blood samples were collected in 3.2 gm% trisodium
citrate as anticoagulant (9 : 1 ratio) and immediately
centrifuged at 3000 rpm for fifteen minutes in a
temperature controlled centrifuge (eppendorf
centrifuge 5804 R) at 40 C. The Prothrombin Time
(Recombiplastin 2G, HemosIL), Activated Partial
Thromboplastin Time (SynthASil, HemosIL),
fibrinogen (PT derived fibrinogen), and d-dimer (DDimer, HemosIL) were evaluated on the ACL Elite
Pro analyzer. Platelet counts were obtained on the
Coulter LH750 fully automated cell counter using
EDTA anticoagulated samples. In house prepared
Pooled Normal Plasma (PNP) from 20 healthy
individuals (equal number of males and females) as
well as commercially available controls were used
daily as internal quality control. A DIC score [6] was
calculated using the ISTH scoring system. Platelet
count more than 100000 per cumm was given a score
of 0, between 50000100000 per cumm score of 1
and less than 50000 per cumm a score of 2. Elevated
D-dimer was given the score 0 if there is no increase,
2 if there is moderate increase (400-800 ng/ml), and 3
if there is strong increase (more than 800 ng/ml).
A prolonged PT with respect to daily control of less
than 3 seconds was given a score of 0, more than 3
and less than 6 seconds was given a score of 1, and
more than 6 seconds was given a score of 2.
Fibrinogen level above 100 mg/dl was given a score
of 0, and level below 100 mg/dl was given a score of
1. The DIC score was calculated by adding scores for
all the four parameters. [6] Statistical analysis: Chi
square and Fishers exact tests were used for analysis
of the data.
547
Nisal et al.,
RESULTS
This was a prospective study carried out in Bharati
Vidyapeeth Deemed Univerisity Medical College,
Pune for a period of one year.
Bleeding (60.0%) was the main clinical feature
observed. It was in the form of either mucocutaneous
bruising, petechiae or purpura. In obstetric
complications, prolonged per vaginal bleeding was
observed post delivery. The cases of hematological
malignancy presented with gum bleeding. Though the
bleeding was the commonest presentation, other
presentations like shock (13.3%), end organ failure in
the form of renal failure (8.3%), hepatic derangement
(8.3%) and respiratory symptoms (6.7%) were also
observed in few cases. CNS (1.7%) and embolism
(1.7%) were rare manifestations. One case of ovarian
malignancy presented with thrombosis.
Fig 1: Cause wise distribution of cases

The commonest cause leading to DIC was found to
be Sepsis (66.7%) followed by Trauma (10.0%),
obstetric causes (8.3%) and malignancy (6.7%).
Hematological malignancies, snake bite and other
causes were rare in this study.
Out of total 60 patients; 40 (66.7%) patients were
found to have an ISTH DIC score of less than 5
which were suggestive of a non-overt DIC or low
grade DIC and 20 (33.3%) patients had a score of
more than 5 which were consistent with an Overt DIC
according to the ISTH scoring criteria.
Nisal et al.,
Fig 2: Schistocytes vs ISTH score

Out of 60 cases, 13 (21.7%) patients showed presence
of schistocytes. Schistocytes were absent in 47
(78.3%) patients. Presence of schistocytes were seen
in 11 patients with high DIC score 5 which is
statistically significant (p value= 0.001). Schistocytes
were seen in only 2 patients with a low DIC score.
Schistocytes were present mainly in the cases of
sepsis (9 out of 13 cases) followed by 2 cases of
eclampsia and one case each of amniotic fluid
embolism and Acute Promyelocytic Leukemia.
Fig 3: Outcome of patients vs ISTH score

There was a significant association of the outcome of
the patients with the ISTH DIC score. A high score
5 was associated with a poor outcome in 9 patients
(45%). Out of these 9 patients; five patients had
underlying cause as sepsis (56%), two patients had
obstetric complications and one patient each with
APML and solid malignancy. Low DIC score was
associated with increased chances of survival (P
value= 0.001). Two out of forty patients who had a
low score succumbed to the disease process itself
rather than to DIC.
The APTT levels were also included in this study
though they were not a part of ISTH score to see their
association with ISTH score. APTT levels were
548
raised in 40 out of total 60 cases. In 18 out of 20

patients of high ( 5) DIC score; APTT levels were
increased which was statistically significant (p
value=0.008). Raised APTT levels showed a good
correlation with high DIC score (5) and thus
patients outcome.
DISCUSSION
The present study International Society on
Thrombosis
and
Haemostasis
Disseminated
Intravascular Scoring System: Is it a good prognostic
indicator in DIC? was carried out for a period of one
year in Bharati Vidyapeeth Deemed University
Medical College, Pune. No single test is sufficiently
accurate to establish or rule out a diagnosis of DIC.
ISTH DIC scoring system for overt DIC uses a
combination of commonly done laboratory tests and
can be used widely in intensive care units. Although
the cases of sepsis dominated the number over all
other predisposing conditions, Obstetric causes,
trauma and malignancy were also seen leading to
DIC. The underlying conditions predisposing to DIC
are frequently unsuspected and are frequently
underestimated. DIC is usually associated with
several predisposing clinical conditions. In our study,
Sepsis (66.7%) was found to be the main underlying
cause leading to DIC. Similar incidence was observed
by Siegal et al and Spero et al. Siegal et al[10] who
have studied the medical records of 118 cases who
met laboratory criteria of DIC. Van Bunderen et al[11]
have studied the association of DIC and solid
tumours. They have found that it is rare but has been
known for decades.
Presence of schistocytes were seen in 11 patients with
high DIC score 5 which is statistically significant (p
value= 0.001). Schistocytes were seen in only 2
patients with a low DIC score. Schistocytes were
present mainly in the cases of sepsis (9 out of 13
cases) followed by 2 cases of eclampsia and one case
each of amniotic fluid embolism and Acute
Promyelocytic Leukemia. In agreement with our
study, schistocytes have been described in most
patients with DIC after septicemia by Levi M et al. [12]
However Visudhiphan et al[13] did not find any
difference between patients of sepsis with or without
DIC. In the present study; there was a significant
association of the outcome of the patients with the
ISTH DIC score (P value< 0.001). A high score 5
was associated with a poor outcome in 9 patients
Nisal et al.,
(45%). Out of these 9 patients; five patients had

underlying cause as sepsis (56%), two patients had
obstetric complications and one patient each with
APML and solid malignancy. Pati et al[14] found that
Survival was better in patients in whom DIC was
precipitated by obstetric causes compared with those
with septicemia (P < 0.01). Spero et al[15] had
analyzed 346 cases of disseminated intravascular
coagulation (DIC). They have found an overall
mortality of 68% which further confirms the dismal
prognosis previously associated with DIC. Bakhtiari
et al[16] analyzed 660 samples from 217 consecutive
patients. The prevalence of DIC was 34%. According
to them, there was a strong correlation between an
increasing DIC score and 28-day mortality.
Limitations of the Study: ISTH recommends daily
scoring in case of overt DIC and repeat scoring 1-2
days for non overt DIC. ISTH DIC scoring system
also recommends follow up scoring and use of
additional tests like Protein C, Antithrombin and
Thrombin anti-thrombin complexes as another
algorithm for non-overt DIC when one gets a DIC
score of less than 5 with the initial scoring done by
using algorithm for overt DIC. These tests were not
done in our study.
CONCLUSION
Disseminated intravascular coagulation is a common
complication secondary to a variety of clinical
conditions. Its early diagnosis is the key for better
outcome of the patient. There is a significant
association of a high ISTH score with the outcome of
the patient. Serial monitoring of ISTH DIC score in
cases of low score i.e. < 5 should be done which will
help in assessing the improvement or worsening of
the patients condition. Presence of schistocytes is not
a clue test for initial diagnostic workup of DIC but, it
might be of clinical value to suggest an associated
thrombotic microangiopathy if found in significant
numbers. In order to improve the prognosis of DIC, it
is important to diagnose the condition accurately and
as early as possible. ISTH DIC scoring criteria more
precisely define clinical and laboratory parameters of
DIC and its clinical utility will help for accurate
diagnosis and monitoring of the patients.
ACKNOWLEDGEMENT
I gratefully acknowledge the guidance by all the
teaching staff and cooperation of all the technical and
549
non technical staff, Department of Pathology, Bharati

Vidyapeeth Deemed University Medical College,
Pune during the course of this study.
11.

REFERENCES
1. Rostom A, Khaled M, Afify M, EL-Sherif A.
Applications of the international scoring system
for Disseminated Intravascular Coagulopathy
(DIC) and its interaction with Sequential Organ
Failure Assessment Score (SOFA) in prediction
of prognosis and final outcome in {ICU}. Egypt J
Crit Care Med. 2013; 1(1):3341.
2. Kumar R, Gupta V. Disseminated Intravascular
Coagulation: Current Concepts. Indian J Pediatr.
2008 Jul 1; 75(7):7338.
3. Bakshi S, Arya L. Diagnosis and treatment of
Disseminated Intravascular Coagulation. Indian
Pediatr. 2003; 40:72130.
4. Kaneko T, Wada H. Diagnostic Criteria and
Laboratory tests for Disseminated Intravascular
Coagulation. J Clin Exp Hematop. 2011; 51(2):
6776.
5. Levi M, Toh CH, Thachil J, Watson HG.
Guidelines for the diagnosis and management of
disseminated intravascular coagulation. Br J
Haematol. 2009; 145(1):2433.
6. Taylor Jr. FB, Toh C-H, Keith Hoots W, Wada
H, Levi M. Towards Definition, Clinical and
Laboratory Criteria, and a Scoring System for
Disseminated Intravascular Coagulation* On
behalf of the Scientific Subcommittee on
Disseminated Intravascular Coagulation (DIC) of
the ISTH. Thromb Haemost. 2001; 86(11):1327
30.
7. Bain B, Bates I, Laffan M, Lewis S.
Investigations of Hemostasis in Dacie and Lewis
Practical Haematology. Eleventh edition. United
Kingdom: Churchill Livingstone; 2012. 393-445
8. Newman D et al. Particle enhanced light
scattering immunoassay. Ann Clin Biochem.
1992; 29:2242.
9. Rossi E, Mondonico P, Lombardi A, Preda L.
Method for determination of functional
(Clottable) fibrinogen by the new family of ACL
coagulometers. Thromb Res. 1988; 52:45368.
10. Siegal T, Selingsohn U, Aghai E, Modan M.
Clinical and laboratory aspects of disseminated
12.
13.
14.
15.
16.
intravascular coagulation (DIC): a study of 118

cases. Thromb Hemostat. 1978; 39(1):12234.
Van Bunderen C, de Weger V, Griffioen-Keijzer
A. Disseminated intravascular coagulation as
clinical manifestation of colorectal cancer: a case
report and review of the literature. J Med. 2014;
72(4):1869.
Levi M, Ten C. Diagnosis of Disseminated
Intravascular Coagulation. N Engl J Med. 1999;
341:58692.
Visudhiphan S, Piankijagum A, Sathaypraseart P,
Mitrchai N. Erythrocyte fragmnetation in
disseminated intravascular coagulationa and other
diseases. N Engl J Med. 1983; 309:113.
Pati HP, Saraya A, Charan V, Sindaram K et al.
Prognostic role of screening tests of haemostasis
and underlying diseases in acute disseminated
intra-vascular coagulation in adults. Clin Lab
Haematol. 1994; 16(1):913.
Spero J, Lewis J, Hasiba U. Disseminated
intravascular coagulation. Findings in 346
patients. Thromb Hemostat. 1980; 43(1):2833.
Bakhtiari K, Meijers J, de Jonge E, Levi M.
Prospective validation of the International
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21.
550
Nisal et al.,
DOI: 10.5958/2319-5886.2015.00106.X

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Health Sciences
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Volume 4 Issue 3
Coden: IJMRHS
th
Research article
Copyright @2015
ISSN: 2319-5886
DEMOGRAPHIC PROFILE OF PEDIATRIC OSTEOSARCOMA IN SOUTH INDIA: A SINGLE

INSTITUTION EXPERIENCE
*Ashok S Komaranchath1, L. Appaji2, K. C. Lakshmaiah3, Mangesh Kamath1, Rekha V Kumar4
1
DM Medical Oncology Resident, 2Professor and HOD, Dept. of Pediatric Oncology, Kidwai Memorial Institute
of Oncology, Bangalore, Karnataka, India
3
Professor and HOD, Dept. of Medical Oncology. Kidwai Memorial Institute of Oncology, Bangalore, Karnataka,
4
Professor, Dept. of Pathology; Kidwai Memorial Institute of Oncology, Bangalore, Karnataka, India
*Corresponding author email: komaranchath@gmail.com
ABSTRACT
INTRODUCTION: Osteosarcoma is the most common primary malignant bone tumor in children and
adolescents, accounting for 4% of all childhood cancers worldwide. In India, the incidence varies from 4.7% to
11.6%, where this malignancy is associated with significant morbidity and mortality. There is paucity of
demographic and clinical data for osteosarcoma in India. Objective: To retrospectively assess the demographic
and clinical profile of pediatric osteosarcoma presenting at a tertiary cancer care centre of South India. Materials
and Methods: From January 2010 to December 2013, all children under the age of 15 years diagnosed with
osteosarcoma on histopathology were retrospectively analyzed for age, gender, rural or urban location, history,
location of tumour, investigations, stage and histopathological subtype. The findings were formulated to chart the
demographic and clinical profile. Results: A total of 37 cases of pediatric osteosarcoma were analyzed. The
median age was 13 years with only three patients under the age of 10 years. There was a slight female
preponderance with male: female ratio of 1:1.3. Most common mode of presentation was with pain and swelling
of local site. Three patients had presented with a pathological fracture. The most common site involved was the
distal femur. Over 90% of the cases were conventional osteosarcoma. Around 32% of patients had stage IV
disease at presentation. Around 37% of patients from rural areas and 20% of patients from urban areas presented
with metastatic disease. Conclusions: The aim of the study was the demographic and clinical description of
osteosarcoma in the pediatric age group. A slight female preponderance was noted. The most common sites were
consistent with western data except for an increased incidence in the fibula. There was an increased incidence of
metastatic disease as compared to western population and a larger proportion of these patients seemed to come
from rural areas.
Keywords: Pediatric Osteosarcoma, Rural population, South India
INTRODUCTION
Osteosarcoma is the most common primary malignant
bone tumor in children and adolescents, accounting
for 4% of all childhood cancers worldwide. In India,
the incidence varies from 4.7% to 11.6%,[1] where
this malignancy is associated with significant
morbidity and mortality. The five year overall
Komaranchath et al.,
survival rate in India is around 44% as compared to

68% in the western countries.[1] The rates of limb
salvage over amputation is also much lesser in India
leading to significant morbidity.[2] There is paucity of
demographic and clinical data for osteosarcoma in
India, especially in the pediatric setting. Our
551
objective was to retrospectively assess the

demographic and clinical profile of pediatric
osteosarcoma presenting at a tertiary cancer care
centre of South India.
Study design: Retrospectively analysis study
Study duration: January 2010 to December 2013
Ethics clearance: The study was approved by the
Institutional Ethics Committee
Inclusion criteria: The medical records of children
under the age of 15 years admitted to Kidwai
Memorial Institute of Oncology, Bangalore with a
diagnosis of osteosarcoma.
Exclusion criteria: All patients more than or equal to
the age of 15 years, Bone tumours other than
osteosarcoma
Methodology: From the Medical record section the
data was retrospectively analyzed for age, gender,
rural or urban location, history, location of tumour,
investigations, stage and histopathological subtype.
The diagnosis was made with histopathological
examination of biopsy specimens. The tumours were
staged using the 7th edition AJCC TNM staging
system.[3] The findings were formulated to chart the
demographic and clinical profile.
Twelve patients (35%) presented in stage 2B and

32% with stage IV disease. Six of the twelve had
pulmonary metastasis, four had skeletal metastasis
and two patients had metastases to both bone and
lungs. More patients from the rural areas (37%)
presented with stage IV disease as compared to those
from urban areas (20%).
Table 1: Baseline Characteristics
PARAMETER
SUBSET
Age
distribution
0-5 years
5-10 years
10-15 years
Male
Female
Rural
Urban
Only pain
Pain and Swelling
Pathological Fracture
Conventional OS
Telangiectatic
sex
Rural/Urban
Presenting
complaint
Type
NUMBER
0
3
34
16
21
27
10
23
11
3
34
8
RESULTS
From January 2010 to December 2013, a total of 37
cases of pediatric osteosarcoma were identified and
analyzed. Most of the patients (73%) were from rural
areas. The median age was 13 years with only three
patients under the age of 10 years and none below the
age of 5 years. There was a slight female
preponderance with 57% girls and 43% boys and a
male: female ratio of 1:1.3. The most common mode
of presentation was with pain and swelling of local
site (62%). There was no significant past or family
history. The most common site for osteosarcoma in
children was the distal femur (46%) followed by the
proximal tibia (24%) and the fibula (11%). Among
the
histopathological
subtypes,
92%
were
conventional osteosarcomas of which 76% were
osteoblastic osteosarcoma, not otherwise specified.
The other subtypes seen were, chondroblastic (14%)
and Fibroblastic (2%). (Fig.2) Telangiectatic
osteosarcoma comprised of 8% of the cases. (Table1).
All patients had high grade osteosarcoma. There were
no cases of parosteal or periosteal osteosarcoma.
Fig 1: Site of Tumour
Fig 2: Subtypes of conventional Osteosarcoma

552
(total 34 cases)
35%
30%
19%
3%
Stage 2A
Stage 2B
Stage 3
Stage 4A
13 %
Stage 4B
Fig 3: Stage at Presentation
17%
33%
Bone
Lung
Both
50%
Fig 4: Sites of Metastasis

DISCUSSION
Osteosarcomas are primary malignant tumors of bone
that are characterized by the production of osteoid or
immature bone by the malignant cells.[4,5,6]
Osteosarcomas are uncommon tumors and are
thought to be derived from primitive bone-forming
mesenchymal cells.[7] Mean annual age-standard
incidence is around 3.8 per million in males and 2.8
per million in females.[8] In India, the incidence varies
from 4.7% to 11.6% of all the childhood
malignancies.[1]
Over a period of four years from January 2010 to
December 2013, a total of 37 cases of pediatric
osteosarcoma were identified and analyzed. Most of
the patients (73%) were from rural areas. The median
age was 13 years with only three patients under the
age of 10 years and on patient less than 5 years of age
{Table 1}. There was a slight female preponderance
with 57% girls and 43% boys and a Male: Female
ratio of 1:1.3(Table 1). Data from the National
Cancer Registry Program of India has suggested that

there is a slightly increased incidence females in
osteosarcoma of the pediatric age group.[1] A similar
sex ratio of 1:1.4 was seen in a Norwegian cohort of
473 osteosarcoma patients.[8] The most common
mode of presentation was with pain and swelling of
local site (62%). Three patients (8%) had presented
with a pathological fracture.(Table-1) This is
consistent with existing literature in which 5-10% of
patients with osteosarcoma present with pathological
fractures.[9,10] There was no patient with any past
history of radiation or retinoblastoma or any
significant family history of malignancy.
Routine laboratory investigations were mostly
normal, except for elevation of alkaline phosphatase
above upper limit of normal in 48% of patients.
Elevation of alkaline phosphatase was seen in around
40% of patients in an American trial conducted to
determine the significance of alkaline phosphatase as
a prognostic marker.[11] The increased incidence in
our series may be due to the higher percentage of
patients presenting with advanced disease and
multiple skeletal metastasis.
The most common site for osteosarcoma in children
was the distal femur (46%) followed by the proximal
tibia (24%) and the fibula (11%).(Fig.1) The first two
most common sites were consistent with that of
western data but incidence of fibular osteosarcoma
was double of that seen in western literature.[8] Over
90% of the cases were conventional osteosarcoma.
Telangiectatic osteosarcoma accounted for 8% of the
cases.(Table 1), (Fig.2) Compared to the SEER
database as well as Norwegian data, there was a
higher incidence of telangiectatic variant of
osteosarcoma and no cases of parosteal osteosarcoma
in our set of patients.[8,12]
All patients had high grade osteosarcoma. The most
common stage at presentation was stage 2B
comprising of 35% of the patients. Only one patient
had stage 3 disease with skip metastasis.(Fig.3)
Twelve patients (32%) had stage IV disease at
presentation out of which six (19%) had lung
metastasis and four (13%) had bone metastasis.(Fig.3
and 4) Two patients out of the thirty-seven had both
lung and bone metastasis.(Fig.4) Only four out of the
seven patients with pulmonary metastasis had
evidence of disease with chest X-ray and for the other
three were evident only with contrast enhanced CT
scan of the chest. This underlines the importance of
553
including a contrast enhanced CT scan of the thorax

as part of the staging work-up of osteosarcoma rather
than just a chest X-Ray.[13] According to the
Childrens Oncology Group guidelines, finding one
or more pulmonary (or pleural) nodules of at least 1cm diameter or three or more nodules of at least 0.5cm diameter generally indicates definite pulmonary
metastases and may not require a biopsy.[14]
Compared to western data, there was a higher
incidence of metastatic disease at presentation (32%
vs. 20%).[15] A larger proportion of patients from rural
areas(37%) presented with metastatic disease as
compared to patients from urban areas (20%).(Table1) This may be a reflection on the fact that, due to
lack of facilities and proper referral centres, patients
from rural areas tend to present at a later stage than
those living in the cities with easier access to quality
health care.
CONCLUSION
The aim of the study was the demographic and
clinical description of osteosarcoma in the pediatric
age group. A slight female preponderance was noted.
The most common sites were comparable with
western data except for an increased incidence in the
fibula. There was an increased incidence of metastatic
disease as compared to western population and a
larger proportion of these patients seemed to come
from rural areas.
ACKNOWLEDGEMENT: NONE
REFERENCES
1. Arora RS, Eden TO, Kapoor G. Epidemiology of
childhood cancer in India. Indian J Cancer. 2009;
46(4):264-73.
2. S Rastogi, V Trikha, SA Khan. Limb salvage in
osteogenic sarcoma : an India perspective. Indian J
Med Paediatr Oncol 2003;24(4):46-50.
3. DeVita VT Jr., Lawrence TS, Rosenberg SA.
Cancer, Principles and Practice of Oncology. 9th Ed.
Philadelphia: Lippincott Williams & Wilkins.
2011;.116: 1583.
4. Huvos A. Bone Tumors: Diagnosis, Treatment,
Prognosis, 2nd Ed., WB Saunders, Philadelphia
1991;130-47
5. Sissons HA. The WHO classification of bone
tumors. Recent Results Cancer Res 1976;(54):104-8
6. McKenna R, Schwinn C, Soong K, et al. Sarcomas

of
the
osteogenic
series
(osteosarcoma,
chondrosarcoma, parosteal osteogenic sarcoma, and
sarcomata arising in abnormal bone): an analysis of
552 cases. J Bone Joint Surg Am 1966; 48:1.
7. Cheung NV, Heller G. Chemotherapy dose intensity
correlates strongly with response, median survival,
and median progression-free survival in metastatic
neuroblastoma. J Clin Oncol 1991;9:105058
8. Berner K; Johannesen TB; Berner A; Haugland HK;
Bjerkehagen B;
Bhler PJ.
Time-trends
on
incidence and survival in a nationwide and
unselected cohort of patients with skeletal
osteosarcoma. Acta Oncol. 2015; 54(1):25-33
9. Bacci G, Ferrari S, Longhi A, et al. Non-metastatic
osteosarcoma of the extremity with pathologic
fracture at presentation: local and systemic control
by amputation or limb salvage after preoperative
chemotherapy. Acta Orthop Scand. 2003;74:44954
10. Bacci G, Longhi A, Versari M, et al. Prognostic
factors for osteosarcoma of the extremity treated
with neoadjuvant chemotherapy: 15-year experience
in 789 patients treated at a single institution.
Cancer. 2006;106:115461.
11. Thorpe WP, Reilly JJ, Rosenberg SA. Prognostic
significance of alkaline phosphatase measurements
in patients with osteogenic sarcoma receiving
chemotherapy. Cancer 1979;43:217881
12. Mirabello L, Troisi RJ, Savage SA. Osteosarcoma
incidence and survival rates from 1973 to 2004:
Data from the Surveillance, Epidemiology, and End
Results Program. Cancer. 2009;115(7):1531-43.
13. Bielack SS, Kampf-Bielack B, Delling G, et al.
Prognostic factors in high grade osteosarcoma of
the extremities or trunk: an analysis of 1,702
patients treated on neoadjuvant cooperative
osteosarcoma study group protocols. J Clin Oncol
2002;20(3):77690.
14. Meyer JS, Nadel HR, Marina N, et al. Imaging
guidelines for children with Ewing sarcoma and
osteosarcoma: a report from the Childrens
Oncology Group Bone Tumor Committee. Pediatr
Blood Cancer 2008; 51:16370.
15. Mialou V, Philip T, Kalifa C, et al. Metastatic
osteosarcoma at diagnosis: prognostic factors and
long-term
outcome--the
French
pediatric
experience. Cancer 2005; 104:1100.
554
DOI: 10.5958/2319-5886.2015.00107.1

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Volume 4 Issue 3
th
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
ESTIMATION OF MALONDIALDEHYDE AND VITAMIN-E LEVELS IN NEONATAL

HYPERBILIRUBINEMIA BEFORE AND AFTER PHOTOTHERAPY
*Sidrah1, Tandrapad Priyanka2, Asiya Naaz3, Lakshmi Chaitanya G4, Sridevi D5
1,2,4,5
Department of Biochemistry, Malla Reddy Institute of Medical Sciences, Hyderabad, India

Department of Biochemistry, SVS Medical College, Mahabubnagar, Telangana, India
*Corresponding author email:sidrah.biochem@gmail.com

ABSTRACT
Background: Hyperbilirubinemia is a common and benign problem in neonates worldwide. It is observed during
the 1st week of life in approximately 60% of term neonates and 80% of preterm neonates. Phototherapy is most
widely used as therapy for unconjugated hyperbilirubinemia. Phototherapy is related to oxidative stress and lipid
peroxidation. The present study is undertaken to establish the relation between anti-oxidant status and the marker
of lipid peroxidation in neonatal hyperbilirubinemia before and after phototherapy. Objectives: To estimate the
levels of MDA (malondialdehyde) and vit-E levels in neonatal hyperbilirubinemia before and after phototherapy.
Material and Methods: A total of 30 patients were eligible for the study who met the inclusion and exclusion
criteria. Blood sample was collected from neonates of preterm/full term age 1 to 10 days with hyperbilirubinemia
undergoing phototherapy. Total Bilirubin, Direct Bilirubin, MDA and vit-E levels were estimated in serum by
thiobarbituric acid (TBA) method and Backer and Franks Method respectively. Results: The present study
showed increase in Total bilirubin, Direct bilirubin, MDA and decrease in vit-E levels before phototherapy when
compared to control group and same subjects after phototherapy showed decrease in Total bilirubin and Direct
bilirubin, MDA and a further decrease in vit-E levels. Conclusion: From these results it is concluded though
phototherapy had a beneficial effect in treatment of neonatal hyperbilirubinemia, supplementation of vit-E is
necessary in addition to phototherapy.
Keywords: Malondialdehyde, Vitamin-E, Neonatal hyperbilirubinemia, Phototherapy.
INTRODUCTION
Hyperbilirubinemia is a common and benign problem
in neonates worldwide. Neonatal hyperbilirubinemia
is observed during 1st week of life in approximately
60% of term infants and 80% of preterm infants. The
incidence of jaundice is higher in breast-fed babies
than in formula-fed ones [1].
Newborns appear jaundiced when the serum bilirubin
level is >7 mg/dL [2]. Although transient, the
condition accounts for up to 75 % of hospital readmissions in 1stweek after birth [3]. However
hyperbilirubinemia in the newborn period can be
associated with severe illness such as hemolytic
disease, metabolic and endocrine disorders, anatomic

abnormalities of the liver and infections [4].
Phototherapy is most widely used as therapy for
unconjugated hyperbilirubinemia [5,6]. It has recently
been reported that phototherapy cause oxidative stress
and lipid peroxidation [7].
The generation of free radicals is a normal
physiological process but increased production of free
radicals acts on lipid to cause lipid peroxidation. The
cells have evolved a number of counter acting
antioxidant defenses [8].
Sidrah et al.,
555
Uncontrolled lipid peroxidation is known to play an

important role in pathogenesis of many neonatal
complications. Excessive generation of free radicals,
depressed antioxidant status or imbalance in
peroxidation and free radical scavenging system
might play an important role in neonatal
hyperbilirubinemia. Antioxidants are compounds that
dispose, scavenge and suppress the formation of free
radicals or oppose their action [9].
The present study is undertaken to establish the
relation between antioxidant status and the marker of
lipid peroxidation in neonatal hyperbilirubinemia
before and after phototherapy.
Malondialdehyde (MDA) is a metabolic product of
polyunsaturated fatty acids widely used as an
indicator of peroxidation [10, 11].
Vitamin-E is most important chain breaking
antioxidant and protects membrane lipids from free
radical damage. The primary function of vitamin- E
as an antioxidant is prevention of non-enzymatic
oxidation of cell components, for example polyunsaturated fatty acids, by molecular oxygen and free
radicals.
Antioxidant activity in the serum of term neonates is
lower than that of adults and is still lower in preterm
and low birth weight babies as compared to term
babies [12]. Supplementation of natural antioxidants
like alpha-tocopherol, ascorbic-acid may be
beneficial in preventing neonatal complications.
Study design & Place: The present case-control
study was carried out in SVS Medical College of
Mahabubnagar district during March 2009 to March
2011.
Ethical approval: After obtaining approval from
institutional ethical committee and informed consent
from parents were taken.
Sample size: 30 in each group.
Inclusion Criteria: Cases: 30Preterm / full-term
Neonates of age 1 to 10 days with hyper
bilirubinemia (>11 mg/dl ) undergoing phototherapy
treatment (before and after phototherapy).
Controls: 30 Preterm / full-term Neonates of age 1 to
10 days with hyperbilirubinemia(<10 mg/dl) and not
advised for phototherapy treatment.
Grouping: Control group ( n=30), Test group(n=30)
Exclusion Criteria: Neonates of diabetic mother.
Sidrah et al.,
Sample Collection: About 4 ml of blood is collected

from peripheral vein by vene puncture under aseptic
conditions into a sterile tube. The serum was
separated by centrifugation at 3000 rpm and stored at
4oc until analysis was carried out.
Methods of estimation: Neonates with bilirubin > 11
mg/dl and those who were recommended for
phototherapy treatment samples were collected. The
following biochemical parameters were analyzed
with their respective methods before and after
phototherapy and compared with the control group
neonates. Estimation of total bilirubin and direct
bilirubin: Bilirubin was estimated by Diazo method
of Pearlman and Lee Method, Bilirubin reacts with
diazotized sulphanillic acid in acidic medium to form
pink coloured azobilirubin with absorbance directly
proportional to bilirubin concentration [13].
Estimation of malondialdehyde: MDA is estimated
by thiobarbituric acid method (TBA). Autoxidation of
unsaturated fatty acids involve the formation of semi
stable peroxides which undergo a series of reaction to
form short chain aldehydes like malondialdehyde.
One molecule of MDA reacts with TBA with the
elimination of two molecules of water to yield pink
crystalline pigments with absorption maximum at 535
nm[14].
Estimation of Vitamin-E: Vit-E is estimated by
Baker and Franks method. Serum tocopherol can
measure by their reduction of ferric to ferrous ions
which then form a red colour complex with , dipridyl. Tocopherols and carotenes are first extracted
into xylene and the absorbance is read at 460nm to
measure the carotenes. A second reading is made
after addition of ferric chloride at 520nm for vitamin
E [15].
Statistical analysis: Data was presented as mean and
standard deviation (mean SD). Means and standard
deviations are compared between control and case
groups by unpaired student ttest and between two
case groups (before phototherapy and after
phototherapy) by paired studentt test. A p-value of
< 0.05 and p<0.001 was considered statistically
significant. Descriptive statistical analysis was carried
out in the present study by using SPSS, 20version
software.
RESULTS
A total of 60 neonates of age 1 to 10 days were
included in the study. Out of these 30 neonates with
556
hyperbilirubinemia
but
not
recommended
phototherapy treatment and 30 neonates with
hyperbilirubinemia
and
recommended
for
phototherapy were included. In Case group blood
samples were collected before and after phototherapy
and results were analyzed. The mean and standard
deviation values of all data and demographic changes
are tabulated. Table-1 the biochemical profile in cases
(before phototherapy and after phototherapy) and
controls.
Total bilirubin levels and Direct bilirubin were
compared between the controls and cases (before and
after phototherapy) showed statistically significant
and the Total bilirubin values and direct bilirubin

values of case group were decreased after
phototherapy.
The mean and standard deviation values of MDA are
shown in table-1 It shows that the MDA levels are
raised in hyperbilirubinemia neonates before
phototherapy when compared with control group
neonates and decreased after phototherapy.
The mean and standard deviation values of Vit-E are
shown in table no-1, it shows that the Vit-E levels are
less in hyperbilirubinemia neonates before
phototherapy when compared with control group
neonates and decreased after phototherapy.
Table1: Comparison of Parameters between control, case before and after phototherapy
After phototherapy
P values$
P values #
Pvalues^
11.612.850
<0.0001
0.004
<0.0001
0.430.18
0.2000.053
<0.0001
0.04
<0.0001
2.4430.203
3.2270.227
2.8600.266
<0.0001
<0.0001
<0.0001
0.2930.104
0.1920.091
0.1330.063
=0.0002
<0.0001
= 0.008
Parameter
Control
Total bilurubin (mg/dl)
9.62.3
Direct bilirubin (mg/dl)
0.230.06
MDA(nmol/ml)
Vit E (mg/dl)
before phototherapy
14.943.2
Comparison between control and before phototherapy, #Comparison between control and after phototherapy, ^
Comparison between before and after therapy * p<0.05 is statistically significant**p<0.001 is highly statistically
significant
DISCUSSION
Neonatal hyperbilirubinemia is a common
complication in newborn and phototherapy is now the
accepted method of treatment which has replaced
exchange transfusion in management of neonatal
hyperbilirubinemia [16].
Bilirubin is generally regarded as a toxic compound
when, in its unconjugated form, it accumulates to
abnormally high concentrations in biological tissues
and is thus responsible for the development of
kernicterus [17,18].
Bilirubin reactions involving free radicals or toxic
oxygen reduction products have been well
documented: Unconjugated bilirubin scavenges
singlet oxygen anions and peroxy radicals and serves
as a reducing substrate for peroxidases in presence of
hydrogen peroxides or organic hydro peroxides [19,20] .
Although phototherapy has been used on millions of
infants worldwide during the past 35 years, in
previous studies it has been reported phototherapy as
a photodynamic process that can cause peroxidative
damage to tissues [21]. It has also been suggested that
hydrogen peroxide and free hydroxyl radical (OH)
are responsible for the photoproducts induced
chromatid changes [22]. .Furthermore, Ostrea et al

reported that the exposure of red cell suspension to
phototherapy light in presence of sensitizer (bilirubin)
resulted in oxidative injury to the red cell membrane
[23]
. Recent studies showed that fluorescent light
decrease ATPase activity in red blood cell [24].
In a healthy human being, formation and inactivation
of reactive oxygen species is balanced at a level at
which the compounds can play their physiological
role without any toxic side effects. This balance can
be unstable in neonatal period following rapid
changes in tissue oxygen concentration immature
antioxidant mechanism and considerable neonatal
developmental changes in antioxidant [25].
As free radicals are potentially toxic, they are usually
inactivated or scavenged by antioxidants before they
can inflict damage to lipids, proteins or nucleic
acids.[26].
In the present study there are significant increased
levels of serum total bilirubin and direct bilirubin in
case group neonates as compared to control group
neonates. Malondialdehyde (MDA) levels have been
increased significantly in serum of test group
neonates (before phototherapy)than in control group
557
Sidrah et al.,
neonates. Rise in MDA shows the increased

generation of reactive oxygen species (ROS) due to
excessive oxidative damage generated in neonatal
hyperbilirubinemia case. These oxygen species in
turn oxidizes many other important biomolecules
including membrane lipids. This raised MDA level
reflects the oxidative injury in neonatal
hyperbilirubinemia, which is attributed to free radical
formation that abstracts hydrogen atoms from
lipoproteins causing lipid peroxidation of which
MDA is the main product.
A significant decrease in the MDA levels was
observed in case group (after phototherapy) as
compared to same test group neonates (before
phototherapy)
In the present study there is a significant decrease in
the level of vitamin-E in test group neonates as
compared to control group neonates. Vitamin-E is an
important chain breaking antioxidant responsible for
scavenging the free radicals and suppression of
peroxidation products in aqueous and lipid region of
the cell [27] .Further significant decrease in vitamin E
was observed in test group neonates after
phototherapy as compared to same test group
neonates before phototherapy. This decrease in the
level of vitamin-E may be to increased turnover, for
preventing oxidative damage in these patients [27] .
CONCLUSION
From these results it can be concluded that neonates
with hyperbilirubinemia above 7 mg/dl and advised
phototherapy showed a significant rise in serum
MDA levels and decrease vitamin-E levels. As
compared to control group of neonates. The same
subjects after phototherapy showed decrease in total
bilirubin, indirect bilirubin, MDA levels and a further
decrease in vitamin-E levels. From the present study
it is concluded though phototherapy had a beneficial
effect in the treatment of neonatal unconjugated
hyperbilirubinemia but supplementation of vitamin-E
and other antioxidants is necessary as secondary
therapy in addition to phototherapy to prevent
oxidative
damage
in
neonates
with
hyperbilirubinemia.
ACKNOWLEDGEMENT: Nil
1. Robert M. Kliegman, MD, Richard E. Behrman,

MD, Hal B. Jenson, MD and Bonita F. Stanton,
MDNelsons Text Book of Pediatrics -18th edition
Elsevier 2008;756.
2. CamiliaR.Martin and John P.Cloherty.Manual of
neonatal care6th Edition Lippincott-Raven,
Philadelphia Wolterskluwer 2008; 18:181.
3. Britton JR, Britton HL , Beebe SA . Early
discharge of the term newborn : a continued
dilemma . Pediatrics. 1994;94(3):291-5.
4. Merdith L, Porter, CPT, MC, USA and
BETHDENNIS. Hyperbilirubinemia in the Term
newborn. American Family Physician : 2002;65:
599-07.
5. Porter ML, Dennis BL. Hyperbilirubinemia in the
term newborn. Am Fam Physician. 2002;65:599
06.
6. Tan KL. Phototherapy for neonatal jaundice.
ActaPaediatr. 1996;85: 2779.
7. Modi.N and KeayA.J. Phototherapy for neonatal
hyperbilirubinemia : the importance of dose
.Arch .Dis.Child. 1983; 58, 406-09.
8. Amithkumar Mani Tiwari, Abbas Ali Mahdi,
Fatima Zahra, Sudarshanachandyan. Evaluation
of Oxidative Stress and Antioxidant status in
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DOI: 10.5958/2319-5886.2015.00108.3

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Volume 4 Issue 3
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Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
th
Revised: 6 May 2015
EFFICACY OF SOFT TISSUE APPLICATION, MANUALLY-THERAPEUTICAL TECHNIQUES FOR

KNEE ARTHROKINEMATICS RECOVERY COMPLEX IN PATIENTS AFTER ARTHROSCOPIC
MENISCECTOMY
*Kostov Rostislav V
Department of Physical medicine, Rehabilitation, Occupational Therapy and Sports, Medical University
Pleven, Bulgaria
*Corresponding author email: rostislav_kostov@abv.bg
ABSTRACT
Introduction: In this article we present the final effect of the application of complex soft tissue manuallytreatment system for recovery of joint kinematics in patients with moderate and minimal protective period of
rehabilitation after arthroscopic meniscectomy. Material and Methods: The study was conducted in 2005-2012
into three medical centers in Bulgaria: Blagoevgrad, Sofia and Pleven. The study included a total of 110 patients
divided into three groups (Control and Experimental I and Experimental Group II) who studied the effect of
topical application of the manual therapeutic techniques compared to traditional rehabilitation methods applied.
For testing the efficacy of a treatment approach in the three groups of patients, the results have processed by the
method of variational analysis. Results: After analysis of results we find significantly more fully and without
residual short violations recovery for all controlled parameters in patients who have implemented comprehensive
manually-therapeutic treatment compared with control group patients. Conclusion: Application of adequate
physiological and pedagogically grounded complex rehabilitation is required in patients after arthroscopic
meniscectomy model with motor deficits in tractable routine rehabilitation. Observations allow us to offer a
methodology for implementation in general practice rehabilitation in patients after meniscal ruptures treated by
arthroscopic meniscectomy and motor deficits, intractable routine rehabilitation.
Keywords: Manually-therapeutic techniques, Rehabilitation, Arthroscopic meniscectomy.
INTRODUCTION
Arthroscopic meniscectomy treated surgical treatment
of patients with the most common intra-articular
damage-meniscus ruptures[1]. This current and
specific surgical approach necessitates adaptation of
complex musculoskeletal physiotherapy to this
modern minimal invasive medical equipment.
In studying and analysis of complex influence
pathogenic factors for the occurrence of primary and
secondary motor deficits, impaired joint kinematics in
the knee is the basis for the formation of secondary
neuro-muscle-skeletal dysfunctions, the kinetic chain
Rostislav
of the entire lower limb[2, 3]. Therefore, the current

trends in contemporary skeletal muscle rehabilitation
impose physiological deterministic resources
influencing both the contractile and non-contractile
pathogenic factors[3,4], mobilizing massage[4] etc., as
well as synergistic interaction between manuallytherapeutic methods and physical healing factors[1, 4].
Significant epidemiological prevalence of meniscus
ruptures[5, 6] motivates us to explore different
pathological mechanisms for disturbance of the joint
mechanics,
approbating
biomechanical
and
560
pathokinesiological manually soft tissue therapeutic

techniques, grouped in an appropriate system that
have not been used in such patients in Bulgaria.
Study design: A randomized study, before starting
rehabilitation all patients are aware of the purpose
and the conduct of scientific research, then manually
signed document certifying the consent for inclusion
in the study. Committed a long-term (7 years) study
the effect of topical application of the manual
therapeutic techniques to overcome musculoskeletal
dysfunctions in patients after arthroscopic
meniscectomy and shaped motor deficits.
The study was conducted in 2005-2012 into three
medical centers in Bulgaria: Blagoevgrad, Sofia and
Pleven.
Grouping:
1. Control group: Numbering 30 patients
predominantly treated with active therapeutic agents
in the concept of classical kinesitherapeutical
procedures applied after arthroscopic meniscectomy
model.
2. Experimental group I: It consists of 30 patients,
treated by the methods of classical kinesitherapeutical
procedures applied after arthroscopic meniscectomy,
supplemented with the use of pain suppressed
proximal, mobilization stretching knee.
3. Experimental group II: It consists of 50 patients,
treated by the methods of classical kinesitherapeutical
procedures applied after arthroscopic meniscectomy,
supplemented with the use of pain suppressed
proximal, mobilization stretching, joint mobilization
techniques Mulligan [7] and Maitland [8] and
analytical, mobilization muscles stretching knee.
Sample size: The total number of patients, forming
our contingent, is 110 people.27.27% from all
patients are included in the control group, 27.27% are
in the experimental group I and45.45% are taken in
the group of Experimental Group II
Inclusion criteria: Diagnosed meniscal lesion with
subsequent arthroscopic meniscectomy model; Full
range of medical documentation concerning the
trauma, imaging results and diagnostic testing
methods and course of the postoperative period;
Pathokinesiologycal preliminary analysis to establish
existing rehabilitation potential; Patients classified as

sub acute clinical function (moderate protective
period of rehabilitation) average 4-6 weeks (in
peripheral meniscal lesions) and 6-8 (with central
damage) after surgery; Patients with existing
indications for physical therapy, but in later stages of
the recovery period with available secondary
complications and unsatisfactory results after
rehabilitation.
Exclusion criteria: Neoplastic processes in injured
knee, an advanced degree of demineralized bone
diseases (severe metabolic osteoporosis, osteopenia
and the like.); Abnormally increased joint mobility
and instability; Unclear clinical and/or functional
diagnosis; Patients with evidence of objective
deterioration following a single application of
approbation methodology, 24 hours after the first
procedure.
Sampling technique: A randomized study was
conducted with patients-contingent of our research is
structured as follows:
Methodology: The patients were randomly selected
and included in the treatment groups defined by
differentiating, pathokinesiological analysis and
according to the indications for use of manuallytherapeutic
techniques
for
recovery
of
arthrokinematic knee complex recovery. We analyzed
the patients by the following individual
characteristics: age; sex; athletes; non-sports people;
after partial meniscectomy model; after total
meniscectomy model (Table. 1).
All patients were diagnosed with meniscus lesion
with subsequent arthroscopic meniscectomy. All
patients have a complete set of medical
documentation concerning trauma, the results of
imaging and diagnostic methods (magnetic resonance
imaging, computerized axial tomography) made
before and after the operation, dataflow
comprehensive set of tests to establish the initial
rehabilitation potential, monitoring the effectiveness
of the applied therapy and the establishment of its
final effect. The study included all patients classified
in clinical functional group II and III by Maitland (i.e.
at the end of moderate-protective recovery period),
where we applied manual techniques which are
appropriate and risk-free.
Table1: Distribution of the contingent of study as defined individual indicators.

Rostislav
561
Gender Age
Athletes Not athletes
173 255
Men
31
32
48
15
Women
25
22
26
21
Total
56
54
74
36
When starting treatment, all patients moved with
facilities and locked protective orthosis of the injured
knee, which is removed during procedures.
The initiation of treatment procedures proceed only
after detailed informing patients about the purpose
and methods of the study, respectively, after
obtaining consent.
Pathokinesiologycal analysis and functional
diagnostics: We applied a complex set of
Pathokinesiologycal and functional diagnostic tests
for establishing the initial rehabilitation potential,
individual indications for including the patients in the
appropriate treatment groups, monitoring the effect of
treatment and establishing the final result. For this
purpose we worked out the required personal
documentation. The selected tools for functional
diagnostics are:
1. Standard algometry movements in the sagittal
plane.
2. Girth measurements of hip circumference
standard in two levels.
3. Manual muscle testing (MMT) to establish the
degree of muscle weakness of the main muscles
in the knee complex.
4. Isometric muscle strength testing, the maximum
analytical directed to individual sections for m.
quadriceps femoris.
5. A complex test of locomotor skills (length of step
with the operated lower limb and the number of
steps) for covering the distance of 5 m., without
facilities.
Statistical analysis: For testing the efficacyof a
treatment approach inthe three groups of patients,
the results have processedby the method of
variation alanalysis. Software packages used
inthe analysisareMS OFFICE 2010 with
application of Excel 2010 and SPSS 17.0. For
this purpose, we introduced data for each of the
three patient groups representing the initial and
final results of the monitored indicators. The
differences between the final and initial data
checked with X.
Rostislav
With partial
With total
Total
meniscectomy meniscectomy
patients
52
11
63
34
13
47
86
24
110
For statistical analysis we determined zero (H0)and
alternative hypothesis (H1).
For selection of statistical methods of research
analysis identified independent samples (choice of
units in a sample is not predetermined by the choice
of units in the other sample)with consistent criteria
for checking the reliability.
The general aggregate of sampling is considered by
the variation analysis; therefore the decisions have
got a probabilistic nature. Therefore, in our study we
determined guarantee probability (P) for each of the
indicators and the level of significance () reflects the
risk of errors in the acceptance of true alternative
hypothesis. We used the standard values for the
guarantee probability (P) and a significance level ():
P=95%, which corresponds to = 0, 05 (5% error
margin).
=99%, which corresponds to =0,01 (1% error
margin).
=99,9%, which corresponds to =0,001 (0,1%
error margin).
For statistical verification of results we use
independent-criteria of Student by some formula.
To determine the tabulated value of criteria (criteria
) it is taken from statistical tables depending on the
degrees of freedom(k)and the level of significance().
For independent samples, we calculated the degree of
freedom by the standard model (k = n1 + n2-2, where
n =the number of compared cases). To establish the
level of significance () we compared calculated tcriteria on standard tabular value. For taking
decisions, we compared the empirical value
(calculated according to data from the sample) with
the tabulated value of the criteria.
RESULTS
1. Measurement the volume of movement in the
knee in the sagittal plane
1.1. Flexion measurement: There is the greatest
improvement in patients EG II (38,60 ), followed by
EG I (27,33 ) and CG (21,83 ). When the results
between CG and EG I are compared, there is a
562
marked improvement in patients from EG I with 5,5

. (t=5, 38; <0,001; P=99,9%). When the results
between CG and EG II are compared, there is a
difference of 16,77 (t = 17,65; <0,001; P=99,9%)
in favor of a approbation complex methodology.
When we compare the results obtained in patients
from EG I and EG II there is a greater improvement
in patients with EG II 11,27 (t=15,72; <0,001;
P=99,9%).
1.2. Measurement of the extension: There is a greatest
improvement in patients from EG II (16,20 ),
followed by EG I (13,83 ) compared to the results in
CG (13). The analysis of the results showed no
significant difference between the results for the
improvement of knee extension in patients CG and
EG I (X = 0,83 ; t = 0,81; > 0,05; P <95%). The
data obtained are illustrated in Figure 1
38.6
40
35
30
25
27.33
21.83
20
13
13.83
16.2
II
15
2.48
2.5
1.95
2
1.5
1.18
I
II
0.5
0
Ist level
IInd level
Fig 2: Average improvement of hip rate (in

centimeters)
3. Manual muscle testing: To verify the degree of
muscle weakness in patients - contingent of study, we
tested the major muscles in the knee complex: mm.
quadriceps
femoris;
tensor
fascialatae;
semimembranosus; semitendinosus et biceps femoris.
We present the data in a tabular form (Table. 2).
Table. 2. Statistical analysis of the results from
MMT of major muscles in the area of the knee, in
patients from all three groups
5
0
Fig1:
Average
improvement
of
angular
movements in the sagittal plane (X) in the three
groups of patients (in centimeters). CG control
group; EG I experimental group I; EG II
experimental group II
2. Hip girth measurement: When we measure hip
circumference of standard I-st level (5-7 cm cranial
from tuberositas as tibiae) it occurs at a distinct
outcome in patients - contingent EG II (1,42 cm),
followed by those in the composition of the EG I
(1,18 cm) compared with CG (1.12 cm).When we
measure hip circumference of standard II-nd level
(10-12 cranial from tuberositas tibiae), we establish
the greatest improvement in patients EG II (2,48 cm),
followed by the results of EG I (2,03 cm) and CG
(1.95 cm). Results of the study are presented in
Figure 2.
1.12
10
Knee extension
1.42
Group
Knee flexion
2.03
CG
Tested muscle
M. quadriceps
femoris
M. tensor fascia
latae
MM.
semitendinosus et
0,60
0,20 16,15
99,9
0,70
0,25 15,38
99,9
0,78
0,25 17,02
99,9
0,75
0,25 16,15
99,9
0,75
0,28
14,35
99,9
0,81
0,20
16,08
99,9
0,88
0,28
17,02
99,9
0,86
0,29
16,26
99,9
1,07
0,17
43,17
99,9
1,07
0,18
43,17
99,9
1,09
0,19
39,72
99,9
1,08
0,18
41,24
99,9
semimembranosus
M. biceps femoris
EG I
M. quadriceps
femoris
M. tensor fascia
latae
MM.
semitendinosus et
semimembranosus
M. biceps femoris
EG II
M. quadriceps
femoris
M. tensor fascia
latae
MM.
semitendinosus et
semimembranosus
M. biceps femoris
4. Testing of isometric muscle endurance

In order to verify the final effect from the treatment,
in respect restore contractile muscle performance in
Rostislav
563
isometric mode, we applied the analytical test of

timing of individual sections for m. quadriceps
femoris. The presented results are synthesized in
Table 3.
DISCUSSION
A significant positive dynamics in the recovery of

angular movements in the knee complex is
established in all patients, contingent of this study.
Table. 3.Statistical analysis of the results obtained after The analysis of the results indicates a most progress
testing the isometric muscular strength in the patients
in Experimental group II, followed by those in
of the three groups
Experimental group I and Control group. With a great
Group Tested muscle
X
S
t
%
statistical reliability, we find also a most significant
M. rectus femoris 25,33 4,90 28,31
99,9
improvement in the other controlled indicators in the
M. vastus
28,83 3,39 46,52
99,9
experimental group II (hip circumference, overcome
medialis
CG
muscle weakness, isometric muscular endurance and
recovery of locomotor skills without facilities).
M. vastus
24,66 3,92 34,42
99,9
Although, in large part approbated manually-therapy
lateralis
techniques aim to restore arthrokinematics of knee
M. rectus
complex, we find significantly better results in regard
29,03 6,40 24,81
99,9
femoris
to contractiles of tissue structures, what we give to
M. vastus
the possibility for an early and full recovery of
EG I
33,4 2,15 84,72
99,9
medialis
muscle function in conditions of various kinetic
M. vastus
chains, corrected joint mechanics and suppressed
31,8 1,76 98,43
99,9
lateralis
pain. In our opinion, the basis of correct and effective
treatment approach is the adjustment of joint
M. rectus
37,04 2,05 127,14 99,9
mechanics, thus there is an opportunity for
femoris
completely stimulation of analytical and complex
M. vastus
EG II
39,58 1,40 199,76 99,9
muscle functions and proprioception from the injured
medialis
knee.
M. vastus
lateralis
38,88 1,76
155,44
99,9
5. Testing gait without facilities

5. 1. Testing the step length of the operated lower
limb to overcome the distance of 5 m
Analysis of results showed the clear positive
dynamics in patients EG II (19,26 cm.), followed by
EG I (12,3 cm.) and CG (10,86 cm.).
5. 2. Testing the number of steps to overcome the
distance from 5 m
Table 4: Statistical analysis of the results of testing
of locomotor abilities to overcome the distance of 5
m. without facilities in patients in all three groups
P%
Indicator Group X
S
t
CG
10,86 1,25
47,53 99,9
Step
EG I 12,3 1,11
60,21 99,9
width
EG II 19,26 1,17
115,93 99,9
CG
2,5
0,50
26,92 99,9
Number
EG I 2,36 0,49
26,44 99,9
of steps
EG II 3,12 0,32
67,20 99,9
Analysis of results showed the greatest improvement
of controlled parameters in patients from EG II (3,12
n.) followed by CG (2,5 n.) and CG I (2,3 n.). The
data are synthesized in Table 4.
Rostislav
CONCLUSION
Based on the results of the study, we present the
following conclusions:
Application of adequate physiological and
pedagogically grounded kinesitherapy (KT) is
required
in patients after
arthroscopic
meniscectomy model with motor deficits
intractable routine rehabilitation.
Each of the three KT complex speeds up recovery
and return patients to everyday life.
The inclusion of a proximal mobilization
stretching (in addition to traditional physical
therapy) accelerates functional recovery of knee
complex (by analytical impact on the truncated
structures).
Most effective is a complex manually-therapeutic
approach, combined with appropriate stretching
techniques and ability to perform in terms of
proximal or distal fixation, for correction of
motor deficit.
Our structured and approved methodology is not
only convenient and easy to use but highly effective
564
in patients after arthroscopic meniscectomy and

shaped motor dysfunction. It reduces arthrogenic
pain, increasing the volume of movement, restoration
of intra-articular and physiological mobility,
stimulating muscle trophicity, improvement isometric
muscular endurance, increasing the muscle strength
and overcoming muscle weakness, arthrokinematic
recovery of knee complex, improving stability gait.
Observations allow us to offer a methodology for
implementation in general practice rehabilitation in
patients after meniscal ruptures treated by
arthroscopic meniscectomy and motor deficits,
intractable routine rehabilitation.
7. Mulligan B. Manual Therapy NAGS,

SNAGS,MWMS ect., Plane View Services. Wellington. New Zealand, 1st edn 1989; 6
8. Maitland G, E. Hengeveld, K. Banks. Maitlands.
Peripheral Manipulation.- Elsevier: Butterworth,
2005;4: 636
ACKNOWLEDGEMENT
The author is thankful to Prof. Troycho Troev, Head
of Clinic in Physiotherapy and Rehabilitation,
Military Medical Academy Sofia and his staff, Prof.
Ivet Koleva, Head of Department of Medical
rehabilitation and ergo therapy, Medical University
Sofia, and Prof. Ivan Topuzov for providing the
necessary facilities for the preparation of the paper.
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injuries: 1. Meniscal injuries in the adult. In
DeLee J (ed.), DeLee and Drezs Orthopaedic
Sports Medicine: Principles and Practice.
Philadelphia: Saunders. 2003; 2:8796.
2. Popov N. Kinesiology and pathokinesiology of
musculoskeletal system. Sofia: NSA-pres,
2009;1:398
3. Popov N. D. Popova. Pain suppressing
mobilization stretching of knee. J. Kinesitherapy,
2002; 3: 10-18.
4. Kraidjikova L. Methods of mobilizing massage in
musculoskeletal
dysfunction
of
knee.J.
Kinesitherapy and rehabilitation, 2009; 4:78-84.
5. Koleva Y. Fundamentals of Physical Therapy and
Rehabilitation (incl. Occupational therapy and
medical SPA). Pleven: Medical University,
2011;317
6. Koleva Y. Short Course of Physiotherapy (for
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course of Medical College, Medical University
Sofia. Sofia: RIK SIMEL, 2008;159
Rostislav
565
DOI: 10.5958/2319-5886.2015.00109.5

&
Health Sciences
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Volume 4 Issue 3
st
Received: 1 Apr 2015
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
EX VIVO STUDY OF GARCINIA MANGOSTANA L. (MANGOSTEEN) PEEL EXTRACT AND

XANTHONES AS ANTI-ADIPOGENESIS IN HEPG2 CELL MODEL
Lusiana Darsono1, Meilinah Hidayat1, Maesaroh Maesaroh2, Nurul Fauziah2, *Wahyu Widowati1
1
Medical Research Center, Faculty of Medicine, Maranatha Christian University, Jl. Prof drg. Suria Sumantri
No.65, Bandung 40164, West Java, Indonesia
2
Biomolecular and Biomedical Research Center, Aretha Medika Utama, Jl. Babakan Jeruk II. No. 9 Bandung
40163, West Java, Indonesia
*Corresponding author email: wahyu_w60@yahoo.com
ABSTRACT
Background: Anti-adipogenesis is one of proposed mechanism for anti-obesity. Adipogenesis regulation of
obesity is important, so identification of anti-adipogenic activity is a potential strategy to find anti-obesity agent.
Aim: The aim of this study is to evaluate the anti-adipogenesis potential of Garcinia mangostana L. peel extract
(GMPE) compared to xanthones in HepG2 cells line as model. Material and Methods: GMPE was performed
based on maceration method using distilated ethanol 70% as the solvent. The level of triglyceride and cholesterol
and the inhibitory activity of triglyceride (TG) and cholesterol (CHOL) in HepG2 cells were assayed and
determined as the anti-adipogenesis parameter. Results: The most active subtance to lower the triglyceride level
was showed by GMPE in every concentration followed by the garcinone-C, -mangostin, garcinone-D and mangostin respectivelly. The highest activity to decrease the cholesterol level was showed by GMPE and
followed by -mangostin, -mangostin, garcinone-c, garcinone-d respectively. Conclusion: GMPE posses the
anti-adipogenesis potential in inhibiting TG and CHOL synthesis was better than any other xanthone (mangostin, -mangostin, garcinone-C and garcinone-D).
Keywords: Obesity, Adipogenesis, Garcinia mangostana L., HepG2, Triglyceride, Cholesterol.
INTRODUCTION
Obesity is one of the most common global metabolic
disorders defined as an excessive body weight in the
shape of fat accumulation.[1] Recently, the metabolic
syndrome including obesity represents one of the
most serious problem worldwide.[2] Obesity has a
strong association with the chronic disease such as
diabetes, cardiovascular diseases, hypertension,
osteoarthritis, some cancer and inflammation-based
pathologies.[3] At the cellular level, obesity is
characterized by an excess accumulation of adipose
tissue is largely comprised of fat cells.[3-5] Obesity
including excessive differentiation and growth of
adipocytes which leads to increase fat cell mass and
number, adipogenesis, lipid accumulation and
lipogenic enzyme expression and surplus energy
accumulation stored as triglyceride (TG) in
Darsono et al.,
adipocytes.[1] Adipose tissue growth involves in

formation of new adipocytes from precursor cells,
further leading to an increase in adipocyte size.[6]
Adipogenesis is a multi step process involving a
cascade of transcription factors and cell cycle protein
regulating gene expression and leading to adipocyte
development.[7] The number of studies to prevent and
treat obesity continues to rise.[8] Developing antiobesity drugs that are efficacious and have minimal
side effects become a pressing need.[1] Antiadipogenesis is one of the proposed mechanisms of
anti-obesity.[9,10] Adipose tissues are specialized for
high capacity to accumulate TG.[11] Human hepatoma
HepG2 cells is the most suitable and accessible
human-derived cells that retain many of the
biochemical functions of human liver parenchymal
566
cells for the ex vivo study including anti-adipogenesis

screening.[12] Subjects with established obesity have
an increased lipogenesis in hepatocytes (not in
adipocyte) that might contribute to develop and/or
retain the excessive fat mass.[13] Excess hepatic lipid
accumulation is associated with nutritional factors,
drugs, and multiple genetic defects in energy
metabolism.[14]
Many
phytonutrients
were
investigated for their potential therapeutic properties.
Some phytochemical bioactive have been shown to
inhibit adipocyte differentiation and induce
adypocyte apoptosis.[15,16] Garcina mangostana L.
(mangosteen) is a tropical fruit originated from
Southeast Asia, has been used in traditional therapy
in the treatment of great variety of medical conditions
for decades.[17] The pericarps of this fruit have been
used for many years as traditional medicine in
treating sicknesses such as trauma, skin infection,
abdominal pain, dysentery, and wounds.[18] The major
bioactive secondary metabolites of G.mangostana
are xanthone derivates.[19] Xanthones could be
isolated from peel, whole fruit, bark, and leaves of
mangosteen.[20] Xanthones were repoted to have a
great variety of pharmacological activities including
antioxidant, antifungal, antibacteria, cytotoxic, antiinflammation, anti-histamine, anti-HIV, and other
activities.[19] The previous study confirm that
mangosteen peel extract contained -mangosteen
(105 ppm), -mangosteen (7.20 ppm), garcinone C
(3.50 ppm), and garcinone D (9.92 ppm) based on
high performance liquid chromatography (HPLC).[21]
Adipogenesis regulation of obesity is important, so
identification of anti-adipogenic activity is a potential
strategy to find anti-obesity agent. Therefore, the aim
of this study is to evaluate the anti-adipogenesis
potential of G. mangostana peel extract (GMPE)
compared to xanthones including -mangostin, mangostin, garcinone-C, garcinone-D in HepG2 cells
by using the inhibitory activity to lower triglyceride
and cholesterol synthesis as the parameters.
The present study was carried out in Biomolecular

and Biomedical Research Center, Aretha Medika
Utama, Bandung, West Java, Indonesia in
collaboration with Medical Research Center, Faculty
of Medicine, Maranatha Christian University. HepG2
cells (human liver hepatocellular carcinoma cell line)
were used as cells model after cells were induced to
differentiate as adipocyte. The laboratory experiment

was performed 4 months.
Plant material preparation and extraction:
Garcina mangostana L. was collected from CisalakSubang, West Java, Indonesia. The plant was
identified by herbarium staff, Department of Biology,
School of Life Sciences and Technology, Bandung
Institute of Technology, Bandung, West Java,
Indonesia. The peel were collected, chopped, and
kept in drier tunnel service. Extraction was performed
based on the maceration method with distilated
ethanol 70% as solvent.[21-23]
HepG2 cell culture and adipocyte differentiation
induction: HepG2 cells (human liver hepatocellular
carcinoma cell line) was cultured in DMEM
(Dulbeccos Modified Eagle Medium, Biowest)
supplemented with 10% FBS (Fetal Bovine Serum,
Biowest) and 100 U/mL penicillin-streptomycin
(Biowest) then incubated for 24 hours at 37C
humidified atmosphere and 5% CO2.[14] After the
cells were confluence, medium was discharged and
cells were harvested after tripsin-EDTA treatment in
2500 rpm centrifuge for 4 minutes, cells then
resuspended by 1 mL new medium and seeded in the
6 well plate (5 x 105 cells/well) with DMEM
supplemented with 10% FBS and 100 U/mL
penicillin-streptomycin then incubated for 48 hours
until confluent. Medium then discharged and
suplemented with starving medium (DMEM + 1%
antibiotic solution) then incubated for 24 hours.
Starving medium then discharged and supplemented
with induction medium (DMEM, 1:2 of 1mM
palmitic acid: 1mM linoleic acid, BSA, and GMPE or
xanthone). Cells then incubated in 37C humidified
atmosphere and 5% CO2.
Cells Lysate: Cell lysate was performed according to
Biorad #163-2068 kit protocol. Confluent cells after
GMPE or xanthone treatment were harvested using
500 g centrifuge for 4 minutes. Supernatan was
discharged and work solution was added into the
pelet cell. The solution briefly was sonicated for 30
second four times. Cells then centrifuge in 16.000
rpm for 30 minutes in room temperature. Supernatant
then transfered to 2 mL ependorf tube and placed in 80C as the sample for the TG and CHOL levels
assay.[24]
Triglyceride (TG) Levels and Inhibitory Activity
Assay: The TG level was measured according to
Randox TR 210 assay kit protocol. Briefly 450L
Darsono et al.,
567
reagent with 5L sample was incubated in 37C for 5

minutes. Double-distiled water (ddH2O) was used as
blank and standard reagent was used as standard.
Seven diferent concentration (2.180; 1.090; 0. 545;
0.273; 0.136; 0.068 and 0.034 mmol/L) were
prepared by serial dilution for standard solution. The
absorbance was measured in 500 nm of wave length.
The TG level was calculated based on the ratio of
sample absorbance to standard absorbance multiplied
by the standard concentration.[25]
Cholesterol Levels and Inhibitory Activity Assay:
The CHOL level assay was measured according to
the Chol Kit Randox CH 200 protocol kit. Briefly
500L mix reagent was added into 24 well plate and
5L sample was added into the sample well. 5L of
ddH2O was used as blank. Seven diferent
concentration (5.170; 2.585; 1.293; 0.646; 0.323;
0.162; and 0.081 mmol/L) were prepared by serial
dilution for standard solution. 5L standard solution
was added into the well as standard. The absorbance
was measured in 500 nm of wave length. The reaction
then incubated at 37C for 5 minutes. The absorbance
was measured in 500 nm of wave length. The
cholesterol concentration was calculated based on the
ratio of sample absorbance to standard absorbance
multiplied by the standard concentration.[26]
Statistical Analysis: The every treatment was done
in three replication. Statistical analysis was conducted
using SPSS software (version 17.0). Significant
differences between the groups were determined
using the Analysis of Variance (ANOVA) and
Tukey Post Hoc Test. Statistical significance was set
at p<0.05. The data were presented as mean
standard deviation.
RESULTS
Table 1: Triglyceride (TG) Level and Inhibition

Activity of GMPE and Xanthones in Various
Concentration
Treatment
-mangostin 250 g/mL

-mangostin 125 g/mL
-mangostin 62.5 g/mL
-mangostin15.625 g/mL
-mangostin 250 g/mL
-mangostin 125 g/mL
Garcinone-C 250 g/mL
Garcinone-C 62.5 g/mL
Garcinone-C15.625g/mL
Garcinone-D 250 g/mL
Garcinone-D 62.5 g/mL
Garcinone-D15.625g/mL
GMPE 250 g/mL
GMPE 125 g/mL
GMPE 62.5 g/mL
GMPE 31.25 g/mL
GMPE 15.625 g/mL
Positive control
Triglyceride (TG)
TG
level
(mg/dL)
24.50 0.26 f
25.68 0.01 f
26.97 0.27 f
46.68 0.74 i
57.60 0.45 lm
35.71 0.11 h
50.70 0.83 jk
54.32 0.37 kl
58.22 0.45 lm
61.14 0.02 m
12.36 0.16 e
12.64 0.02 e
32.68 0.11 gh
33.14 1.11 gh
31.75 3.78 g
34.19 0.06 gh
50.02 0.66 ij
55.50 0.54 l
60.48 0.16 m
75.44 1.54 n
0.00 0.01 a
0.00 0.02 b
0.00 0.01 c
0.00 0.18 d
26.64 1.70 f
89.20 0.69 o
TG inhibition %
72.54 0.29 k
71.22 0.01 k
69.76 0.30 k
47.68 0.83 h
35.420.5cde
59.97 0.12 i
43.160.93 fg
39.100.40 ef
34.720.51cd
31.46 0.03 c
86.15 0.18 l
85.84 0.02 l
63.37 0.11 ij
62.851.24 ij
64.41 4.24 j
61.67 0.07 ij
43.930.74gh
37.780.60de
32.19 0.17 c
15.42 1.73 b
100 0.01 p
100. 0.02 o
100 0.01 h
100 0.20 m
70.14 1.91 k
0.00 0.00 a
*The data are presented as mean standard deviation.

Different letters in the same column (a-o and its
combination) indicate significant differences among the
means of groups (GMPE and xanthones in various
concentrations) based on Tukeys pos hoc comparison
(P<0.05).
Table 1 showed that GMPE and xanthones including

-mangostin,
-mangostin,
garcinone-C
and
garcinone-D have lower TG levels in HepG2 cells
compared to the triglyceride level in the cell lysate
without GMPE and xanthones. The most active
substance was showed by GMPE in every
concentration followed by the garcinone-C, mangostin,
garcinone-D
and
-mangostin
respectivelly showed by the TG inhibitory activity.
High plasma TG is associated with obesity.[27]
The highest CHOL inhibition activity was showed by

GMPE followed by -mangostin, -mangostin,
garcinone-C and garcinone-D respectivelly. GMPE
showed CHOL inhibition activity over 50% in the
concentration above 15.625 g/mL (Table 2).
Morever, GMPE showed 100% CHOL inhibition
activity. Obesity has an association with decreasing
high-density lipoprotein cholesterol (HDL-C)
level.[28]
Darsono et al.,
568
Table 2: Cholesterol (CHOL) Level and Inhibition

Activity of GMPE and Xanthones in Various
Concentrations
Samples
-mangostin 250 g/mL

-mangostin 125 g/mL
-mangostin15.62g/mL
-mangostin 250 g/mL
-mangostin 125 g/mL
-mangostin31.25g/mL
-mangostin15.62g/mL
Garcinone-C31.25g/mL
Garcinone-15.625g/mL
Garcinone-D31.25g/mL
Garcinone-15.625g/mL
GMPE 250 g/mL
GMPE 125 g/mL
GMPE 62.5 g/mL
GMPE 31.25 g/mL
GMPE 15.625 g/mL
Positive control
Cholesterol (CHOL)
CHOL
level CHOL
(mg/dL)
inhibition (%)
8.60 0.50 ab
93.71 0.37 kl
10.91 0.03 bc
92.02 0.02 k
27.96 0.04 ef
79.540.03 ghi
42.27 0.08 gh 69.07 0.06 ef
44.82 0.08 gh 67.21 0.06 ef
23.10 0.15 cde 83.11 0.11 ij
29.00 0.10 ef
78.790.08 ghi
29.05 0.57 ef
78.750.42 ghi
35.05 0.28 efg 74.360.20 fgh
36.89 0.33 fg
73.01 0.25 fg
24.91 1.48 def 81.78 1.07 hi
25.93 0.03 def 81.03 0.01 hi
52.94 0.28 h
61.27 0.21e
68.86 0.24 i
49.62 0.18 d
100.00 0.04 k
25.85 0.04 b
26.26 1.16 ef
80.790.85 ghi
45.44 0.11 gh 66.76 0.08 ef
74.72 0.01 i
45.33 0.01 d
78.13 4.15 ij
42.84 3.04 cd
87.94 1.03 j
35.66 0.75 c
0.00 0.34 a
100.000.25 m
0.00 0.21 a
100. 0.14 lm
13.55 0.13 bcd 90.09 0.10 jk
29.03 1.41 ef
78.771.03 ghi
107.81 12.39 k 21.12 9.07 b
136.68 8.20 l
0.00 0.00 a
* The data are presented as mean standard

deviation. Different letters in the same column (a-o
and its combination) indicate significant differences
among the means of groups (GMPE and xanthones in
various concentrations) based on Tukeys pos hoc
comparison (P<0.05).
DISCUSSION
Obesity is a risk factor for severe disease such as
diabetes, atherosclerosis, coronary heart disease and
several cancer.[29] Primarily, obesity is a disorder of
lipid metabolism and the enzyme involved in this
process could be selectively targeted to develop antiobesity drugs.[8] Different parts of medicinal plants
like stem, flower, seed, root, fruit, etc. are used to
obtain pharmacologically active metabolite.[30]
Mangosteen peel has been used in medicinal in both
Chinese and Ayurvedic. The yellow exudate from
mangosteen peel contain xanthone as the major class

of compounds including -mangostin, -mangostin,
-mangostin, garcinone-C and garcinone-D along
with mangostinone, tanins, and flavonoid called
epicatechin.[31] In this ex vivo study, we evaluated the
anti-adipogenesis potential of GMPE and xanthones
on HepG2 cells. Our results demonstrated that GMPE
and xanthones exhibited potential to decrease the
level of CHOL and TG as anti-adipogenesis
parameters compared to the cell without GMPE or
xanthones treatment.
In present study, GMPE could lower TG level
compared to the cells without GMPE or xanthones
treatment. Inhibition of TG metabolism was mediated
by decreasing of gene expression of FAS (Faty Acid
Synthase), ACC(Acetyl-CoA Carboxylase), and
malic enzyme, among other factors.[32] The decrease
of TG level may result from decreasing lipid
synthesis.[33] Excess fat is stored as TG in the adipose
tissue.[34] The decreased levels of TG indicated the
adipogenesis inhibition. The potential for suppresing
of adipogenesis and reducing lipid accumulation in
cells mode were showed by some xanthones
including -mangostin and -mangostin.[35] mangostin also showed its ability to inhibit FAS
correlated with intracellular lipid accumulation in
differentiating adipocytes and stimulated lipolysis in
mature adipocytes.[18]
Adipocyte normaly contain free CHOL and will
redistributed from the plasma membran to the lipid
droplet as TG storage increase. Adipocyte CHOL
levels will increase in proportion to TG level.[34,35]
GMPE also showed have CHOL inhibitory activity in
concentration dependent maner showed by the
decrease HOL level compared to the cell without
GMPE or xanthones treatment as anti adipogenesis
parameters. Previous study also found that
G.mangostana posses the CHOL level reduction.[36]
The GMPE showed anti-adipogenic activity through
suppressing proliferator-activated receptor gamma
(PPAR) expression and FAS activity.[37] Maria et al
(2007) study observed that diets supplemented with
mangosteen positively affect plasma lipid levels and
plasma antioxidant activity in rats fed cholesterolcontaining diets.[38] Based on our ex vivo study, we
recommended the GMPE have beneficial effects as
anti-adipogenesis agent was comparable with
xanthones through the inhibition activity on CHOL
and TG synthesis in HepG2 cells model.
\
569
Darsono et al.,
CONCLUSSION
The GMPE posses the anti-adipogenesis potential on
decreasing CHOL and TG levels in HepG2 cell
better than xathones (-mangostin, -mangostin,
garcinone-C and garcinone-D). However, in vivo test
in an animal model still needed to confirm the antiadipogenesis activity of the GMPE and xanthones.
8.
9.
ACKNOWLEDGMENT
We gratefully acknowledge the financial support of
the Research Center and Service Commuinity,
Maranatha Christian University for research grant
2014. We are thankful to Pande Putu Erawijantari
from Biomolecular and Biomedical Research Center,
Aretha Medika Utama, Bandung, Indonesia for her
valuable assistance. This research was also supported
by Biomolecular and Biomedical Research Center,
Aretha Medika Utama, Bandung, Indonesia for
research method and laboratory facilities.
10.
11.
12.
13.

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ISSN: 2319-5886
PATTERN OF EXTRA-PULMONARY TUBERCULOSIS IN PATIENTS WITH HUMAN

IMMUNODEFICIENCY VIRUS INFECTION, AT A TERTIARY CARE CENTRE IN EASTERN INDIA
*Mathew Ninan1, Doye George1, P Sukumaran1, Gunanidhi Sahu2, RN Mania2, Philip Mathew3
1
Department of Pulmonary Medicine,3Department of Community Medicine, Pushpagiri Medical College,

Tiruvalla, Kerala, India
2
Department of Pulmonary Medicine, SCB Medical College, Cuttack, Orissa, India.
*Corresponding author email: communitymedicine@pushpagiri.in
ABSTRACT
Introduction: Tuberculosis and Human Immunodeficiency Virus (HIV) co infection is becoming one of the most
important public health issues in India. In some developing countries, 40% of all tuberculosis cases are attributed
to HIV infection and in more than 50% of cases, tuberculosis is the first manifestation of HIV infection.
Materials and methods: A cross sectional study was done among the in-patients of a tuberculosis ward in a
tertiary care hospital in Eastern India. Fifty patients with HIV and tuberculosis infection of an extra pulmonary
site, were included in the study. Results: A vast majority of the participants were young males. More than 80%
were using intoxicants like alcohol or tobacco, 76% admitted exposure to commercial sex workers and 12% were
intravenous drug users. Twenty five (50%) of the participants had disseminated tuberculosis, that is tubercular
infection of more than one anatomical site. Maximum (62%) participants had tubercular infection of lymph
nodes, followed by pleura, abdomen and central nervous system. Discussion and conclusions: The sociodemographic correlates of participants were similar to that seen in similar studies in other parts of the world. But
in our study, tubercular lymphadenitis was the most common extra-pulmonary manifestation and proportion of
disseminated tuberculosis cases was as high as 50%. Both these findings are different from studies from other
parts of the world. These findings warrant a larger research study and programmatic changes to address issues of
HIV/TB co infection.
Keywords: Tuberculosis epidemiology, Tuberculosis diagnosis, Risk factors, HIV Infections/epidemiology
INTRODUCTION
Tuberculosis in India is characterised by high
incidence and prevalence of both tuberculosis
infection and disease. Different disease surveys have
yielded varied results, the prevalence of smear
positive pulmonary tuberculosis ranged from 0.6-7.6
per 1000 population while that of culture positive
tuberculosis was in the range 1.7-9.8 per 1000
population.(1)There is also a problem of high
transmission rates and is indicated by a very high
Annual Risk of Tuberculosis Infection (ARTI) of
around 1.5%.[1] Fittingly, India is classified along
with sub-Saharan countries in terms of the

effectiveness of tuberculosis control. [2] Even though
some studies have demonstrated a small downward
trend over a long period of time, especially after the
launch of the Revised National Tuberculosis Control
Programme (RNTCP), most of the tuberculosis
researchers and epidemiologists refuse to believe that
the peak of infection has already been crossed.[2]
According to a global consensus effort by World
Health Organisation (WHO) in 1997, the estimated
number of new cases for the year was around 8
572
Ninan et al.,
million and more than 50% of it was contributed by 5

countries of the WHOs South East Asian Region
(SEARO). They concluded that the burden of
tuberculosis is remaining so enormous, as the control
efforts have failed in South-Asia, sub-Saharan Africa
and Eastern Europe. [3] Since the global consensus
effort by WHO, Indian health authorities have
stepped up efforts to expand the RNTCP programme.
By 2005, RNTCP was successful in initiating
treatment for over 3.5 million patients and avoided
premature mortality in 600,000 patients. [4] Though
this renewed interest in tuberculosis control and
heavy investment in Directly Observed Treatment,
Short course (DOTS) strategy has yielded results in
urban and rural areas of the country, the tuberculosis
situation in Tribal areas have worsened over the last
few years. [2] In a state like Orissa, where Scheduled
Tribes (STs) form over 22% of the total population,
the ARTI was estimated to be 1.7% to 1.8%, much
above the national average rates. [5] The reasons for
this high infection load can be many; the poor
penetration of healthcare delivery infrastructure
coupled with low awareness levels among people can
be the main ones. In a study done in Orissa, only 16%
of the respondents knew about the cause of
Tuberculosis while 31% knew the correct mode of
spread of the disease. This is much lower than the
statistics from other parts of the country, derived
from similar surveys. [6]
Human Immunodeficiency Virus (HIV) infection is
an independent risk factor for tuberculosis and is
considered as a cause for the resurgence of
tuberculosis in many industrialised countries. The
high incidence of tuberculosis (almost 290/100,000
population) in Africa is also attributed to the high
prevalence of HIV infection in that part of the world.
[7]
In some countries like Tanzania, over 40% of the
tuberculosis cases are attributed to the presence of
HIV infection in those patients. [8] In most of the
developing countries, Tuberculosis is considered as
the most serious opportunistic infection among those
people with HIV infection and is the first
manifestation of Acquired Immunodeficiency
Syndrome (AIDS) in over 50% of the cases. [9] India
has the third most number of HIV patients with 2.1
million people living with HIV/AIDS. Though the
HIV epidemic in India is slowing down with a 57%
reduction in new HIV infections over a period of
2000 to 2011, it has the potential to explode into
gargantuan proportions. These points towards the

need for sustained investment in research activities on
HIV and allied subjects. [10] Diagnosis of tuberculosis
among HIV affected individuals pose a dilemma for
clinicians as most of the cases tend to be extrapulmonary and the routine sputum testing may prove
to be ineffectual. [11] The sites for extra-pulmonary
tuberculosis among HIV infected individuals seem
varied; according to a study from New Delhi,
abdomen (70%) is the most common site for extrapulmonary tuberculosis, followed by lymph nodes
(22%), pleura (15%) and Central Nervous System
(3%). [11] Another study from Shimla, Himachal
Pradesh showed that CNS tuberculosis constituted
over 33% of the extra-pulmonary tuberculosis cases
in HIV patients and was followed by abdominal
tuberculosis (26%). The study also found out that
disseminated tuberculosis was seen only in patients
with CD4 count of less than 200/cmm. [12] In a study
from Gujarat, it was found that among HIV patients,
60% of the tuberculosis disease was extra-pulmonary
in site and mediastinal tuberculosis constituted about
34% of them, followed by extra-thoracic
lymphadenopathy in 18% of the patients. [13]
Conversely, the prevalence of HIV infection among
those with tuberculosis is also much higher when
compared to general population. In a study done in
Agra, Uttar Pradesh, it was observed that the 4.3% of
the adults and 8.5% of the children attending a
tuberculosis clinic were HIV infected. [14, 15]
Tuberculosis and HIV are converging dual epidemics
and constitute the greatest public health challenge of
our time. The diagnosis and treatment of HIVTuberculosis co-infection is a difficult task and
understanding the pattern of disease in such cases
may help to identify patients who may need treatment
for both infections. [16]
Study design: Cross-sectional, observational study
Inclusion criteria: Patients admitted with a diagnosis
of tuberculosis of any site were screened for HIV
infection using Enzyme Linked Immunosorbent
Assay (ELISA). Those patients, who had positive
results on ELISA, were tested and confirmed using
Western Blot technique. [17] All the patients who had
a positive test in ELISA and Western Blot, and
having suspicion of extra-pulmonary tuberculosis
were included in the study.
573
Ninan et al.,
Exclusion criteria: Those patients admitted in

intensive care units or on assisted ventilation were
excluded from the study
Ethical approval: Clearance was obtained from the
Institutional Review Board of SCB Medical College,
Cuttack, before the start of the study. Also, written
informed consent was obtained from all participants.
Duration of study: January 2004 to December 2005.
Methodology: Extra-pulmonary tuberculosis was
confirmed by mycobacterial and histopathological
examination of the relevant samples. For lymph node
tuberculosis, a lymph node biopsy or a Fine Needle
Aspiration Cytology (FNAC) was done to obtain
samples. Ascitic fluid or pleural fluid was taken to
confirm pleural or abdominal tuberculosis
respectively. Cerebro-Spinal Fluid (CSF) was taken
and examined to diagnose Central Nervous System
tuberculosis. Radiological investigations like contrast
enhanced CT scans were used for diagnosis when
mycobacterial and histopathological examination was
inconclusive or when appropriate samples were not
obtained.
The participants were administered a standardised,
pilot-tested questionnaire to find out their sociodemographic correlates, risk factors, symptoms, past
history and treatment history. A focussed clinical
examination was done to elicit all the associated
clinical signs. A sputum sample was collected for
Ziehl-Neelsen(ZN) staining [18], to rule out pulmonary
tuberculosis and a venous blood sample was collected
for measuring CD4 (Cluster of Differentiation 4)
counts, which is a surrogate marker for disease
activity
in
HIV
infection[19].
Table 1: Baseline characteristics (n=50)

Attribute
Age
(in Years)
Sex
Marital
Status
Occupational
status
HIV status of
spouse(n=41)
Characteristics
15-24
25-34
35-44
45 and above
Male
Female
Married/ Unmarried/
Separated/Divorced
Spouse died
Manual labourer
Driver
Small business
Army/Police
Housewife
Others
Positive
Negative
Unknown
%
6%
44%
44%
6%
92%
8%
82%
18%
0
54%
22%
10%
2%
8%
4%
58.5%
29.2%
12.2%
A vast majority of the participants were using

intoxicants regularly. Forty four (88%) of the
participants admitted using Alcohol, 40(80%) said
they use tobacco in some form and 4(8%) used
cannabis/cannabinoids. Among the other social risk
factors for HIV infection, 35(70%) had history of
migration from the place of birth and 5(10%) had
history of imprisonment or incarceration by a court of
law. A vast majority of the participants (38, 76%)
admitted having contact with commercial sex workers
and 6(12%) have used intravenous drugs in the past.
Interestingly, none of the participants were
homosexuals [Table 2]
Table 2: Social risk factors for HIV infection
(n=50)
Risk factor
Use of intoxicants
RESULTS
A total of 50 patients with HIV/TB co-infection and
with extra-pulmonary manifestation of tuberculosis
infection, were included in the study. The maximum
numbers of participants were from the age group 2534 years (44%) and 35-44 years (44%). Males
constituted 92% of the total study participants and
82% of the participants were married. Most of the
study participants were manual labourers (54%) and
drivers (22%). Twenty four (58.5%) of the currently
married participants had their spouses with a positive
HIV status, while it was negative for 29.2% and
unknown for 12.2%. [Table 1]
Number
3
22
22
3
46
4
41
9
0
27
11
5
1
4
2
24
12
5
History of Migration
History of
Imprisonment
Contact with
Commercial Sex
Worker
Men Having Sex with
Men (MSM) (n=46)
Intravenous drug user
Characteristic
Alcohol
Tobacco
Cannabis
Hallucinogens
Yes
No
Yes
No
Yes
No
Number(%)
44(88%)
40(80%)
4(8%)
6(12%)
35(70%)
15(30%)
5 (10%)
45(90%)
38(76%)
12(24%)
Yes
No
Yes
No
0
46(100%)
6(12%)
44(88%)
Twenty five (50%) of the participants had

disseminated tuberculosis, that is tuberculosis
infection of more than one anatomical site. The rest
of the participants had tuberculosis limited to one
extra-pulmonary site. Lymph nodes were the most
574
Ninan et al.,
common site (62%) followed by pleura (32%),

abdomen (22%), Central Nervous System (8%) and
bone marrow (2%). None of the participants had
tuberculosis infection of the spine or the
mediastinum. [Table 3]
Table 3: Pattern of Extra-pulmonary tuberculosis
(n=50)
Extra-pulmonary site
Number Percentage
Lymph node(s)
31
62%
Pleura
16
32%
Abdomen
11
22%
Central Nervous system 4
8%
Bone marrow
1
2%
Spine
0
0
Mediastinum
0
0
Among the 25 participants with disseminated
tuberculosis infection, 7(28%) had infection of lymph
nodes and lung, 5 (20%) had infection of lymph node
and pleura and 4(16%) had infection of lymph nodes
and abdomen. One (4%) participant each had
tuberculosis infection of bone marrow and miliary
tuberculosis. [Table 4]
Table 4: Pattern of Disseminated tuberculosis
(n=25)
Sites of Dissemination
Lymph node and lung
Lymph node and pleura
Lymph node and abdomen
Lymph node, pleura and abdomen
Lymph node and CNS
Lymph node, CNS and lung
Pleura and abdomen
Pleura and lung
Abdomen and lung
Bone marrow
Miliary
Number
7
5
4
1
1
1
2
1
1
1
1
%
28%
20%
16%
4%
4%
4%
8%
4%
4%
4%
4%
Among the 25 participants where the tuberculosis

infection was limited to one extra-pulmonary site,
12(48%) had infection of the lymph nodes, 8(32%)
had infection of the pleura, 3(12%) had abdominal
tuberculosis and 2(4%) had Central Nervous system
tuberculosis. [Table 5]
Table 5: Pattern of isolated extra-pulmonary
tuberculosis (n=25)
Extra-pulmonary site
Number Percentage
Lymph node
12
48%
Pleura
8
32%
Abdomen
3
12%
Central Nervous System
2
4%
DISCUSSION
The study reveals a familiar trend seen in other parts
of the world, where HIV infection and tuberculosis
are generally considered as a disease of social
inequities. The participants in our study are from
occupations which are relatively lowly paid, a large
number of them are migrants, and the use of
intoxicants is very high. A similar finding is seen in
studies done in other parts of the world, especially
South Asian nations and African countries. [20] A vast
majority (76%) of the participants reported exposure
to commercial sex workers and this may be the
reason behind the baseline demographic correlates
being skewed in favour of young males. This finding
is also in concordance with the results of similar
studies from other parts of the country which states
that commercial sex is the primary mode of HIV
infection for males and sexual relations with the
infected husband is the most important mode of
acquiring infection for females. [21]
In our study, the most common site of extrapulmonary tuberculosis infection was lymph nodes,
followed by pleura and abdomen. Tubercular
lymphadenitis was seen commonly in disseminated
tuberculosis infection as well as in isolated extrapulmonary tuberculosis. This finding is quite
different from the findings in other parts of the
country which stated that infection of the abdomen
and Central Nervous System were the most common
extra-pulmonary sites in HIV/TB co infection. The
proportion of disseminated tuberculosis cases among
all the extra-pulmonary tuberculosis patients was as
high as 50%, and this is much higher when compared
to data from other parts of the world. [22] This may be
due to the late diagnosis of tuberculosis and HIV
infection, Orissa being a relatively backward state in
socio-economic progress and healthcare delivery
indicators.
CONCLUSION
This study points towards the need to do more
extensive research in HIV/TB co-infection. All the
stakeholders of the Revised National Tuberculosis
Control Programme (RNTCP) and the National Aids
Control Programme (NACP) needs to be sensitised
on the need to screen for the other infection when one
of it is diagnosed in a patient. Since the diagnosis of
extra-pulmonary tuberculosis is difficult and
575
Ninan et al.,
expensive, it poses a clinical dilemma to the treating

doctors. Diagnostic protocols and cost-effective
techniques need to be formulated in diagnosing and
treating these conditions when both the infections
occur together.
10.
11.
ACKNOWLEDGEMENT
The authors would like to thank the patients and staff
of SCB Medical College and Hospital, Cuttack for all
their cooperation and help.
12.

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DOI: 10.5958/2319-5886.2015.00111.3

&
Health Sciences
www.ijmrhs.com
st
Research article
Copyright @2015
ISSN: 2319-5886
Accepted: 23rd May 2015
SERUM GAMMA-GLUTAMYL TRANSPEPTIDASE AND LIPIDS IN YOUNG ADULTS WITH

UNCOMPLICATED ESSENTIAL HYPERTENSION
*Rajarajeswari D1, Ramalingam2, Sharmila T3, Prasad Naidu M4, Naidu JN5
Dept of Biochemistry, Narayana Medical College and Hospital, Chinthareddy Palem, Nellore, Andhra Pradesh,
India.524003
*Corresponding author email: bio.rajeswari@gmail.com
ABSTRACT
Introduction: Gamma-glutamyl transpeptidase initially used as an indicator of alcohol ingestion is now viewed
as a sensitive marker of sub clinical inflammation. Recent clinical studies have shown its association with blood
pressure and lipid profile. As GGT degrades glutathione, an antioxidant GGT can be considered as a pro
inflammatory marker playing a role in atherogenesis and hypertension. Materials and methods: 65 subjects with
Essential hypertension and 50 age and sex matched healthy controls both male and female between 18-50 years
of age were recruited from General Medicine department of Narayana Medical college and Hospital, Nellore,
A.P. Serum GGT was measured by calorimetric kinetic assay. Fasting Serum Triglycerides, Total Cholesterol
and HDL cholesterol by standard enzymatic procedures and LDL cholesterol by Friedwald equation. Results:
GGT is significantly elevated in hypertensive subjects (MeanSD 64.218.62IU/L) compared to controls
(MeanSD 26.208.91IU/L) (P value <0.001). GGT is significantly correlated with systolic BP (r= 0.26 p<0.01)
and diastolic BP(r= 0.28 p<0.01). Conclusion: Our findings suggest that elevated GGT in young adults may
contribute to their susceptibility to hypertension and provide an additional evidence of novel role of GGT in
cardiovascular risk evaluation.
Keywords: Serum -glutamyl transpeptidase, Lipid profile, Young adults, Essential Hypertension.
INTRODUCTION
Essential Hypertension is increasing rapidly among
young generation due to changes in dietary habits and
lifestyle modifications [1] Hypertension is one of the
major risk factor for cardiovascular diseases [2]
.Premature death in young adults is mainly due to
cardiovascular diseases. Therefore it is very essential
to diagnose and treat hypertension as early as
possible[3] . Recent research has revealed that GGT is
a proinflammatory marker involved in atherosclerosis
and cardiovascular risk. Gamma-glutamyl transpeptidase , initially used as an indicator of liver
function is now found to be elevated in other
metabolic disorders like
non-insulin diabetes
mellitus, Hypertension etc[4]. GGT is a glycoprotein

consisting of two polypeptide chains [5,6]. GGT is
mainly originated from liver but also seen in other
organ tissues like kidney, lung , pancreas and blood
vessels[7,8].Glutathione is a tripeptide with Glutamic
acid , Glycine and Cysteine in its structure[9,10] .
Glutathione is an important anti oxidant and exits
both in its reduced and oxidised forms within the cell.
the main function of GGT is to degrade glutathione
to form a dipeptide which act as a reducing agent and
forms free radicals[11] . LDL in the vascular
endothelium is oxidised by the action of free radicals
and forms a plaque lining the blood vessels.
578
Rajarajeswari et al.,
Progressive formation of plaque results

atherosclerosis leading to hypertension[12].
in

Present study is a case control study, 65 subjects with
Essential hypertension and 50 age and sex matched
healthy controls both male and female between 25
and 60 years of age were recruited from General
Medicine department of Narayana Medical College
and Hospital, Nellore, A.P. The present study is
approved by institutional ethical committee
Sample size: 65 subjects with Essential hypertension
and 50 age and sex matched healthy controls both
male and female between 25 and 60 years of age
were recruited from General Medicine department
of Narayana Medical college and Hospital, Nellore,
A.P. were included in the study.
Exclusion criteria: Pregnant, lactating women and
patients with diabetes, liver disease and patients on
drugs which might influence the serum levels of
lipids and GGT were excluded from the study.
Methodology: Blood pressure (BP) was measured by
a physician. Patients who were found to have Systolic
Blood Pressure (SBP) higher than 140 mmHg and/or
Diastolic Blood Pressure (DBP) higher than 90
mmHg on three consecutive days were considered as
hypertensive [13]. Blood samples 5ml were collected
under aseptic conditions from all the study
participants for estimation of serum GGT. All
samples were centrifuged and analyzed within 3
hours of sample collection. Serum GGT was
measured by calorimetric kinetic assay [16].. Serum
total cholesterol, triglycerides, HDL and LDL
cholesterol are analyzed by Friedwald equation [21]
Statistical analysis: all data were analysed by SPSS13 version. P< 0.01 were considered as significant
years for controls. Table 1 shows that GGT is

significantly elevated in hypertensive subjects
(MeanSD 64.218.62 IU/L) compared to controls
(MeanSD 26.208.91IU/L) (P value <0.001). Fig1
illustrates the MeanSD values of variables between
cases and controls. Fig2 shows that GGT is
significantly correlated with systolic BP (r= 0.26
p<0.001). Diastolic BP is also significantly correlated
with serum GGT(r= 0.28 p<0.01). Serum total
cholesterol, triglycerides and LDL cholesterol are
significantly elevated in subjects with essential
hypertension.HDL cholesterol is significantly
decreased compared to controls (p<0.001).fig3 shows
significant negative correlation is observed between
serum GGT and HDL cholesterol (r=-0.29)
Table 1: Main characteristics of hypertensive
cases and controls
Biochemical
parameters
GGT (IU/L)
Cholesterol
(mg/dl)
Triacyl glycerol
(mg/dl)
HDL(mg/dl)
LDL(mg/dl)
SBP(mm/Hg)
DBP(mm/Hg)
Cases
n=65
70.1025.08
359.3570.13
Controls
n=50
23.348.42
148.2528.60
p-value
238.87110.1
5
35.696.52
160.3915.48
148.06 9.23
115.31 7.61
79.1328.88
<0.0001
75.1218.32
89.2620.34
93.814.56
75.29 3.78
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
Data are expressed as MeanSD. P<0.05 are

significant.
RESULTS
A total of 115 blood samples were collected and
grouped into normal subjects and subjects with
essential hypertension. MeanSD of age for cases is
38.71 8.48 years as compared with 32.72 11.28
Fig 1: Shows main characteristics of hypertension

cases and controls
579
large Finnish group in both sexes, demonstrated that

serum GGT is independently linked to alocohol
ingestion and as a prognostic marker for myocardial
ischemia. recent insights into the role of serum GGT
in the development of hypertension and some
metabolic disorders like type 2 diabetes increases our
understanding of the evolution of these diseases and
to stage the disease for better treatment of the disease
[19, 20, 21]
CONCLUSION
Fig 2: Negative correlation between HDL and GGt
levels(r=0.29821)
Our findings suggest that elevated GGT in young

adults may contribute to their susceptibility to
hypertension and provide an additional evidence of
novel role of GGT in cardiovascular risk evaluation.
ACKNOWLEDGMENT
Our
acknowledgements
to
department
of
Biochemistry, NMCH, and Nellore for their constant
support thought our research work.
REFERENCES
Fig 3: Positive correlation between the systolic BP
and GGT levels (r=0.120707)
DISCUSSION
The present study indicates that GGT level is elevated
in hypertensive patients compared with their
normotensive subjects. Our results demonstrated a
positive association between higher serum GGT level
and clinical hypertension. These results are in
agreement with previous studies that reported a
positive association between higher serum GGT level
and clinical hypertension [13,14,15,16]. In the present
study the age of the patients in hypertensive group
was 38.71 8.48 years as compared with 32.72
11.28 years in the normotensive group. The
male:female ratio between the two groups did not
show any significant statistical difference . The
present study suggests that serum GGT levels are
elevated in hypertensive patients as compared with
their age and sex matched normotensive subjects (P <
0.001). Our results are in agreement with the current
role of GGT in the development of hypertension [17,18].
Previous study conducted by Ruttmanns etal in a
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and critical care medicine. 2009;179(3): 228-34.
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Atar, Asli I., et al. "Association between gammaglutamyltransferase
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Shao, Na, et al. "Design of bis-spiropyran ligands
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Mao Yu. Serum gamma-glutamyl transferase: A
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"Inflammation and atherosclerosis." Circulation.
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Kotani K, Shimohiro H, Adachi S, Sakane N. The
association between an increased level of gamma
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Shankar A, Li J. Association Between Serum
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Lee DS, Evans JC, Robins SJ, Wilson PW,
Albano I, Fox CS, et al. Gamma Glutamyl
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comparison of the effects of Gamma
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K, Ulmer H. Gamma-glutamyltransferase as a
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581
DOI: 10.5958/2319-5886.2015.00112.5

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Copyright @2015
th
th
Research article
ISSN: 2319-5886
RETROSPECTIVE STUDY OF RELAPAROTOMY IN DEPARTMENT OF OBSTETRICS,

GYNAECOLOGY AND FAMILY PLANNING IN, RURAL TERTIARY CARE HOSPITAL, ANDHRA
PRADESH, INDIA
*Uma Thombarapu1, Prabha Devi Kodey2, GangadharaRao Koneru3
1
Assistant Professor,2Professor & Head of the Department, 3Professor, Department of Obstetrics & Gynaecology,
NRI Medical College & General Hospital, Chinakakani, Guntur, Andhra Pradesh, India
*Corresponding author email: umathombarapu@gmail.com
ABSTRACT
Introduction: Relaparotomy is biggest dilemma to the surgeon and critical to the patient to undergo second
surgery within short span of time .It is challenging both physically and mentally to the patient. Aim: Aim of the
study was to determine incidence of relaparotomy and its indication, management and outcome in the department
of Obstetrics, Gynaecology and Family Planning (OBGYN & FP) in NRI Medical College & General Hospital at
Guntur District. Materials and Methods: It is a retrospective observational study for the duration of 3 and
years. Total number of surgeries -7, 718. Total number of relaparotomy- 27 which include referral cases. Results:
Incidence for relaparotomy was 0.34%. Most important cause for relaparotomy was haemorrhagic causes
(44.4%), followed by burst abdomen (33.3%). Relaparotomy can increase morbidity, mortality (14.8%) of
patients with increased hospital stay on an average of 27 days including Intensive Care Unit, further increasing
the financial burden to the patient. Conclusion: Emergency relaparotomy is a life saving procedure. Good
expertise in selection of primary surgery and right surgical technique, intra operative hemostasis, control of post
operative infection can avoid relaparatomy
Keywords: Relaparotomy, Haemorrhage, Burst Abdomen, Hemoperitoneum, Hemostasis.
INTRODUCTION
Relaparotomy is original Greek word with three
components re - repeated, Laparostomach and tomie cut. If laparotomy done within 60 days of primary
surgery for the original disease it is called
relaparotomy[1]. Early relaparotomy is one that is
done, within 21 days of the primary surgery. If the
laparotomy is done which is planable, repeated and
multiphasic to complete the primary surgery is not
considered as relaparotomy. The main purpose for
relaparotomy in OBGYN & FP is to achieve
haemostasis, to control sepsis and repair burst
abdomen[2]. The decision to perform and manage
relaparotomy should be done by senior consultant, as
it is associated with considerable surgical and
anaesthetic complications as patient may be in

shock[3], sepsis and multi organ dysfunction. It is a
difficult decision requiring good clinical judgement[4].
Even some times inappropriate decision may be
deleterious to the patient leading to increased
morbidity and mortality. Increasing trends in
caesarean sections [5] performing surgeries at the
peripheral centers where non availability blood bank
facilities, intensive care units still increases the
complications leading to relaparotomy. Aim of the
study is to identify incidence, risk factors of
relaparotomy and analyse all cases for its indications,
management and outcome.
Uma et al.,
582
RESULTS
Type of study: A retrospective observational study.

The study was done during the time period of 3
years from January 2011 to June 2014. The study was
conducted at NRI Medical College & General
Hospital which is a tertiary care centre catering high
referral services to surrounding 5 districts mostly
rural areas.
Ethical approval: Ethical Clearance was obtained
from Institutional Research Committee.
Inclusion criteria: Laparotomies that were done
within 60 days of primary surgery whether it is from
the institute or referred from other center for the sake
of complications of the primary surgery of the
primary surgery. The indication for the primary
surgery selected was following obstetrics,
gynaecology and tubectomy.
Exclusion criteria: Relaporatomies that were done
after 60 days of the primary surgery for the
completeness and the indication for primary surgery
were non obstetric, gynaecological and tubectomy
were excluded from the study.
Data was collected from Medical Records.
Demograhic data like age, socioeconomic status,
booking status of the patient were taken .During this
period the total number of Obstetric surgeries were
4,540, which include both emergency and elective.
Total numbers of gynaecological surgeries were
1,116 (elective - 995; emergency -121). Total number
of family planning surgeries -2,062. In this study we
included even referral cases where primary surgery
was done at other hospitals. Indication for
relaparotomy and the procedure done, time interval
between primary and secondary surgery noted.
Number of blood components transfusion, Intensive
Care Unit stay, complications developed and total no
of hospital stay were analysed.
Statistical analysis: Data collected was tabulated and
analysed using percentages and average.
Table 2: Year wise distribution of cases in Obgyn-Fp
Total number of relaparotomy - 27 (3 years)

The Demographic Analysis was done for 27 cases.
Table 1: Age wise distribution
Age (years)
No of Patients n =27
20-30
17
31-40
41-50
51-60
61-70
>70
Among 27 cases, 21 patients belong to low socio

economic status.10 booked, 13 unbooked and 4
referral cases. Total number of surgeries done during
3 years - 7,718.Among them 27 relaparotomy, the
incidence being 0.34%.
The incidence of
relaparotomy was 0.18% from our institutional
primary surgeries. Out of 7,718 surgeries 4,550
obstetrics cases. Among which 13 relaparotomy, 5
(0.11%) institutional and 8 referral. The incidence for
total obstetric relaparotomy was 0.28%. This
constitutes 48% of total relaparotomy. Among 13
cases of obstetric relaparotomy, 9 were following
emergency and remaining 4 following elective
caesarean section. Out of 1,116 gynaec surgeries,
relaparotomy was done in 12 cases. 9 (0.8%)
institutional and 3 referral. The incidence of
relaparotomy for total gynec cases was 1.07%
constitutes 44.4% relaparotomy. Total number of
family planning surgeries was 2,062, from our
Institute, none needed relaparotomy. The 2 cases of
relaparotomy were referral following tubectomy
outside
Year
No. of gynaecological
surgeries
2011
313
1251
No of family
planning
surgeries
755
2012
355
1420
623
2013
284
1279
490
2014
164
590
194
total
1116
4540
2062
No of
relaparotomy
No of obstetric
surgeries
No of
relaparotomy
No. of
relaparotomy
-
583
Uma et al.,
The most common indication for relaparotomy hemorrhagic causes in 12 (44.4%) burst abdomen 9
cases accounting for 33% , followed by sepsis 2
(7.4%), bladder injury (1), to remove big ovarian
mass (1), recurrent ovarian cyst (1), 3.7% each. Only
one patient had hemoperitoneum, sepsis and multi
organ dysfunction. Out of 12, Hemorrhagic causes
include Hemoperitoneum 8, Rectus sheath hematoma
3, and retroperitoneal bleed after total abdominal
hysterectomy for broad ligament fibroid 1.
Among 27 cases, 13 had caesarean section as primary
surgery, 4 elective and 9 emergency. 12 out of 27
relaparotomy, 10 elective gynaecological surgeries, 2
emergency and 2 patients following tubectomy.
Table 3: Time interval between primary surgery
&relaparotomy
Time interval
No of cases
<12 hrs
12-24 hrs
1-7 days
>7-14 days
>14-30 days
>30days up to 6 weeks
9 (1/3 rd) patients required relaparotomy in first week

following primary surgery. For all relaparotomy
cases
high risk consent was taken, required
investigations done which include USG abdomen, CT
and MRI abdomen and pelvis where ever required,
coagulation profile, arranged blood and blood
products , called for help from other speciality
consultants.
Out of 27 cases 11 were given general anaesthesia
and 16 were given spinal anaesthesia. The type of
incision was vertical in 16 and transverse in 11 cases.
The most common procedure that was done during
relaparotomy was repair of burst abdomen - 9cases
(33%) 8-gynec and 1- LSCS. Risk factors for these
Diabetes -3, HIV-2 cases, BMI> 25 in 8. 7 with
vertical incision, 2 with previous surgical incisions.
One patient needed Colostomy at the time of
relaparotomy. Total blood components given 158
units, 52 Whole blood, 34 Packed Red Blood Cells,
35 Fresh Frozen Plasma, 37 Platelets for all
relaparotomy patients.
Uma et al.,
Table 4: Procedures done during relaparotomy

No
of
Type of procedure done
patients
Repair of burst abdomen
9 (33%)
Rectus sheath hematoma

evacuation
3 (11%)
Evacuation of hemoperitoneum
3 (11%)
Subtotal Hysterectomy
2 (7.4%)
Bladder injury
1 (3.71%)
Haemorrhage control + fibroid

removal + internal iliac artery
1
ligation (very huge fibroid
attempted surgery outside)
Ovarian cystectomy(post LSCS)
Removal of abdominal pack (kept

for control of retroperitoneal
1
haemorrhage following broad
ligament fibroid)
Mesosalpinx
tear
suture
1
(tubectomy)
Infundibulo
pelvic
ligament
bleed+rectus sheath hematoma 1
evacuation+ pre vesical ooze
Evacuation of pus
Control of haemorrhage of tubo

ovarian ligament tear ( following 1
tubectomy)
Huge ovarian cyst removal , not
able to achieve haemostasis
abdominal pack kept insitu then 1
this was proceeded by third
laparotomy
Out of 27 patients 11 needed ICU care.
Complications that developed in these patients: 6
required mechanical ventilation, 10 were treated for
hemorrhagic shock and hypotension, 2 patients
required dialysis, 1 lung collapse, 1 faecal fistula, 1
tracheostomy, 1 patient developed seizures, 1 MODS,
4 secondary suturing, 4 required repeat relaparotomy
(second relaparotomy), 3 patients developed
Disseminated Intravascular Coagulopathy.
Out of 27 cases 4 required second relaparotomy
(14.8%) within 12 hours. Among them 3 were
obstetric cases and 1 was gynec case. All 4 cases
were referred from other hospitals.
584
Out of 27 cases 4 deaths (14.8%), 2 obstetric and 2

gyneac. Out of these 4 deaths 3 were repeat
relaparotomy (second relaporatomy).
Average
duration of stay in hospital was 27 days.
Fig 1: Dysgerrmminoma in pregnancy undergone

relaparotomy on post op days 2of LSCS
Fig 2: Evacuated clots from hemoperitonem

DISCUSSION
In this descriptive study we collected all the data
from the patient records and audit was done. 62.9%
of the patients fall into the group of 20 30 years of
age. 21 (77.7%) patients belong to income group of <
Rs.20, 000.
Total number of surgeries done was 7,718 which
include both elective and emergency. 27 needed
relaparotomy out of 7,718 (0.34%). 14 (0.18%) were
institutional and 13(0.16%) were referral cases.
Study of Faridpur, Bangladesh of 1 year where 1,864
surgeries were done, 15 cases needed relaparotomy
(0.80%)[6]. Incidence of our study is less than the
Faridpur study.
In our study 13 patients needed relaparotomy out of
4,540, elective and emergency cases following
caesarean section (0.28%), 5 (0.11%) institutional
and 8(0.17%) referral. In study conducted at teaching

hospital at Dhaka out of 3,830 caesarean sections 24
relaparotomy (0.62%) , 4 (0.1%) were institutional
and 20 (0.52%) were referral[3]. A study conducted at
teaching hospital at Kolkota for 3 years, the incidence
of relaparotomy after caesarean section was 0.5%[7].
Another study at teaching hospital Ghana incidence
of relaparotomy was 0.7% [8]. Our institutional
relaparotomy incidence 0.11% after caesarean section
was less than compared to other studies.
The most common indication for relaparotomy in our
study was hemorrhagic cause 12 (44.4%) followed by
burst abdomen 9(33%), sepsis 7.4 %. In another
similar study conducted at Firadpur, Bangladesh most
common cause was hemoperitoneum (39.8%). The
most common procedure done during relaparotomy
was repair or Burst abdomen in 9 cases (33%),
bladder injury repair in 1, removal of ovarian cyst in
post-operative day 2 following LSCS done outside
center latter it came as Dysgerrmminoma(Fig :1).
Evacuation of pus in 2. One case was attempted
primary surgery outside for huge Ovarian cyst,
patient admitted to us within a month with abdominal
distension and proceeded for relaporatomy ovarian
cystectomy attempted difficult
to achieve
haemostasis, abdomen packing done with mops,
second relaparotomy done for removal of pack 24
hours latter patient developed Disseminated Intra
vascular Coagulation and died(Fig :2).
Among hemorrhagic causes, most common procedure
rectus
sheath
hematoma
drainage
11%,
hemoperitoneum drainage in 11%. 2 patients were
subjected sub-total hysterectomy (7.4%) and drainage
of pus in two cases (7.4%). The most common
procedure done was subtotal hysterectomy at
Firadpur study 26.4% and Dhaka study 50%.
Decision for relaporatomy taken by senior consultants
and attended by team of surgeons from the
department and other specialities where ever
required. Most of the relaparotomy done between
days 1 7
On an average each patient required 5.8 units blood
and blood products. Out of 27, 11 required ICU care.
Out of 27 patients 10 (37%) of patients had
hypotension due to shock. 6 patients required
mechanical ventilation.
In our study 4 (14.8%) required third laparotomy,
among them 3 were obstetric cases and 1 gynec. All
585
Uma et al.,
cases that underwent third surgery were referral. In

Firadpur study the incidence of third laparotomy was
13.3%, in Kolkota study in 19.6%.
Maternal mortality in our study was 14.8%, 2
obstetric and 2 gynec cases, among them 3 referral.
The mortality in Firadpur study 6.6%, Kolkota study
12.1%, 9.09% in Ghana study, 25% in Dhaka study.
In another study conducted in India mortality was
12.76% [9].
Risk and possible complications of Exploratory
Relaparotomy depends upon the reason for surgery.
The most common indications are bleeding, infection,
poor healing, and damage to internal organs. Even
though relaparotomy is unavoidable in some cases,
several measures such as careful surgical techniques,
meticulous
haemostasis,
aseptic
conditions,
experienced surgeon even at the time of primary
surgery are essential to avoid relaparotomy to
maximum extent. Relaporotomy is a challenge for
attending surgeon and anaesthesiologist, and it is
much more if the surgeon is same for relaporatomy.
Timely
done
relaparotomy
is
lifesaving.
Relaporotomy creates psychological trauma, financial
constraint to patients and relatives.
CONCLUSION
Emergency relaparotomy life saving procedure.
Interval between primary operation &relaparotomy is
one of the most important significant factors for
outcome. Every surgeon should be well versed with
surgical techniques. To avoid relaparotomy correct
documentation, timely required investigations and
meticulous surgical techniques during primary
surgery are essential. Referral may be needed even
for primary surgery, multi-disciplinary approach;
decision for relaparotomy should be taken promptly
without undue delay may reduce morbidity and
mortality.
2. Erdal SM, Abdulkadir T, Siddik EM, Ender SH,

Ali O, Sibel S, et al. Relaparotomy after initial
surgery in obstetric and gynecologic operations:
analysis of 113 cases. Ginekol Pol. 2012; 83:42932
3. Rouf S, Sharmin S, Dewan F, Akhter S.
Relaparotomy after Cesarean Section :
Experience from a Tertiary Referral and
Teaching Hospital of Bangladesh . Bangladesh J
ObstetGynaecol. 2009; 24(1):3-9.
4. Anita K, Kavita W. Emergency Obstetric
Hysterectomy. J ObstetGynaecol India 2005;
55(2):132-134.
5. AkhterR,Hossain T, RashidM. Relaparomy after
caesarean delivery: Aprospective study. J Dhaka
Med Coll.2011; 20(1);57-626.
6. IP Allam,Mahabuba,SR Das. Relaparotomy in
Obstetrics and Gynaecology Department of
FiradpurMedical College Hospital Experience
in One Year.Firadpur med coll j.2012; 7(2):7578.
7. Seal SL, Kami l ya G, Bhat t acharyya SK,
Mukherj i J, Bhattacharyya AR. Relaparotomy
after Cesarean Delivery: Experience from an
Indian Teaching Hospital. J ObstetGynaecol Res.
2007; 33(6):804-9.
8. Seffah JD - Re-laparotomy after Cesarean
section; Int J Gynaecol Obstet. 2005 ; 88(3):2537.
9. Dasgupta S, Pratim SP, Aradhana K, Partha M,
Karti M. Early re-operations after gynecological
and
obstetrical surgery a five years. J
ObstetGynecol India 2010; 60(6):507 10.
ACKNOWLEDGEMENT: Nil
REFERENCES
1. Unalp HR, Kamer E, Kar H. Urgent abdominal
re-explorations. World J Emerg Surg. 2006; 1:
10.
586
Uma et al.,
DOI: 10.5958/2319-5886.2015.00113.7

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th
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
nd
PREVALENCE OF CHRONIC DISEASES IN INDIVIDUALS ASSISTED BY THE FAMILY HEALTH

PROGRAM IN NITEROI, BRAZIL: EVALUATION OF SELECTION BIAS AND PROTOCOL
*Rosa Maria Luiza G1, Mesquita Evandro T2, Jorge Antonio Jos L2, Correia Dayse MS3, Lugon Josemir R2,
Kang HC4, Yokoo EM5, Wahrlich V5.
1
Professor of the Department of Epidemiology and Biostatistics. Universidade Federal Fluminense, Niteri, Rio de
Janeiro, Brazil
2
Professor of the Department of Clinical Medicine Universidade Federal Fluminense, Niteri, Estado do Rio de
Janeiro, Brazil
3
Professor of the Department of Nursing Fundamentals and Administration, Universidade Federal Fluminense, Niteri,
Rio de Janeiro, Brazil
4
Professor of the Department of Pathology, Universidade Federal Fluminense, Niteri, Rio de Janeiro, Brazil
5
Professor of the Department of Social Nutrition, Universidade Federal Fluminense, Niteri, Rio de Janeiro, Brazil
*Corresponding author: Maria Luiza Garcia Rosa Rua Marques de Paran 303, Centro Niteri, Rio de Janeiro,
Brasil CEP 24033-900 - e-mail: mluizagr@gmail.com
ABSTRACT
Background: The strategy of the Family Health Program has been used as an alternative scenario for prevalence
studies. This study intended to present the protocol of the Digitalis Study (DS), prevalence study of chronic
diseases, to assess sources of possible selection bias and estimate their impact on the prevalence of self-reported
hypertension, diabetes, and myocardial infarction. Methods: Randomization was performed between 38 160
registered individuals with 45 to 99 years by the Family Health Program .Differences between the sources of
selection bias (non-acceptance, non-attendance, substitutions) were observed for gender and age. Results: Of the
1,190 residents contacted, 67.1% agreed to participate. There were 144 residents who were not randomly selected
but whose participation was confirmed (substitutes). Women and individuals in the intermediate age groups and
the prevalence of hypertension were higher among substitutes compared with the randomly selected individuals.
Conclusion: The approach of the DS was adequate for the purposes of estimating prevalences, but there was a
significant percentage of non-participation. The randomization strategy did not assume outdated records;
alternative schedules for visits were not provided for; follow-up at the invitation stage was not sufficient to
prevent substitutions and the inclusion of substitutes with a higher prevalence of hypertension.
Keywords: Epidemiological Methods, Epidemiology of chronic non communicable diseases, Kidney disease,
Heart Failure.
INTRODUCTION
Few national surveys have been conducted in Brazil,
and studies involving regional or citywide samples
are scarce. During the last few years, performing such
studies has become even more difficult due to
problems associated with urban violence.[1]
Rosa Maria Luiza et al.,
The Family Health (FH) strategy has been adopted in

many countries worldwide and is considered an
alternative strategy for prevalence studies.[2-4] This
strategy is currently being used in a large number of
Brazilian municipalities and is conducted by
587
multidisciplinary teams, which keep records of all the

residents in the areas covered by each unit. Physical
facilities in these areas are available.[5-7] Health
professionals who are known in the area have access
to residents, which allows them to make invitations
and provide visits close to the participants' places of
residence, thus increasing adherence.
Prevalence studies rely on the voluntary participation
of selected individuals and are more subject to
selection bias. Non-participation in cross-sectional
studies can reach over 50% and tends to increase.[8]
Moreover, there is evidence that health professionals
tend to favor individuals with the greatest need in
clinical trials, thus violating the randomization
strategy.[9] Drawing a parallel with studies in the
Family Health Program, it can concluded that this
alternative is more subject to selection bias, given
that healthcare professionals have close ties with the
local residents, thus making it more difficult to
respect the impartiality of the rules of random
selection even after training.
The objective of the present study is primarily to
assess sources of possible selection bias and ascertain
their impact on the prevalence of self-reported
hypertension, diabetes, and myocardial infarction in
the DIGITALIS STUDY, which was conducted using
the population assisted by the Family Health Program
[Programa Mdico de Famlia (PMF)] in Niteri.
Secondarily the the present study describes the study
design, protocol, and the strengths and limitations of
the strategy used in the DIGITALIS STUDY.
The digitalis study
Objective: The study was primarily designed to
estimate the prevalence of heart failure and chronic
kidney disease.
Design: This was a cross-sectional study that
included 38 160 residents of both genders between 45
and 99 years of age who were registered at the Family
Health Program in Niteri, State of Rio de Janeiro,
Brazil.
Sample calculation: The sample size was calculated
to estimate the prevalence and the measures of
association, taking into consideration the feasibility
of the study.[10] The calculations predicted that it
would be possible to assess 600 individuals, plus 10%
for incomplete assessments (losses), in 18 months. It
was assumed that low prevalences, corresponding to a
prevalence of less than 5% (heart failure, chronic
kidney disease and diabetes), intermediate
prevalences (microalbuminuria, obesity), and high

prevalences, corresponding to a prevalence of more
than 50% (hypertension), would be found.[11-14] Thus,
the maximum relative accuracy would be 50%, 8%,
and 8%, respectively. With respect to the estimation
of the measure of association for the low (6%) and
high prevalences (60%) of disease in the exposed
group, the minimum prevalence ratios (PR) were 3
and 1.2, respectively. All calculations were performed
at a 5% level of significance and 80% power using a
two-tailed hypothesis test.
Procedures for selecting participants: The units
(sectors) to be included in the study were randomly
selected from the official list of PMF sectors. For
each sector, approximately 80 individuals of both
genders between 45 and 99 years of age were
randomly selected from the records of residents kept
by the program. It was anticipated that 30
examinations would be conducted per visit. Thus, it
was recommended that 50 residents be invited to
account for non-attendance and that another 30 names
be collected for possible substitutions. In the training
for the professionals from the randomly selected
sectors, the following items were clarified: a) the list
should be followed until the end, including the order
of the names; b) individuals with medical conditions
that prevent them from attending the health facility
should not be invited (exclusion); c) if the study
failed to achieve the 50 participants using the names
on the random selection list, the study coordinators
could invite residents who were not on the list as long
as they invited the occupant of the residence who was
closest to the gender and age (+/- 5 years) of the
randomly selected resident being replaced. Substitute
alternatives were
requested
by the PMF
professionals to speed up the process of inviting
residents. In some sectors, primarily due flooding by
rain, families moved and records had not yet been
updated. During the training, the invitations were
delivered, which included scheduling of the date and
time of the visit for each resident to be completed by
the professional in that sector. The invitation
contained information about the examinations that
would be performed during the visit, instructions as
to fasting and what to hear, and a request for the
participants to bring prescriptions and the packaging
of the drugs/remedies they were using. Prior to the
visits, the sectors returned the lists, which included
information regarding the substitutions made and the
588
reason the randomly selected residents could not

participate. Based on the list sent by the sectors, the
field materials were prepared and personalized for
each resident. The reasons for non-attendance or noncompletion of the assessment were not collected.
Visit procedures: On the day of the visits, which
were always held on Saturdays beginning at 7 am,
the assessment was started after the resident read and
signed the informed consent form. The first step was
to collect blood, and the participant was then given a
balanced snack selected by nutritionists, with options
for diabetics. The evaluation consisted of the
following procedures: blood and urine collection,
resting electrocardiogram, resting tissue echoDoppler, clinical consultation with a physical
examination, nursing consultation, anthropometric
evaluation (weight, height, and waist circumference),
completing a food frequency questionnaire, and
completing the DIGITALIS questionnaire. On the day
of the visit, the participants were scheduled to take a
bone densitometry test (Dual-energy X-ray
absorptiometry - DXA) at the Laboratory of Nutrition
and Functional Assessment (Lanuff) of the
Fluminense Federal University (Universidade Federal
Fluminense) .
The researchers received training based on
procedures developed for the study and tested in the
pilot study using a PMF unit not included in the
study. The results of all of the examinations
performed were forwarded to the family doctor of
each of the individuals in envelopes addressed to the
participants.
The blood tests included complete blood count,
glucose, urea, creatinine, TSH, lipid profile, vitamin
D, uric acid, brain natriuretic peptide (BNP). In
addition, the levels of albumin, creatinine, sodium,
and alkali reserve as well as the pH were measured in
the urine samples collected during the visit. Blood
was collected, labeled, centrifuged, stored at
approximately 4 C and sent to the Sergio Franco
Laboratory [Laboratrio Srgio Franco (LSF)]. The
urine was collected, labeled, stored at 4 C and also
sent to the LSF. The samples were delivered to the
laboratory within 5 hours after collection following
each visit. The analyses were performed using the
LSF methodologies (www.lsf.com.br). All of the
information that was collected was compiled into a
single database, prepared and analyzed using SPSS
software, versions 17 and 21.
Table 1 presents the primary and secondary

objectives, examinations, and instruments used to
diagnose or classify each disease, factor, or risk
marker.
METHODS
Assessment of the magnitude of selection bias: The
DIGITALIS STUDY has three potential sources of
selection bias: non-confirmation, non-attendance, and
substitutions. To examine the differences between
these three sources, we used the variables gender and
age, which are available in the Brazilian census and
in the PMF registration records. Data from the 2010
Brazilian census for the population between 45 and
99 years of age residing in the neighborhoods where
randomly selected sectors are located were compared
to the data for the randomly selected individuals. The
objective was to identify possible differences due to
chance, given that the reference population would be
the group of randomly selected individuals.
We used hypertension (high prevalence), type 2
diabetes (intermediate prevalence), and self-reported
acute myocardial infarction (low prevalence) to
determine the impact of substitutions in the
estimations of prevalence.
The chi-square test was used to assess differences in
proportions, and the Yates' correction for continuity
was used in the case of 2x2 tables. Student's t-test
was used to test for differences between means. The
level of significance was set at 5% and a power of
80% using the two-tailed hypothesis test.
RESULTS
The stages of participant selection are presented in
Figure 1. In total, 26 sectors and 1,894 individuals
were randomly selected. Overall, 1,190 residents
were contacted, including 798 (67.1%) who agreed to
participate and whose participation was confirmed by
the PMF and 392 (32.9%) individuals who were not
found or refused to participate. Of the 1,894
randomly selected residents, 704 did not need to be
contacted. Of the 942 individuals invited to
participate and confirmed by the sectors, 144 (15.3%)
residents were not randomly selected and were
designated as substitutes. Of the 633 residents who
attended and completed the cardiac assessment, 106
(16.7%) were substitutes. Considering the 942
individuals invited, 67.2% attended and completed
589
the cardiac assessment. In total, 66.0% of the

randomly selected individuals and 73.6% of the
substitutes participated in the study. Non-participation
reached 56% among the 1,190 randomly selected and
contacted residents, including 392 residents who

could not be found or refused to participate and 271
who did not attend or did not complete the cardiac
assessment.
*704 names on the random selection were not used, #798 accepted the invitation, &392 were not found or did not accept the
invitation, Completed the heart failure assessment.
Fig 1: Flow chat of participant selection

Table: 1. Primary and secondary objectives of the Digitalis study, measurement instruments, and
classification criteria
Objectives
Measurement instruments
Classification
Primary
Prevalence of heart failure (CF): stages Clinical history, analysis of the prescription and Stages of HF
and phenotypes
physical examination, electrocardiogram, tissue echo- HF with reduced ejection fraction
HF with normal ejection fraction
Doppler, BNP.[15]
Prevalence of chronic kidney disease Clinical history, glomerular filtration, urinary albumin
CKD and stages
(CKD) by stage
excretion, prevalence of a history of urolithiasis.[16]
Secondary
Prevalence of hypertension
Clinical history, use of antihypertensive medication,
Hypertension
blood pressure measurements: three measurements,
right arm, sitting position, 1-minute interval between
measurements, taking the average of the 2nd and 3rd
measurements. Device: OMRON 711 HC.[17]
Prevalence of type 2 diabetes
Clinical history, analysis of prescriptions, fasting
Type 2 diabetes
glucose, fasting insulin.[18]
Prevalence of obesity
BMI waist circumference.[19]
Prevalence of subclinical thyroid TSH.[20]
disease
Prevalence of osteoporosis/sarcopenia Dual-energy X-ray absorptiometry: iDXA (General
Osteoporosis; sarcopenia
Electric
Company/Lunar
Prodigy,
Madison,
WI), software enCORE 2010 - version 13.40.[21]
Prevalence of mood disorders
PHQ-9.[22]
Prevalence of cognitive disorders
Mini Mental.[23]
Prevalence of sleep disorders
Berlin Questionnaire.[24]
Prevalence of nursing diagnoses
NANDA .[25]
Association with risk factors/markers
Lifestyle
Eating habits
Food frequency questionnaire (FFQ).[26]
Physical activity
Short International Physical Activity Questionnaire
(IPAQ).[27]
Tobacco use
Specific questions.[28]
Alcohol consumption
Specific questions.[29]
Health-related quality of life assessment SF36 .[30]
590
Some participants failed to submit urine samples,

which reduced the number of patients who completed
the renal assessment to 602. The analysis of bias for
this objective was not addressed in this article.
Of the 392 unconfirmed residents, 215 (54.8) had
moved to a different address, 77 (19.6) had no reason
recorded for non-attendance, and 54 (13.8) had died,
the highest percentage of whom were women. Illness,
travel, work, alcoholism, prior commitments,
healthcare plan, and a birth date outside the preset age
group were less common reasons.
When comparing both genders, a greater number of

women were confirmed and attended the assessment
(p = 0.03 and <0.01, respectively). The mean age for
both genders was lower among those who were
confirmed compared with those who were
unconfirmed (p <0.01 among women) and slightly
higher among those who attended and completed the
cardiac assessment compared with those who did not
attend or did not complete the cardiac assessment (p
= 0.04 among men) (Table 2).
Table 2: Difference in the percentages according to gender* and mean age by gender# and according to the
confirmation and completion of the assessment for both the randomly selected individuals and the substitutes
Confirmed
Unconfirmed
p-value
Attended and
Did not attend or did not
p-value
n(%)
n(%)
completed the cardiac
complete the cardiac
assessment n(%)
assessment n(%)
Men
384 (67,4)
186 (32,6)
0.03
243 (63,3)
141 (36,7)
<0,01
Women
558 (73,0)
206 (27,0)
390 (69,9)
168 (30,1)
Total
942 (70,6)
392 (29,4)
633 (67,2)
309 (32,8)
MeanSD
MeanSD
MeanSD
MeanSD
Men
59.4210.4
60,6612,0
0,23
60,2210,6
57,910,0
0,04
Women
58.9010.7
62,9613,8
<0,01
59,1110,2
58,4111,9
0,52
Total
59.1110.7
61,8713,0
<0,01
59,5910,4
58,1411,00
0,08
*Difference tested using the chi-square test, with correction for continuity
# Difference tested using Student's t-test
As expected, there were no statistically significant
differences for the distribution by gender or age
group according to census [A] or random selection
[B] . The results of the analysis for each group, i.e.,
randomly selected individuals and substitutes, are
presented in Table 3. The largest differences between
the percentages of women occurred between the
randomly selected individuals [B] and the confirmed
substitutes [D] (66%) as well as the substitutes who
attended and completed the cardiac assessment [F]
(69.8%) (p = 0.01 and p <0.01). The distribution
pattern per age group was similar between both the
group of randomly selected individuals and the group
of substitutes. Among the confirmed randomly
selected individuals and those who attended and
completed the cardiac assessment, there was an
overrepresentation of individuals between 50 and 59
years of age and an underrepresentation of
individuals between 70 and 84 years of age (p =

0.061 and p = 0.014, respectively, when compared
with the randomly selected individuals [B]). Among
the confirmed substitutes and those who completed
the cardiac assessment, there was a higher percentage
of individuals in the 50-54 and 65-74 age groups than
in the 60-74 age group (p = 0.364 and p = 0.053,
respectively, compared with the randomly selected
individuals [B]).Among those who did not participate
(both those who were unconfirmed and those who did
not complete the cardiac assessment among the
randomly selected individuals), there was a slight
overrepresentation of individuals between 45 and 54
years of age, an underrepresentation of those between
55 and 64 years of age, and no significant differences
in the other age groups (p = 0.36 and p = 0.05,
respectively, compared with the randomly selected
individuals [B]).
591
Table 3: Distribution by gender and age group& according to the census, random selection, confirmation by
the PMF, and completion of the cardiac assessment
Census
[A]
N
(%)
Randomly
selected
individuals
[B]
Confirmed
randomly
selected
individuals#
[C]
(%)
assessment [E]
Substitutes:
Noncompleted the
Participa-tion
cardiac
@$
assessment
[G]
[F]
(%)
(%)
(%)
27440 (42,5)
Women 37049 (57,5)
Total 64489 (100,0)
Age (years)
842
1052
1894
(44,5)
(55,5)
(100,0)
335 (42,0)
463 (58,0)
798 (100,0)
49
95
144
(34,0)
(66,0)
(100,0)
211
316
527
(40,0)
(60,0)
(100,0)
32
74
106
(30,8) 310
(69,8) 353
(100,0) 663
(46,76)
(0,53)
(100,0)
45-49
50-54
55-59
60-64
65-69
70-74
13559
12672
10661
8396
6184
4987
(21,0)
(19,6)
(16,5)
(13,0)
(9,6)
(7,7)
385
359
308
232
170
154
(20,3)
(19,0)
(16,3)
(12,2)
(9,0)
(8,1)
161
170
144
113
69
58
(20,2)
(21,3)
(18,0)
(14,2)
(8,6)
(7,3)
24
24
24
22
14
16
(16,7)
(16,7)
(16,7)
(15,3)
(9,7)
(11,1)
97
100
111
82
46
41
(18,4)
(19,0)
(21,1)
(15,6)
(8,7)
(7,8)
13
16
20
13
13
14
(12,3)
(15,1)
(18,9)
(12,3)
(12,3)
(13,2)
143
137
94
68
63
45
(21,57)
(20,66)
(14,18)
(10,26)
(9,50)
(6,79)
75-79
80-84
85-89
90-94
95-99
Total
3797
2437
1253
455
118
64519
(5,9)
(3,8)
(1,9)
(0,7)
(0,2)
(100,0)
129
78
45
22
12
1894
(6,8)
(4,1)
(2,4)
(1,2)
(0,6)
(100,0)
46
19
11
5
2
798
(5,8)
(2,4)
(1,4)
(0,6)
(0,3)
(100,0)
15
3
0
2
0
144
(10,4)
(2,1)
(0,0)
(1,4)
(0,0)
(100,0)
28
10
7
4
1
527
(5,3)
(1,9)
(1,3)
(0,8)
(0,2)
(100,0)
13
2
0
2
0
106
(12,3)
(1,9)
(0,0)
(1,9)
(0,0)
(100,0)
51
30
17
7
8
663
(7,69)
(4,52)
(2,56)
(1,06)
(1,21)
(100,0)
Sex
Men
(%)
Confirmed
substitutes
[D]
Randomly selected
individuals:
completed
the
cardiac
N (%)
Differences were tested using the chi-square test. Differences between genders were tested using the correction
for continuity. For the age categories, the last two age groups were combined. *p-value for the comparison
between A and B for gender = 0.098 and age = 0.153. #p-value for the comparison between B and C for gender =
0.255 and age = 0.061. p-value for the comparison between B and D for gender = 0.015 and age = 0.364. pvalue for the comparison between B and E for gender = 0.07 and age = 0.014. p-value for the comparison
between B and F for gender = 0.005 and age = 0.053. @p-value for the comparison between B and G for gender =
0.319 and age = 0.660. $Randomly selected individuals who were not found or did not accept as well as
individuals who were confirmed but did not attend or did not complete the cardiac assessment.
Table 4: Prevalence of hypertension, diabetes and infarction* in individuals who completed the cardiac
assessment for the randomly selected individuals and substitutes
Hypertension
pvalue DM2#
pvalue Infarction
pvalue
Randomly
Substitutes
Randomly selected Substitutes
Randomly Substitutes
n
(%)
selected
n
(%)
n (%)
individuals n (%)
selected
individuals
individuals
n (%)
n (%)
378 (71,3) 82 (79,6) 0,09
137 (25,8)
23 (22,3) 0,54
23 (4,3)
*Difference tested using the chi-square test with correction for continuity
The prevalence of hypertension, type 2 diabetes, and
self-reported myocardial infarction among those who
completed the cardiac assessment and were either
randomly selected or substitutes is presented in Table
4. The prevalence of hypertension was higher among
4 (3,9)
0,83
the substitutes for both genders (p = 0.09). The

opposite was true for type 2 diabetes and selfreported acute myocardial infarction, although the
trend was not statistically significant.
592
DISCUSSION
The DIGITALIS STUDY examined 633 individuals
registered in 26 sectors of the PMF in an effort to
estimate the prevalence of heart failure, reaching the
number of individuals required based on the sample
size calculation. The performance of numerous
evaluations using qualified and previously trained
researchers suggested the possibility of assessing
different associations, allowing one to construct
hypotheses for different health fields.
The decision to perform the examinations at the PMF
units near the residences of participants proved to be
feasible for all procedures performed. We conclude
that the approach used in this study was positive,
although the approach needs to be refined to increase
the internal validity of future studies.
Non-participation results from the inability to recruit
sampled individuals, and as non-participation
increases, a study's vulnerability to selection bias also
increases.
However, approximately 50% of
epidemiologic studies published in renowned journals
do not report the percentage of non-participation.[8]
Analysis and reporting of sources of potential bias
allow for a more careful interpretation of prevalence
results.
As expected, there were no significant differences
between the distribution by gender and age group
between the census and the randomly selected
individuals. More women were confirmed and
attended/completed the cardiac assessment, i.e., men
had higher rates of non-participation; this is a
common finding in the literature.[30-35] The reasons for
non-participation were studied in an Australian
cohort study on osteoporosis in which there was
greater participation of women.[32] As a reason for
non-participation, more men than women claimed to
have time constraints. For other less-cited aspects,
including disinterest, illness, travel, fear of
examination results, and a lack of comprehension,
there were no differences between genders, or the
reasons were more cited by women. In a Chinese
survey published in 1997, two years of demographic
and mortality data were compared between those who
agreed to participate and those who did not. There
was greater participation of women and younger
people, and the mortality in this population group was
lower.[31] In two Swedish surveys, the percentage of
participation among selected individuals was higher
for women and those with higher levels of education

and income.[33-34] In the study presented here,
although the residents who confirmed their
participation were younger than those who did not
confirm their participation (p <0.01), the opposite
was true for attendance/completion of the cardiac
assessment (p = 0.08).
The DIGITALIS STUDY has some aspects that differ
from those of other studies that discuss the
characteristics of non-participation. The target
population is less economically privileged than the
average Brazilian population. Although everyone had
access to primary care services, some blood tests as
well as echo-Doppler and DXA were not always
available. Participation in the study would allow such
tests to be performed and medical reports to be
available in a short time period. This information was
provided in the invitation. Perhaps this scenario
increased the participation of less healthy people,
both among the confirmed individuals and among
those who attended and completed the study.
A similar trend was observed between the distribution
by gender and age groups within the groups of
randomly selected individuals and the substitutes for
the confirmation and completion of the cardiac
assessment. This result indicates that the randomly
selected individuals and substitutes were different
groups. The participation of women was higher
among
the
substitutes,
which
was
also
overrepresented by individuals in the intermediate
age groups, especially among those who completed
the assessment, although these age differences were
not statistically significant.
Given that the prevalence of hypertension is higher
among women and increases with age (data not
shown), it appears that the characteristics of the
substitutes contributed to this increased prevalence.
Among the randomly selected individuals, there was
an overrepresentation of individuals in the younger
age groups, which may have reduced the impact that
the substitutes had on the increased prevalence of
hypertension. This evidence indicates that most
hypertensive patients were invited as substitutes.
This trend was reversed in the case of type 2 diabetes
and self-reported myocardial infarction, i.e., the
prevalence among the substitutes was lower than the
prevalence among the randomly selected individuals.
This result could contradict the hypothesis suggested
above. However, these two diseases have very low
593
prevalence, making it difficult to include affected

individuals among the substitutes.
The assessment into possible sources of bias
performed here exhibited several limitations. Only
the influence of gender and age were evaluated,
which limited our understanding of the reasons for
non-participation and thus the possible interventions
needed to reduce non-participation. Although training
has been conducted and there has been a commitment
on the part of PMF professionals to respect the rules
for substitutions, the results indicate that this did not
happen. Substitutes attended the assessment more
than those who were selected randomly and had a
higher prevalence of hypertension.
Was the larger attendance observed because the
substitutes were sicker or because they were simply
more cooperative with PMF professionals? This is a
question that remained unanswered.
CONCLUSION
Using the results of all the examinations performed,
the approach of the DIGITALIS STUDY proved to be
adequate for the purposes of estimating prevalence,
achieving the number of participants indicated by the
sample size calculation. However, there was a
significant percentage of non-participation. The
randomization strategy did not presuppose outdated
records; alternative schedules for visits were not
provided for individuals who worked on Saturday
mornings; and follow-up at the invitation stage was
insufficient to prevent substitutions and the inclusion
of substitutes with a higher prevalence of
hypertension. An evaluation of the selection bias will
allow for a better interpretation of the estimated
prevalences.
Acknowledgments: The present work was developed
during the pos-doctoral stage of the first author, in the
Department of Epidemiology of the Faculty of Public
Health of the Universidade de So Paulo, realized
between 01st October 2013 to 30th September 2014
Competing interests: The authors have no
competing interests to declare.
Funding:
DIGITALIS
STUDY
received
scholarships and work-study grants for undergraduate
students were granted by the National Council of
Technological and Scientific Development [Conselho
Nacional de Desenvolvimento Cientfico e
Tecnolgico (CNPq)], the Rio de Janeiro Research

Foundation [Fundao de Amparo Pesquisa do
Estado do Rio de Janeiro (FAPERJ)], and UFF. A
portion of the examinations was funded by the LSF.
Ethical approval: Digitalis study was conducted
according to the principles established by Resolution
196/96 of the National Research Ethics Committee
[Comisso Nacional de tica em Pesquisa (CONEP)]
and approved by the Research Ethics Committee of
the Antonio Pedro University Hospital, Fluminense
Federal University [Universidade Federal Fluminense
(UFF)] (CAAE:0077.0.258.000-10).
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February 2015].
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Guidelines for Data Processing and Analysis of
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30. Laguardia J, Campos MR, Travassos CM, Najar
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Epidemiology 2010;
596
DOI: 10.5958/2319-5886.2015.00114.9

&
Health Sciences
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Volume 4 Issue 3
Coden: IJMRHS
rd
Research article
Copyright @2015
ISSN: 2319-5886
Accepted: 23rd Jun 2015
EVALUATION OF AGGREGATIBACTER ACTINOMYCETEMCOMITANS LEVELS IN LOCALIZED

AGGRESSIVE PERIODONTITIS BEFORE AND AFTER PERIODONTAL SURGERY
*Saranyan Ravi, Priya Kesavan, Manovijay Balagangadharan, Raja Arasapan, Nisha N, Ann Joseph Anthraper
Department of Periodontics, Vinayaka Missions Dental College and Hospital, Salem, Tamilnadu, India
*Corresponding author: ravisaranyan@gmail.com
ABSTRACT
Background The role of microorganisms in the etiology of periodontal disease is well understood. The
association of specific organisms in the pathogenesis of periodontal disease was established by the specific plaque
hypothesis. This study examined the effects of periodontal surgery on Aggregatibacteractinomycetemcomitans
(Aa) levels in localized aggressive periodontitis before and after periodontal surgery. Method: A clinical study
was done on 24 male and 16 female patients who underwent surgical periodontal therapy. Bacterial counts were
assessed from the plaque samples and gingival specimens. Results: Mean reduction of pre and post operative
bacterial counts were statistically significant at 1%.COnclusion: A reduction of bacterial count was observed in
plaque and gingival tissue samples after surgery.
Keywords: Aggregatibacteractinomycetemcomitans, Aggressive periodontitis, Periodontal surgery, Bacterial
count, Biopsy
INTRODUCTION
Aggressive periodontitis is a disease of the
periodontium caused by specific microorganism that
differs from other forms of periodontitis in clinical,
microbiological and histo-pathological features[1].
Aggressive periodontitis is a disease of the
periodontium occurring in an otherwise healthy
adolescent which is characterized by a rapid loss of
alveolar bone about more than one tooth of the
permanent dentition[2].
Localized aggressive periodontitis (LAP) have
genetic factors that play an important role in the
pathogenesis of the disease[3].However it is uncertain
how the genetic factors are expressed. The increased
incidence of localized aggressive periodontitis would
suggest an x- linked inheritance with reduced
penetrability[4,5]. While the precise etiology of LAP is
uncertain, microbial sampling of the affected sites
point towards the relationship with aggregate
bacteractinomycetemcomitans (Aa). Aa is a
Ravi et al.,
facultative, gram negative, capnophilicnon motile

coccobacilli. Slots 1976, Newman and Socransky
1976[6,7] identified Aa from the sub gingival flora of
patients with LAP. Over 90% of patients with LAP
have elevated levels of serum antibodies to Aa in
both gingival crevicular fluid and gingival
tissues[8,9].These antibodies may modulate the disease
process by influencing the colonization and
proliferation of the bacterium in the periodontal
pocket.
Moreover Aa produces a number of virulent factors
that can penetrate the gingival tissues and plays a role
in the pathogenesis of the disease (Zambon
1983)[9].Factors that promote Aa colonization in the
oral cavity include adhesions, bacteriocins, and
invasions. Aa can interact and adhere to all
components of the oral cavity, tooth surface,
epithelial cells and intercellular matrix. It is also able
to elicit its own uptake into epithelial cells and its
597
spread to adjacent cells usurping normal epithelial

function. It also enhances its own survival by
elaborating factors that interfere with the host's
defense system and pass through the epithelial cell
barrier causing periodontal destruction thus migrating
to deeper tissues[10].
The study was conducted according to the university
norms of Annamalai University. Written informed
consent of the patients was obtained. This study was
conducted at division of periodontia Rajah Muthiah
Dental College and Hospital, Chidambaram
Annamalai University Tamilnadu India. The period
of study was three months.
Inclusion criteria: Both male and female patients
were included for the study. 24 male and 16 female
patients who attended the dental op, diagnosed as
aggressive periodontitis in the age group of 15-25
years were selected for the study.
Patients chosen were free from any systemic diseases,
have not undergone oral prophylaxis or any other
periodontal treatment and not taken antibiotics 6
months prior to the study.
Exclusion criteria:
Pregnant and lactating women, smokers, patients with
immune-modulatory therapy, systemic diseases were
excluded from the study.
Grouping:
Patients were divided into 2 groups randomly. Group
1 received surgical treatment and systemic
tetracycline. Group 2 received nonsurgical treatment
and systemic tetracycline.
Sample collection: A baseline microbial sampling
was done from each subject .The sample sites chosen
were first molar and central incisor of the same side.
The selected teeth were isolated with sterile cotton
rolls and the loosely adherent plaque in direct
proximity to the sample site was carefully removed
using sterile cotton gauze.
Methodology: A sterile paper point was introduced
into the mesiobuccal interproximal pocket until
resistance was met11. It was kept in place for 10
seconds and after removal the collected sample was
then transferred into a test tube containing sterile
saline. The bacterial deposits were dispersed in a
vortex mixture for 60 seconds.
From the above solution one loopful of plaque
suspension was placed over a glass slide and
smeared. It was then air dried, heat fixed and stained

by gram staining to confirm that the organism was
Aggregatibacteractinomycetemcomitans It was then
sent for culture to estimate the culture count [11].
Inflammed granulation tissue and the diseased soft
tissue wall of the pocket was taken for biopsy and
sent for histopathological examination, swell as to
look for Aa in the gingival tissues(fig:1, fig:2,fig:3).
It was expressed as scanty-25-50CFU, moderate-50100 CFU and abundant-100-150CFU, where CFU
stands for colony forming units. The subjects were
examined after a period of 3 months.
Fig 1: Gram stained smear of Aa revealing

cocobacillary& pleomorphic forms x120
Fig 2: ulcerated membrane showing inflammatory

infiltration of cells in the submucosax100
RESULTS
Gingival biopsy of group 1 patients showed a
complete elimination of microorganisms where as in
group 2 there was a reduction but not complete
elimination of the organisms. As shown in table 1
colony forming units of Aa were measured following
pre and post operative treatment.
598
Ravi et al.,
Table 1: colony forming units of Aa were

measured following pre and post operative
treatment
Patient
Pre
CFU
Bacterial count
treatment Post treatment CFU
4
4
12
25-50
ND
Group -1 50-100
100150
8
Group - 50-100 25-50
II
12
10050-100
150
Group I p<o.o1 highly significant. Group 2 p<0.001
not significant, ND:
Fig 3: Mean CFU of A.a in group 1 and group 2

pre and post treatment
DISCUSSION
Most forms of periodontal disease are apparently
caused by specific bacterial species, and the treatment
should be directed towards the elimination of
periodontal pathogens. Various clinical data are
available indicating that periodontal surgery alone or
in combination with systemic antimicrobial treatment
can promote healing in LAP.
Suppression of Aa cannot be satisfactorily obtained
using only nonsurgical clinical regimens where as it
can be obtained with surgical therapy and systemic
tetracycline therapy[12] as it has been well established
that Aa invades the gingival tissue[13]. The findings of
the present study is in concurrence with the above
study as shown in Table 1
Furthermore surgical procedures suppress Aa only
from the clinically treated sites, where as systemic
tetracycline therapy can remove the organism both
from the infected sites and buccal mucosa. Studies of
Lindhe (1984)[14] demonstrated that treatment of LAP

involving removal of subgingival plaque and
inflammed periodontal tissue resulted in resolution of
gingival inflammation and gain of clinical
attachment.
Slots and Rosling (1983)[12] showed systemic
administration of tetracycline suppressed Aa infection
and its effects were observed in deep pockets and
total number of Aa recovery per site were markedly
reduced following antibiotic therapy. The results of
the present study coincides with the above study
Christersson et al 1985[15] reported that surgical
elimination of granulation tissue either by curettage
or in conjunction with modified widman flap resulted
in suppression of Aa often to undetectable levels and
concluded that substantial supression of Aa can be
accomplished by the surgical removal of periodontal
tissues. The result of the present study is in
concurrence with the above study.
Kornman and Robertson [16] and kunihara 1985[17]
showed that periodontal surgery combined with
systemic tetracycline has been shown to reliably
suppress subgingival Aa which in accordance with
the present study.
Though various antibiotic regimens are available
antiobiotic sensitive test was done which showed that
Aa was highly sensitive to tetracycline. Though Aa
strains showed resistance to tetracycline, modified
forms of tetracycline derivatives (doxycycline) are
widely continued to be used in the management of
LAP.
Inspite of various treatment modalities available for
the management of LAP it has been established that
surgical therapy in conjunction with systemic
antibiotic therapy prove to be the most effective
method in obtaining periodontal health along with
suppression of Aggregatebacteractinomycetemcomitans.
CONCLUSION
Within the limitations of the present study, it can be
concluded that there was a reduction in
Aggregatibacteractinomycetemcomitans in both the
groups. However the reduction in the bacterial count
was more evident in the surgical group than the nonsurgical group.
599
Ravi et al.,
ACKNOWLEDGEMENT: Dr.R.Mythili, Dean,

Rajah Muthiah Dental College and Hospital,
Annamalai University
13.

REFERENCES
1. Newman MG, Socransky SS. Studies of
microbiology of periodontosis. Journal of
periodontology1976; 47: 373.
2. Baer P.N. The case of periodontosis as a clinical
entity.Journal of clinical periodontology.1971 42:
516.
3. Benjamin SD, Baer PN. Familial patterns of
advanced alveolar bone loss in adolescent
.Journal of periodontology1967; 5: 82.
4. Meknick M; Shields C.D. Periodontosis a
phenotyphic and genotyphic analysis. Oral
surgery, Oral Medicine 1976; 42:32.
5. Spektor MD, Page RL. Clinical studies of one
family manifesting rapidly juvenile periodontitis.
Journal of periodontology 1985; 56:93.
6. Slots J. The predominant cultivable organisms in
juvenile periodontitis.Scandinavian Journal of
Dental Research 1976;84:1.
7. Haffajee AD, Socransky SS. Clinical,
microbiological,
immunological
features
associated with treatment of active periodontal
lesions. Journal of clinical periodontology
;1984;11:600.
8. Genco RJ, Zambon JJ. Serum and gingival fluid
antibodies as adjuncts in the diagnosis of
Actinobacillus associated periodontal disease.
Journal of periodontology;1985 56:41.
9. Zambon JJ, Slots J, GencoRJ. Serology of oral
Aa and serotype distribution in human
periodontal disease. Infection and Immunity
1983;41:19.
10. Meyer DW, Mirtz KP, Brissete C. Article name.
Perio 2000 1999;20 136-67.
11. Slots J, Reynold HS, GencoRJ. Actinobacillus in
human periodontal disease a cross sectional
microbiological examination. Infection and
Immunity. 1980; 29: 1013.
12. Slots J, Rosling BG. Suppression of
periodontopathicmicroflora in LAP by systemic
14.
15.
16.
17.
tetracycline. Journal of clinical periodontology.

1983 10: 465.
Saglie FR, Carranza FA. Identification of tissue
invading bacteria in human periodontal disease.
Journal of periodontal Research; 1982 17:425.
Lindhe J, Liljenberg Treatment of LAP. Results
after 5 years. Journal of clinical periodontology
1984; 11:399.
Christersson L.A. Microbiological and clinical
effects of surgical treatment of LAP. Journal of
clinical periodontology 1985; 12:465.
Kornman KS, Robertson PB. Clinical and
microbiological evaluation of therapy of juvenile
periodontitis. Journal of periodontology1985;
56:443.
Kunihara DM., Caine FA A clinical trial of
phenoxy methyl pencillin for adjunctive
treatment of juvenile periodontitis. Journal of
periodontology 1985; 56:352.
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DOI: 10.5958/2319-5886.2015.00115.0

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
Coden: IJMRHS
th
Received: 28 April 2015
Revised: 23rd May 2015
Research article
Copyright @2015
ISSN: 2319-5886
Accepted: 18th June 2015
A STUDY TO EVALUATE THE ABNORMAL MENSTRUAL PATTERNS AMONG ADOLESCENT

GIRLS IN BAREILLY
*Chauhan Sandhya1, Kariwal Peeyush2, Kumari Anita1, Vyas Shaili3
1
Associate Professor, Department of Pediatrics, SRMSIMS, Bareilly, Uttar Pradesh, India

Associate Professor, Department of Social and Preventive Medicine, RIMSR, Saifai, Uttar Pradesh, India
3
Assistant Profession, Department of Social and Preventive Medicine, HIMS, Dehradun
2
*Corresponding author email: drsandhyapedia@gmail.com

ABSTRACT
Background: Menstrual cycle abnormalities have been considered a common occurrence during puberty.
Numerous earlier studies have analyzed the various patterns seen in the epidemiological data regarding menstrual
cycle of adolescent girls. But there is dearth of data from Rohilkhand region of central UP state. Aims: To find
out the abnormal menstrual patterns among adolescent girls in Bareilly city. Materials and Methods: A cross
sectional study conducted by presenting a pre-designed and pre-tested self administered questionnaire, containing
questions pertaining to menstrual practices and knowledge to 994 adolescent girls of urban schools in Bareilly
city. Results: Mean age of menarche was 13.110.95 years. 6.07% girls in the study reported cycles of abnormal
lengths. This subset also had significantly higher prevalence of irregularity. Presence of irregular menses beyond
3 gynaecological years diminished the possibility of their regularization in near future. Among the girls with
excessive dysmenorrhea, nearly 1/3rd had associated menstrual disturbances also. Conclusion: Incidence of
irregular menstrual cycle was 20.75% and it was significantly more in oligomennorheic cycles. Among normal
menstrual cycles also, presence of IMC was significantly more in adolescents having mild oligomennorhea.
6.07% of girls reported abnormal cycle lengths. 77.70% reported dysmennorhea. Chances of menstrual cycles
getting regularized after 5 years of menarchy are minimal. Adequate knowledge regarding abnormal variations in
the menstrual cycle pattern during adolescence may permit implementation of strategies for preventing potential
reproductive and other health morbidities in adulthood.
Keywords: Mild oligomennorhea, Polymenorrhea, Menarche, Menstrual cycle, Adolescence.
INTRODUCTION
Menstrual cycle abnormalities have been considered a
common occurrence during puberty and constitute a
common complaint with which adolescents girls
present in pediatric clinics. According to District
Level Household and Facility Survey 3 (DLHFS)
nearly 25% of the adolescent girls complain of some
problem related to menstruation.[1] Menstrual
disturbances in adolescents are often explained by
immaturity of the hypothalamic-pituitary-gonadal
axis. Certain prospective follow up studies on the
menstrual pattern of adolescent girls have shown that

few conditions, which present in the first few years of
menarche viz oligomenorrhea and/or irregular
menstrual cycles may be related to polycystic ovarian
disease(PCOD) later on.[2,3] Similarly, though
diseases like endometriosis often begin in
adolescence, they are most often recognized after
many years.[4] Thorough evaluation of menstrual
cycle disorders in adolescence provides a window of
opportunity for early diagnosis and treatment of
601
Sandhya et al.,
conditions affecting HPO axis. Delay in the

evaluation and treatment of disordered menses in
some cases may contribute to reduced bone density
also.[5]
Adequate knowledge regarding abnormal variations
in the menstrual cycle pattern during adolescence
may permit early identification of the potential
reproductive and other health morbidities for
adulthood. This can help in screening susceptible
individuals in early adolescence after the onset of
menarche and timely intervention may be sought if
required. Extensive search failed to reveal any related
data from the study region. This study presents the
findings of first epidemiological survey regarding
menstrual patterns from Rohilkhand region of central
Uttar Pradesh state in India.
Aim and objective:
1. To present the epidemiological data regarding
patterns of the menstrual cycle in a representative
sample of Indian girls.
2. To analyze the abnormal variations in the
menstrual cycle patterns in this study sample.
Study type: A cross sectional, questionnaire based
study.
Sampling technique: These were selected by
convenience sampling.
Sample size: Considering the national incidence of
menstrual disorders[1] among adolescents to be 2030%, the sample size was calculated using the
formula n=4pq/L with a 10% relative precision and
95% confidence interval to be 933 girls aged <18
years. [p=approximate prevalence rate of menstrual
disorders in adolescents; q=1-p ; L= permissible error
in the estimation of p i.e. 10% of 30%]
Ethical approval: Written consent was taken from
the head of respective school for conducting the
study. Verbal consent of the study subjects was also
taken after explaining the aims and need of this study.
Ethical committee of our institute cleared the study
protocol.
Duration of study: This study spanned over a time
period of 6 months from October 2013 to April 2014.
Data was collected simultaneously from all study
schools on same day.
Inclusion criteria: The study subjects were all the
unmarried girls studying in standard(s) 9th,10th,11th or
12the and who were physically present in the school
on the day of the study. The study was conducted in 3

girls Government school from urban city located in
Uttar Pradesh.
The girls were approached during the school hours
after obtaining the permission and written consent of
the school authorities.
Exclusion criteria: Respondents were excluded from
the study if they did not attain menarche or they
failed to complete the given questionnaire.
Methodology: A pre-designed and pre-tested self
administered questionnaire was prepared in the local
language Hindi. This was used for data collection.
Copies of the questionnaire were distributed and
filled by the adolescents in classrooms, following an
anonymous respondent approach immediately after
an explanation of its content by the principal author.
The questionnaire included details of menstrual cycle
pattern (cycle length, regularity and duration of flow),
hygienic practices and self- reported reproductive
morbidities.
The following definitions are used in the study[6]:
Gynecological age: The number of years since
menarche.
Oligomennorhea: Infrequent and irregular bleeding at
>45days interval.
Polymennorhea: frequent regular or irregular
bleeding at <21days interval.
Irregular menses: Bleeding at varying intervals, 21
days but <45 days interval.
Dysfunctional uterine bleeding: Prolonged and
excessive menstrual bleeding associated with
irregular periods.
Statistical analysis: Statistical analysis was done
using GraphPad 5 software.
RESULTS
All the girls in the study sample were unmarried and
aged 12-17 years (Table 1). All together 994 girls
were given the questionnaire. Response rate was
100%. 88(8.8%) girls had not attained menarche.
Remaining 906(91.14%) subjects filled the
questionnaire completely and only these subjects
were included in the further statistical analysis for
menstrual patterns.
The mean age of the girls included in the study was
14.891.41 years and the mean age of menarche was
13.110.95 years. The median age for menarche was
13 years with a range of 10-17 years. 219(24.01%)
girls had menstruated by the time they were 12 years
602
Sandhya et al.,
of age. All the girls attained menarche by the

maximum age of 17 years in the study population.
Among 88/994(8.85%) girls who had not attained
menarche, 23/88(26.14%) girls were in the age group
of 14-16 years. (Table 1)
Table 1: Characteristics of menstrual cycle in study
subjects
Gynaecological age (n=906)
1st year
340 (37.28%)
nd
2 year
264 (28.95%)
rd
3 year
175 (19.18%)
4th year
51 (5.59%)
th
5 year
32 (3.51%)
Dont remember
44 (4.86%)
Cycle length (n=906)
No of study subjects (%)
<21 days
34 (3.75%)
21-35days
738 (81.46%)
36-45days
84 (9.27%)
46-90days
9 (0.9%)
>90 days
12 (1.3%)
Dont remember
29 (3.2%)
Duration of menstrual flow (n=906)
7 days
840 (92.71%)
>7 days
48 (5.29%)
Dont remember
18 (1.98%)
Regularity of menstrual cycle (n=906)
Regularity
683 (75.38%)
Irregular
188 (20.75%)
Couldnt specify
35 (3.86%)
Table 2 describes menstrual patterns in study
subjects as per their gynaecological age. In the study
sample, 822/906(90.72%) girls had a normal cycle
length of 21-45 days. Abnormal cycle lengths were
reported by 55/906(6.07%) girls. Among these,
34/906(3.95%) girls reported polymenorrhea and
21/906(2.41%) girls reported oligomenorrhea.
With increasing gynaecological age statistically
significant increase in regularity of menses was seen.
Similarily irregular menses also showed a statistically
significant decreasing trend with progressive
gynaecological age till 3rd gynaecologic year.
However presence of irregular menses from 4th
gynaecological year onwards diminishes the

possibility of their regularization in near future as per
the present study.
704/906(77.70%) girls reported dysmenorrhea with
102/906 (11.26%) girls reporting excessive pain
which resulted in restriction of day to day activities
and absenteeism at school. Mild to moderate
dysmenorrhea showed a significantly decreasing
trend with increasing gynecological age. But the trend
of excessive dysmenorrhea definitely showed an
increase with increasing gynecological age. Among
the menstrual disturbances associated with excessive
dysmenorrhea, 35/102 (34.31%) girls had irregular
menstrual cycles, 16/102 (15.68%) had cycles of
abnormal lengths, 74/102 (72.55%) reported vaginal
discharge, 33/102(32.36%) had burning micturition
and 29/102 (28.43%) reported both. 8/906 (8.8%)
girls reported excessive dysmenorrhoea along with a
cycle duration of >7days.
The proportion of both short and long cycles showed
no significant increasing or decreasing trend with the
increasing gynecological age. Noteworthy the
menstrual length of menstrual cycle normalized in all
the study subjects by 5th gynaecological age.
Out of 48 girls with a cycle duration of >7 days,
14(29.16%) had abnormal cycle lengths, 14(29.16%)
had irregular menstrual cycle(IMC) and 8(16.66%)
had excessive dysmenorrhea. Such girls can be
considered to be suffering with dysfunctional uterine
bleeding(DUB).
For further comparison of the menstrual cycle length
and regularity of menstrual cycles (Table 3), the 822
girls with a cycle length of 21-45 days were further
divided into 2 subgroups on the basis of cycle length;
21-35 days cycle length and 36-45 days cycle length.
Significantly more girls in the latter subgroup
reported IMC (p=0.0001).
As per table 4, prevalence of IMC was significantly
more in cycles of abnormal lengths. The incidence of
IMC was 38.23% in the polymenorrheic group
(p=0.0205) compared to 51.43% in oligomenorrheic
group (p=0.0001).
603
Sandhya et al.,
Table 2: Relationship of gynaecological age with various patterns of menstrual cycle and dysmennorhea:
Menstrual characteristics
Gynaecological age
P value
1st (n=340)
2nd (n=264)
3rd (n=175)
4th (n=51)
5 (n=32)
Regular menstrual cycle(n=683) 257 (75.58%)
197(74.62%) 143(81.71%) 41(80.93%) 23(71.87%) 0.0016
Irregular menstrual cycle(n=188) 71 (20.88%)
60 (22.72%) 29 (16.57%) 9 (21.95%) 8 (25%)
0.0136
Duration of menstrual flow >7 15 (4.41%)
21 (7.95%)
5 (2.85%)
5 (9.8%)
1 (3.12%)
0.2424
days (n=48)
Mild dysmennorhea (n=357)
174 (51.17%)
105(39.77%) 69 (39.43%) 7 (13.72%) 2 (6.25%)
0.0136
Moderate dysmennorhea (n=245)
101 (29.70%)
88 (33.33%) 42 (24%)
10 (19.6%) 4 (12.5%)
0.0143
Severe dysmennorhea (n=102)
28 (8.23%)
30 (11.36%) 18 (10.28%) 26(50.98%) 0
0.6169
Menstrual cycle length of 21-45 308 (90.58%)
252(95.45%) 165(94.28%) 50 (98%)
32 (100%)
0.0012
days (n=822)
Polymennorhea (n=34)
14 (4.12%)
10 (3.78%)
10 (5.70%)
0
0
0.0587
Oligomennorhea (n= 21 )
18 (5.29%)
2 (0.75%)
0
1 (1.96%)
0
0.4026
Menstrual cycle length of 46-90 8 (2.35%)
1 (0.04%)
0
0
0
0.2243
days (n=9)
Menstrual cycle length of >90 10 (2.94%)
1 (0.04%)
0
1(2%)
0
0.2463
days (n=12)
Table 3: Comparison of various abnormal menstrual cycle patterns in girls having cycle length of 21-35
days with girls having cycle length of 36-45 days as per their gynaecological age:
Gynaecologi
21-35 DAYS CYCLE
36-45DAYS CYCLE
c age
No of study Irregular
Duration
of No of study Irregular
Duration
of
subjects (n)
menstrual
menstrual flow of subjects (n)
menstrual
menstrual flow of
cycle (%)
>7days (%)
cycle (%)
>7days (%)
1
268
34 (12.68)
7 (2.61)
40
22 (55)
3 (7.50)
2
226
41 (18.14)
17 (6.34)
26
12 (46.15)
2 (7.69)
3
154
24 (15.58)
3 (1.94)
11
5 (45.45)
0
4
44
17 (38.63)
5 (11.36)
6
3 (50)
0
5
31
10(32.25)
1(3.22)
1
1 (100)
0
Total
723
126(17.4)
33 (4.62)
84
43 (51.19)
5 (5.95)
Table 4: Correlation of regularity of Menstrual cycle with length of menstrual cycle.
TOTAL(n=906)
RMC(n=683)
IMC(n=188)
CYCLE LENGTH
<21days
(n=34)
21
(61.67%)
13 (38.23%)
21-35days
(n=738)
616
(83.47%)
122 (16.53%)
36-45days
(n=84)
38
(45.24%)
43 (51.19%)
>45days
(n=21)
10
(47.62%)
11 (52.38%)
DISCUSSION
Age of menarche in current study was similar to
recent Indian studies.[7-10] The mean age of menarche
is typically between 12-13 years.[11] Age of menarche
is determined by general health, genetic, socioeconomic and nutritional factors. Chronic disease,
malnutrition and high level of physical activity can
delay menarche.[12]
Prevalence of different patterns of Menstrual Cycle:
In the present study, 2.31% girls reported

oligomenorrhea. Other population based studies on
adolescent girls have reported a prevalence ranging
from 2.5%-22%.[7,8,13,14] The
proportion
of
adolescents with oligomenorrhea declined after the
second gynaecologic year in the present study. This
is in accordance with other studies which suggest that
in most of the adolescents, oligomenorrhea present
604
Sandhya et al.,
with the onset of menarche changes to a normal

pattern, especially with in the first 2 years after
menarche.[3,15-17]
The incidence of polymenorrhea in the present study
was 3.75% which is similar to past studies from
HongKong, Italy and India, which reported a varied
range from 3.0 to 8.0%.[7,14-16,18]
The incidence of irregular menstrual cycles in the
present study is 20.75% . Past studies from India
have reported irregularity in upto 30% of study
subjects.[7,14]
Across the gynaecological ages, the incidence of IMC
showed little fluctuation. This was different from
findings of Chan et al which showed a decreasing
trend in the prevalence of IMC with increasing
gynaecological ages.[15] The present study shows that
the incidence of IMC was much higher in cycles of
abnormal length, more so in oligomenorrheic cycles.
Past studies suggest that the risk of cycles remaining
oligomenorrheic can be predicted by IMC.[3,19]
Also the risk of cycles becoming oligomenorrheic
can be predicted by IMC , particularly the cycles
with an average length of 35-45 days.[3] The POMP
study had specified the group with 36-45 days cycle
length
and
irregular
menses
as
mild
[3]
oligomennorhea. Further this study showed that
about 34% girls with Mild oligomennorhea went on
to develop oligomenorrhea as compared to only 4%
girls having regular menstrual cycles.[3] According to
the available prospective studies, in such girls the
first symptoms of PCOS (polycystic ovarian
syndrome) may appear in the perimenarchal period.
Similarly a Swedish prospective study of menstrual
disturbances in adolescents demonstrated that after 6
years of follow-up, irregular menstruation was still
present in 62%.[2] Further, 59% of those with
persisting irregular menstruation fulfilled the criteria
for diagnosis of PCOS.[2] Since PCOS is the most
important cause of anovulatory infertility,
observations of the present study are extremely
relevant with respect to the fertility concerns for these
young women in the future. On the basis of the above
mentioned available evidence, we can conclude that
adolescents presenting with IMC since the beginning
of menarche and mild oligomenorrhea (i.e. cycle
length of 36-45 days) should be counseled
accordingly and as far as possible kept under close
follow up for years.
Dysmenorrhea is the most common gynecologic

complaint among adolescent and young adult
females.[20] Due to the relative immaturity of the
hypothalamic pituitary-ovary axis in the first 2 years
following menarche, more than half of the menstrual
cycles are anovulatory. Initial anovulatory cycles tend
to be pain free, although heavy menstrual loss can
result in an element of dysmenorrhoea. When regular
ovulatory cycles commence, the periods often
become more painful due to the increased levels of
circulating prostaglandins.[17] The majority of
dysmenorrhea in adolescents and young adults is
primary (or functional), is associated with no pelvic
pathology, and has a clear physiologic etiology.[20]
According to DLHS 3 about 79.5% of adolescent
Indian girls complain of dysmenorrhea.[1] In the
present study, 77.70% of girls reported to be suffering
with variable degree of dysmenorrhea with 11.26%
girls having excessive dysmenorrhea leading to
restriction of daily activities and school absenteeism.
Among the
girls with excessive dysmenorrhea,
nearly 1/3rd had associated menstrual disturbances
also. Parker et al reported that 93% of girls suffered
menstrual pain and approximately 25% of the sample
had marked menstrual disturbance.[21] Another study
from Turkey also showed a highly significant
correlation of pain with menstrual disturbances in
approximately 25% of teenagers.[22] This suggests
that association of dysmenorrhea with other
menstrual disturbances should prompt the treating
physician for earlier investigation and search of an
underlying
pathological
disorder
such
as
endometriosis. It is likely that many cases of
endometriosis in young women may have been
misdiagnosed as spasmodic dysmenorrheal.
Endometriosis has always been assumed to present
many years after menarche but studies have shown it
to occur prior to menarche and between 16 months
after the onset of menarche.[4] According to The
Endometriosis Association, 66% of adult women with
endometriosis report the onset of pelvic symptoms
prior to age 20. Most importantly, patients who report
symptoms as teens visit an average of 4 or more
physicians before receiving a correct diagnosis.[4]
Abnormal uterine bleeding may occur in women of
all ages, and it is a particularly common issue for
adolescents.[22] In the present study 5.29% girls
presented with prolonged vaginal bleeding and nearly
1/3rd of them had associated features in the form of
605
Sandhya et al.,
abnormal cycle lengths, 1/3rd had irregular cycles and

nearly 20% had excessive dysmenorrhea. Although
the majority of adolescents presenting with abnormal
vaginal bleeding do, in fact, have anovulatory
bleeding caused by an immature HPO axis, one must
consider other severe causes in abnormally prolonged
bleeding depending upon the associated features
present viz. endometritis or endometriosis if
associated with significant dysmenorrhoea and PCOS
in adolescents who continue to have abnormal
vaginal bleeding 3 to 4 years after menarche. Other
causes may include abnormalities of the uterus or
vagina, endocrine or other systemic disorders and
coagulopathies.[23]
CONCLUSION
Abnormal cycle lengths were reported by 6.07% of
girls. Among these, 3.95% girls reported
polymenorrhea
and
2.41%
girls
reported
oligomenorrhea. The incidence of irregular menstrual
cycles in the present study is 20.75%. In the present
study 5.29% girls presented with prolonged vaginal
bleeding and nearly 1/3rd of them had associated
features in the form of abnormal cycle lengths, 1/3rd
had irregular cycles and nearly 20% had excessive
dysmenorrhea. 77.70% girls reported dysmenorrhea
with 11.26% girls reporting excessive pain which
resulted in restriction of day to day activities and
absenteeism at school. While mild to moderate
dysmenorrhea showed a significantly decreasing
trend with increasing gynecological age the trend of
excessive dysmenorrhea showed an increase with
increasing gynecological age. The incidence of IMC
increases with oligomennorhea. Noteworthy in the
normal cycles also, incidence of IMC was
significantly more in mild oligomennorheic group.
Adolescents having mild oligomennorhea (i.e. cycles
of 36-45 days duration) along with IMC require
regular and intense observation for first 5 years after
menarche.
Suggestions: Considering the complex psychology
and high risk behavior of adolescents, an appropriate
anticipatory guidance should be given to the
adolescents regarding what is normal or abnormal for
their reproductive health at every contact point. Also
thorough evaluation and prompt identification of mild
oligomennorhea throughout adolescence may help the
health care providers in early identification of
potential health concerns for adulthood especially

infertility.
Acknowledgements: Authors will like to thank the
management of SRMSIMS, Bareilly for help in
collecting data. We also thank Dr (Prof) Pratik
Gahalaut for critical editing of this manuscript.
Conflict of interest: All the authors declare that they
dont have conflict of interest.
Funding: None
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R, Shekhar TV. International Institute for
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October 25). Available from: http://www.rchiips.
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2. Wiksten AM, Hirschberg AL, Hagenfeldt K.
Prospective follow-up of menstrual disorders in
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3. Van Hooff MH, Voorhorst FJ, Kaptein MB,
Hirasing RA, Koppenaal C, Schoemaker J.
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mass index, hormone levels and polycystic
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age 18 years. Hum Reprod 2004;19:383-92.
4. Dessole M, Melis GB, Angioni S. Endometriosis
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DOI: 10.5958/2319-5886.2015.00116.2

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
Received: 25th Apr 2015
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
SERUM LEVELS OF HIGH SENSITIVITY C REACTIVE PROTEIN AND MALONDIALDEHYDE IN

CHRONIC KIDNEY DISEASE
*Rakshitha Gowda B.H1, Meera K.S 1, Mahesh E2
1
Dept of Biochemistry, 2Dept of Nephrology, M. S. Ramaiah Medical College, MSR Nagar, Bangalore,
Karnataka, India
*Corresponding author email: rakshitha28282@yahoo.com
ABSTRACT
Background: Chronic kidney disease cases are at increased risk for progression to end stage renal disease and
accelerated atherosclerosis, with premature cardiovascular morbidity and mortality being the more frequent
outcome. Aim: The study was taken up to find if there is any association between nontraditional cardiovascular
risk markers like high sensitivity C reactive protein (marker of inflammation) and malondialdehyde (marker of
lipid peroxidation) with the progression of chronic kidney disease. Methodology: The study included 44 pre
dialysis chronic kidney disease cases and 44 healthy controls. Serum levels of creatinine, high sensitivity C
reactive protein and malondialdehyde were estimated in both groups. The mean estimated glomerular filtration
rate(eGFR) in chronic kidney disease patients was calculated by the MDRD formula. Results: The mean eGFR in
cases was found to be 23.65 14.99 ml/min by MDRD formula. The serum hsCRP and malondialdehyde levels in
cases was 11.8 7.24 mg/L and 3.02 1.24 nmol/ml respectively. Conclusion: There was a significant negative
correlation (p<0.001) between high sensitivity C-reactive protein and malondialdehyde with eGFR. A highly
significant positive correlation was found between serum hsCRP and malondialdehyde (p<0.001) in chronic
kidney disease underlining the synergism between oxidative stress and inflammation, perpetuating to further
deterioration of renal function and enhancing the predisposition to cardiovascular risk with the progression of
chronic kidney disease.
Keywords: Chronic kidney disease, Estimated glomerular filtration rate, High sensitivity C reactive protein,
Inflammation, Malondialdehyde, Oxidative stress.
INTRODUCTION
Chronic kidney disease has gained attention as a
public health problem worldwide with the increase in
incidence and prevalence of the disorder. The Kidney
Diseases Outcomes Quality Initiative (K/DOQI)
defines chronic kidney disease (CKD) as kidney
damage or glomerular filtration rate (GFR) less than
60 ml/min/1.73 m2 for a period of three months or
more, irrespective of cause [ 1] . The glomerular
filtration rate (GFR) is the amount of plasma that is
filtered by the glomeruli per unit time and is a reliable
measure of the functional capacity of the kidneys.
Based on the GFR, CKD is divided into five stages
with stage 5 being end stage renal disease having a
GFR of <15ml/min. Chronic kidney disease cases

with end stage renal disease ultimately undergo renal
replacement therapy either in the form of dialysis or
renal transplantation. The measurement of GFR is
very useful in monitoring the progression of CKD,
targeting treatment and predicting renal replacement
therapy.
However, cardiovascular morbidity and mortality due
to accelerated atherosclerosis is encountered more
frequently in CKD patients. CKD is considered an
independent risk factor for the development of
cardiovascular disease [2]. There is increase in
cardiovascular risk whilst there is decline in
608
Rakshitha et al.,
glomerular filtration rate below 70 ml/min[3, 4]. The

traditional risk factor for development of
atherosclerosis includes increase in age, predilection
to male gender, hypertension, smoking, diabetes
mellitus, dyslipidemia and others[5]. However, these
conventional factors are unable to entirely explain the
mechanism for the increased risk for atherosclerosis
in the CKD population. In some of the CKD cases
there is increase in serum triglyceride with associated
decrease in serum high density lipoprotein and with
no alteration in other fractions of lipoproteins[6].
The uremic milieu of CKD patients contains high
amounts of proinflammatory proteins and cytokines
like C reactive protein, interleukin 6 and others[7].
Atherosclerosis too is an inflammatory condition of
the arteries and C reactive protein (CRP) which is
produced chiefly in the hepatocytes under the
influence of interleukin 6 (IL-6) and IL-1 is an
important inflammatory mediator. Among the various
mechanisms responsible for increase in oxidative
stress in uremia, activation of reduced nicotinamide
adenine dinucleotide oxidase which can be stimulated
by angiotensin II is a very important one[8].
There are studies to demonstrate the role of increased
levels of CRP and reactive oxygen species in end
stage renal disease and patients undergoing dialysis [9,
10]
. However, the role of inflammatory protein CRP
and oxidative stress markers like malondialdehyde in
the progression of renal dysfunction and accelerated
atherosclerosis in predialytic patients is not very
clear. The study was intended to determine the levels
of high sensitivity C reactive protein (hsCRP) as a
marker of inflammation and malondialdehyde
(MDA), a lipid peroxidation product as a marker of
oxidative stress in predialytic renal disease patients
and to decipher if there is any association between
serum hsCRP and MDA levels with the progression
of kidney disease.
Study design: Cross sectional , case control study
Ethical approval: The study was approved by
Institutional Ethics review board; an informed
consent was taken from the patients before the
collection of blood sample.
Sample size: The study population included 44
healthy control subjects and 44 pre dialytic
nephropathy cases who attended the outpatient clinic
of the Department of Nephrology of our college.
Inclusion criteria: Clinically diagnosed chronic

kidney disease patients with serum creatinine level
greater than 1.5mg/dL were chosen. The estimated
GFR (eGFR) was calculated based on MDRD
formula [11] [186 (S.creatinine mg/dl)1.154 x
(age)0.203 x (0.742 if female)] mL/min/1.73 m2 ]
Patients were grouped into:
Stage III CKD eGFR : 30-59 ml/min
Stage IV CKD eGFR : 15-29 ml/min
Stage V CKD eGFR : <15ml/min
Exclusion criteria: Patients with active infection or
chronic
ongoing
inflammation,
history
of
cardiovascular disease, autoimmune disorders, renal
transplant and chronic kidney disease on dialysis,
drug supplementation such as steroids, immuno
suppressants, non steroidal anti - inflammatory drugs,
oral contraceptives, hormone replacement therapy,
statins, niacin and fibrates were excluded from the
study.
Grouping: The participants were divided into two
groups:
Group1: Control (Healthy volunteers who visited the
hospital for routine health check up and were willing
to be part of the study were taken as controls. The
control subjects had the same exclusion criteria as
CKD cases.)
Group 2: Chronic kidney disease(CKD) cases.
Methodology
5 ml of venous blood sample was collected under
aseptic conditions from the study groups in BD
vacutainer. The sample was allowed to clot. The
serum was separated from the sample at the earliest
after centrifugation and used for the estimation of
serum creatinine, high sensitivity C - reactive protein
and malondialdehyde. Serum creatinine was
estimated on Roche/Hitachi COBAS c-501
autoanalyzer by Buffered Kinetic Jaffe reaction
without deproteinization (ID-MS traceable method).
hsCRP in serum was measured by a turbidimetric
immunoassay. The assay is based on a latex-enhanced
turbidimetric
immunoassay
method.
The
agglutination that occurs between CRP in a sample
and anti-CRP antibody which has been sensitized to
latex particles, is detected as an absorbance change
(570 nm), with the magnitude of the change being
proportional to the quantity of CRP in the sample.
The actual concentration is then determined by
interpolation from a calibration curve prepared from
calibrators of known concentration. MDA was
measured by the Thiobarbituric acid reactive
609
Rakshitha et al.,
substances (TBARS) method as described by Wilbur

et al [12].
Statistical analysis: The results were expressed as
mean SD. Significance was assessed at 5% level of
significance. Student t test (two tailed,
independent) and analysis of variance (ANOVA) was
used to find the significance of study parameters.
Pearson correlation was used to study the relation
between the various parameters. Statistical analysis
was performed using SPSS 15.0 software.
RESULTS
The study population involved 44 predialytic CKD
patients whose average age was 55
13 yrs. The
serum creatinine level in controls were within
physiological range and in CKD cases the mean
serum creatinine level was 4.07 2.78 mg/dl .The
eGFR ranged from 4.48 ml/min to 60.61ml/min in
CKD cases with an average of 23.6514.99 ml/min.
In controls the eGFR was found to be 152.33
41.51ml/min (Table 1). The serum hsCRP levels in
cases was 11.8 7.24 mg/L with values ranging from
1.9 mg/L to 26.5 mg/L. The hsCRP level was found
to be fivefold higher in cases as compared to controls
(p<0.001) (Table 1). The serum malondialdehyde
level was significantly higher in chronic kidney
disease cases (Table 1). The serum levels of
inflammatory marker, hsCRP and oxidative stress
marker, MDA was five times higher in CKD cases as
compared to healthy controls.
The result was analysed by comparing eGFR, Serum
hsCRP and MDA in stage III, IV, V CKD (Table 2).
There is a gradual increase in serum creatinine from
stage 3 to stage 5 CKD. There is doubling of serum
hsCRP in stage 4 as compared to stage 3 and three
fold increases in stage 5 as compared to stage 3. The
hsCRP in stage V is 18.61 3.86 mg/L as compared
3). With the progression of CKD, the rise in serum

MDA, a marker of oxidative stress indicates
intensification of inflammation.
Table 1: Comparison of eGFR and biochemical
parameters in controls and CKD cases
Biochemical
parameters
Controls (n=44)
Mean SD
Cases(n=44)
Mean SD
S. Creatinine
(mg/dL)
eGFR
(ml/min)
0.59 0.10
4.07 2.78
**
152.33
**
S. hsCRP
(mg/L)
S. MDA
(nmol/mL)
2.43 0.74
23.65
14.99
11.8 7.24
0.55 0.24
3.02
**
41.51
p value
**
1.24
Table 2: Comparison of biochemical parameters in

different stages of CKD.
Parameters
Stage III
CKD(n=16)
Stage IV
CKD(n=12)
eGFR
(ml/min)
40.72
20.46 3.78
8.7
Stage
VCKD
(n=16)
8.97
p
value
-
3.1
3.11
7.08
2.41
<0.001**
3.77
11.21 6.23
18.61
<0.001**
1.34
2.92
3.86
3.71
0.7
<0.01
S.
Creatinine
(mg/dL)
S. hsCRP
(mg/L)
1.8
3.01
5.44
S. MDA
(nmol/mL)
2.39
1.28
Table 3: Pearson correlation of S. creatinine, hsCRP

and MDA with eGFR.
Pair
Cases
r value
p value
S. Creatinine v/s eGFR
-0.797
**
S. hsCRP v/s eGFR
-0.742
**
S. MDA v/s eGFR
-0.389
to stage IV were it is 11.21 6.23 mg/L. There is

significant rise in hsCRP with the progression of
CKD. There is also a gradual rise inS. MDA with the
progression of the disease (Table 2).
There was a significant correlation between S.
creatinine and eGFR (Table 3). A highly significant
negative correlation was also found between S.
hsCRP and S. MDA with eGFR (Figure 1, 2). With
the deterioration of kidney function, there is increase
in serum creatinine, hsCRP and MDA level.
There was a highly significant positive correlation of
serum MDA with hsCRP (r=0.642, p<0.001),(Figure
610
Rakshitha et al.,
Fig 1: Scatter plot depicting Pearson correlation

between MDA (nmol/mL) and eGFR(ml/min)
Fig 2: Scatter plot depicting Pearson correlation

between hsCRP(mg/L) and eGFR (ml/min)
Fig3: Scatter plot depicting Pearson correlation

between hsCRP (mg/L) and MDA (nmol/mL)
DISCUSSION
With the rise in both incidence and prevalence of
diabetes mellitus in India and the growing inclination
towards earlier diagnosis of chronic kidney disease,
there is an increase in the number of predialytic CKD
patients [13]. CKD patients exhibit an augmented risk
for the development of cardiovascular morbidity and
mortality which cannot be entirely substantiated by
the traditional Framingham risk factors such as age,
gender,
hypertension,
diabetes
and
hypercholesterolemia. Furthermore, the lipid profile
in CKD patients is not very significant as some cases
show hypertriglyceridemia which has no definite
relation with the various stages of CKD and could be
misleading. The uremia related risks include
proteinuria, increased reninangiotensin system
activity, chronic volume overload, altered calcium

and phosphorus metabolism, inflammation, infection,
anemia, malnutrition, oxidative stress, elevated levels
of homocysteine, uremic toxins and thrombogenic
factors[14].
Oxidative stress and inflammation have received
increased importance as nonconventional risk factors
of cardiovascular morbidity and mortality in CKD [
15]
. Oxidative stress is defined as the tissue damage
resulting from an imbalance between an excessive
generation of oxidant compounds and insufficient
antioxidant defense mechanisms. Evaluation of
oxidative stress is difficult because free radicals have
very short half-lives. However there are more stable
marker molecules that have longer half-lives, ranging
from hours to weeks, which can be used to assess
oxidative stress. Malondialdehyde, a water soluble,
three carbon, low molecular weight reactive aldehyde
is one such molecule which is a product of the lipid
peroxidation and has been studied as an indicator of
oxidative stress. Serum MDA levels in CKD cases
was found to be significantly higher as compared to
healthy controls (p<0.001) (Table1). This finding is
in agreement with studies by Oberg et al [15], who also
reported increase in oxidative stress in CKD patients.
Increased generation of MDA as well as its decreased
renal clearance due to compromised renal function
leads to increased concentration of lipid peroxidation
products in circulation in renal failure patients.
The mechanisms of oxidative stress in uremia
involves activation of reduced nicotinamide adenine
dinucleotide (NAD(P)H) oxidase, xanthine oxidase,
myeloperoxidase (MPO), mitochondrial oxidases and
uncoupling of endothelial nitric oxide synthase
(eNOS) [16]. Furthermore, in CKD there is activation
of the renin angiotensin aldosterone axis which leads
to the stimulation of NADPH oxidase by angiotensin
II [17]. NADPH oxidase is the most important source
of reactive oxygen species in the systemic as well as
renal vasculature. Angiotensin II activates
NADH/NADPH oxidase and protein kinase C activity
in vascular cells thereby increasing superoxide ion
production and decline in nitric oxide (NO)
availability which can lead to endothelial dysfunction
in CKD. The reactive oxygen species along with
inflammation can cause increase in cardiovascular
risk and further deterioration of renal function. The
production of reactive oxygen species (ROS) which
can occur at constitutive levels in nonphagocytic cells
(e.g., glomerular cells and tubular epithelial cells) for
611
Rakshitha et al.,
physiological purposes gets deranged and can lead to

loss of redox homeostasis and oxidative stress which
can contribute to proinflammatory and profibrotic
pathways in the kidney. Peroxidation of lipids brings
about changes in the molecular structure of the lipids
and these changes becomes more marked when the
damaged lipids are the constituents of the biological
membrane disrupting the cohesive lipid layer
arrangement and structural organization. The lipid
peroxides in general, enhances prostaglandin
synthesis which is another source of free radical and
associated decrease in NO production are well known
risk factor for atherosclerotic complication.
Formation of ROS is evident in many areas of the
kidney, predominantly in the renal cortices, sparing
the medulla which is susceptible to hypoxia and less
ROS production under physiologic conditions. The
substantial generation of ROS is all the more
damaging because CKD patients have a weaker
antioxidant system which also can be attributed to
intake of diet low in antioxidant nutrients. The
disturbances in cellular oxidant and pro-oxidant status
can cause detrimental effect by altering cellular
signaling process and perpetuating renal cell
apoptosis and senescence [16]. Due to increased
production of reactive oxidants there is predisposition
to enhancement in the peroxidation of lipids and
lipoproteins. In the study, a graded increase in serum
MDA levels are observed with the progression of
renal failure. A significant negative correlation
between serum MDA levels and eGFR is found in the
CKD cases (p<0.001) (table 3).
Serum
malondialdehyde (MDA) a three carbon compound
reflects both autoxidation and oxygen mediated
peroxidation of poly unsaturated fatty acids in
particular. It reflects the oxidative status of the
biological system. MDA causes damage to low
density lipoproteins (LDL) which in turn can be taken
up by macrophages via scavenger receptors and form
foam cells. Due to increased production of ROS and
increased oxidative stress, lipid peroxidation products
are found to be elevated in chronic kidney disease
cases. Oxidative stress promotes vascular smooth
muscle cell proliferation, hypertrophy and collagen
deposition, leading to thickening of the vascular
media and narrowing of the vascular lumen.
Increased oxidative stress, can initiate further damage
to the endothelium thereby causing impairment of
endothelium-dependent vascular relaxation and
increases vascular contractile activity.
The augmented oxidative stress in CKD leads to a net

deficiency of NO. Nitric oxide production and/or
bioavailability in the vascular endothelial cells
involves normal functioning of endothelial nitricoxide synthase (eNOS), and optimal concentrations
of the substrate L-arginine and the cofactor 5,6,7,8tetrahydrobiopterin (BH4). The physiological actions
of NO include the regulation of vascular tone and
blood pressure, prevention of platelet aggregation and
inhibition of vascular smooth muscle proliferation.
NO can inhibit the activation of xanthine oxidase and
NADPH oxidase. Under pathological conditions
such as CKD there is uncoupling of endothelial NO
synthase enzyme wherein, electrons are transferred
to molecular oxygen instead of L-arginine to produce
superoxide rather than NO and also inactivation of
nitric oxide that is already available by the increased
oxygen free radicals [ 18]. These oxygen free radicals
additionally work on LDL cholesterol molecules
leading to their oxidation. Oxidized LDL attack the
arterial intima and triggers an inflammatory response
in the vessel wall.
The causes for inflammation in CKD are
multifactorial. Several markers are studied as
inflammatory markers in CKD. These markers can be
used to predict future risk of CVD in CKD patients.
In the study C reactive protein level, a marker of
inflammation in stage III, IV and V of CKD and
healthy controls was estimated. hsCRP is an acute
phase reactant, produced chiefly in the hepatocytes
and its synthesis is transcriptionally driven by
interleukin 6 with synergistic enhancement by
interleukin 1. CRP is released in response to
inflammation produced by numerous external and/or
internal stimuli and increases dramatically after
severe trauma, bacterial infection, inflammation,
surgery or neoplastic proliferation. It is found to bind
to the fc 1 and 2 receptors on the surface of
phagocytic cells and helps in the clearance of the
apoptotic and necrotic cells behaving as an opsonin.
In an apparently normal individual with no other
signs of infection, inflammation or trauma, the basal
levels of CRP are detected by a more sensitive assay,
as high sensitivity CRP (hsCRP). Serum hsCRP is a
well studied marker in inflammation and has
advantage for the detection and predictor of
inflammation. The mean hsCRP level in CKD cases
obtained in the study was 11.8 7.24 mg/L which is
comparable to earlier studies by G Abraham et al
[19]
612
Rakshitha et al.,
The hsCRP levels have increased by almost up to five

times in nephropathy patients as compared to
controls, but did not show any significant correlation
with age of the patients studied. Underlying etiology
of CKD, such as diabetes or hypertension is by itself
a major contributory factor to the existing
inflammation. Payson et.al [ 15] have reported on the
renal related causes of inflammation in CKD such as
retention of uremic toxins, sympathetic over activity
and fluid overload. hsCRP has been well studied as a
biomarker indicating increased cardiovascular risk,
wherein levels >3mg/L indicate high risk and levels
<1mg/L indicate low cardiovascular risk [20]. Raised
levels of serum hsCRP between 1-3 mg/L is an
indicator of chronic low level inflammation probably
arising out of the endothelial dysfunction ,
predisposing to the pathogenesis of atherosclerosis.
High concentration of the protein CRP and its mRNA
has been noted in the plaque, as compared to its
concentration in the plasma, which could well
contribute to the proinflammatory and proatherogenic
effects of the protein. Devaraj et al [ 21] have outlined
the active role of CRP in the pathogenesis of
atherosclerosis wherein they have demonstrated that
CRP can also be synthesized by macrophages,
smooth muscle cells and endothelial cells, by noting
the presence of both protein as well as its mRNA in
the atherosclerotic plaque. CRP can no longer be
considered an innocent bystander or biomarker of
cardiovascular risk, but is an actively proatherogenic
molecule which induces cell adhesion molecules,
plasminogen activator inhibitor-1, complement
activation and attenuates nitric oxide production. This
leads to a state of endothelial dysfunction which is a
well known precursor of cardiovascular morbidity
[21]
. Inflammation, oxidative stress and endothelial
dysfunction represent a key triad for the development
and progression of atherosclerosis.
The study showed a significant graded increase
(p<0.001) in the levels of hsCRP in stages III, IV and
V CKD. hsCRP levels have more than doubled and
tripled in stage IV and V CKD as compared to stage
III underlining the highly significant correlation
(p<0.001) between hsCRP and eGFR. hsCRP can
also predispose to inflammation through binding with
lipoprotein and activation of the complement system
[22]
. The rising inflammation may lead to deteriorating
renal function which in turn could lead to further
increase in inflammation setting up a vicious cycle.
hsCRP has been reported to induce adhesion
molecule expression in human endothelial cells

favoring the involvement of hsCRP in the
atherosclerotic process.
The present study shows a significant positive
correlation between oxidative stress marker MDA
and hsCRP, a marker of inflammation in CKD.
Nguyen et al [23], have also found similar results.
Nuclear factor kappa B is a redox sensitive
transcription factor that leads to induction of genes of
various proinflammatory cytokines and adhesion
molecules, links oxidative stress with inflammation.
Inflammation and ROS together lead to increased
propensity for atherogenesis in CKD as reviewed by
Cachofeiro et al [24]. hsCRP has been shown both as
marker and mediator of atherosclerosis. Patients with
CKD have increased mortality which cannot be
explained by traditional risk factors like diabetes
mellitus, hypertension and others but by
nontraditional factors like inflammation, malnutrition
and predisposition to infection which can culminate
in increased cardiovascular risk in these cases. hsCRP
can also be considered as a potent independent
predictor of cardiovascular mortality as well as
malnutrition. Inflammation per se can contribute to
increased cardiovascular risk but there can be
increased
predisposition
to
cardiovascular
complication in CKD cases due to the inflammatory
response.
CONCLUSION
Inflammation and oxidative stress in chronic kidney
disease sets in much before dialysis, and furthermore
the hsCRP and MDA levels rise significantly with the
fall in eGFR. In CKD patients inflammation and
production of reactive oxygen species are linked in a
vicious cycle which in turn can cause damage to both
the glomerular filtration membrane as well as
vascular endothelium, worsening the progressive loss
of nephrons as well as triggering atherosclerosis in
this population. Better insight into the role of the
nontraditional cardiovascular risk factors in various
stages of CKD is warranted. Levels of these markers
can be used not only for risk stratification but also for
management wherein levels can be monitored pre and
post therapeutic interventions. Prospective studies
involving serial measurements of these biomarkers
and cardiovascular outcomes in chronic kidney
disease would be more valuable in better patient care.
613
Rakshitha et al.,
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DOI: 10.5958/2319-5886.2015.00117.4

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
Coden: IJMRHS
th
Research article
Copyright @2015
ISSN: 2319-5886
ARE ACCREDITED SOCIAL HEALTH ACTIVIST WORKERS AWARE OF THEIR ROLES AND
RESPONSIBILITIES
*Kohli C1, Kishore J2, Sharma S3, Nayak H4

1, 3,4
Resident, 2Professor, Department of Community Medicine, Maulana Azad Medical College, New Delhi, India
*Corresponding author email: kohlicdoc17@gmail.com

ABSTRACT
Introduction: The role of Accredited Social Health Activist (ASHA) workers is vital in public health delivery
system in India. The study was planned with objective to assess the socio-demographic profile of ASHA workers,
awareness and practices of their roles and responsibilities and difficulties faced while working in north-east
district of Delhi, India. Materials and methods: A descriptive cross sectional study was conducted in north east
district of Delhi among 55 ASHA workers after taking written informed consent. Data was collected using a pre
tested semi-structured questionnaire consisted of items on socio-demographic profile of ASHA workers,
knowledge and practices about their roles and responsibilities and difficulties faced in community. The data was
analyzed by using SPSS software version 17. Qualitative data was expressed in percentages and quantitative data
was expressed in mean + SD. Results: Mean age (+SD) of ASHAs was 31.84 + 7.2 years. Most of them were
married (96.4%), Hindu (85.5%) and were catering to a population of 1000-2000 (87.3%). Most of the ASHA
workers were aware of their work of maternal and child health services. However lesser numbers were aware of
their role in registration of births and deaths and to treat minor ailments. 96.4% reported that they maintain family
planning register, only 51 (92.7%) reported that they maintain antenatal register. 10 (18.2%) ASHAs reported that
they face problems in coordination with Auxiliary Nurse Midwife (ANMs). Conclusion: ASHAs performance is
impacted by their limited orientation towards their roles and responsibilities. Training should provide complete
knowledge about the same.
Key words: Community health workers, Roles, ASHA, Delhi
INTRODUCTION
The role of community health workers (CHWs) in
healthcare delivery system is widening as they are
considered inevitable to meet the universal healthcare
provision and the millennium development goals.[1]
The CHWs enable access to and utilization of health
services and inculcate health promotive behaviours
among the people in the community. They are
deployed to cater to the demand of underutilized
services, unmet health behaviours and underserved
populations.[2] The National Rural Health Mission
(NRHM) was launched by the Government of India
in 2005 to strengthen the healthcare delivery system
and to provide comprehensive integrated health care

services to people in rural area and recently urban
area also included. One of core strategies was to
recruit and train female Accredited Social Health
Activist (ASHA) workers in each village to act as
interface between community and public health
system. They are given the task of providing basic
preventive and curative services, promoting use of
existing health services and encouraging dialogue on
social health issues.[3] ASHA is a female volunteer
honorary worker selected by the community,
deployed in her own village (one in every 1000
616
Kohli et al.,
population) after a short training on community

health. She is preferred to be between 25 and 45 years
old, with a minimum formal education of 8 years and
demonstrable leadership qualities. They are provided
with performance based incentives for their
services.[4] There are presently a total of 866726
ASHAs selected across the country. The proposed
total is 908281, of which 95.42% have been selected.
Most ASHAs have completed the first four rounds of
training and in states that had initiated this, the fifth
round of training as well.[5] Since the success of
NRHM depends hugely on performance of ASHA
workers, it is important to assess their perception
regarding the tasks that they have to perform in the
community and difficulties faced by them while
working. The rationale of such research is to
contribute to the literature on design and support to
ASHA program to maximize their impact to improve
the health indicators. Keeping in view the above
aspect, this study was planned with objective to
assess the socio-demographic profile of ASHA
workers, awareness and practices of their roles and
responsibilities and difficulties faced while working
in north-east district of Delhi, India.
Study design: A descriptive cross sectional study
was carried out on ASHA workers recruited under
NRHM, Delhi covering a population of
approximately 1, 10,000. Study area was chosen by
using convenience sampling method.
The study was conducted over a period of three
months from October to December 2014. Written
informed consent was taken from the study subjects.
Ethical clearance was taken from department before
start of study. The option to opt out of the study was
kept open without any clause. The data was kept
confidential and was used for research purpose only.
Inclusion criteria: A total of 55 ASHA workers
who provide services to Gokalpuri, Chandu Nagar
and adjoining areas constituted the study population.
ASHA workers who gave consent for the study were
included in the study. There were no specific
exclusion criteria for study. All ASHA workers in the
study area were approached for participation in the
study voluntarily.
Methodology
Data was collected using a self designed pre-tested
semi-structured questionnaire prepared in English and
translated in local language. Questionnaire consisted
of items on socio-economic and demographic profile

of ASHA workers like age, educations status, income,
religion etc. Questions on knowledge and practices
about roles and responsibilities of ASHA workers
like maternal health, child health, control of common
communicable diseases etc. were included.
Perception of ASHAs about their training and
working conditions was also assessed. The
questionnaire was pilot tested before start of study for
its reliability and validity. Cronbachs alpha which is
coefficient of reliability was calculated to be 0.81.
The content validity of the tool was established by
giving it to experts in the relevant field. Most of the
experts agreed on most of the items and necessary
modifications were made as per their suggestions.
Statistical Analysis: The data was entered in MSExcel and analyzed by using SPSS software version
17. Qualitative data was expressed in percentages and
quantitative data was expressed in mean + SD.
RESULTS
Socio demographic profile
Table 1 shows socio demographic profile of ASHA
workers. Mean age (+SD) of ASHAs was 31.84 + 7.2
years. Maximum 31 (56.4%) belonged to age group
of 25-35 years. 47 (85.5%) were Hindu and 8 (14.5%)
ASHAs were from Muslim community. Majority
(61.8%) of ASHAs were educated up to or above
senior secondary school and most of them were
married (96.4%). 16 (29.1%) belonged to scheduled
caste (SC), 16 (29.1%) to other backward classes
(OBC) and 21 (38.2%) to general category. Majority
(87.3%) were catering to a population of 1000-2000.
Mean (+SD) population catered was 1891.85
(+384.27).
All ASHAs (100%) were provided with ASHA diary.
All were aware about the incentives they were
entitled to for their work. 53 (96.4%) ASHAs
reported that they did not get any money in advance
for providing services in emergency. Only 24
(43.6%) reported that they receive their incentives on
time. Monthly income (+SD) of ASHAs was Rs.
2117.39 + 796.95. When inquired about the last
ASHA training session attended, 37 (67.3%) reported
that they attended 0-3 months back, 8 (14.5%)
attended 3-6 months back and 10 (18.2%) 6 months
1 year back.
617
Kohli et al.,
Table 1: Socio-demographic profile of ASHA

workers
Characteristic
Frequen
cy
%(N= 55)
Age(in years) <25 yrs
8
14.5
25-35 yrs
31
56.4
35-45 yrs
16
29.1
Religion
Hindu
47
85.5
Muslim
8
14.5
Education
Middle
5
9.1
Secondary
16
29.1
Senior secondary
34
61.8
and above
Marital status Unmarried
2
3.6
Married
53
96.4
Caste
Scheduled casts (SC)
16
29.0
Scheduled tribe (ST)
2
3.6
Other backward
16
29.1
Classes (OBC)
General
21
38.2
Population <1000
4
7.3
served
1000-2000
48
87.3
>2000
3
5.5
Table 2: Awareness of ASHAs about their roles
and responsibilities
Responsibility
Positive
%
response (N= 55)
Mobilize children for immunization
54
98.2
Provide counseling for family 53
96.4
planning
Bring pregnant women for check up 52
94.5
to health centre
Accompany pregnant women to 49
89.1
hospital during delivery or
complications
Distribute Iron folic acid (IFA) 47
85.5
tablets to pregnant women
Counsel the mother regarding
43
78.2
child nutrition
Registration of births and deaths
29
52.7
in the area
Can act as DOTS provider
43
78.2
Give treatment for minor ailments
16
29.1
Table 3: Perception regarding training of ASHAs

Training
aspect
Complete
(%)
Incomplet
e (%)
Need to be
repeated
(%)
Too much
information
being given
Pregnancy
and child
birth
Newborn
care
Child
health
Family
planning
Common
diseases
Nutrition
Use
of
medicines
Roles and
responsibili
ties
of
ASHA
33(60.0)
5 (9.1)
11
(20.0)
6 (10.9)
36 65.5)
5 (9.1)
4 (7.3)
10 (18.2)
33 60.0)
7 (12.7)
9 (16.4)
6 (10.9)
43 78.2)
8 (14.5)
1 (1.8)
3 (5.4)
36 65.5)
9 (16.4)
7 (12.7)
3 (5.4)
37 67.3)
35 63.6)
8 (14.5)
10
(18.2)
10
(18.2)
6 (10.9)
6 (10.9)
4 (7.3)
4 (7.3)
4 (7.3)
4 (7.3)
37 67.3)
Fig 1: Duties performed by ASHAs in their

respective areas
Fig 2: Time since last ASHA training received
618
Kohli et al.,
Fig 3: Problems faced by ASHAs in maintaining

registers
Awareness and practices about responsibilities
ASHAs were asked if they were aware of their roles
and responsibilities as shown in Table 2. Most of the
ASHAs workers were aware of their work of
mobilizing children for immunization, providing
counseling for family planning, bringing mothers for
ANC and companying them for hospital for delivery.
However lesser numbers were aware of their role in
distribution of tablet Iron and Folic acid, registration
of births and deaths, and DOTS. Few were aware of
their role to treat minor ailments.
The number of hours a day an ASHA used to work
was 4.8 + 1.4. All ASHAs reported that they were
actively involved in spreading awareness about health
in their areas. Only 7 (12.7%) reported that they acted
as DOTS provider for any patient. Figure 1 shows
duties performed by ASHAs in their respective areas.
18 (32.7%) reported that they always organize
monthly meeting of adolescent girls for promoting
menstrual hygiene. 19 (34.5%) reported that they
always inform about the births and deaths in their
area to the health centre. Only 18 (32.7%) reported
that they always inform about any unusual health
problems/disease outbreaks in their community to the
health centre.
A majority of ASHAs (96.4%) reported that they
maintain family planning register, only 51 (92.7%)
reported that they maintain antenatal register, 54
(98.2%) maintain immunization register, 36 (65.5%)
maintain birth and deaths register and 34 (61.8%)
used to maintain household survey register. Only 40
(72.7%) ASHAs said that they knew about TB
patients in their area.
Figure 2 show that 67.3% ASHAs received basic
training in last 3 months. 1 (1.8%) ASHAs received
training up to 4th module while 12 (21.8%) and 42
(76.4%) received up to 6th and 7th module
respectively. When asked about if they were able to

understand the module, 47 (85.5%) responded they
always understand the module, 5 (9.1%) said that she
sometimes understand the module and 3 (5.5%) said
they never understand the module.
Data was collected about the problems faced by
ASHAs during training. 7 (12.7%) reported that
training sessions were overcrowded. ASHAs were
asked about their perception regarding training
aspects. The responses were collected on four
aspects; training is complete, incomplete, need to be
repeated or too much information is imparted.
Responses are shown in Table 3. Data was collected
from ASHA workers about the duties performed by
them in their respective areas. 18 (32.7%) ASHA
workers reported that they always organize monthly
meeting for adolescent girls for promoting menstrual
hygiene, 6 (10.9%) reported only sometimes and 31
(56.4%) said they never organize any such meetings.
About their responsibility of informing births and
deaths in the area to the health centre, 19 (34.5%)
reported that they always inform the same, 9 (16.4%)
said only sometimes while 27 (49.1%) reported they
never inform. One of the responsibilities of ASHA
workers is to inform about any unusual health
problems/disease outbreaks in the community to the
health centre, 18 (32.7%) ASHAs reported always, 16
(29.1%) sometimes while 21 (38.2%) ASHAs
answered they never report about the same.
Difficulties faced by ASHAs
When asked if they face any problems in maintain
registers, 11 (20.0%) reported that they face
difficulties in the same. 7 (12.7%) said that they find
registers very difficult to understand, 3 (5.5%) said
too much information have to be filled in the registers
while 5 (9.1%) reported that they do not get enough
time for completing registers. The various problems
are shown in Figure 3.
10 (18.2%) ASHAs reported that they face problems
in coordination with Auxiliary Nurse Midwife
(ANMs). Only 30 (54.5%) ASHAs perceived that
community had faith in them. 37 (67.3%) ASHAs
reported that they face barriers while working in the
community. The common barriers faced by ASHA
workers while working in the community were
language problems as reported by 9 (16.3%),
religious and socio cultural barrier (7.3%) and
casteism as reported by 6 (10.9%). Only 11 (20.0%)
reported that they have been given ASHA kit. Out of
619
Kohli et al.,
these 11, only 8 reported that medicines were refilled

regularly in kit.
DISCUSSION
The present study showed that although most of the
ASHAs belong to age group of 25-45 years, 8
(14.5%) were below 25 years of age and 2 (3.6%)
was unmarried, which is contrary to guidelines of
ASHA workers selection. Similarly 3 (5.5%) ASHAs
were serving more than 2000 population which is
more than recommended.5 These figures are less than
reported by Mahyavanshi DK et al where higher
number of ASHA workers were recruited against
selection criteria.[6] The positive findings were good
representation of all caste of community, education
status of ASHAs and the fact that all ASHA belonged
to local community. Most of the ASHAs were serving
a population of 1000-2000 as revealed by other
studies also.[7]
Almost all ASHA workers were aware about their
roles and responsibilities regarding maternal and
child health services. Similar results were showed by
a study conducted by Gosavi SV et al in Wardha
where all ASHAs knew about their role in
immunization and antenatal services.[8] Not all
ASHAs were aware about their responsibility for
birth and death registration in their areas and to act as
DOTS provider. This is evident by the fact that only 7
(12.7%) reported that they acted as DOTS provider
for any patient. This lack in knowledge for content of
responsibility significantly affected their practices in
community. Only 29.1% ASHAs were aware that
they can give treatment for minor ailments in present
study. Similar results were shown by another study
where only twenty three percent ASHAs were aware
that they should also give medical care for minor
ailments.[9] This is an important role that should be
communicated to them well during training. This
shows that training session should focus on all
aspects, not merely focusing on maternal and child
health.
All ASHAs were aware about the incentives they are
entitled to for their work. Only 24 (43.6%) reported
that they receive their incentives on time. These
findings are consistent with results showed by Singh
M et al where payments were delayed for about a
month.[10] All ASHAs reported that they are actively
involved in spreading awareness about health in their
areas. Family planning and immunization register
was maintained by majority of ASHAs but not all
Kohli et al.,
reported maintaining antenatal register, birth and

deaths register and household survey register.
Another study by Garg PK et al also reported similar
findings in which not all ASHA workers were
maintaining records.[11]
Efficiency of training was assessed by asking ASHAs
about their perception regarding training where 5
(9.1%) reported to have understood the module only
sometimes and 3 (5.5%) said they never understand
the module. On almost all the topics of training,
significant percentage of ASHAs thought discussion
was incomplete or needs to be repeated. Similar to
our study findings, inefficient training have been
reported by others authors also where ASHAs were
reported to be unable to retain all the functions and
responsibilities told to them. They were also not
aware of compensation provided under various
schemes.[12] This shows that refresher training should
be integral part of ASHA workers skill building
schedule.
A number of problems were reported by ASHAs in
maintaining registers, coordination with ANMs,
language problems etc while performing their duties.
Lack of coordination among community health
workers is a cause of concern for success of NRHM
which heavily lay upon their role. This incoordination leads to lack of supervision and thereby
de-motivating the workers.[13] It is responsibility of
ANMs to help and guide ASHA workers to maintain
registers and provide supportive supervision.
CONCLUSION
The study found some gaps in knowledge and
practices about the roles and responsibility among
ASHA workers. Not all of them were aware of their
role in areas like antenatal care and child care which
are crucial for success of NRHM. Certain barriers
faced by them were socio cultural, language and
coordination with supervisors.
Recommendations: Guidelines should be followed
strictly in recruitment and selection of ASHA
workers. Present study showed that ASHAs
performance is impacted by their limited orientation
towards their roles and responsibilities. Training
should provide complete knowledge about the same.
Measures should be taken to address the problems
faced by ASHA workers. Limitations: Major
limitation of the study is small sample size. However,
important issues of concern have been pointed out
well.
620
ACKNOWLEDGMENT
The authors are grateful to study participants for their
contribution.
REFERENCES
1. World Health Organization. Health systems
financingthe path to universal coverage.
Geneva: World Health Report, 2010;
2. Gopalan SS, Mohanty S, Das A. Assessing
community health workers performance
motivation: a mixed-methods approach on India's
Accredited Social Health Activists (ASHA)
programme. BMJ Open 2012; 2:001557.
3. Ministry of Health and Family Welfare,
Government of India. National Rural Health
Mission Document. New Delhi, 2005
4. Ministry of Health and Family Welfare,
Government of India. Accredited Social Health
Activist (ASHA) guidelines, National Rural
Health Mission. New Delhi: 2005. Available
from
http://nrhm.gov.in/communiti
sation/
asha/about-asha.html.
5. Kishore J. National rural health mission: in
National health programs of India. 11th ed. New
Delhi: Century publications; 2014; 374.
6. Mahyavanshi DK, Patel MG, Kartha G, Purani
SK, Nagar SS. A cross sectional study of the
knowledge, attitude and practice of ASHA
workers regarding child health (under five years
of age) in Surendranagar district Health line.
2011;2(2):50.
7. Bhatnagar R, Singh K, Bir T, Datta U, Raj S,
Nandan D. An assessment of performance based
incentive system for ASHA Sahyogini in
Udaipur, Rajasthan. Indian J Public Health. 2009;
53(3):166-70.
8. SV Gosavi, AV Raut, PR Deshmukh, AM
Mehendale, BS Garg. ASHAS' Awareness &
Perceptions about their roles & Responsibilities:
A Study from rural Wardha. J Mahatma Gandhi
Ins Med Sci. 2011; 16(1):1-8.
9. Kumar S, Kaushik A, Kansal S. Factors
influencing the work performance of ASHA
under NRHM a cross sectional study from eastern
Uttar Pradesh. Indian J Commun Health 2013;
24(4):325 31.
10. Singh M, Kandpal SD, Negi KS, Shikha D. An

assessment of performance based incentive
system of ASHA in Doiwala block, district
Dehradun. Indian J Prev Soc Med.
2011;42(4):399-02.
11. Garg PK, Bhardwaj A, Singh A, Ahluwalia SK.
An evaluation of ASHA workers awareness and
practice of their responsibilities in rural Haryana.
Natl J Commun Med. 2013; 4(1):76-80.
12. Jain N, Srivastava NK, Khan AM, Dhar N,
Manon S, Adhish V et al. Assessment of
functioning of ASHA under NRHM in Uttar
Pradesh. Health Pop: Perspectives Issues. 2008;
31(2):132-40.
13. Sharma R, Webster P, Bhattacharyya S. Factors
affecting the performance of community health
workers in India: a multi-stakeholder perspective.
Glob Health Action. 2014;7:25352.
621
Kohli et al.,
DOI: 10.5958/2319-5886.2015.00118.6

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
nd
Received: 2 May 2015
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
th
Revised: 10 Jun 2015
SELF WOUND MANAGEMENT PRACTICES BEFORE ATTENDING ANTIRABIES VACCINE

CLINIC
* Amit Kumar Mishra1, Smita Panda2, Prakash Chandra Panda3
1
Assistant Professor, Department of Community Medicine, Pondicherry Institute of Medical Sciences,

Pondicherry
2
Associate Professor, Department of Community Medicine, 3Associate Professor, Department of Pediatrics
V.S.S.M.C.H, Burla
*Corresponding author email: dramitvss@gmail.com
ABSTRACT
Introduction: In INDIA almost 20000 people die (40% of world death) each year from rabies. Most of these
deaths could be prevented by post exposure prophylaxis with wound washing, rabies immunoglobulin &
vaccination. Local wound management alone can reduce viral load by up to 80%. Objective: To study selfwound management practices in animal exposure patients before attending a tertiary level ARV clinic.
Methodology: Data regarding wound management was collected by individual interview of patients attending the
ARV clinic during OCT 2011 to MAR 2012. The data collected in the form of a questionnaire. Analysis of data
was done in the Department Of Community Medicine, V.S.S. Medical College, Burla. Results: Total 493 cases of
animal exposure were attended during the study period. Most common biting animal was dog (94.5%). 31% of
cases were under the age of 10 years & 23% belongs to the age of 10-19 years. Male to female ratio was 3:1. Most
of the cases (91%) were of category III exposure. Immediate management of wound was practiced by 63-77% of
cases before visiting ARV clinic; only 2% wash the wound with running water & soap for 15 minutes. 39% of
cases applied Dettol/savlon at the wound side & other 38% applied turmeric, red chilli, kerosene, Band-Aid &
ghee locally. Most cases (61%) reported to ARV clinic within 24hours.
Keywords: Animal Exposure, Post Exposure Prophylaxis, Wound Management, Self-Management Practices.
INTRODUCTION
Worldwide each year 50 000 people die from rabies,
with India carrying the greatest burden of more than
20 000 deaths (40% of world death) each year from
rabies. [1] Deaths are due to improper wound
management & delay in reporting in clinics. Most of
the patients first opt for available local remedies on
the wounds before reporting which became fatal for
them. Improper wound management helps the rabies
virus to grow & penetrate the nerves. Deaths could be
prevented by Post Exposure Prophylaxis which
includes wound management, vaccination &
immunoglobulin. Local wound treatment alone can
reduce the chances of developing rabies by up to 80%

by reducing viral load at local sites. 40% of people
who are bitten by suspect rabid animals are children
under 15 years of age. [2] On this background the
above study was conducted in an Antirabies Vaccine
(A.R.V.) Clinic of a tertiary hospital to assess the
self-wound management practices in animal exposure
patients before attending the clinic for treatment.
Amit et al.,

Study Design: A cross sectional study
622
RESULTS
During the study a total of 493 patients were included
in the research. Out of the 493, 368 (74.6%) patients
were male; with the male to female ratio of 2.9:1.
Most of the subjects (153, 31%) were in the age
group 0 9 years followed by 10 19 years age
group (113, 23%).
Table 1: Distribution of subjects as per the animal
exposure
Animal Exposure
No. of Patients
Percentage
Dog
466
94.5
Cat
Monkey & Others
Total
15
12
493
3.0
2.5
100
Table 2: Distribution of subjects as per the

category of exposure
Category of
No. of
Percentage
[3]
Exposure
Patients
Category I
11
2.2
Category II
37
7.5
Category III
445
90.3
Total
493
100
Out of the 493 cases attended the ARV clinic for
treatment, 90.3% (445) subjects were of Category III
exposure to different animals and they took both the
vaccine and immunoglobulin with the wound
dressing as the treatments. Only 37 cases were of
category II exposure. Dog was the most common
animal that causes the injury among 94.5% (466) of
subjects followed by Cat (3%).
Table 3: Immediate Pre-treatment after Animal
Exposure
Self-Treatment Before
No. of
Percentage
Attending the Clinic
Patients
Not washed
183
37
Washed with water
167
34
Washed with running

89
18
water without soap
Washed with running
water and soap for
44
09
< 15min
Washed with running
water and soap for
10
02
> 15 min
In this research 37% of the subjects not washed their
wounds irrespective of the duration of exposure to the
animal. Only 2% of them washed their wound as per
the guidelines by using a running water source with
soap.
113
(23%)
NO
Local Application
Study Duration & Place: Study was carried out

from October 2011 to Mar 2012 in A.R.V. Clinic of
V.S.S. Medical College, Burla.
Inclusion Criteria: All patients exposed to animal
bites and attending the A.R.V. Clinic of V.S.S.
Medical College, Burla were included in the study. In
case of paediatric patientsattending the A.R.V. Clinic
for vaccination, the data were collected from the
accompanying person.
Exclusion criteria: Patients, who were not willing to
participate, were excluded from the study.
Ethical clearances for the study was collected from
the Ethical committee of the medical college and
consent to participate in the study were collected
from each study individuals before collection of data.
Sample size: All the animal exposure cases reported
within the study period and willing to participate in
the study were included in the study. A total of 493
cases were included in the study within the study time
period.
Methodology: A pre-designed and pre-tested
structured questionnaire was used for collection of
information from cases of animal exposure. In case of
paediatric cases exposed to animal bites and reported
in the clinic for vaccination, the questionnaire were
asked to the accompanying person and data were
recorded for analysis. The questionnaire was prepared
to collect information regarding socio-demography,
type of animal exposure, cause of bite (provoked or
unprovoked), history of previous exposure, any selfmanagement practice before attending the ARV
clinic. Collected data were analysed in SPSS V.16
software.
17 (3.4%)
13 (2.6%)
GHEE
RED CHILLI
49 (10%)
33 (6.7%)
76
(15.3%)
BAND AID
KEROSENE
TURMERIC
192
(39%)
DETTOL/
0
100
200
Number of Patients
Fig 1: Distribution of cases as per types of local

application before reporting to clinic
623
Amit et al.,
Out of the 493 subjects, 39% applied local

antiseptics, 38% applied local remedies like turmeric,
red chilli, kerosene, ghee etc. in their wound before
coming for treatment. 23% of subjects came to the
ARV clinic without any application in their wounds.
16%
19%
Same day
3rd day
4%
61%
Next day
3rd day onwards
Fig 2: Interval between animal exposure and

reporting at A. R. V. Clinic
In the present study 61% of the subjects reported to
the clinic for treatment on the same day and 19% of
the subjects reported to the clinic after 3rd day of
exposure irrespective of the type and site of animal
bites.
DISCUSSION
In the present study majority of the cases were
between the age group 0-9 years followed by 10-19
years. This finding is similar to that reported by Vyas
et al,[4] Bedi et al,[5] Williams et al, [6]Sharma et
al,[7]Hanspal et al,[8]Shetty et al [9]where the maximum
number of cases were <15 years. The above finding is
contradictory to the findings of TR Behera et al [10]
where most of the cases were in the productive age
group > 18 years.
Out of the 493, 74.6% (368) cases were male. In the
present study the male to female ratio is 2.9:1. A
study by Vyas et al [4] reported the male to female
ratio of 3:1 and Shetty et al [9] reported the ratio of
1.98:1 in a study at Pune. The higher male proportion
in this study corroborates with other studies by
Hanspal et al [8] at Jamnagar, Sudarshan et al [11] in
Bangalore & Khokhar et al [12] in Delhi.
In the present study, dog was found to be the most
common biting animal as 94.5% cases were bitten by
them. This is similar to findings of Vyas et al,[4]
Williams et al, [6]Sharma et al, [7]and Shetty et al.[9]In
others studies like Bedi et al,[5] Shetty et al[9]&Behera
et al,[10]from different parts of India also had reported
dog bite as the most common animal exposure among
the cases reported to ARV clinics. Majority of cases

(90.3%) in the present study had category III bites
and this finding is similar to the study findings by
Vyas et al,[4] Bedi et al,[5] & Behera et al.[10] Only
2.2% cases belonged to Category I indicating that
awareness of community about Category I exposure
is poor.
Out of 493 cases reported to ARV clinic, 34% of
cases washed their wound before coming to the clinic
but only 2% of cases washed their wound as per the
guidelines with soap and running water for more than
15 minutes. In the study by Sharma et al [7] only
23.5% cases were washed their wound with soap and
water.
Immediate pre-treatment at the wound site was done
by 380 (77%) of the cases. Out of the 380 cases who
had applied something at the site of bite, highest i.e.
39% had applied Dettol/Savlon followed by 15.3%
who applied turmeric paste at the site. Vyas et al [4]in
their study reported that 72% cases applied some
local treatment in their wounds before coming to
ARV clinic. In the present study only 23% of cases
did not apply anything on the wound before seeking
the treatment. Sharma et al [7] in his study reported
that 44.3% of reported cases did not apply anything
on the wound before coming to ARV clinic.
Majority of cases (61%) reported to the clinic on the
same day and received treatment including antirabies
vaccine & immunoglobulin whenever required.
Almost all cases (96%) reported within first three
days after animal bite with highest percentage 61%
reporting on the same day. In a study by Vyas et al
[4]
22.5% of cases reported on the same day and
maximum 42.5% reported on second day. Sharma et
al [7] reported that majority of cases of animal bite did
not report immediately to heath centre for treatment
after dog bite. Sharma et al [7] & Shetty et al [9]
reported that maximum number of cases reported
within 24 hours of the animal bite. Behera et al [10]
reported that in their study maximum subjects
reported to health centre within 24 to 48 hours of
animal bite.
CONCLUSION
In this study, majority of the animal exposure cases
were of Category III exposure and only few cases
were practiced the proper method of wound washing
and on time reporting. The application of different
materials on wound before attending the clinic for
624
Amit et al.,
treatment and the higher proportion of late reporting

suggests that there is lack of awareness among people
regarding the fatality of rabies.
Suggestions: As most of the people are practicing
local treatment before attending health centre for
treatment, this signifies that the population are not
aware of the risk of rabies and what to do in case of
an animal bite. Health educational programs are
needed to create awareness among people regarding
the benefits of proper wound washing and the dangers
of inadequately managed animal bite wounds.
Acknowledgement: Authors would like to thank all
the participants who consented to participate in the
study.
Conflict of Interest: None declared
REFERENCES
9.
10.
11.
12.
Rabies Clinic of GGS Hospital, Jamnagar

(Gujarat); APCRI Journal 2007;8 (2).16-18.
Shetty R A, Chaturvedi S, Singh Z, Profile of
animal bite cases in Pune. J Commun Dis. 2005;
37(1):66-72.
BeheraTR, SatapathyD M, Tripathy RM, Sahu A.
Profile of animal bite cases attending the ARC of
M.K.C.G. Medical College, Berhampur (Orissa).
APCRI journal 2008;9(2):56-9.
Sudarshan MK, Mahendra BJ, Narayan DH. A
Community survey of dog bites, anti rabies
treatment, rabies & dog population management
in Bangalore city J Commun Dis, 2001 33 (4):
245-51.
Khokhar A, Meena GS, Mehra M. Profile of dog
bite cases attending M.C.D. Dispensary at Alipur,
Delhi.
Indian
Journal
of
Community
Medicine.2003; 28 (4):157-9
1. Voelker R. Global effort takes aim at rabies.

JAMA, 2007; 298: 1749-50.
2. Rabies. Fact sheets. WHO 2012: http://
www.who.int/mediacentre/factsheets/fs099/en
(Accessed on 21 Apr 2012)
3. Rabies. Media centre: WHO. http://www.
who.int/mediacentre/factsheets/fs099/en/
(Accessed 25 Sep 2011)
4. Vyas S, Gupta K, Bhatt G, Tiwari H. Animal bite
management practices: Study at three municipal
corporation hospitals of Ahmedabad. National
Journal of Community Medicine. 2010:1(2):75-8.
5. Bedi R, Bedi DK, Tankha A, Choudhary V,
Matoria RS. Profile of animal bite cases attending
Anti Rabies Clinic of JLN Medical College &
Hospital, Ajmer. APCRI Journal. 2006:
8(1):2830.
6. Williams RF, Logaraj M. A Community based
Study on Dog Care, Dog Bite and AntiRabiesTreatment in a Selected Rural Block in
Tamil Nadu. Indian Journal of Rural health care.
2012:1(2):147-50.
7. Sharma A L, Bhuvar P A, Bhavalkar J S, Pawar
SN. Profile of management of animal bite cases
among rural population in district Pune,
Maharashtra. Indian J Public Health. 2007;
51(1):62-3.
8. Hanspal JS, Bhanderi D, Nagar S. A review of
attendance trend of animal bite cases in the Anti
625
Amit et al.,
DOI: 10.5958/2319-5886.2015.00119.8

&
Health Sciences
www.ijmrhs.com
Copyright @2015
th
th
Revised: 12 June 2015
Research article
ISSN: 2319-5886
Accepted: 23rd June 2015
EFFECT OF EXERCISE TRAINING ON C-REACTIVE PROTEIN LEVELS: A FOLLOW UP STUDY

*Rathod Shivanand S1, Sagdeo Mohan M2, Date Anjali A2, Nagose Vaishali B3, Ankur1, Praveen Kumar
Kodumuri1
1
Department of Physiology, 3Department of Pathology, Mamata Medical College, Khammam, Telangana

2
Department of Physiology, NKP Salve Institute of medical sciences and research centre, Nagpur
*Corresponding author email: shivanandrathod@gmail.com
ABSTRACT
Context: It is widely believed that physical fit individuals are at lower risk of developing coronary disease.
Physical activity may exert some of its beneficial effect by reducing levels of inflammation. C-reactive protein
(CRP), an inflammatory marker, is believed to be a mediator of atherogenesis & predictor of cardiovascular
disease. Exercise training is suggested to reduce the levels of CRP levels. We evaluated effect of exercise
training on levels of CRP by measuring and comparing these levels at regular intervals throughout the length of
study. Materials and Methods: 40 male students of Physical education aged between 18 to 25 years, were
followed up for the period of nine months with CRP levels being measured at the interval of every 3 months. The
subjects underwent regular planned physical training for about 3 hours daily (1 hours in the morning and 1
hours in the evening with resting period according to individual needs), five days a week. Results: CRP levels
significantly decreased from 3.73 mg/l to 3.11mg/l at the end of first 3 months. These levels further decreased to
2.72 mg/l at 6 months and 2.10 mg/l at 9 months (P<0.0001).Conclusion: The present study suggests that with
increase in the duration of exercise training period, CRP levels reduce significantly.
Keywords: C-reactive protein, Exercise, Inflammation, Atherosclerosis
INTRODUCTION
Regular physical exercise reduces the risk of agerelated diseases. Epidemiologic evidence suggest that
chronic diseases, such as heart disease and diabetes
type 2 can be prevented with regular physical
exercise.[1] It has also been shown that inflammatory
factors like C-reactive protein(CRP) is an important
risk factor for atherosclerosis & cardiovascular
disease events.[2,3,4] Elevated plasma levels of CRP
have been associated with an increased risk of
coronary heart disease, while regular physical
exercise plays an important role in lowering CRP and
in turn decreasing the risk of coronary heart
disease.[2,5]Data
from
epidemiological
and
longitudinal studies suggest that increasing physical
activity is an effective means of lowering systemic
levels of inflammation [6,7] and this anti-inflammatory

effect of physically active life style is irrespective of
age.8 Some studies implicate that high initial CRP
levels in sedentary healthy adults are also reduced in
response to exercise training. [6,7,8] On an average,
high levels of CRP are seen in individuals who
smoke, have high blood pressure, are overweight, and
fail to exercise. [9]
Earlier study by Laka TA et al. has shown the
beneficial effect of exercise in lowering CRP levels
after 20 weeks of follow up. A cross sectional study
by Kasapis C et al. reported inverse relationship
between physical activity and CRP levels.[5] The
present follow up study was undertaken to assess the
effect of 9 months exercise training on CRP levels. In
626
Shivanand et al.,
this study, with the first reading taken as baseline,

CRP is compared every 3 months for the period of 9
months duration of exercise training
The present study is Longitudinal; constituting 40
newly admitted first year male students of physical
education. Before starting the study work, permission
was obtained from institutional ethics committee,
NKP Salve Institute of medical sciences and research
centre, Nagpur. All participants were given detailed
information about the study and informed consent
was obtained.
All the participants lie in age group between 18 - 25
years and were not previously involved in regular
physical exercise. They were willing to start regular
physical activity and continue it at least for the period
of 9 months and follow up of 9 months. Subjects with
history of asthma and/or autoimmune disease, those
on steroids therapy/ immunosuppressive therapy and
those with chronic diseases like diabetes,
hypertension were excluded.
Exercise pattern: All the subjects were doing a
regular planned exercise, about 3 hours daily, five
days a week, about 1 hours in the morning and 1
hours in the evening. The type of exercise was
aerobic with a daily schedule. Enough time was given
for the students to adapt in initial 3 weeks. During
this period intensity of training was increased
gradually. .
1. First week
10 minutes: warm up
15 minutes: jogging
10 minutes: stretching
2. Second week
15 minutes: warm up
15 minutes for jogging and running each
3. Third week
15 minutes: warm up
30 minutes for jogging and 15 running.
The general scheme for exercise after 4th week, in the
morning as well as in the evening, was as
follows:
15 minutes: Warm up exercises
30 minutes each for running and jogging with

resting period of one hour, divided according to
individual needs.
Registration of subject for the study was followed by
the detailed recording of medical history, personal
history and family history. Also a detailed exercise
history, other than mentioned above, if any, including
place, daily hours, daily schedule and type of exercise
and other exercise related activities like participation
in other sports were obtained.
At the time of admission these students were not
involved in exercise training, at this point of time the
measured parameter (CRP) was considered as
baseline measurement (B). Then these students were
followed up for the period of 9 months with CRP
being measured at the interval of 3 months; i.e. at 3
months (3M), 6 months (6M) and 9 months (9M)
after commencement of exercise training period.
Under all strict aseptic precautions, blood sample was
collected from each subject and CRP was assessed
with the help of quantitative turbidimetric test by
using C-reactive protein Turbilatex kit (Euro
Diagnostics Pvt. Ltd., Chennai.)
RESULTS
All the observations were compared by using one
way analysis of variance with repeated measures (one
way ANOVA with repeated measures, Tukeys
Multiple Comparison Test and unpaired t-test) with
the help of software Graph Pad Prism.
Table 1: showing levels of C-reactive protein at B,
3M, 6M and 9M.
Basal
3M
6M
C-reactive 3.7 0.75 3.1 0.78 2.70.67

Protein
(mg/l)
Significance HS ( P<0.0001)
9M
2.1 0.67
Table 1 shows the mean C-reactive protein (CRP)

levels before (B), 3 months (3M), 6 months (6M) and
9 months (9M) of exercise training period. From the
above table it is evident that the levels of CRP
reduced significantly with increase in the duration of
exercise training period with high statistical
significance. The observations were also compared
between all the groups. The results are as follows
627
Shivanand et al.,
Table 2: showing comparison of CRP between all

the groups.
Tukeys Multiple
Comparison
Test
B vs 3M
B vs 6M
B vs 9M
3M vs 6M
3M vs 9M
6M vs 9M
Mean
Diff.
Significant? 95% CI
P < 0.05? of diff
0.6215
1.005
1.625
0.3833
1.004
0.6203
Yes
Yes
Yes
Yes
Yes
Yes
0.5063- 0.7367
0.8895-1.120
1.510-1.740
0.2680- 0.4985
0.8883- 1.119
0.5050- 0.7355
Table 3: comparison of CRP between all the

groups with p value
Unpaired t test
P value
B vs 3M
B vs 6M
B vs 9M
3M vs 6M
3M vs 9M
6M vs 9M
0.0006
P<0.0001
P<0.0001
0.0212
P<0.0001
P<0.0001
From table 2 and 3, and graph 1 it can be seen that

after comparing the CRP levels between all the
groups, the levels of CRP decreased in response to
increase in the duration of exercise training period.
The results were found to be statistically significant.
5
4
3
2
1
on
th
s
m
9
on
th
s
m
6
m
3
on
th
s
on
th
s
C-reactive protein (mg/l)
Repeated measures one-way ANOVA data
Duration Of physical training
Fig1: Comparison of CRP levels between all

groups with standard error of mean
DISCUSSION
In this 9 month long study we assessed the effect of
physical training on CRP; a complex protein that our
bodies produce when exposed to infection or trauma,
also an important marker of inflammation.[ 9] Amount
of CRP produced depends on many different factors.
Higher levels of CRP are observed in smokers,
hypertensive subjects, overweight individuals and

people with sedentary lifestyle. [9] In our study we
observed that the levels of CRP decreased in response
to exercise training.
Present study is important from public health point of
view. Large number of data suggest that individuals
with high levels of inflammation are at increased risk
of cardiovascular and metabolic diseases.1, 2, 3, 4
Measurements of inflammatory markers like CRP,
serum amyloid A and interleukin-6(IL-6) are highly
predictive of future risk of atherosclerosis and
cardiovascular disease events, stroke and the
development of peripheral arterial disease.9, 10 This
possible inflammation lowering effect of exercise
training can explain the benefits of regular physical
exercise for preventing and treating cardiovascular
and metabolic diseases.
The important aspect of this study is regular follow
up of participants for 9 months and comparing the
measurements at every 3 months interval. CRP levels
started to decrease as early as at first 3 months. These
levels further reduced at 6 month and 9 month
interval, suggesting that, regularity as well as
duration of physical training plays important role in
reducing CRP levels.
How exercise training reduces inflammation and
suppresses CRP levels is not well defined. Some
studies suggest that adipocytes produce the
inflammatory cytokines (IL-6, interleukin-1 (IL-1) &
Tumour necrosis factor- (TNF- ) and these
cytokines lead to increased production of CRP by
liver.[10, 11, 12] Thus higher levels of CRP are
associated with adiposity caused by sedentary
lifestyle. Long term physical exercise attenuates the
production of atherogenic cytokines from monocytes
and leucocytes and ultimately reduces the CRP
production from liver. [13]
Leptin, a hormone secreted by adipocytes, is also
believed to increase the levels of CRP. [14] Physically
active individuals have less adipocytes, so less
secretion of leptin and this may lead to decreased
stimulation of hepatic CRP production. [15]
Romano M et al. suggested that oxidant injury causes
vascular endothelial cell inflammation by increasing
production of cytokines and elevating CRP
production in liver; [16] physical activity is believed to
counteract this effect by up regulating antioxidant
enzymes and increasing defence against oxidant
injury, leading to reduction in the production of
628
Shivanand et al.,
cytokines and CRP. [17] However, exact mechanism

by which increase in physical activity reduces CRP
and inflammation is not clear. Further studies are
needed to explore these mechanisms.
8.
CONCLUSION
Finding in our study suggest that, regular physical
exercise has a possible anti-inflammatory effect as
the levels of CRP decreased in response to exercise
training. This decrease in the levels of CRP is found
to be proportional to the duration of exercise training
period.
Along with this future studies are needed to see
whether these beneficial effects of physical activity
on serum levels of CRP remain or revert back
completely or partially to their previous levels after
stopping the exercise or after a significant gap.
Conflict of Interest: Authors declare no conflict of
interest.
9.
10.
11.
12.
REFERENCES
1. Woods JA, Vieira VJ, Keylock KT. Exercise,
Inflammation, and Innate Immunity. Neurol Clin
2006;24 58599.
2. Kondo N, Nomura M, NakayaY, Ito S, Ohguro T;
Association of Inflammatory Marker and Highly
Sensitive C-Reactive Protein With Aerobic Exercise
Capacity, Maximum Oxygen Uptake and Insulin
Resistance in Healthy Middle-Aged Volunteers.
Circ J 2005; 69: 452 57.
3. Ross R. Atherosclerosisan inflammatory disease.
N Engl J Med 1999; 340: 11526.
4. Pearson TA, Mensah GA, Alexander RW,
Anderson JL, Cannon RO, Criqui M et al. Markers
of inflammation and cardiovascular disease:
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Centers for Disease Control and Prevention and the
American
Heart
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2003;107:499 11.
5. Kasapis C, Thompson PD; The Effects of Physical
Activity on Serum C-Reactive Protein and
Inflammatory Markers; JACC 2005; 45(10): 1563
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6. Michael Gleeson. Immune function in sport and
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7. Lakka TA, Lakka HM, Rankinen T, Leon AS, Rao
DC, Skinner JS et al; Effect of exercise training on
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17.
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Activity Status, But Not Age, Influences
Inflammatory Biomarkers & Toll-Like Receptors.
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Paul M Ridker, C-Reactive Protein A Simple Test
to Help Predict Risk of Heart Attack and Stroke.
Circulation 2003; 108;81-85.
Ridker PM, Hennekens CH, Buring JE, Rifai N; C reactive protein And Other Markers Of
Inflammation In The Prediction Of Cardiovascular
Disease In Women. N. Engl. J Med 2000; 336:342.
Nicklas BJ, You T, Pahor M; Behavioural
treatments for chronic systemic inflammation:
effects of dietary weight loss and exercise training.
CMAJ 2005; 172 (9):1199-09.
Yudkin JS, Stehouwer CD, Emeis JJ, Coppack SW.
C-reactive protein in healthy subjects: association
with obesity, insulin resistance and endothelial
dysfunction: a potential role for cytokines
originating from adipose tissue? Arterioscler
Thromb Vasc Biol 1999; 19:972-78.
Smith JK, Dykes R, Douglas JE, Krishnaswamy G,
Berk S. Long term exercise and atherogenic activity
of blood mononuclear cells in person at risk of
developing ischemic heart disease JAMA
1999;281:1722- 27.
Shamsuzzaman AS, Winnicki M, Wolk R,
Svatikova A, Phillips BG, Davison DE et al.
independent association between plasma leptin and
C-Reactive protein in healthy humans; Circulation
2004; 109:2181-85.
Tomaszewski M, Charchar FJ, Przybycin M,
Crawford L, Wallace AM, Gosek K et al Strikingly
Low Circulating CRP Concentrations in
Ultramarathon Runners Independent of Markers of
Adiposity How Low Can You Go? Arterioscler
Thromb Vasc Biol 2003; 23; 1640-44.
Romano M, Sironi M, Toniatti C, Polentarutti N,
Fruscella P, Ghezzi P et al. Role of IL-6 and its
soluble receptors in induction of chemokines and
leucocyte recruitment Immunity 1997;6:315-25.
Taddei S, Galetta F, Virdis A, Ghiadoni L, Salvetti
G, Franzoni F, et al. Physical activity prevents age
related impairement in nitric oxide availability in
elderly athletes; Circulation 2000; 101:2896-01.
629
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DOI: 10.5958/2319-5886.2015.00120.4

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
th
Research article
Coden: IJMRHS
Revised: 5th Jun 2015
Copyright @2015
ISSN: 2319-5886
ASSOCIATION OF VISCERAL FAT WITH DETERIORATED PULMONARY FUNCTION IN NEWLY

DIAGNOSED HYPOTHYROID PATIENTS
*Sudhir Modala1, Usha Dhar2, K V Thimmaraju3, B J Pradeep Kumar4, Bandi Hari Krishna5
1,
Ph.D, scholar, Department of Physiology, Santosh medical college, Ghaziabad, UP, India
Professor & Head, Department of Physiology, Santosh medical college, Ghaziabad, UP, India
3
Professor & Head, Department of Biochemistry Rohilkhand Medical College, Bareily, UP, India
4
Ph.D, scholar, Department of Biochemistry, Santosh medical college, Ghaziabad, UP, India
5
Assistant Professor, Dept of Physiology, Narayana Medical College, Nellore, Andhra Pradesh, India.
2
*Corresponding author email: sudhirmodala@gmail.com.

ABSTRACT
Objectives: To assess the pulmonary function and its association with visceral fat in newly diagnosed
hypothyroid patients. Materials & methods: The study group subjects were 37 females and the control group
subjects (n=37) were age and gender matched healthy volunteers. Pulmonary functions were assessed by
computerized spirometer. The readings for Forced vital capacity (FVC), Forced expiratory volume in the
first second (FEV1), Forced expiratory volume percent (FEV1 /FVC%) and Peak expiratory flow (PEF) were
noted for participants in both the groups. Body fat assessment was done with Omron HBF 375, a body fat
analyzer. Results: The baseline parameters like mean age and height for each group are comparable and there
was a significant difference in weight and BMI between the groups (p = 0.000). The pulmonary function test
parameters were significantly less in hypothyroid patients when compared to controls (p = 0.000). Further,
association between visceral fat pulmonary function test parameters showed negative correlation for (FVC (L): r =
- 0.888; p = 0.000), (FEV1 (L): - 0.811; p = 0.000) and (FEV1/FVC (%): r = 0.430; p = 0.008). Conclusion: It
is concluded that deteriorated pulmonary function in hypothyroidism is associated with increased visceral fat.
Therapeutic interventions like diet, exercise, yoga to reduce visceral fat should be incorporated as part of
treatment to improve the pulmonary function.
Keywords: Body fat distribution, Thyroid hormones, Visceral fat, Pulmonary function
INTRODUCTION
Hypothyroidism is a clinical state resulting from
decreased secretion of thyroid hormone from thyroid
gland due to functional or structural impairment of
production of thyroid hormone and affects most the
organ systems. Major clinical findings are weakness,
fatigue, coarseness and dryness of the skin,
intolerance to cold, poor concentration and memory,
weight gain, constipation, paraesthesis, menorrhagia,
disorders of hearing, bradycardia, delayed relaxation
of tendon reflexes etc., [1] Most of these signs and

symptoms may recover after thyroid hormone
replacement therapy [2]. Earlier reports showed that
both myxedema and hypothyroid states induces
depression of hypoxic ventilatory drive that is
responsive to thyroid hormone replacement therapy.
This alteration of ventilatory control may contribute
to the hypoventilation seen in myxedema and
hypothyroidism [3] Hypothyroidism affects respiratory
630
Sudhir et al.,
muscle strength which is linearly related to the

thyroid hormone levels. Respiratory muscle weakness
is present in both Inspiratory and expiratory muscles
and is reversible with treatment [4] . Studies showed
that sleep apnea episodes are common in patients
with untreated hypothyroidism even with normal
respiratory function. Thyroxin replacement therapy
decreases apnea frequency, even without change in
body weight [5].
However the information about deteriorated
pulmonary function in newly diagnosed hypothyroid
patients is scanty. Further the association of visceral
fat levels with deteriorated pulmonary function was
not studied in detail.
Therefore, the present study was designed to
determine the pulmonary function and its association
with visceral fat in newly diagnosed hypothyroid
patients.
MATERIALS & METHODS
Study design
The study was approved by Institute Ethics
Committee. This study was carried out in the
Department of Physiology in our college.
Inclusion criteria: The study group subjects were 37
females between 25 30 years. The control group
subjects (n=37) were age, gender and BMI matched
healthy volunteers. Patients with serum T4<5.53
g/dl and
serum
TSH>4.68
mIU/L
by
[5]
Chemiluminiscence technique
were taken as
hypothyroid.
Exclusion criteria: Participants with any systemic
disease or with any organ system dysfunction were
excluded.
Sample size: N=74 (Control n=37, Test group n=37)
Methodology:
Pulmonary functions were assessed by using
computerized spirometer (Spirowin Version 2.0 of
Genesis Medical systems pvt. Ltd) which gives ERS93 predicted values at BTPS conditions. The test
procedure was explained to the participants and a
demonstration of the test procedure was given. The
participants were allowed to sit quietly for 10 minutes
to become mentally and physically relaxed prior to
testing. Participants were asked to inspire as much as
possible and hold the sterile mouth piece in the mouth
with the lips forming a tight seal around the mouth
piece and expire rapidly and forcefully through the
mouth piece.
After preliminary trials, the test was performed three

times and the best recording was taken. The readings
for Forced Vital Capacity (FVC), Forced Expiratory
Volume in the first second (FEV1), Ratio between
FEV1/ FEVC%, Peak Expiratory Flow (PEF) were
noted for participants in both the groups.
Assessment of Total body fat % (TBF %), Sub
Cutaneous Fat (SCF %), Visceral Fat (VF%) was
done by Omron HBF 375, a body fat analyzer
working under principle of bio impedance analysis
(BIA) method[6].
Statistical analysis: Data were expressed as mean +
SD. Unpaired t test was used to compare between
group parameters and Pearsons correlation was used
to study the association between visceral fat and
pulmonary function parameters.
RESULTS
Seventy four participants were included in this study:
37 newly diagnosed hypothyroid patients and 37
apparently healthy individuals. The baseline
parameters like mean age and height for each group
are comparable and are summarized in Table 1. and it
shows the significant difference in weight and BMI
between the groups (p = 0.000).
Table 2. Shows the between group differences of
body fat distribution. As shown in Table 3. The
pulmonary function test parameters were significantly
less in hypothyroid patients when compared to
controls (p = 0.000).
Analysis of data for association between visceral fat
pulmonary function test parameters showed negative
correlation for (FVC (L): r = - 0.888; p = 0.000),
(FEV1 (L): r = - 0.811; p = 0.000) and (FEV1/FVC
(%): r = 0.430; p = 0.008).
Table 1: Baseline characteristics of control and
hypothyroid subjects
Parameter Control
Hypothyroid
P
group
group
value
Age (years) 30.30 + 29.43 + 4.41
0.360
3.61
Height
158.43 + 159.76 + 5.62
0.308
(cms)
5.47
Weight (kg) 57.59 + 68.03 + 6.53
0.000
3.47
BMI
22.98 + 26.70 + 2.79
0.000
2
(Kg/m )
1.58
Values are expressed as mean + SD.
631
Sudhir et al.,
Table 2: Body fat distribution of control and

hypothyroid subjects.
Parameter
TBF %
SCF %
VF %
Control
group
(n=37)
25.63
+
2.92
18.32
+
3.36
6.85 + 1.63
Hypothyroid
group
(n=37)
28.02 + 3.98
P
value
20.53 + 5.40
0.038
10.65 + 3.89
0.000
0.004

Table 3: Pulmonary function test parameters in
control and hypothyroid groups
Parameter
FVC (L)
FEV1 (L)
FEV1/FVC (%)
PEF (L/sec)
Control
group(n=37)
2.85+ 1.64
2.34+ 0.34
82.26+ 11.16
5.19+ 1.00
Hypothyroid
group (n=37)
2.29 + 0.36
1.37 + 0.34
59.21 + 8.78
3.02 + 0.72
P
value
0.000
0.000
0.000
0.000

Table 4: Pulmonary function test parameters and
its association with visceral fat in hypothyroid
patients
Visceral fat %
Parameter
FVC (L)
FEV1 (L)
FEV1/FVC (%)
Hypothyroid group
r value
p value
- 0.888
- 0.811
- 0.430
0.000
0.000
0.008
DISCUSSION
The key findings of this study were that in newly
diagnosed hypothyroid patients, the pulmonary
functions were deteriorated. Further, this deteriorated
pulmonary function was correlated with visceral fat.
Earlier studies reported a significant reduction in
FVC, FEV1, FEV1/FVC % in hypothyroidism when
compared to control subjects [7].
However, the information about pulmonary function
in newly diagnosed hypothyroid patients and its
association with visceral fat is scanty. This study is
distinct that, we reported the pulmonary function in
newly diagnosed hypothyroid patients and it
association with visceral fat. The components of
respiratory system (respiratory centre, upper airway
and lower respiratory system) can be affected by
deficiency in body hormones as well as excess
hormonal secretion [8,9] . Both expiratory and
Inspiratory respiratory muscles are weakened in
hypothyroidism in association with the thyroid

hormone levels and it may be reversible with
thyroxine therapy[4]. Furthermore, thyroid deficient
muscles have impaired free fatty acid utilisation,
which enhances their glycogen consumption, thereby
reducing skeletal muscle endurance. One of the
major Inspiratory muscles that are involved in
hypothyroidism is the diaphragm. Diaphragm
weakness can be very severe and associated with
hypoventilation [10,11]. In this study, we found that
the values for FVC and FEV 1 were significantly
lower in recently diagnosed hypothyroids as
compared to controls. These findings are supported
by studies by other researchers [6,7]
The changes observed in our spirometric findings
can be explained on the basis of earlier reports by
other researchers which suggests that depression of
respiratory centre, interference of neuro muscular
junction and nerve conduction to the muscles of
respiration and respiratory muscles diseases in
hypothyroidism
may
leads
to
alveolar
hypoventilation which in turn affect central
ventilatory control and impairs ventilation[12]. In
addition, in hypothyroid state, reduction of
surfactant, surfactant phospholipid, phosphatidyl
glycerol and phosphatidic
acid along with
increase in surface active lipids phosphatidylserine
and phosphatidylinositol in alveolar epithelium may
decrease alveolar septation and decreased lung
compliance and absorption of adsorption. [12,13]
Moreover, deposition of mucopolysaccharides in the
lungs leads to fibrosis and alveolar wall thickening
with loss of elastic tissue and may increase the work
of breathing.
All above modifications reduces
ventilatory lung functions [14,15] . Infections of
respiratory system
are
very
common
in
hypothyroidism than healthy individuals which
may be a reason for low PFT parameters[16].
Further, in this study the association between visceral
fat and deteriorated pulmonary function can be
explained as that patients with visceral obesity had
lower ERV than patients with subcutaneous obesity
and same body mass index levels. The reduction of
ERV was associated with a reduction of the arterial
oxygen tension[17] and the decrease of ERV and FVC
associated with
abdominal obesity has been
attributed to a mechanical effect played by
accumulation of visceral fat. The raised intraabdominal pressure observed in visceral obesity is
632
Sudhir et al.,
able to pump upwards the diaphragmatic muscle,

parenchymal compression, particularly at the basal
regions. Moreover, the over-stretching of the
diaphragmatic muscle fibres due to the elevation of
the diaphragmatic domes produced by visceral fat can
decrease the contractile efficiency of the
diaphragmatic muscle [18]. Therefore it is possible that
the improvement of pulmonary function in
hypothyroid patients can be achieved by reducing the
visceral fat. Limitations: Further studies are required
to test this hypothesis directly.
CONCLUSION
7.
8.
9.
From this study, it is concluded that deteriorated

pulmonary function in hypothyroidism is associated
with increased visceral fat. Therapeutic interventions
like diet, exercise, yoga to reduce visceral fat should
be incorporated as part of treatment to improve the
pulmonary function.
10.
Acknowledgment: We sincerely acknowledge HODs

of
Departments of Physiology, biochemistry,
Medicine and Endocrinology for their continuous
support during the study.
Conflict of Interest: Nil.
12.
11.
13.
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1. Kek PC, Ho SC, Khoo DH. Subclinical thyroid
disease. Singapore Med J. 2003; 44(11):595600.
2. Larsen PR, Davies TF. Hypothyroidsm and
thyroiditis. In: Larsen PR, Kronenberg HM,
Melmed S, Polonsky KS (eds). Williams
Textbook
of
Endocrinology.
10th
ed.
Philadelphia: Saunders. 2003: 42356.
3. Zwillich CW, Pierson DJ, Hofeldt FD, Lufkin
EG, Weil JV. Ventilatory control in myxedema
and hypothyroidism. N Engl J Med. 1975;
292(13):6625.
4. Siafakas NM, Salesiotou V, Filaditaki V,
Tzanakis N, Thalassinos N, Bouros D.
Respiratory muscle strength in hypothyroidism.
Chest. 1992;102(1):18994.
5. Fletcher P, Andrew KN, Calokerinos AC, Forbes
S, Worsfold PJ. Analytical applications of flow
injection with chemiluminescence detection--a
review. Lumin J Biol Chem Lumin.
2001;16(1):123.
6. Chintala KK, Krishna BH, N MR. Heart rate
variability in overweight health care students:
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15.
16.
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correlation with visceral fat. J Clin Diagn Res

JCDR. 2015;9(1):068.
Cakmak G, Saler T, Salam ZA, Yenign M,
Demir T. Spirometry in patients with clinical and
subclinical hypothyroidism. Tberkloz Ve
Toraks. 2007;55(3):26670.
Koral L, Hekimsoy Z, Yildirim C, Ozmen B,
Yorgancioglu A, Girgin A. Does thyroid
replacement therapy affect pulmonary function
tests in patients with subclinical hypothyroidism?
Saudi Med J. 2006 ;27(3):32932.
Saaresranta T, Polo O. Hormones and breathing.
Chest. 2002; 122(6):216582.
Saaresranta T, Polo O. Sleep-disordered
breathing and hormones. Eur Respir J.
2003;22(1):16172
Laroche CM, Cairns T, Moxham J, Green M.
Hypothyroidism presenting with respiratory
muscle weakness. Am Rev Respir Dis.
1988;138(2):4724.
Wilson WR, Bedell GN. The pulmonary
abnormalities in myxedema. J Clin Invest. 1960
Jan;39:4255.
Ruel J, Coulombe P, Dussault JH. Thyroid
hormones, malnutrition, and biochemical
composition of developing rat lung. Am J
Physiol. 1982;242(6):37883.
Ksenzenko SM, Davidson SB, Saba AA, Franko
AP, Raafat AM, Diebel LN, et al. Effect of
triiodothyronine augmentation on rat lung
surfactant phospholipids during sepsis. J Appl
Physiol Bethesda Md. 1997 ;82(6):20207.
Harrison RN, Tattersfield AE. Airway response
to inhaled salbutamol in hyperthyroid and
hypothyroid patients before and after treatment.
Thorax. 1984;39(1):349.
Rajagopal KR, Abbrecht PH, Derderian SS,
Pickett C, Hofeldt F, Tellis CJ, et al. Obstructive
sleep apnea in hypothyroidism. Ann Intern Med.
1984;101(4):4914.
Enzi G, Vianello A, Baggio MB et al. Respiratory
disturbances in visceral obesity. In: Oomura Yet
al. eds. Progress in Obesity Research 1990.
London: John Libbey & C, 1990; 33539.
Sharp JT, Druz WS, Kondragunta VR.
Diaphragmatic responses to body position
changes in obese patients with obstructive sleep
apnea. Am Rev Respir Dis. 1986;133(1):327.
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Sudhir et al.,
DOI: 10.5958/2319-5886.2015.00121.6

&
Health Sciences
www.ijmrhs.com
Copyright @2015
st
th
Received: 1 June 2015
Research article
ISSN: 2319-5886
Accepted: 22nd June 2015
DEGREE AND ONSET OF MYDRIASIS CAUSED BY MYDRIATIC AGENTS WITH SPECIAL

REFERENCE TO TOBACCO ADDICTION
*Henal Javeri1, Tulsi Thampan1, Krusha Shah1, Neeta Misra2, Rahul R Kunkulol3
1, 2
Department of Ophthalmology, 3Coordinator, Directorate of Research, Rural Medical College, Pravara Institute
Of Medical Sciences(DU), Ahmednagar, Maharashtra
*Corresponding author email: hjjaveri20@gmail.com
ABSTRACT
Introduction: The quality of an intra-ocular examination depends on adequate pupil dilation (mydriasis).
Magnitude of dilation depends on sphincter and dilator muscles of pupil. Most frequently used drugs in
ophthalmology for mydriasis are parasympathetic antagonists (Tropicamide), sympathetic agonist
(Phenylephrine) and combination of Phenylephrine + Tropicamide. This study was planned to evaluate and
compare onset and degree of mydriasis achieved by the above drugs and to study changes in the same in tobacco
addicts. Aim: To evaluate and compare the onset and degree of mydriasis achieved by Tropicamide (1%),
Phenylephrine(10%), Tropicamide (0.8%) +Phenylephrine (5%) combination. To compare the changes in onset
and degree of Mydriasis in tobacco addicts. Materials & Methods: This is a descriptive cross sectional study
carried out in the ophthalmology department of PRH, Loni. Total of 52 patients were enrolled for the study and
grouped according to the mydriatics used into Group 1 (n=25) Tropicamide (1%) ,Group 2(n=18) Phenylephrine
(10%) Group 3(n=20) Tropicamide (0.8%) + Phenylephrine (5%) combination and each group were evaluated for
onset and degree of dilatation. Each group was further divided into tobacco and non-tobacco addicts. Results &
Conclusion: The combination of Tropicamide (0.8%) and Phenylephrine (5%) have the fastest onset of
mydriasis, and achieved the highest dilation in 60 min. as compared to Tropicamide (1%) and Phenylephrine
(10%) alone. Tobacco addicts in each group were observed to have lesser magnitude of dilation than non tobacco
addicts.
Keywords: Mydriasis, Degree and onset of mydriatic agents; Tobacco addicts, Tropicamide, Phenylephrine
INTRODUCTION
Mydriasis is the dilation of the pupil, usually defined
as when having a non-physiological cause, but
sometimes defined as potentially being a
physiological pupillary response. 1,2 The excitation of
the radial fibres of the iris which increases the
pupillary aperture is referred to as mydriasis. More
generally, mydriasis also refers to the natural dilation
of pupils, for instance in low light conditions or under
sympathetic stimulation. 2
A mydriatic ocular examination may be necessary to
detect some eye diseases as it allows better
Javeri et al.,
visualization of all structures posterior to the pupil

and is necessary for examination of the peripheral
ocular fundus. It is also necessary for examination
techniques such as binocular indirect ophthalmoscopy
and scleral indentation. 3
The ideal mydriatic agent should show a rapid onset
of action, achieve adequate mydriasis, permit a quick
recovery and not cause discomfort or side effects.
The quality of an intra-ocular examination depends
on adequate pupil dilation.4 Magnitude of dilation
depends on the sphincter muscle of the pupil,
634
controlled by parasympathetic nerves and on the

dilator muscle of the pupil, controlled by sympathetic
nerves.5
The most commonly used drugs include the
parasympathetic
antagonists
like
Atropine,
Homatropine, Cyclopentolate, Tropicamide and
sympathetic agonists like Phenylephrine. The
parasympathetic antagonists act by contracting the
iris sphincter muscle (circular muscles of the iris)
producing passive mydriasis whereas the sympathetic
agonists act by stimulating the iris dilator muscle
resulting in active mydriasis.6, 7, 8 Atropine,
Homatropine and Cyclopentolate produce mydriasis
along with cycloplegia and have duration of action of
1-2 weeks, 2-3 days, and 24 hours respectively.6
Therefore, they are not suitable for routine refraction
purposes. Tropicamide produces mydriasis within 2040 minutes of instillation and (weak) cycloplegia
maximal within 30 minutes. Both effects usually last
up to 6 hours. It is available in 0.5% and 1%
concentrations.3
Phenylephrine stimulates post-synaptic alpha
receptors which constrict the dilator muscles, hence
causing mydriasis.9 It causes mydriasis by affecting
the iris dilator muscle. Maximal mydriasis occurs in
10-90 minutes with recovery after 5-7 hours without
cycloplegia. It is available as 2.5% and 10%
concentrations.3
Commonly used drugs in ophthalmology practice for
mydriasis are Tropicamide (1%), Phenylephrine
(10%), Tropicamide (0.8%) either alone or in
combinations.
Taking into consideration the varied use of mydriatic
agents for various ophthalmological examination it
was thought prudent to evaluate and compare the
degree of mydriasis produced by Tropicamide,
Phenylephrine and their combination at the
ophthalmology OPD of PRH, Loni, in terms of onset
and degree of mydriasis and to study changes in the
same in tobacco addicts.
AIM:
1. To evaluate the degree of mydriasis achieved by
Tropicamide (1%), Phenylephrine (10%),
Tropicamide (0.8%) +Phenylephrine (5%)
combination.
2. To find out the onset of action of the above drugs.
3. To compare the changes in onset and degree of
Mydriasis in tobacco addicts .
MATERIALS & METHODS

Study design: This is a Descriptive cross sectional
study carried out on individuals who came to the
O.P.D for routine refraction in the ophthalmology
department of PRH, Loni.
Ethical approval: Institutional Ethical Committee
approval was obtained, written informed consent was
taken from all the participants
All the patients satisfying the following inclusion
exclusion criteria were enrolled for the study:
Inclusion Criteria: Any patient of age more than 18
years coming for any ophthalmological examination
receiving mydriatic agent, patients with baseline
pupil size 2 0.5 mm and patients of either sex,
Exclusion Criteria: Patients with history of any
ocular infections, patients with posterior synechiae,
shallow anterior chamber, patients with history of
liver and cardiac disorders, patients on any
antihypertensive medications or any relevant drug
history of symphathomimetics or cholinergic drugs,
Patients with congenital ocular disorder, and any
ocular trauma or undergone any ocular surgery
Grouping & sample size: Total of 63 patients (126
eyes) were enrolled for the study. Patients were
randomly grouped according to the mydriatics used
into 3 groups:
Group 1(n= 25): Tropicamide (1%)
Group 2(n= 18): Phenylephrine (10%)
Group 3(n= 20): Tropicamide (0.8%) +Phenylephrine
(5%)
Each group was further divided into tobacco and
non-tobacco addicts depending upon the personal
history of tobacco addiction given by them.
Procedure:
After the enrolment of patients, the baseline pupil
size was measured and according to the grouping
schedule the patients were instilled the mydriatic
agent (total three drops of drug) in the lower
conjunctival fornix,each at the interval of 10min up
to 30 min. Patients were directed to keep their eyes
closed to prevent loss of medication through the
punctum in the conjunctival sac. Measurement of
pupil diameter was done using pupil gauge.10 Five
repeated measurements were taken at an interval of
15 minutes (from 0min -60mins).
635
Javeri et al.,
RESULT
MEAN PUPIL DIAMETER (in

mm)
A total of 63 patients were included in the study. Of

these 36 (57.14%) were males and 27 (42.86%) were
females.
6
4
No. of subjects
13
11
Non-Tobacco
addicts
8
6
5.48
3.25
3.1
with
7.17
6.71
Group
2-
7.92
8.08
7.25
7.28
5.19
MEAN PUPIL DIAMETER

(in mm)
6.56
TOBACCO ADDICTS
NON-TOBACCO ADDICTS
2.58
10
TROPICAMIDE
PHENYLEPHRINE
TROPIC.+PHENYL.
TOBACCO ADDICTS
NON-TOBACCO ADDICTS
Fig 5: Pupil Diameter Group 3 (n= 20):

Tropicamide (0.8%) +Phenylephrine (5%)
TIME (IN MIN)
DISCUSSION
Fig 2: Comparison of Mydriasis Achieved At

Every 15 Min. Interval
8
5.39
6
mm)
6.56
4.28
Fig 4: Pupil Diameter

Phenylephrine (10%)
Fig 1: Distribution of Tobacco and Non-Tobacco

Addicts
3.02
5.97
6.89
Group 1 n Group 2 (n Group 3 (n

= 25)
= 18)
= 20)
4.72
6.89

mm)
12
Tobacco
Addicts
6.75
2.66
6.94
7.35
5.7
6.34
7.44
6.73
3.83
TOBACCO ADDICTS
Fig 3: Pupil Diameter with Group 1-Tropicamide

(1%)
The baseline pupil diameter was average 2 0.5 mm

before the instillation of the mydriatic agent. On
instillation of mydriatic agent in the patients of group
1 [Tropicamide(1%) n=25] the mean pupillary
diameter was 2.69 mm and pupil dilated upto 7 mm at
the end of 60 mins, whereas those in group 2
[Phenylephrine (10%) n=18] achieved a mean
pupillary diameter of 2.72 mm at instillation and
dilatation was 6.71 mm at the end of 60 min and in
group 3 [Tropicamide(0.8%)+Phenylephrine(5%)
n=20] achieved a significantly higher mean pupillary
diameter of 3.73 mm at instillation and dilated upto
7.69 mm at the end of 60 min. Thus the combination
of Tropicamide (0.8%) +Phenylephrine (5%) (group
3) had a faster onset and maximum degree of dilation.
On comparing the drugs in term of rapidity of action
it was seen that the adequate diameter of around 6.5
mm which is required for ophthalmologic testing, was
achieved at 30 min with the combination, whilst it
636
Javeri et al.,
was achieved at the end of 45 min in patients of

group 1 [Tropicamide(1%) n=25]
and 2
[Phenylephrine (10%) n=18].
Further, on comparing the degree of mydriasis
achieved between tobacco and non-tobacco addicts,
amongst each group, a mean pupillary diameter at
instillation, was 3 mm, 2.66 mm and 3.1 mm in
tobacco addicts of groups 1, 2,and 3 respectively,
while an average diameter of 3 mm, 3.02 mm and
3.25 mm respectively in non tobacco addicts.
At the end of 60 min, the degree of mydriasis of
tobacco addicts in group 1, 2, 3 were 6.73 mm, 6.56
mm, 7.28 mm respectively thus indicating a slower
and lesser degree of dilation than non tobacco addicts
who showed dilation of 7.44 mm, 6.89 mm and 8.08
mm respectively.
Another similar study by O Majid et al concluded that
by using 0.75% Tropicamide and 2.5% Phenylephrine
pupil successfully dilated to 7 mm within 40 minutes
after initial eye drop application in 77% of patients
enrolled in the study.10 Our study was comparable
with a research that suggested that the use of a
mixture of 0.75% Tropicamide and 2.5%
Phenylephrine is a superior dilating mixture
compared to the application of Tropicamide (1%) and
Phenylephrine (10%) alone.11A study by Keimyung
university showed that the combination of 1%
Tropicamide and 2.5 % Phenylephrine was better
than 1% Tropicamide and 2.5% Phenylephrine eye
drops alone.5
The pupil is under the control of the autonomic
nervous system. Parasympatholytic as well as
sympathomimetic drugs have been used to dilate the
pupil. The parasympathetic regulation dominates over
the sympathetic effect in the control of the pupil.12
.Therefore application of only the sympathomimetic
drug is usually inadequate to sustain the pupil dilation
in bright light during indirect ophthalmoscopy.
However, Parasympatholytic agents alone may not
provide sufficient dilation. Combination of both
drugs offers better pupil dilation than single drug
use.13 Also; the combination has a lower
concentration of each drug, thereby reducing the risk
of cardiovascular side effects of Phenylephrine.
In a rural setup, where tobacco consumption is
largely prevalent, suboptimal dilation was recorded in
tobacco addicts on comparison with non-addicts in all
three groups. Tobacco addicts in each group were
observed to have lesser magnitude of dilation as
Javeri et al.,
compared to non-tobacco addicts of the same group at

the end of 60 minutes. However, tobacco addicts in
group 3 [Tropicamide(0.8%)+Phenylephrine(5%)
n=20] achieved maximum mydriasis compared to
tobacco addicts of group 1 [Tropicamide(1%) n=25]
and group 2 [Phenylephrine (10%) n=18] .
Widespread tobacco addiction is mainly caused by
nicotine which is the only active pharmacological
agent in tobacco. The effects of nicotine are initiated
by binding to nicotinic cholinerginic receptors in
autonomic
ganglia,
adrenal
medulla,
and
neuromuscular junctions as well as in the central
nervous system. Binding of nicotine to cholinergic
receptors causes the release of a number of vasoactive
catecholamines
and
neuroactive
peptides.14
Parasympathetic activity thus decreases the
parasympatholytic effects of the mydriatic agents and
hence acheiving less magnitude of dilation of pupil.
The limitations of our study include the crude
methods of pupil size measurement and the lack of
regular monitoring of pulse and blood pressure in
patients who were administered Phenylephrine only.
CONCLUSION
The combination of Tropicamide (0.8%) and
Phenylephrine (5%) have the fastest onset of
mydriasis, and achieved the highest dilation in 60
min. as compared to Tropicamide (1%) and
Phenylephrine (10%) alone. Tobacco addicts in each
group were observed to have lesser magnitude of
dilation than non tobacco addicts.
Acknowledgements: We would like to thank the
Directorate of Research, PIMS, Loni, for their
guidance and support, the entire staff of the
Department of Ophthalmology, who helped us carry
out the research and all the subjects who were a part
of the research, for their co-operation.
REFERENCES
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Mydriasis http://medical-dictionary.The free

dictionary. com/mydriasis Date accessed- April 2,
2015.
2. Mydriasishttp://en.wikipedia.org/wiki/Mydriasis#
cite_note-1 Date Accessed- April 2, 2015.
3. Wallis M, Clinical Guidelines-Pupil Dilation.
http://www.optometrists.asn.au/media/274917/
clinical_guideline_pupil_dilation.pdf#page=1&zo
om=auto,-107,848 . Accessed- April 20, 2015.
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Jethani J, Solanki H, Nayak A. Effect Of the

single-drop mydriatic combination of 0.8%
Tropicamide with 5%phenylephrine with
multiple applications of the same. Indian J
Opthalmol.2011;59(4):323-5.
Park JH, Lee YC, Lee SY.The Comparison of
Mydriatic Effect between Two Drugs of Different
Mechanisms.Korean
J
Ophthalmol.2009;23(1):40-42
Commonly Used Dilating drops (mydriatic
medications). http://www.pgcfa.org/kb/entry/166/
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Mydriasis
http://www.drsynapse.co.in/2013/10/1.html
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. Date accessed- May 19, 2015.
O Majid, R Tabassum, M Keng, S Zahoor, I
Shamas. Effective pupil dilatation with a mixture
of 0.75% Tropicamide and 2.5% Phenylephrine:
A randomised controlled trial. The Internet
Journal of Ophthalmology and Visual Science.
2009; (8): 1.
Trinavarat A, Pitulsung A. Effective pupil
dilatation with a mixture
of
0.75%
Tropicamide and 2.5% Phenylephrine: A
randomised controlled trial. Indian J Opthalmol
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Kardon R. The pupil.In: Kaufman PL, Alm A,
editors.Adlers physiology of the eye. St. Louis:
Mosby; 2003. .713-43.
Eyeson-Annan ML, Hirst LW, Battistutta
D,Green A. Comparative pupil dilation using
Phenylephrine alone or in combination with
Tropicamide.Ophthalmology 1998; 105;726-32.
Uzeyir Erdem, Fatih C. Gundogan, Umut Ash
Dinc, Umit Yolcu, Abdullah ilhan, and Salih
Altun. Acute Effect of Cigarette Smoking on
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2015, Article ID 625470, 5 pages
638
Javeri et al.,
DOI: 10.5958/2319-5886.2015.00122.8

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
th
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
th
ANTIDIABETIC AND HYPOLIPIDEMIC ACTIVITY OF GYMNEMA

DEXAMETHASONE INDUCED INSULIN RESISTANCE IN ALBINO RATS
SYLVESTRE
IN
*Hemanth Kumar V 1, Nagendra Nayak IM 2, Shobha V Huilgol 3, Saeed M Yendigeri 4, Narendar K5

1,5
Lecturer, 3Professor, Department of Pharmacology, Al-Ameen Medical College, Vijayapura, Karnataka

Associate Professor, Department of Pathology, Al-Ameen Medical College, Vijayapura, Karnataka
2
Professor of Pharmacology, KS Hegde Medical Academy, NITTE University, Mangalore, Karnataka
4
*Corresponding author email: hems286@gmail.com

ABSTRACT
Background: Gymnema sylvestre plant was widely used for medicinal purpose. The plant leaves were
traditionally used to treat diabetes. Aim: To determine the antidiabetic and hypolipidemic activity of Gymnema
sylvestre in dexamethasone induced insulin resistance in Albino rats. Objectives: The present study was
undertaken to evaluate antidiabetic and hypolipidemic activity of Gymnema sylvestre leaf aqueous extract against
dexamethasone induced insulin resistance in Albino rats. Materials and Methods: Animals were divided into
five groups. Normal control and diabetic control group received gum acacia (2%) orally for 12days, and normal
saline (i.p.), dexamethasone (8mg/kg/i.p.) from day 7- day12 respectively. Two test groups (Gymnema sylvestre
leaf aqueous extract 2 and 4gm/kg/p.o./12days) and standard control received metformin (2gm/kg/p.o./12 days).
The two test groups, standard control group received dexamethasone (8mg/kg/i.p) from day 7- day 12
respectively. The antidiabetic and hypolipidemic activity was estimated by measuring serum glucose, insulin,
lipid levels and histopathological evaluation of liver tissue. Results were analyzed by using one way ANOVA
followed by Scheffes multiple comparison test. Results: Treatment with aqueous extract of Gymnema sylvestre
(2 and 4gm/kg/p.o) significantly (p<0.01) altered the elevated glucose, lipid, insulin levels and also improved the
histopathology of liver in dexamethasone induced insulin resistance rats. Conclusion: Treatment with aqueous
extract of Gymnema sylvestre improved the altered glucose, insulin and lipid profile in insulin resistance rats.
Keywords: Gymnema sylvestre, Glucocorticoids, Insulin Resistance, Diabetes, Dyslipidemia.
INTRODUCTION
Gymnema sylvestre belongs to family asclepidaceae.
It is a native plant in south west of India, Australia
and Africa. It is also known as Meshashringi in
sanskrit,
in
Hindi:
Gurmar,
Kannada:
Sannagerasehabmu and Telugu: Podapatri. From
ancient times it is used to treat diabetes,
hypercholestrolemia, asthma, eye complications and
inflammation.[1]
The major pathogenic factor in the development of
type -2 diabetes is the cellular resistance of muscle,
liver and or fat cells to the action of insulin.[2]
Glucocorticoids were widely used for several clinical

conditions.
With
prolonged
exposure
of
glucocorticoids, insulin secretion will increase to
compensate for the excess of glucose in blood due to
development of insulin resistance in peripheral
tissues.[3] Insulin resistance is associated with
development of various complications such as
diabetes, cardiovascular events and hepatic steatosis
etc.[3- 5]
Since ages Gymnema sylvestre plant preparation was
used in different formulations for the treatment of
639
Hemanth et al.,
diabetes. Chewing the leaves of Gymnema sylvestre

was reported to suppress the sweet taste.[6] Gymnema
sylvestre plant leaves improve the enzyme activity
responsible for glucose uptake and utilization and
also increases the permeability of cells to insulin.[6] It
was also used in the treatment of obesity and
hypercholesterolemia.[7]
Dexamethasone is a potent Glucocorticoid with
minimal mineralocorticoid action. Several studies
were conducted with the use of dexamethasone for
induction of insulin resistance. It induces insulin
resistance in rodents in a relatively short period of
time. [8, 9] Hence, dexamethasone was selected in
present study for induction of insulin resistance.
There is scarce data on insulin sensitizing action of
Gymnema sylvestre plant leaves. Hence, the present
study was undertaken to investigate protective role of
aqueous extract of Gymnema sylvestre on serum
glucose, insulin and lipid profile in dexamethasone
induced insulin resistant rats.
Study design: Experimental animal based study
Ethics approval: Institutional animal ethics
committee permission was taken prior to study.
Raring of animals: The present study was conducted
on male albino Wistar rats (weight 230-300gms).
Rats were obtained from central animal house of the
institution. Animals were maintained under standard
conditions, as provided by Committee for the Purpose
of Control and supervision on experimental animals
(CPCSEA), at temperature (232)o C, humidity
505%, 12:12hr light-dark cycles. Animals were
maintained in polypropylene cages (UN Shah
Manufacturers), rat pellets (Hindustan lever ltd,
Mumbai) and water were given ad-libitium.
Drugs and Chemicals: Dexamethasone injection
was obtained from Zydus pharmaceuticals, Mumbai.
Metformin was obtained from USV Limited,
Mumbai, India. Ketamine injection was obtained
from Neom laboratories limited, Mumbai, India.
Reagents and kits: The biochemical parameters
glucose, lipid profile were measured by using
commercially available kits (Erba Mannheim,
Transasia Biomedicals LTD.). Serum Insulin levels
were estimated by using Ultra sensitive rat insulin
ELISA kit from Gen X Bio Health Sciences private
limited, New Delhi.
Collection of plant material: Leaves of Gymnema

sylvestre were obtained from the local market in
Vijayapura. The leaves were identified and
authenticated by Dr. Sunil Kumar KN, Senior
Research officer, Pharmacognosy, SDM Centre for
research in Ayurveda & Allied sciences, Udupi.
Preparation of the aqueous extract of Gymnema
sylvestre: Weighed accurately 450gms of the
Gymnema sylvestre leaves and kept in a round bottom
flask. 500ml of distilled water was added and allowed
to stand for 24hours. The contents were filtered and
the extract was concentrated by distillation. The
solvent was removed by evaporation on a water bath.
It was completely dried under vacuum. Around
120gms of dried extract was obtained. [10]
Phytochemical analysis: Gymnema sylvestre
aqueous extract was subjected to preliminary
phytochemical analysis for detection of major
chemical constituents such as alkaloids, phenols,
resins, tannins, terpenoid and saponins etc. [11-13]
Grouping:
Animals were divided into 5 groups (n=6). Study was
conducted for a period of 12 days. Pilot study was
conducted initially to determine the antidiabetic doses
of Gymnema sylvestre.
Group I served as normal control
Group II diabetic control, received gum acacia (2%
p.o.)
Group III received 2gm/kg/p.o., and group IV
4gm/kg/p.o. of Gymnema sylvestre aqueous extract
for 12days respectively.
Group V rats were treated with a standard drug
metformin (2gm/kg/p.o/12days).
All the groups except normal control group received
dexamethasone (8mg/kg/i.p.) from day7- day 12. [1415]
At the end of the study period, fasting blood was
collected by retro-orbital sinus puncture for
estimation of biochemical parameters. Later rats were
anesthetized with intra peritoneal ketamine injection,
[16]
sacrificed by cervical dislocation and liver tissues
were collected for histopathological investigations.
Biochemical estimation: Blood samples were
centrifuged at 2000RPM and serum was separated.
Serum was used for following biochemical
investigations.
Serum glucose levels were measured by glucose
oxidase and peroxidase (GOD-POD) method, [13, 17]
triglycerides by Glycerol Phosphate Oxidaseperoxidase (GPO-POD), [13, 17] Total Cholesterol
640
Hemanth et al.,
(CH), High density lipoproteins (HDL) and Low

density lipoproteins (LDL) - Cholesterol by OxidasePeroxidase (CHOD-PAP) methods. [13, 17-20] Very low
density lipoproteins (VLDL) cholestrol was estimated
by using the formula, VLDL = triglycerides / 5. [17-20]
All the biochemical investigations were done by
using fully automated analyzer (ERBA-EM 200). [1720]
Serum Insulin levels were estimated by Enzyme

Linked Immunosorbant Assay (ELISA) method by
using ELISA reader. [20]
Histopathology: A small portion of liver was fixed in
formalin (10%) solution. Liver sections were made
5m thick, and stained with hemotoxylin and eosin
(H&E) stain. After staining, the sections were
observed under microscope at 10X and photographs
were taken.
Statistical analysis: The data was presented in Mean
SEM. Results were analyzed by one-way ANOVA
followed by Scheffe multiple comparison tests using
SPSS software. Statistical significance was assumed
if p < 0.01.
RESULTS
Phytochemical analysis:
The preliminary
phytochemical analysis of Gymnema sylvestre
aqueous extract showed presence of alkaloids,
phenols, saponins, tannins, and terpenoids.
Biochemical
parameters:
Treatment
with
dexamethasone (8mg/kg/i.p.) significantly increased
the serum glucose, insulin, total cholesterol, TGs,
LDL, VLDL and decreased HDL levels in
comparison to control group. It indicates the
dexamethasone induced diabetes, dyslipidemia due to
insulin resistance. [Table 1]
Treatment of rats in group III, IV with aqueous
extract of Gymnema sylvestre significantly reduced
the elevated serum glucose, insulin, total cholesterol,
TGs, LDL, VLDL and raised the HDL levels in
comparison to dexamethasone (8mg/kg/i.p) treated
group. [Table 1] There was no significant difference
in serum glucose, insulin and lipid profile between
Metformin treated rats and Gymnema sylvestre
(2,4gm/kg/p.o.) aqueous extract treated rats. [Table 1]
Table: 1 showing antidiabetic and hypolipidemic activity of Gymnema sylvestre leaf aqueous extract
Groups
Glucose
Insulin
HDL
LDL
VLDL
TGS
CH
(mg/dl)
(ng/ml)
(mg/dl)
(mg/dl)
(mg/dl)
(mg/dl)
(mg/dl)
Group-I
96.831.22
3.00.18
26.501.75 14.351.25 10.270.25 51.391.26 84.111.85
Group- II
272.251.82*
19.460.33* 6.770.30*
98.781.17* 32.670.36* 163.361.79* 196.711.61*
Group-III
146.20 3.16
7.6 0.68 20 1.39
48.4 3.24 16.180.77 80.93 3.85 118.55 2.44
Group-IV
143.82 2.41
7.25 0.53 20.1 1.30 46.31 2.14 15.790.64 79.0 3.22 119.63 2.76
Group-V
137.542.23
5.11 0.31 24.01 1.63 42.732.27 14.550.50 72.762.55 110.892.58
Values are expressed in mean SEM, *p< 0.01 vs. normal control, p<0.01 vs. diabetic control
Histopathological observations:
The control group and metformin treated groups
showed normal hepatocytes, hepatic parenchyma. All
the sinusoids appear normal. Periportal (Zone 1), mid
zone (Zone 2) and centrilobular area (Zone 3) appears
normal. [Fig 1 and Fig 2 respectively]
The histopathological examination of dexamethasone
8mg/kg/i.p. treated group showed increase in the size
of hepatocytes, cytoplasm is vesicular to clear. Fat
deposition was observed in Zone 2, Zone 3. [Fig 3]
The liver sections of rats treated with aqueous extract
of Gymnema sylvestre (2gm/kg/p.o) showed normal
architecture of liver, dilation and congested vein. Fat
deposition was observed only in zone 3. [Fig 4]
Fig 1: Gum acacia treated rats shows normal

hepatic architecture [H&E 10X]
641
Hemanth et al.,
Fig 2: Treatment with Metformin (2gm/kg/p.o.)

shows normal hepatic architecture[H& E10X]
Fig 5: Treatment with Gymnema sylvestre

(4gm/kg/p.o.) shows complete improvement. [H&
E 10x]
DISCUSSION
Fig 3: Dexamethasone (8mg/kg/i.p.), shows fat

deposition in liver [H& E 10x]
Fig 4: Treatment with Gymnema sylvestre

(2gm/kg/p.o.) shows partial improvement. [H& E
10X]
High doses of Gymnema sylvestre (4gm/kg/p.o.)
completely restored the pathological changes induced
by dexamethasone. Rat livers showed normal
hepatocytes, sinusoids. There were no apparent signs
of fat deposition, liver appear normal. [Fig 5]
Several theories were postulated for the development

of adult onset of diabetes, one of the possible
mechanisms is the Insulin resistance. Dexamethasone
induces insulin resistance which leads to the
development of hyperinsulinemia, hyperglycemia,
dyslipidemia and hepatic steatosis. Insulin resistance
develops before the clinical appearance of
complications. In several clinical conditions, higher
doses of dexamethasone is used.
Prophylactic
treatment of insulin resistance can prevent the
development of secondary complications. [21]
In current study, dexamethasone administration
resulted in insulin resistance and leading to diabetes,
dyslipidemia and hepatic steatosis. Treatment with
aqueous
extract
of
Gymnema
sylvestre
(2,4gm/kg/p.o.) significantly reduced the elevated
serum glucose, insulin, lipid levels and also improved
the liver pathology changes induced by
dexamethasone.
Glucocorticoids (GCs) in general increases blood
glucose levels by various mechanisms; increased
hepatic glucose production (gluconeogenesis),
decreased peripheral glucose uptake into muscle and
adipose tissue, breakdown of muscle and fat to
provide additional substrates for glucose production.
[22]
Prolonged GC exposure is associated with
development of severe insulin resistance and
metabolic dysfunction; however the precise
molecular mechanism is not clearly defined. [22] The
present study also confirms same findings, as
dexamethasone significantly increased the serum
glucose, insulin levels in insulin resistant animals.
642
Hemanth et al.,
According to review of literature Gymnema sylvestre

plant leaves were helpful in treatment of diabetes.
Several studies have been conducted to identify the
possible mechanisms of Gymnema sylvestre in
improvement of hyperglycemia. It was reported that
Gymnema sylvestre induces hypoglycemia. [1] Leaves
of Gymenma sylvestre improve the glucose utilization
by increasing the activity of enzymes responsible for
glucose utilization by insulin dependent pathways. It
increases phosphorylase activity, decrease in
gluconeogenic enzymes and sorbitol dehydrogenase
enzyme. [1, 23] Our study supports the earlier findings
that the aqueous extract of Gymnema sylvestre leaves
significantly decreased elevated serum glucose levels.
It is believed that one of saponin form of Gymnema
sylvestre plant leaves, Gymnemic acid was
responsible for development of hypoglycemia.
Gymnemic acid molecules by filling the receptors on
absorptive layers of intestine decreases absorption of
glucose, resulting in low blood sugar levels. [24]
In the present study aqueous extract of Gymnema
sylvestre (2 and 4gm/kg/p.o.) significantly decreased
the elevated insulin levels. Dexamethasone
administration led to increase in insulin level in
comparison to control group due to insulin resistance.
The
possible
mechanism
responsible
for
improvement in insulin resistance by Gymnema
sylvestre may be due to improvement in the
permeability of cells to the insulin action. [23] This
might result in significant decrease in the circulating
insulin levels. Further studies are required to know
the exact mechanism by which it improves insulin
resistance.
In the current study, treatment with aqueous extract
of Gymnema sylvestre significantly improved the
altered lipid profile. As per review of literature,
glucocorticoids administration leads to lipid
disturbances. It elevates triglycerides, total
cholesterol and LDL cholesterol levels, and reduces
HDL levels which may be secondary cause of
dyslipidemia. The Mechanisms responsible for
glucocorticoid induced dyslipidemia could be
impaired catabolism of LDL, increase in the activity
of lipoprotein lipase and subsequent increase in LDL
and VLDL levels due to increased plasma insulin. [25]
Gymnema sylvestre plant leaves were helpful in
treatment of obesity and hypercholesterolemia.
Studies were conducted for the possible role of
Gymnema sylvestre plant leaves in dyslipidemia. In
the present study, it significantly decreased

circulating cholesterol, triglycerides, LDL and VLDL
levels. It promotes the fecal excretion of cholesterol
and cholic acid derived bile acids. It also decreases
serum triglycerides and cholesterol and improves
hypertriglyceridemia and hypercholesterolemia.
These actions might be due to phytochemical
constituent saponins. [26] Isolation of specific
phytochemical constituent, was not possible hence,
the whole leaf aqueous extract were used in present
study. One of the possible mechanism for
improvement in serum cholesterol by Gymnema
sylvestre may be due to increase in fecal fermentation
leading to production of organic acids, propionic
acids, acetic acid. [26] Positive correlation was found
between propionic acid and serum cholesterol.
Injection of propionic acid decreased blood
cholesterol in rats. [27]
Gymnema sylvestre (2,4gm/kg/p.o.) significantly
decreased elevated CH, TGs, LDL, VLDL and
increased HDL level. It decreases the activity of
hormone sensitive lipase (converted neutral fats into
free fatty acids) and decrease of cholesterogenisis and
fatty acid synthesis. [27] In the present study Gymnema
sylvestre increased the HDL level, which is
correlating with other studies. [6] These increased
HDL levels will be helpful in preventing various
cardiovascular complications due to dyslipidemia.
Presence of sitosterol in leaf of aqueous extract might
also be responsible for lipid lowering action. It
decreases the absorption of lipids. [28]
Glucocorticoids can contribute to fatty liver
production, through a combination of increased fatty
acid synthesis and decreased fatty acid oxidation in
liver. [29] Raised free fatty acid levels have been
associated with the development of hypertension,
skeletal muscle insulin resistance, and fatty liver, this
last being considered as the hepatic consequence of
the metabolic syndrome due to specific hepatic
insulin resistance. [30] Present study supports the
earlier finding that, the dexamethasone (8mg/kg/i.p)
administration resulted in development of hepatic
steatosis due to insulin resistance. Fat deposition was
observed in zone 2, zone 3. [Fig 3] Treatment with
aqueous extract of Gymnema sylvestre in high doses
(4gm/kg/p.o.) significantly improved pathological
changes in liver (Fig 5). This might be due to the
decrease in circulating fatty acid levels, leading to
643
Hemanth et al.,
decrease in the fat deposition in liver induced by

dexamethasone.
CONCLUSION
Dexamethasone is used in high doses in several
clinical conditions. This might lead to development of
Insulin resistance leading to various complications.
Pretreatment with Gymnema sylvestre significantly
prevented the development of insulin resistance and
associated complications diabetes, dyslipidemia and
hepatic steatosis.
Identification of cellular mechanism of insulin
resistance and, Isolation of specific constituent
responsible for hypoglycemic effect of extract will be
more helpful, which remains the limitations of
present study.
Acknowledgement: We are sincerely thankful to the
Dr. Sucheta shetty and members of Central research
laboratory, NITTE University, Mangalore for their
great support in estimation of insulin levels by ELISA
method.
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Glucocorticoids and insulin resistance: old
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Gloria A and Marco B. Pathophysiology of
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Neuroendocrinology. 2010; 92(1):86-90.
6. Grijesh kumar M, Pankaj Kishor and Veeru

Prakash. Antidiabetic and Hypolipidemic activity
of Gymnema sylvestre in alloxan induced diabetic
rats. Global Journal of Biotechnology and
Biochemistry. 2009; 4(1): 37-42.
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hypoglycemic on oxidative stress and antioxidant
status in diabetic rats. The Open Diabetes
Journal. 2009; 2:48-52.
8. Korach-Andre M, Gao J, Gounarides J, Deacon
R, Islam A, Laurent D. Relationship between
visceral adiposity and intramyocellular lipid
content in two models of insulin resistance. Am J
Physiol Endocrinol Metab. 2005; 288:E106
E116.
9. Okumura S, Takeda N, Takami K, Yoshino K,
Hattori J, Nakashima K et al. Effects of
troglitazone on dexamethasone-induced insulin
resistance in rats. Metabolism. 1998; 47:351
354.
10. Grijesh kumar M, Pankaj kishor M and Prakash
V. Antidiabetic and hypolipidemic activity of
Gymnema sylvestre in alloxan induced diabetic
rats. Global journal of biotechnology&
biochemistry. 2009;4(1): 37-42
11. Harbone JB. Phytochemical methods, A guide to
modern techniques of plant analysis. 2nd ed.
Chapmanand Hall, London; 1984:84-88.
12. Kokate CK, Purohit AP and Gokhale SB.
Pharmacognosy, Nirali Prakashn. 21st edn, Pune;
2002: pp 105-106, 111-113
13. Jamkhande PG , Patil PH and Surana SJ.
Evaluation of N-Butanolic fractions of Butea
monosperma flowers on Dexamethasone induced
Hyperglycemia and Hyperlipidemia in mice
International journal of phyto pharmacy research
2010; 1(1):5-10
14. Hemanth kumar V, Nagendra Nayak IM, Shobha
Huilgol V, Saeed Yendigeri M, Narendar K and
Rajasekhar Ch. Dose dependent hepatic and
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15. Mathai P, Nayak N, Rao M, Bhat GM, Vinodraj
K, Chandralekha N, Rajesh D, Chethan TK.
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29. Rucha Patel, Monika Patel, Ricky Tsai, Vicky
Lin, Angie Bookout L and Yuan Zhang et al.
LXR is required for glucocorticoid-induced
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journal of clinical investigation. 2011; 121(1):
431-41.
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DOI: 10.5958/2319-5886.2015.00123.X

&
Health Sciences
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Volume 4 Issue 3
th
Research article
Coden: IJMRHS
Revised: 19th June 2015
Copyright @2015
ISSN: 2319-5886
OUTCOME OF INTERTROCHANTERIC FRACTURES TREATED WITH SHORT FEMORAL NAIL

Yadkikar Shriniwas V1, Yadkikar Vishnu S2, Patel Mayank3, Dhruvilkumar Gandhi3, Kunkulol Rahul4
1
Assistant Professor, 2Prof. & Unit In charge, 3Resident, Department of Orthopaedics, Rural Medical
College, Pravara Institute of Medical Sciences (DU), Loni, Maharashtra

4
Professor, Department of Pharmacology, Rural Medical College, Pravara Institute of Medical Sciences (DU),
Loni, Maharashtra
*Corresponding author email: yadkikarpushkar@yahoo.co.in
ABSTRACT
Aim: To study the functional and anatomical outcome of Inter trochanteric fractures of femur treated with Short
femoral nail. Method: This was retrospective study carried out in which 60 patients (50 Male & 10 Female) of 5th
to 8th decade of life who underwent Short femoral nail fixation for both Stable & unstable Inter Trochanteric
fractures. From the records each patient data was assessed for time required for mobilization, average fracture
healing time, degree and grade of hip range of movements, complications, anatomical reduction achieved using
Short femoral nail fixation. Results: 55 cases achieved Anatomical reduction. Good to Excellent Hip range of
Motion was in 55 (90 %) cases. Fracture union was seen in all cases. No evidence of Z Effect, AVN of femoral
head, Implant failure, Fracture of femoral shaft below the Nail tip was seen in any case, However Reverse Z
Effect was seen in 4 & shortening of less than 2 cm was seen in 2 cases, External rotation of 10 degree was seen
in1 case. Average fracture Union time was 14 weeks. Conclusion: Short femoral nail appears to be better
implant for fixation of both Stable & unstable Inter Trochanteric fractures as it fulfills the biomechanical
demands being minimally invasive, less blood loss , it prevents excessive varus collapse at fracture site, produces
less stress riser effect below the nail tip, Short operative time, Facilitates early mobilization & functional
recovery of patients. But Anatomical fracture reduction & optimal implant placement are absolutely must for
better results.
Keywords: Short femoral nail, trochanteric fractures
INTRODUCTION
Intertrochantric fractures are common in 5th 8th
[1,2].
decade of life
Conservative treatment is poorly
tolerated by elderly patients making operative
treatment as treatment of choice [1-4]. Dynamic hip
[1, 4, 5]
screw is time tested extrmedullary device
. But
it has disadvantages like larger soft tissue dissection,
more blood loss, medialization of distal fragment,cut
[3]
out of screw & excessive collapse at fracture site .
To avoid these complications intramedullary devices
are developed which are minimally invasive. Gamma
Shriniwas et al.,
nail first of its generation has provision of only single

[3,4]
screw placement which can cutout
. To overcome
this complication Proximal femoral nail was
developed by AO/ASIF in 1997 which has provision
of two proximal screw placement making construct
biomechanically stable & suitable even for Unstable
[4],.
trochnateric fractures
Gamma nail & Proximal
femoral both have larger proximal diameter for
average Indian femur [1,2,3], which can cause
[3].
splintering of bone
This led to invention of Short
646
femoral nail with length of 180mm & proximal

diameter of 15mm. This enables easy insertion &
[3].
reduces chances of fracture of femur
This nail has
longitudinal slot throughout its length which helps in
accelerating the endosteal bone healing process [3,4]. It
also has 6 degrees of medio lateral angle & flutted
distal tip which facilitates easy insertion, reduces
stress concentration below the nail tip[3] In this series
we have reviewed records of 60 patients with both
stable & unstable trochanterric fractures treated with
short femoral nail.
Aim: To study the functional and anatomical
outcome of Inter trochanteric fractures of femur
treated with Short femoral nail
Objective:
1. To study the time required for mobilization of
Inter trochanteric fractures of femur treated with
Short femoral nail fixation
2. To determine the average fracture healing time of
Inter trochanteric fractures of femur treated with
Short femoral nail
3. To find out degree and grade of hip range of
movements achieved with Inter trochanteric
fractures of femur treated with Short femoral nail
4. To study the complications if any with the Inter
trochanteric fractures of femur treated with Short
femoral nail
5. To assess the anatomical reduction achieved
using Short femoral nail fixation
MATERIAL & METHOD
Study design: Retrospective analysis. Records of
60cases of Inter Trochanteric fractures treated with
Short femoral nail from Dec 2012 to April 2015in our
department.
Ethical approval: The study was started after taking
permission from Ethical committee.
Inclusion criteria: Patients of both sexes with both
Stable & unstable intertrochanteric fractures from of
5th to 8th decade of life who underwent Short femoral
nail fixation.
Exclusion criteria: 1. Patients with intertrochanteric
fractures with subtrochanteric extensions. 2. Patients
with intertrochanteric fractures with ipsilateral
femoral shaft fractures fixation. 3. Those patients
whose detail records were incomplete
Methodology:
Shriniwas et al.,
Hospital records of total 60 cases of intertrochanteric

fractures treated with Short Femoral Nail fixation
from Dec 2012 to April 2015 in the department of
Orthopaedics, Rural medical college, Loni.
The following parameters were assessed form the
records:
Age, Gender, Mode of Injury (Domestic Fall /High
velocity trauma), Stable or unstable intertrochanteric
fractures pattern on X rays ( stability of fracture was
decided by presence or absence of posterio medial
cortex integrity of proximal femur, Fractures were
classified by AO/ASIF Classification), Duration
between admission & operation, Type of reduction
obtained Closed or Open, Time required for
mobilization following fracture fixation, Fracture
healing time in weeks, Assessment of Functional
recovery by hip range of motion by Harris Hip Score
& ability to do activities of daily routine like
climbing stairs, squatting, walking, cross leg sitting,
Occurrence of
Complications following Short
femoral nail fixation like Reverse Z ,Z effect, implant
failure, infection, AVN femoral head, Non union,
Anatomical reduction achieved following fracture
fixation by restoration of posterior & medial cortex
integrity & restoration of Neck shaft angle.
RESULTS
Amongst 60 cases 50 were Male & 10 Females.
Majority of patients were from 5th 8th decade of life
(Mean age 67yrs) [Table 1]. Most common
mechanism (in 50 cases) of injury was Domestic fall
while in 10 cases it was High velocity trauma.
AO/ASIF Classification was used [Table 2]
Table 1: Age wise distribution Number of patients
Age range Male Female
Total
Percentage
50-59
15
2
17
28%
60-69
25
4
29
48%
70-79
8
3
11
18%
80-89
2
1
3
5%
Table 2: Fracture Pattern
Number Percentage
Fracture Pattern
Stable fractures A1
21
35
Unstable fractures A2 24
40
Unstable fractures A3 15
25
Stable fracture pattern was seen in 35 % cases,

while unstable pattern was seen in 75 %
Per operatively patients were above knee skin traction
on Thomas splint. Average duration between
admission & operation was 8 days. No pre operative
647
CT scan was done in any case. IV antibiotics

(Intravenous Injection Cefotaxim 1 gm twice a day)
were given up to 5 days post operatively.
Patients were operated in supine position on fracture
table with affected limb given adduction & traction.
Closed Anatomical reduction was achieved in 55
cases while 5 cases required minimal open reduction
[Fig 1].
Fig 3: Case2 Preoperative and postoperative X
ray showing Implant in situ in reduced fracture
(AP view)
Fig 1 : Type of reduction

In 55(92%) cases closed Anatomical reduction was
achieved
Anatomical reduction was assessed on image
intensifier in AP & Lateral view by restoration of
Posterior & medial cortex continuity. Approximately
5 cm incision was given from Tip of trochanter &
extended up wards. Under image intensifier control
Guide wire was passed through the tip of trochanter
across the fracture site. Short Femoral nail was
mounted over Jig & it was inserted across the fracture
site over guide wire. Depending up on neck shaft
angle of reduced fracture nail of 135 degree was used
in 55 while that of 130 degree used in 5 cases. Then
over the guide wire for proximal locking screw
6.4mm Antirotation screw & then 7.9mm cervical
screw inserted through Jig in to the femoral head their
central position was confirmed on image intensifier in
both AP & Lateral view [Fig 2, 3, 4, 5].
Fig 2: Case 1 Preoperative and postoperative X

(AP view)
Shriniwas et al.,
Fig 4: Case 3, Preoperative and postoperative X

(AP view)
Fig 5: Case 4, Preoperative and postoperative X

(AP view)
Depending upon the stability of constructs Dynamic
or static distal locking was done. Wound was closed
in layers. As per pain tolerance active knee &hip
mobilization was started by post op day 4 -5.
Average operation time was 60 minutes. Average
hospital stay was 3 weeks. As per pain tolerance
active quadriceps strengthening & knee mobilization
exercises started by post op day 4 -5. Non weight
bearing mobilization with walker was started by 2
weeks, while full weight bearing was permitted only
when complete Radiological & Clinical evidence of
complete fracture union [Fig 6].
648
Fig 6 : Time required for non weight bearing

mobilization
Average fracture union time was 14 weeks. Fracture
union was seen in all cases. Commonly used nail
diameter was 10mm. Average follow up period was
1 year. Recently done 5 more cases are still under
follow up. Functional recovery of patients was
assessed by Harris Hip Score & ability to perform
activities of daily routine like walking, squatting,
cross leg sitting, stair case climbing was done. Good
to excellent results were seen in 55 (90%) cases
[Fig7,8]
Fig 7 : Hip range of motion as per harris hip score
Fig 8: Functional recovery assessment

Few complications were seen in our series. But none
of the mentioned complications hampered functional
recovery of patient. There was no evidence of Z
Effect , non union, implant failure , fracture shaft
femur below the nail tip, no revision surgery
required, no evidence of Avascular necrosis of
femoral head seen in any case in this series.[Fig .9]
Shriniwas et al.,
Fig 9 : Post operative complication

Reverse Z effect was seen in 4 cases , shortening
of less than 2 cm was seen in 2 cases,.No
evidance of Z effect, AVN, Implant failure was
seen in any case
DISCUSSION
Inter Trochanteric fractures are very frequently faced
by orthopaedic surgeons worldwide [4]. Increase in the
incidence of these fractures are seen 5th decade of life
onwards [1,4,5]. Age of patient, osteoporosis, general
health, associated co morbidities are some of the key
factors to be considered for the successful treatment
of these fractures[3,4]. Conservative treatment is
poorly tolerated by elderly patients & it is also
associated with complications like Decubitus ulcers,
Deep vein thrombosis, and Aspiration pneumonitis [4].
There for surgical treatment is preferred option of
treatment as it facilitates early mobilization &
functional recovery [1,4-9]
Dynamic hip screw is time tested extramedullary
load bearing device used for fixation of trochanteric
[1,4,10-13].
fractures
It works on the principal of
controlled concentric collapse at fracture site. But it
also has disadvantages like it requires larger surgical
exposure leading to more blood loss, devices causes
excessive collapse at fracture site, possibilities of cut
out of lag screw from femoral head if not placed
[3,4]
properly . In osteoporotic bones & unstable
fractures complications like fracture instability,
excessive medialization fracture fragment may lead to
pain & deformity [3]
The Cephalomedullary nails with Trochanter tip as
entry portal are Load sharing implants, as they are
placed close to the mechanical axis of femur, they
have short lever arm. In unstable trochanteric
fractures control of axial load transmission &
rotational stability are important factors which are
effectively managed by intramedullary devices[3]. So
649
they are considered biomechanically stronger than

[3,4].
extramedullary devices
Identification of Tip of
trochanter is easy & requires less soft tissue
dissection [3,4]
Gamma nail the original design of cephalomedullary
nail has provision of single screw placement in the
femoral head. But single screw construct was
[3,4],
considered unstable for trochanteric fractures
which led to introduction of Proximal femoral nail in
1997by AO/ASIF which has provision of two screw
placement in the femoral head. This system of
Antirotation screw & cervical load bearing screw in
this nail makes this construct biomechanically very
[3,4,9]
stable
. As compared to Gamma nail there is
less incidence of fracture of femoral shaft below the
nail tip[4,9] But Proximal femoral nail may also have
complications like Reverse Z effect, Z effect, lateral
wall of trochanter fracture in osteoportic bone,
incidence of fracture of femoral shaft below the nail
[4,9]
tip cannot be ruled out12
Both Gamma & Proximal femoral nail has proximal
diameter of 17mm which can be large of average
[1,3].
Indian femur13
Thus it can lead to widening &
[3]
eventual fracture of trochanter
Standered Proximal femoral nail has 250mm length,
it crosses femoral isthmus &sometimes it can abut
against the femoral shaft leading anterior thigh pain.
Fixation below the level of isthmus is not required in
most of intertrochanteric fractures.If there is
mismatch between nail size & femoral curvatures it
can lead to cortex penetration & splintering [3,4]. This
can happen due to over sized implant which is
manufactured
as
per
western
population
[3].
anthropometric parameters
Short femoral nail is designed to have length of
180mm with proximal diameter of 15mm. It also has
tip of trochanter as entry portal but due to its small
[3]
size it reduces stress concentration at this site . It
doesnt cross femoral isthmus, it prevents
[3][
metallization of femoral shaft Fig8.1,8.2] . It also
causes efficient load transfer than extramedullary
devices by acting like internal buttress [3]. Small
diameter of nail may also prevent splintering of
femoral shaft & fracture below the nail tip[3]
[
Fig9.1,9.2].
In our assessment it was observed that majority of
patients were from 5th 7th decade of life. Average
time of non weight bearing mobilization of patient
Shriniwas et al.,
following short femoral nail fixation was 2weeks,

Good to excellent functional recovery assessed by
Harris Hip Score & ability to perform activities of
daily routine like walking, squatting, cross leg sitting,
climbing stairs was seen in 90 % cases, it is
minimally invasive so it is better tolerated by elderly
patients. Closed anatomical reduction which achieved
by restoration of integrity of posterior & medial
femoral cortex seen on AP & Lateral view of X-rays
was seen in 92% cases. Average fracture union time
was also 14 weeks. Fracture fixation with short
femoral nail was also observed with fewer
complications. None of the observed complications in
the assessment was hampering functional recovery of
patients.
CONCLUSION
Short femoral nail appears to be better implant for
fixation of both Stable & unstable Inter Trochanteric
fractures as it fulfills the demands being minimally
invasive, less blood loss prevents excessive varus
collapse at fracture site, produces less stress riser
effect below the nail tip, Short operative time,
Facilitates early mobilization of patients which is
very helpful in elderly patients. But it also important
that Anatomical or Near Anatomical fracture
reduction & optimal implant placement are absolutely
must for better results.
Acknowledgement: Special thanks to Dr K Vilas
Babu Prof. & Head and sincere thanks supporting
staff of Operation Theater & Department of
Orthopaedics, Rural Medical College, Loni
Conflict of interest: None declared
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DOI: 10.5958/2319-5886.2015.00124.1

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Health Sciences
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Volume 4 Issue 3
th
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
th
BACTERIOLOGICAL PROFILE OF NEONATAL SEPTICEMIA: A RETROSPECTIVE ANALYSIS

FROM A TERTIARY CARE HOSPITAL IN LONI
*Sneha Ann Oommen1, Santosh Saini2, Kunkulol Rahul R3
1
UG Student, Rural Medical College, Pravara Institute of Medical Sciences, Loni, Maharashtra, India
Professor & HOD, Department of Microbiology, Rural Medical College, PIMS, Loni
3
Coordinator, Directorate of Research, PIMS-DU, Loni
2
*Corresponding author email: sneha_ann_oommen@yahoo.co.in

ABSTRACT
Background: Septicemia continues to be a major cause of neonatal mortality and morbidity worldwide.
Epidemiology and surveillance of neonatal sepsis helps in implementation of rational empirical antibiotic
strategy. Objectives: To study the bacteriological profile and antibiotic sensitivity pattern and types of neonatal
septicemia among the suspected blood & CSF samples coming to Microbiology Department of Pravara Rural
Hospital. Methodology: All the reports fulfilling the eligibility criteria were studied for Percentage of neonatal
sepsis in suspected blood and CSF samples, Percentage of bacteriological sepsis (Gram-positive and Gramnegative sepsis), Bacterialogical profile, Percentage of EOS (Early Onset Sepsis) and LOS (Late Onset Sepsis) in
confirmed cases, Antibiotic Sensitivity Tests. Result: The study showed a culture positivity of 259 blood culture
samples (23.31%) for bacterial growth out of 1111 and 36 (5.99%) positive out of 601 C.S.F culture samples.
Early onset sepsis (92.54%) was found to be predominant with Klebsiella the predominant EOS pathogens and
Coagulase Negative Staphylococci (CONS) as predominant LOS pathogen. Gram Negative sepsis (59.32%)
predominated in this study. In this study the predominant organisms were found to be Klebsiella (28.81%).The
antibiotic sensitivity testing showed that gram negative isolates were sensitive to Meropenem and gram positive
isolates to Linezolid, Netilmicin and Chloramphenicol . Conclusion- The most common bacteria causing neonatal
sepsis was found to be Klebsiella and the gram negative samples showed highest sensitivity for Meropenem and
Cefazolin was found to be most resistant. The gram positive samples showed highest sensitivity for Linezolid and
Penicillin and Ampicillin were found to be most resistant.
Keywords: Neonatal Sepsis, Rural, Antibiotic Sensitivity,Culture ,Bacteriological Profile
INTRODUCTION
Neonatal septicemia refers to a generalized bacterial
infection that has been documented by positive blood
culture or CSF culture during the first 28 days of life
and it is an important cause of morbidity and
mortality in neonates[1]. In common clinical usage,
neonatal sepsis specifically refers to the presence in a
neonate of a bacterial blood stream infection, such as
meningitis,
pneumonia,
pyelonephritis
or
gastroenteritis in the setting of fever.
Sneha et al.,
According to the World Health Organization (WHO)

estimates, there are about 5 million neonatal deaths a
year, 98% occurring in developing countries with
neonatal infection being one of the major causes,
causing about 1.6 million deaths annually [2]. Sepsis
and meningitis are responsible for most of these
deaths.
Neonatal septicemia is one of the four leading causes
of morbidity and mortality among neonates in India
652
[3]
.The neonatal period accounts for 38 % of all deaths

in children younger than 5 years. The National
Neonatal Perinatal Database (NNPD) reported an
incidence of 8.5 per 1000 live births for blood culture
proven sepsis [4] .The incidence of sepsis in the
neonate is greater than at any other period of life and
varies from hospital to hospital depending on the
rates of Prematurity, Prenatal care, conduct of labour
and environmental condition in nurseries.
Neonatal sepsis may be classified according to the
time of onset of disease as Early Onset Septicemia
(EOS) i.e. from birth to 7 days and Late Onset
Septicemia (LOS) i.e. from 7 days to 28 days. The
clinical relevance is that the EOS is mainly due to
bacteria acquired before or during delivery while
LOS is from nosocomial or community sources.
The EOS infections are caused by organisms
prevalent in the maternal genital tract or in the
delivery area. The predisposing factors include low
birth weight (LBW), prolonged rupture of
membranes; foul smelling liquor, multiple per
vaginum examinations, maternal fever, difficult or
prolonged labour and aspiration of meconium.
The LOS infections are caused by the organisms
thriving in the external environments of the home or
the hospital. The infection is often transmitted
through the hands of the care-providers. The
predisposing factors include LBW, lack of
breastfeeding, poor cord care, superficial infection
(pyoderma, umbilical sepsis), aspiration of feeds, and
disruption of skin integrity with needle pricks and use
of intravenous fluids.
In the literature, however there is little consensus as
to what age limits apply, with EOS ranging from 48
hours to 7 days after delivery. This makes it difficult
to compare studies where cases are grouped into EOS
and LOS without further details.
Neonatal meningitis occurs in 2-4 cases per 10000
live births and contributes significantly to mortality
from neonatal sepsis; it is responsible for 4% of all
neonatal deaths .Since studies have shown the
presence of neonatal meningitis in the absence of
bacteremia it becomes significant to also examine the
CSF culture to exclude the presence neonatal
meningitis [5] .
A wide variety of bacteria may cause neonatal sepsis,
to compound the problem, regional and temporal
differences in etiological agents exist. The
uncertainty surrounding the clinical approach to the
treatment of neonatal septicemia can be minimized by

periodic epidemiological surveys of etiological agents
and their antibiotic sensitivity patterns leading to
recognition of the most frequently encountered
pathogens in a particular setting.
The epidemiological data from other developing
countries shows important differences in the
incidence, risk factors, pattern and antimicrobial
sensitivities of the pathogens and mortality from that
of developed countries. Group B streptococci disease
is the most important cause of neonatal sepsis in
Europe and North America, but there is a
preponderance of gram negative organisms in tropical
and developing countries like ours [6].
As neonatal septicemia is a life-threating emergency
and delays in diagnosis and treatment with
appropriate antibiotics may have devastating
consequences, surveillance is needed to identify the
common signs and the pathogens of neonatal
septicemia in a particular area.
The purpose of this research is to give an overview
of the burden of bacterial sepsis and meningitis in the
newborn population in a rural setup. The focus will
be on the pathogens mostly implicated and their
antibiotic susceptibility pattern, as knowledge of the
bacteriological profile of the etiologic agents would
help to reduce the associated mortality in neonatal
septicemia.
This is retrospective study at a rural tertiary hospital
to determine the bacteriological profile of neonatal
septicemia and the antibiotic sensitivity patterns of
the isolated bacteria from the suspected blood and
CSF samples. A retrospective analysis of 1111 blood
samples and 601 CSF samples showing clinical
picture suggestive of neonatal sepsis was done for a
period of 2 years.
Aims & Objectives:
1. To find out the percentage of neonatal septicemia
among the suspected blood & CSF samples coming to
Microbiology Department in a rural tertiary hospital.
2. To determine the percentage of early onset sepsis
& late onset sepsis in confirmed cases.
3. To determine the bacteriological profile of neonatal
septicemia in a rural tertiary hospital.
4. To determine the antibiotic sensitivity of the
isolated bacteria.
METHODOLOGY
653
Sneha et al.,
Study design: This is a retrospective observational

review of the reports of blood cultures & CSF
cultures of all suspected cases of neonatal septicemia
in a tertiary rural hospital for a period of two years.
Ethical consideration: Approval from Institutional
Ethical Committee was dully taken and study was
done after ethical clearance. Data collected from
Medical records
The reports were obtained from the records of
Microbiology department and were subjected to the
following eligibility criteria:
Inclusion criteria: All the blood and C.S.F. samples
of suspected cases of neonatal septicemia were
considered. Sepsis was suspected if the mother
showed evidence of chorioamniotis, prolonged
rupture of membranes, diarrhea, fever or urinary tract
infection and the neonate manifested systemic signs
such as lethargy, chest retraction, grunting,
abdominal distension, tachycardia, hypothermia etc.
Exclusion criteria: Cases of incomplete reporting
were excluded from consideration. Aerobic spore
bearers, wherever grown, were regarded as
contaminants and excluded.
All the reports fulfilling the above eligibility criteria
were studied for
1. Percentage of neonatal sepsis in suspected blood
and CSF samples
2. Percentage of bacteriological sepsis (Grampositive
and
Gram-negative
sepsis),
Bacterialogical profile
3. Percentage of EOS (Early Onset Sepsis) and LOS
(Late Onset Sepsis) in confirmed cases
4. Antibiotic Sensitivity Tests
Statistical analysis: Data was tabulated by using the
Microsoft excel and data was presented as
percentage.
RESULTS
(i) Percentage of neonatal sepsis in suspected
blood and CSF samples : During the study period of
2 years, 1712 neonates were clinically diagnosed as
having sepsis of which 259 (23.31%) out of 1111
were confirmed by blood culture and 36 (5.99%) out
of 601 were confirmed by CSF culture.
(ii) Percentage of Gram-positive and Gramnegative sepsis: Of the 295 organisms isolated 175
(59.32%) were gram negative and 120 (40.67%) were
gram positive. The predominant gram negative
organism isolated were Klebsiella (48.57%) and
Pseudomonas (26.85%) followed by Acinetobacter

and E.coli (both 10.28%) as shown in Fig.1 The
predominant organisms isolated among the gram
positive were CONS (44.16%) and Staph.aureus
(37.50%) as shown in Fig 2.
(iii) Percentage of EOS (Early Onset Sepsis) and
LOS (Late Onset Sepsis) in confirmed cases :
Among the positive cases 273 (92.54%) were EOS
and 22 (7.45%) were LOS as shown in Fig.3.The
predominant organisms in EOS were Klebsiella
(29.30 %) followed by CONS (17.21%),
Pseudomonas (16.84%) and Staph.aureus (15.38%) as
shown in Fig.4.The predominant organisms in LOS
were CONS (27.27%),Klebsiella (22.72%) and
Staph.aureus (13.63%) as shown in Fig.5.
(iv)Bacteriological profile of neonatal sepsis : In
this study the predominant organisms were found to
be Klebsiella (28.81%) followed by CONS (17.96%),
Pseudomonas (15.93%) and Staph.aureus (15.25%) as
shown in Fig. 6.
(v) Antibiotic Sensitivity testing : The antibiotic
sensitivity testing showed that most of the gram
negative isolates were sensitive to meropenem
followed by chloramphenicol, ciprofloxacin ,
gentamicin and amikacin. They were not sensitive to
the commonly used antibiotics like penicillin,
ampicillin etc as shown in Fig.7.
The antibiotic sensitivity testing of the gram positive
isolates showed that they were maximally sensitive to
Linezolid, Netilmicin and Chloramphenicol as shown
in Fig.8.
Gram-Negative isolates (175)

85
47
18
18
Fig 1: Gram Negative Organisms isolated
654
Sneha et al.,
53
Gram-Positive isolates (120)
1%
1%
1%
1%
45
Klebsiella
6%
6%
18
29%
CONS
6%
2
Pseudomonas
2
15%
Staph.aureus
18%
E.coli
16%
Fig 2: Gram Positive Organisms isolated
Fig 6: Bacteriological profile of Neonatal Sepsis

Antibiotic sensitivity of Gram negative
% of EOS and LOS in confirmed cases

7%
EOS
LOS
sensitivity
100
80
60
40
20
0
resistance
93%
Fig 3: Percentage of EOS (Early Onset Sepsis) and

LOS (Late Onset Sepsis) in confirmed cases
Early Onset Sepsis (273)

1
3
2
2
17
20
40
60
80
100
Fig 4: Organisms isolated in Early Onset Sepsis
Late Onset Sepsis (22)

Citrobacter
1
1
Proteus
Acinetobacter
Staph.aureus
2
2
3
Klebsiella
5
0
6
6
Fig 5: Organisms isolated in Late Onset Sepsis
Linezolid
Netilmicin
80
Ampicillin
Klebsiella
Ciprofloxacin
47
46
Erythromyc
CONS
Amikacin
42
16
17
Gentamicin
Acinetobacter
50
Penicillin
Enterococcus
Sensitivity
Resistance
Cotrimoxaz
Micrococci
100
Chloramph
Enterobacter
Fig 7: Antibiotic Sensitivity of Gram negative

organisms
Antibiotic sensitivity of Gram positive
Fig 8: Antibiotic Sensitivity of Gram positive

organisms
DISCUSSION
Clinical recognition of neonatal sepsis is not always
straight-forward .Appropriate intervention requires an
early etiological diagnosis. For effective management
of neonatal sepsis cases , study of the bacteriological
profile with their antibiotic sensitivity pattern plays
an important role .Several studies on neonatal sepsis
have documented the diversity of bacteria and their
temporal variability. The present study reiterates the
earlier findings and emphasizes the importance of
655
Sneha et al.,
periodic surveys of microbial flora encountered in

particular neonatal settings to recognize the trend.
The culture positivity of the blood samples were
23.3% and the CSF samples were 5.99% .Similar
blood culture positivity of around 24.88% was
reported by Mathur et al in their study[7]. But a
significantly higher blood culture positivity rate of
42% was reported by Ghanshyam et al in their study
in a tertiary care hospital in India [8] and a lower
blood culture positivity rate of 13.7% was reported by
Kaistha et al in their study [9].The CSF culture
positivity rates in the study by Katiyar et al was
2.06% which is lower than the findings in our study
[10]
.
The low culture isolation rate in this study might be
due to several reasons like administration of
antibiotics before blood or CSF collection either to
the mother or to the infant .Also the possibility of
infection with anaerobes cannot be ruled out
.Negative culture does not exclude sepsis as cases
with negative blood culture have been reported with
fatal illness and post-mortem evidence of infection
.Chow et al reported that 26% of all neonatal
septicemia was caused by anaerobes [11].
Early onset sepsis was found in majority of the
confirmed cases rather than late onset sepsis .The
percentage of EOS in this study was found to be
92.54% with Klebsiella, CONS, Pseudomonas and
Staph. aureus being the predominant pathogens
causing EO. The LOS was reported in 7.45%
cases,with CONS, Klebsiella and Staph.aureus
causing the LOS. Higher culture positivity rates in
LOS were reported by P. Jyothi et al (25.2%) and
Kaistha et al (14.18%) which differs from this study
[12]
.All the other studies reported higher culture
positive rates in EOS except in a study by Ahmed et
al in Bangladesh which reported the positivity rates of
EOS as low as 26%. They reported that the higher
mortality rates in early onset cases to be the reason
for this discrepancy [13] .
In this study, Gram negative organisms were isolated
in about 59.32% of the cases with Klebsiella
(48.57%) and Pseudomonas (26.85%) being the
predominant ones among the gram negative
organisms. Similar findings was reported by
Ghanshyam et al with 60% gram negative isolates
with Klebsiella as the predominant gram negative
isolate [8].Comparatively a higher incidence of gram
negative isolates was found in the study of Mathur et

al (87.1%) [7].
In this study Gram Positive were isolated in 40.67%
cases with CONS and Staph.aureus being the
predominant pathogen. In the study by P.Jyothi et al,
they also found CONS to the predominant Gram
positive organism [12] but majority of the other studies
reported Staph. aureus to be the predominant Gram
positive organism over CONS [8, 9, 14] .Various studies
have shown that in the last two decades, the isolation
of Gram positive organisms has increased
significantly [9].But nevertheless the predominance of
gram negative corroborates with the findings of other
studies done in the Indian context. Many studies have
shown the preponderance of gram negative organisms
in tropical and developing countries also [13, 15].
Klebsiella was found to be the predominant pathogen
causing neonatal sepsis which is in accord with many
studies like that of Ghanshyam et al (33.8%) ,
Zakariya et al (66%) , Mathur et al (38.5%) ,Kaistha
et al (28.3%) and P.Jyothi et al (31%) which were
done in India .Other studies from developing
countries also found Klebsiella as the common
organism. But studies done by Ahmed et al, Agnihotri
et al and Bhat et al showed the predominant pathogen
to be E.coli (30%),Staph.aureus (35.3%) and
Pseudomonas (33.2%) respectively .
Group B Streptococcus (GBS) was not isolated in this
study , unlike western , developed countries where it
is the major agent of neonatal septicemia This may be
attributed to low prevalence of GBS colonization of
pregnant women in this area or possibly , to the
presence of strains with low virulence [13].Since a
sizeable number of culture specimens were negative
by aerobic culture , the possibility of infection by
anaerobes must be entertained and anaerobic culture
can be performed routinely in cases of neonatal sepsis
[8]
. However, the feasibility, logistics and costeffectiveness of routine anaerobic culture for neonatal
sepsis need to be explored further.
The antibiotic sensitivity testing showed that most of
the gram negative isolates were sensitive to
meropenem
followed
by
chloramphenicol
,ciprofloxacin ,gentamicin and amikacin .They were
not sensitive to the commonly used antibiotics like
penicillin , ampicillin etc .The antibiotic sensitivity
testing of the gram positive isolates showed that they
were maximally sensitive to linezolid ,netilmicin and
cholramphenicol .This is comparable to the study
656
Sneha et al.,
done by P. Jyothi et al in which maximum sensitivity

was observed in imipenem and linezolid [12]
.Netilmicin and amikacin was found to be highly
sensitive in the study done by Agnihotri et al for
Staph.aureus and gram negative isolatesrespectively
[1]
.While the study by Mathur et al showed
Gentamicin to be sensitive in gram negative cases
[7]
.Many studies also reported cefotaxime to have
shown maximum sensitivity [7] but it could not be
compared as some of the samples from this study had
not undergone the sensitivity testing for cefotaxime .
Besides the antimicrobial sensitivity patterns differs
in different studies as well as at different times in the
same hospital.This is because of emergence of
resistant strains as a result of indiscriminate use of
antibiotics.Thus ,Meropenem and Linezolid were
found to the most sensitive drugs for gram negative
and gram positive respectively , but these two drugs
should not be used indiscriminately and kept as
reserve drugs , otherwise resistance to these drugs
may develop , thereby threatening the treatment .
Therefore, to conclude, an effective infection control
programme which will among others ensure good and
effective
hand
washing,
regular
antibiotic
susceptibility surveillance and evaluation, and the
enforcement and periodic review of the antibiotic
policy of the hospital as well as the encouragement of
rational antibiotic use will reduce the rates of
acquiring neonatal infections and development of
bacterial resistance.
CONCLUSION
Klebsiella was found to be the most common agent
causing the neonatal sepsis followed by CONS,
Pseudomonas, Staph.aureus and E.coli .Gram
Negative sepsis predominated in this study. Early
onset sepsis was found to be the major burden of
neonatal sepsis in the rural setting.
Meropenem was found to the most sensitive drug for
gram negative and Linezolid for gram positive. In the
gram negative samples Cefazolin was found to be
most resistant drug and Penicillin and Ampicillin in
the gram positive.
Acknowledgement: We acknowledge all the faculty
members of the Department of Microbiology for their
help and cooperation for this study.
Conflict of interest: None declared
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Ritu Garg and Jagdish Chander, Neonatal
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10. Katiyar R and Bose S, Bacteriological profile of
neonatal septicemia in Pravara Rural hospital ,
Pravara Med review 2012, 4(2):4-6
11. Chow A W, Leake, R. D. and Terry Y, The
significance of anaerobes in neonatal bacteremia :
analysis of 23 cases and review of the literature,
Pediatrics ,1974, 54:736-745
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Basavaraj, Bacteriological profile of neonatal
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Chowdhury, Mahbul Hoque and Gary L.
Darmstadt, Clinical and Bacteriological Profile of
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14. Zakariya B P, Bhat V, Harish BN, Arun Babu T
and Joseph NM, Neonatal sepsis in a tertiary care
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658
Sneha et al.,
DOI: 10.5958/2319-5886.2015.00125.3

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
th
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
th
ECTOPIC PREGNANCY AFTER SEQUENTIAL EMBRYO TRANSFER: REVIEW OF 22 CASES

Nadkarni Purnima K, Nadkarni Kishore, Singh Pooja P, Singh Prabhakar , Nadkarni Aditi A , *Agarwal Neha R
21st century Hospital & Test Tube Baby Centre, Surat, Gujarat, India
*Corresponding author email: nehaagarwal1827@gmail.com
ABSTRACT
Objective: To assess the prevalence of ectopic pregnancy among women who conceived with assisted
reproductive technology and to see if there is increased risk after sequential embryo transfer. Methods: The
ectopic pregnancy rate for ART pregnancies was calculated among women who conceived and had ectopic
pregnancy after ICSI followed by Sequential embryo transfer from an ART centre. Variation in ectopic risk by
patient and ART treatment factors was assessed including Sequential transfer, risk factor for ectopic pregnancy,
tubal infertility and previous ectopic pregnancy. Results: Of 1960 women, who underwent ICSI, 1047(53.41%)
had positive pregnancy, 22(2.1%) women had an ectopic pregnancy out of which 2(9%) had heterotrophic
pregnancy. The mean age was 30.44.33. Tubal factor contributed to 31.81% cases and 27.27% had previous
history of ectopic pregnancy. Sequential transfer was done in all the patients with more than one embryo
transferred. There was no significant increase in the ectopic pregnancy when Sequential embryo transfer was
done. Conclusion: Tubal factor infertility and history of previous ectopic pregnancy contributed to risk factor for
ectopic pregnancy in our study. Sequential transfer did not increase the risk of ectopic pregnancy.
Keywords: Ectopic Pregnancy, Assisted Reproductive Technology, In Vitro Fertilisation, Intracytoplasmic Sperm
Insemination.
INTRODUCTION
Ectopic pregnancy is an important cause of maternal
morbidity and is one of the recognised complications
of In Vitro Fertilisation In Vitro Fertilisation (IVF).
Extra uterine Pregnancy is the implantation of the
embryo anywhere except in the endometrial lining of
the uterine cavity [1]. The aetiology of ectopic
pregnancy is not well understood. However, multiple
risk factors have been associated with ectopic
pregnancy [2]. Other etiological risk factors are
tubal/pelvic surgeries, endometriosis, exposure to
diethylstilbestrol in utero, chromosomally abnormal
embryo, use of progesterone-only pills, cigarette
smoking, conception following induction of ovulation
and in vitro fertilization and number of embryo
transfer (assisted reproductive technology), history of
previous abortion, previous ectopic pregnancy,
history of infertility, race, and age above 35 years.

However, ectopic pregnancy (EP) can also occur
without any obvious risk factors[3,4].
In the results generated from the American society for
reproductive Medicine in 2001, there were 560
ectopic pregnancies, representing 1.8% of all
pregnancies following IVF [5]. The incidence of
ectopic pregnancy after IVF ranges from 2.1% to
9.4% of all clinical pregnancies [6-10]. Multiple embryo
transfer has always been associated with increased
risk of EP with transfer of two or less embryos
carrying lower risk than after three or more. [12] The
current guidelines on the number of embryos to
transfer, which are based on the maternal age, could
therefore decrease the incidence of EP following IVF
Neha et al.,
Int J Med Res Health Sci., 2015;4(3):659-661
659
especially in younger patients with a single embryo

transfer [13].
The purpose of our study was to see the incidence of
ectopic pregnancy after sequential embryo transfer
and effects of various factors (tubal, previous ectopic,
number of embryos transferred) on it.
Study design: Retrospective study
Study Duration: Study done over a period of 2 years
2013-2014 at 21st century infertility centre, Surat.
Ethics approval: Study was approved by the Ethics
Review Board
Inclusion criteria: Patients who underwent infertility
treatment in the form of ICSI (Intracytoplasmic
Sperm Insemination) but had Ectopic Pregnancy were
included in this study.
Methodology:
Total 1960 patients underwent Assisted Reproductive
Technology (ART),
(Intracytoplasmic Sperm
Insemination) for varied infertility reasons.
Information was retrieved from the ART centres
medical files.
Age of the patients was noted. All the associated risk
factor for ectopic pregnancy was noted this included
previous history of ectopic pregnancy, tubal factor
previous operative history of myomectomy or
endometriosis, and pelvic infection. In our study
patients had Sequential Embryo Transfer on day2/3
and day5/6. Our study was compared to a similar
study Monteral QC 2011(8) but they did not
Sequential Transfer done.
Statistical analysis: Done by using descriptive and
inferential statistics using Chi square test. The
software used in the analysis was Graph Pad Prism
5.0 and p<0.05 is considered as level of significance.
RESULTS
Table 1:
Cases
Age
Incidence
Tubal factor
Our
Monteral
Study QC Study[8]
30.44.3 34.21
2.1%
4.9%
31.8%
33.3%
P-value
0.056
0.44
1.00
Previous ectopic
Previous
myomectomy
27.27%
9%
16.6%
5.6%
0.12
0.59
Previous endometriosis
operated
22.72%
22.2%
1.00
9%
5.6%
0.59
Pelvic infection
Neha et al.,
In our study, all the patients had sequential transfer

on day 2/3 and day 5/6. Out of total1047 positive
patients, 1013 had 2+1 transfer rate (i.e 2 embryos on
day2/3 and 1 on day5/6) and 34 had 2+2, these were
the patients who previous more than 2 IVF failures or
with had advanced maternal age. In our study 20
patients had 2+1 embryo transfer and only 2 had 2+2.
The incidence of ART ectopic pregnancy was 2.1%.
Mean age was 30.3914.33.
Various factors for infertility and its association with
ectopic pregnancy rate were seen. There was
increased incidence of ectopic pregnancy in patients
having tubal factor as one of the cause for infertility
and ectopic pregnancy. It was also noted that 27.27%
of the patients had previous history of ectopic
pregnancy. Heterotrophic pregnancy was seen among
2 patients of 22 cases. There was no statistical
difference between two groups. It was seen that
sequential Transfer of more than one embryo did not
increase the ectopic pregnancy rate.
DISCUSSION
Despite the fact that IVF allows direct implantation of
embryos with the complete bypass of the fallopian
tubes, extra uterine pregnancies are more common
following IVF than natural conceptions and have
been a well recognised complication since the first
occurrence reported in 1976[12].
The incidence of ectopic pregnancy in our centre was
2.1% this was less as compared to a study in
Montreal[8] which had an rate of 4.9% still it was not
statistically significant . The incidence was similar to
a natural ectopic pregnancy rate of 2.1% in Nigeria
[14]
. The mean age of patients with ectopic was
30.314.33 this was correlating with Montreal study
[8]
which had an mean age of 34.21.
The incidence of ART ectopic varies from 1.8% to
11%among those with tubal factor infertility [6]. The
rate of ectopic pregnancy was seen more with tubal
factor infertility which was around 31.81% and this
was not statistically significant as compared with
Montreal group. This was shown by Strandell et.al &
Mohammed malak et al, that tubal factor one of the
risk factor for infertility [7,8]. These authors reported
an incidence of 4% of ectopic pregnancy, which was
higher compared to our study.
History of previous ectopic pregnancy was also
increase the risk for ectopic pregnancy [9]. Our study
showed 27.27% of patients with ectopic pregnancy
660
had a previous history of ectopic pregnancy this was

higher as compared to 16.6% [8] but had no statistical
significance.
Previous history of surgeries for endometriosis [15] and
myomectomy also increase the risk for ectopic
pregnancy. Our study also had patients with previous
history of endometriosis and myomectomy but the
number of patient in the study group was small.
All the patients with ectopic pregnancy had
sequential transfer with more than one embryo
transferred .There was no significant difference with
number of embryos transferred and the ectopic
pregnancy rate. This matched with Zhonghua Fu
Chan Ke Za Zhi[12]. Whereas compared to Chai [16].
Our study did not correlate as they found increase in
rate of ectopic pregnancy with increase in number of
embryos transferred which was not increased in our
study.
CONCLUSION
Tubal factor contributes to one of the risk factor for
ectopic pregnancy. Sequential transfer of more than
one embryo did not increase the risk of ectopic
pregnancy in our study.
REFERENCES
1. Clayton HB, Schieve LA, Peterson HB, Jamieson
DJ, Reynolds MA, Wright VC. Ectopic
pregnancy risk with assisted reproductive
technology
procedures.
obstet.
Gynecol.
2006;10:595-604.
2. Abdul FI. Ectopic pregnancy in Ilorin: a review
of 278cases Med 2000;9(3):92-96.
3. Gharoro EP, Igbafe AA. Ectopic pregnancy
revisited in Benin City, Nigeria: analysis of 152
cases.
Acta
Obstet
Gynaecol
Scand.
2002;81(12):1139-43.
4. Drife JS. Tubal pregnancy; rising incidence,
earlier
diagnosis,
more
conservative
management. B J.1990;301:1057-58
5. Society for Assisted Reproductive Technology;
American Society for Reproductive Medicine.
Assisted reproductive technology in united states:
2001 results generated from the American
Society for Reproductive Medicine /Society for
assisted reproductive tehnology registry. Fertil
Steril 2007;87:1253-66.
6. Ggunby J, Bissonnette F, Librach C, Cowan L.
IVF Directors Group of the Canadian Fertility
and Andrology Society . Assisted reproductive

technologies (ART) in Canada :2004results from
the Canadian ART Register. Fertil Steril
2008;89:1123-32.
7. Strandell A , Thorburn J, Hamberger L . Risk
factor for ectopic pregnancy in assisted
reproduction. Fertil Steril 1999;71:282-6
8. Mohammed Malak, Tawfeeq, hananel Holzer,
Togas Tulandi. Ectopic pregnancy, in vitro
fertilization.
J
Obstet
Gynaecol
Can
2011;33(6):617-619.
9. Murray H, Baakdah H Bardell T, Tulandi T.
Diagnosis and treatment of ectopic pregnancy.
CMAJ2005;173:905-12.
10. Azem F, Yaron Y, Botchan A. Ectopic pregnancy
after in vitro fertilization embryo
transfer(IVF/ET): the possible role of the ET
technique .J Assist Reprod Genet 1993;10:302-4.
11. Steptoe PC, Edwards RG.1976.Reimplantation of
a human embryo with subsequent tubal
pregnancy. Lancet. 1976;55: 880-882.
12. Zhonghua Fu Chan Ke Za Zhi. Ectopic
pregnancy after in vitro fertilization and embryo
transfer clinical analysis of 17 cases:
200;36(7):411-3.
13. Pandian Z, Marjoribanks J, Ozturk O, Serour G,
Bhattacharya S. Number of embryos for transfer
following in vitro fertilisation or intracytoplasmic sperm injection. Cochrane Database
Syst Rev. 2013; 7: Article ID CD003416
14. Osaheni L Lawani, Okechukwu B Anozie, Paul O
Ezeonu. Ectopic pregnancy: a life threatening
gynaecological emergency. International Journal
of Womens health 2013:5;515-521.
15. Li C, Meng CX, Zhao WH, Lu HQ, Shi W,
Zhang J. Risk factors for ectopic pregnancy in
women with planned pregnancy: a casecontrol
study. Eur J Obstet Gynecol Reprod Biol. 2014;
181:176-82.
16. Chai J, Yeung TW, Lee VC, Li RH, Lau EY,
Yeung WS et al.. Live birth rate, multiple
pregnancy rate, and obstetric outcomes of
elective single and double embryo transfers:
Hong Kong experience. Hong Kong Med J.
2014; 20:102-6.
661
Neha et al.,
DOI: 10.5958/2319-5886.2015.00126.5

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
th
Received: 5 Jun 2015
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
HYPOXIC STATUS AND ITS PROGNOSIS IN PATIENTS WITH HEAD INJURY

*Sangle Ankita1. Rahul Kunkulol R2. Shaikh Meena3, Ameya Sangle4
1
IIIrd MBBS Student, Rural Medical College, PIMS-DU, Loni, Maharashtra

Coordinator, Directorate of Research, PIMS-DU, Loni, Maharashtra
3
Professor, Department of Surgery, Rural Medical College, PIMS-DU, Loni, Maharashtra
2
*Corresponding author email: ankita_sangle@yahoo.co.in

ABSTRACT
Objectives: The study was undertaken to investigate the effect of decreased oxygenation status in a patient of
head injury and its outcome in terms of prognosis of patients with regards to neurological damage. The objective
was to find out a co-relation between the SpO2 levels in a patient on admission and the severity of head injury.
Methods: Prospective observational study was carried out after the approval of Institutional ethical committee.
Patient with head injury coming to the emergency department of Pravara Rural hospital were assessed for the
severity using Glasgow Coma scale (GCS) and hypoxic status by using pulse oximeter with bedside monitors.
The SpO2 levels were monitored at 2hrs, 4hrs, 8hrs, 24hrs and 48hrs by pulse oximeter. At the time of discharge
outcome was assessed again by Glasgow Outcome Scale (GOS) and the treatment that followed it. Results &
Conclusions: Oxygenation status of patient on admission affects the prognosis of the patient in terms of residual
neurological deficit as assessed by the Glasgow Outcome scale. There is significant co-relation between the
Oxygen saturation at the time of admission and the outcome at the time of discharge. Therefore the SpO2 values
are significant in patients with head injury as a prognostic factor.
Keywords: Hypoxia, Glasgow Outcome Scale, Glasgow Coma scale, SpO2, pulse oximeter.
INTRODUCTION
Traumatic brain injury (TBI) is the result of an
external mechanical force applied to the cranium and
the intracranial contents, leading to temporary or
permanent impairments, functional disability, or
psychosocial maladjustment.[1,2]
TBI is the major cause of death related to injury. The
risk of TBI peaks when individuals are aged 15-30
years. The risk is highest for individuals aged 15-24
years. Men are approximately twice as likely as
women to sustain a TBI.[3]Among children aged 0-14
years. An estimated 475,000 TBIs occur each year.[4]
TBI is usually classified based on severity,
anatomical features of the injury, and the mechanism
(the
causative
forces).[5]
Mechanism-related
classification divides TBI into closed and penetrating
Ankita etal.,
head injury. [6] A closed (also called non-penetrating,

or blunt) [7] injury occurs when the brain is not
exposed[8]. A penetrating, or open, head injury occurs
when an object pierces the skull and breaches the
dura mater, the outermost membrane surrounding the
brain[8].
Medical complications like hypotension (low blood
pressure), hypoxia (low blood oxygen saturation),
lower cerebral perfusion pressures and longer times
spent with high intracranial pressures are associated
with a bad prognosis[9,10]. Prognosis worsens with the
severity of injury[11]. Most TBIs are mild and do not
cause permanent or long-term disability; however, all
severity levels of TBI have the potential to cause
significant, long-lasting disability[12]. Permanent
662
disability is thought to occur in 10% of mild injuries,

66% of moderate injuries, and 100% of severe
injuries[13].
The brain needs a continuous supply of oxygen to
survive. About 20% of the body's oxygen intake is
utilized by the brain, more than any other organ.
Metabolism in the brain relies on glucose as the main
energy source and oxygen is required for the
metabolic process. Interruption of the oxygen supply
can lead to the impairment of the functioning of the
brain and can cause irreversible injury. A complete
interruption of the supply of oxygen to the brain is
referred to as cerebral anoxia. Partial or inadequate
supply of oxygen to the brain is known as cerebral
hypoxia.
SpO2 stands for Peripheral capillary oxygen
saturation. It is an estimation of the oxygen saturation
level of the blood. Oxygen saturation is a term
referring to the concentration of oxygen in the blood.
It measures the percentage of hemoglobin binding
sites in the bloodstream occupied by oxygen. The
range for normal oxygen saturation in the blood is 95100 percent.
In assessment of severity of brain injury the Glasgow
Coma Scale (GCS) is the most common scoring
system used whereas Glasgow Outcome Scale (GOS)
is used for assessing global outcome following a
brain injury.
Various studies have shown the effects of multiple
factors on the prognosis of patients of severe
traumatic brain injury. The prognostic outcomes were
strongly correlated with GCS score, age, pupillary
response and size, hypoxia, hyperthermia, and high
intracranial pressure (ICP). These findings indicate
that prevention of hypoxia may be useful means for
improving the outcome of patients with severe head
injury[14].
In view of the aforementioned use of assessment
scales it was thought prudent to evaluate the
traumatic brain injury patients coming to the PRH for
their hypoxic status and the final outcome.
OBJECTIVES:
1. To study severity of head injury and SpO2
levels at the time of admission.
2. To observe the outcome of head injury in
the patients in relation to SpO2 status at
discharge.
Ankita etal.,
3. To study the oxygen saturation level at

admission and the outcome at discharge.
MATERIALS AND METHOD
Study design: This was a prospective observational
study done in collaboration with Department of
Surgery.
Ethics approval: The Institutional ethical committee
approval was obtained before the initiation of the
study, inform consent was obtained from each patient
Inclusion criteria: 1. Patients with Traumatic Brain
Injury, above 18 years of age of Patients of either sex,
Patients in whom assessments feasible.
Exclusion criteria: Pregnancy or breast feeding,
Patients with history of cerebro-vascular accident,
epilepsy, psychological illness, Patients addicted to
alcohol, drugs or on any other medication, Patients
receiving cardio pulmonary resuscitation.
Study period: 6 months
Sample size: 25 patients were included according to
inclusion and exclusion criteria.
Methodology: Patients satisfying the inclusion
criteria were assessed till discharge. Total of 25
patients admitted in the ICU following RTA/Head
Injury were monitored for SpO2 levels at the time of
admission and at the intervals of 2hrs, 4hrs, 8hrs,
24hrs, 48hrs and at discharge with regards to the vital
status of the patient.
1. The severity of head injury was evaluated by
Glasgow Coma Scale (GCS) at the time of
admission.[15]
2. Regular follow up of the patient condition was
monitored up to the discharge of the patient from
hospital.
3. CNS assessment of the patient was done on
discharge to assess for residual disease.
4. Vitals of patients were monitored during
treatment to assess the prognosis of the patient.
5. Glasgow Outcome Scale (GOS) was used to
exam the outcome of patients at the time of
discharge.
6. Outcome of patients with score <5 on the GOS
was considered not favorable.
663
Evaluation of severity of head injury:

The severity of head injury was assessed by Glasgow coma scale.[15]
Glasgow Coma Scale (GCS)
1
2
3
4
Eye
Does not
Opens eyes in response
Opens eyes in
Opens eyes
open eyes
to painful stimuli
response to voice
spontaneously
Verbal
Makes no
Incomprehensible
Utters inappropriate
Confused,
sounds
sounds
words
disoriented
Motor
Makes no
movements
Extension to painful
Abnormal flexion to
stimuli (decerebrate
painful stimuli
response)
(decorticate response)
Evaluation of outcome of the treatment with
regard to residual neurological deficit:
Outcome of patients was assessed by Glasgow
Outcome Scale (GOS).[16]
Glasgow Outcome Scale (GOS)
Scale
Description
5 (Good
outcome)
Resumption of normal life; there may be

minor neurological and/or psychological
deficits.
Able to work in a sheltered environment and
travel by public transportation.
Dependant for daily support by reason of
mental or physical disability or both.
Unresponsiveness and speechless for
weeks or months until death.
4 (Moderately
disabled)
3 (Severely
disabled)
2 (Persistent
vegetative
state)
1 (Death)
Not applicable.
Statistical analysis: The data was collected, pooled,
subjected to appropriate statistical analysis and
conclusions were drawn.
RESULTS:
8%
4%
ROAD TRAFFIC ACCIDENT

88%
FALL FROM HEIGHT
ASSAULT
Fig 1: Distribution of patients according to the

cause of head injury.
Ankita etal.,
Flexion / Withdrawal
to painful stimuli
5
N/A
6
N/A
Oriented,
converses
normally
Localizes
painful stimuli
N/A
Obeys
commands
Fig 2: Age wise distribution of patients with head

injury.
Table 1: Distribution of patients according to their
gcs score at the time of admission.
GCS SCORE NO. OF PATIENTS %
3/15
0
0
4/15
0
0
5/15
0
0
6/16
1
4
7/15
2
8
8/15
1
4
9/15
1
4
10/15
0
0
11/15
1
4
12/15
0
0
13/15
4
16
14/15
4
16
15/15
11
44
Total
25
100
664
68%
80
60
40
N 20
o 0
8%
GOS 5; good
outcome; no
neurological
deficit
less than 95
more than 95
20%
4%
Poor outcome;
neurological
deficit or death
Fig 3: Distribution of patients according to their

spo2 level at the time of admission.
By applying Z test of difference between two
proportions there is a significant difference between
proportions of SpO2 level at the time of admission
less than 95 and more than 95 in GOS 5 and poor
outcome (i.e. p<0.05).
Fig 4: Distribution of patients according to their

glasgow outcome scale score
DISCUSSION
This study was undertaken to investigate the effect of
decreased oxygenation status in a patient of head
injury and its outcome, and examines the prognosis of
patients with regards to neurological damage.
Maximum cause of head injury that is 88% is due to
road traffic accident whereas it is 8% due to fall from
height and only 4% due to assault (graph 1). The age
wise distribution of patients with head injury shows
about 40% of the patients belong to age group of 1827yrs. The total of 4% patients belonged to age group
of 78-87yrs. 24% patients belonged to both age
groups of 28-37yrs and 38-47yrs. Total of 8%
patients were in age group of 58-67yrs (graph 2).
Distribution of patients according to their Glasgow
coma scale (GCS) score at the time of admission
shows maximum of 44% patients had GCS score of
15/15; while each 13/15 and 14/15 GCS score had
Ankita etal.,
16% patients at the time of admission (table 1).

Distribution of patients according to their SpO2 level
at the time of admission showed 68% patients with
SpO2 level more than 95 and GOS 5 while 8%
showed SpO2 level less than 95. 20% patients with
poor outcome, neurological deficit or death GOS <5
had SpO2 more than 95 while 4% had less than 95. Z
test applied on the observed data proved the
significance of co-relation between the SpO2 levels
and the prognosis (graph 3). Outcome of patients with
head injury at the time of discharge evaluated by the
Glasgow outcome scale (GOS) shows 76% of the
patients had a good outcome with a GOS score of 5
while 16% of total were moderately disabled and
needed assistance and 8% of patients died during the
course of treatment even though oxygenation
saturation was ensured and further hypoxia was
prevented (graph 4).
This study showed that in spite of high GCS score
and near normal SpO2 levels, mortality in patients
was observed. The study sample was male
dominated. The results of the study are in accordance
with the studies conducted previously although there
are differences to the experimental design. Firstly, in
this study, hypoxia was measured with pulse
oximetry and was not validated with arterial blood
gas analysis. Thus, it is possible that episodes of
hypoxia were not detected. Secondly, the study
populations were different. Here the sample
population, whose median age was 40 years, was
older than those from the Chestnut et al study in
Traumatic Coma Data Bank with (median age, 25
years) and the average admission GCS was higher (14
Vs 3). Further investigation into the effects of
hypotension and hypoxia on patients with head injury
is warranted since the physiological effects of these
secondary insults may be different in different
population age groups. Consideration of other insults
and other parameters simultaneously could be
considered to have a better reliable prognosis as all
such variables are not independent of each other. The
study concludes that the oxygenation status of the
patient at the time of admission affects the prognosis
in a patient with head injury and that early oxygen
support and maintenance of SpO2 levels could ensure
a good prognosis in patients.
665
CONCLUSION
This study arrives at a conclusion that oxygenation
status of patient on admission affects the prognosis of
the patient in terms of residual neurological deficit as
assessed by the Glasgow Outcome scale. There is
significant co-relation between the Oxygen saturation
at the time of admission and the outcome at the time
of discharge. Therefore the SpO2 values are
significant in patients with head injury as a prognostic
factor.
Limitations: Consideration of other parameters
simultaneously with SpO2 levels could be a better and
more reliable prognostic factor as all such variables
may not be independent of each other. O2 levels could
be monitored by ABG analysis with provide a more
accurate value than oximetry and episodic hypoxia
could also be noted.
Acknowledgement: Authors dually acknowledge the
ICMR for selecting this research project for the short
term studentship, the valuable contribution of the
department of surgery, directorate of research and all
the patients for their support and participation in this
study.
REFERENCES
1. Steyerberg EW, Mushkudiani N, Perel P.
Predicting outcome after traumatic brain injury:
development and international validation of
prognostic scores based on admission
characteristics. PLoS Med. 2008;5(8):e165.
2. Niedzwecki CM, Marwitz JH, Ketchum JM.
Traumatic brain injury: a comparison of inpatient
functional outcomes between children and adults.
J Head Trauma Rehabil. 2008;23(4):209-19.
3. Kraus JF, Black MA, Hessol N. The incidence of
acute brain injury and serious impairment in a
defined
population.
Am
J
Epidemiol.
1984;119(2):186-201.
4. Langlois JA, Rutland-Brown W, Thomas KE.
The incidence of traumatic brain injury among
children in the United States: differences by race.
J Head Trauma Rehabil.2005;20(3):229-38.
5. Saatman KE; Duhaime AC Workshop Scientific
Team Advisory Panel Members et al.
Classification of traumatic brain injury for
targeted therapies". Journal of Neurotrauma
2008;25 (7):71938.
Ankita etal.,
6. Maas AI, Stocchetti N, Bullock R. Moderate and

severe traumatic brain injury in adults. Lancet
Neurology. 2008;7 (8): 72841
7. Blissitt PA. Care of the critically ill patient with
penetrating head injury". Critical Care Nursing
Clinics of North America 2006;18 (3): 32132
8. Hannay HJ, Howieson DB, Loring DW, Fischer
JS,
Lezak
MD.
Neuropathology
for
neuropsychologists". In Lezak MD, Howieson
DB,
Loring
DW.
Neuropsychological
Assessment. Oxford [Oxfordshire]: Oxford
University Press.2004;15862. ISBN 0-19511121-4.
9. Maas AI, Stocchetti N, Bullock R. Moderate and
severe traumatic brain injury in adults". Lancet
Neurology.2008; 7 (8): 72841.
10. Frey LC . Epidemiology of posttraumatic
epilepsy: A critical review". Epilepsia.2003; 44
(S10): 1117.
11. Rao V, Lyketsos C. Neuropsychiatric sequelae of
traumatic brain Injury". Psychosomatics.2000;41
(2): 95103.
12. Brown AW, Elovic EP, Kothari S, Flanagan SR,
Kwasnica C. Congenital and acquired brain
injury. 1. Epidemiology, pathophysiology,
prognostication, innovative treatments, and
prevention". Archives of Physical Medicine and
Rehabilitation.2008; 89 (3S1): S38.
13. Zink BJ. Traumatic brain injury outcome:
Concepts for emergency care. Annals of
Emergency Medicine. 2001; 37 (3): 31832.
14. Ji-Yao Jiang, Guo-Yi Gao, Wei-Ping Li, MingKun Yu, and Cheng Zhu. Journal of
Neurotrauma. 2002, 19(7): 869-74.
15. Encyclopedia of Clinical Neuropsychology 2011,
pp 1149-1150 Glasgow Coma Scale, Jerry
Wright.
16. Encyclopedia of Clinical Neuropsychology 2011,
pp 1150-1152 Glasgow Outcome Scale, Jerry
Wright.
666
DOI: 10.5958/2319-5886.2015.00127.7

&
Health Sciences
www.ijmrhs.com
Volume 4 Issue 3
th
Research article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
th
ANTICANCER EFFECTS OF CARICA PAPAYA IN EXPERIMENTAL INDUCED MAMMARY

TUMORS IN RATS
*Gurudatta M1, Deshmukh YA2, Naikwadi A A3

1
Lecturer, 3Professor, BLDE Universitys Shri B. M. Patil Medical College Hospital & Research, Centre, Bijapur,
Karnataka, India
2
Professor & Head Department of Pharmacology, MGM Medical College, Kamothe, Navi Mumbai, Maharashtra,
India
*Corresponding author email: gurudatta.ph@gmail.com
ABSTRACT
Objective: To evaluate the anticancer effect of Carica papaya in DMBA induced mammary tumors in rats.
Methods: Wistar rats were divided in to five groups (n=6), Group-I (Normal control) administered vehicle olive
oil, Group-II, Group-III ,Group-IV and V induced mammary tumors by administering single dose of DMBA (7,12
Dimethyl benz(A)anthracene) orally 65 mg/kg. Group-III was administered aqueous leaf extract of Carica papaya
(ALQECP) in a dose of 200 mg/kg body wt for a period of 3 months, group-IV has given ALQECP 200 mg/kg
body wt for a period of 21 days post 3 months of tumor induction, group-V rats were administered a small dose of
Carica papaya extract intra tumor locally in the region of tumor. Results: Values of CA15-3 were increased in
group-II rats (tumor control) significantly, whereas in group-III (prevention group) the levels of CA15-3 were
found to be reduced substantially and the P value < 0.001. Similarly, CA-15-3 levels were reduced significantly
in group-IV (treatment group)and P<0.005. The levels of LDH were seen to be increased in group-II, where as in
group-III LDH levels were decreased and P<0.001.similarly group-IV LDH levels also reduced significantly but
not to the level of group-III. Conclusion: Among the various markers for the detection of cancer antigen15(CA15-3) and lactate dehydrogenase (LDH) are important biochemical parameters that give a clear
understanding of the progression and proliferation of cancer cells. In this study it was found that there is increase
in the levels of markers such as CA15-3 and LDH and also the tumor volume in tumor control, these marker
levels were decreased by the administration of aqueous leaf extract of Carica papaya in a dose of 200 mg/kg body
wt. ALQECP not only prevented the progression of cancer growth but also has significant effect in reducing the
both CA15-3 and LDH levels in treatment group.
Keywords: Carica papaya, DMBA, Wistar rats
INTRODUCTION
Breast cancer is one of the principal cause of cancer
related deaths in women worldwide and it accounts to
the tune os 3,27,000 deaths every year and one in
every 10 newly detected cancer cases each year[1,2]. In
India breast cancer is second leading cause of cancer
deaths among womens[3]. Breast cancer development
Gurudatta et al.,
is associated with alterations of the delicate balance

between cell proliferation and apoptotic cell death,
cellular redox status, deregulation of cellular
differentiation and endocrine derangement [4, 5].
Despite advances in understanding the molecular
basis, diagnosis and treatment of this fatal disease
667
over the past decades, this malignancy remains

elusive. Therefore, the identification of new and
efficient anticancer drugs has always been a focal
point in cancer research [6, 7]. In India breast cancer is
second leading cause of cancer deaths among
womens.
Carica papaya is known by many other names such
as papaya, papaw, pawpaw, mamao and melon tree
[8]
. It is cultivated for its young leaves, shoots and
fruits which are cooked as a vegetable or for its ripe
fruit which is consumed as a beverage [9]. Literature
reported that, papaya had positive effect against
bacterial various infections [10]. Treatment with carica
papaya improved efficiency of phagocytic cells that
destroy bacteria [11]. In vitro studies conducted on
extracts from skin, flesh, and seeds of both ripe and
unripe Carica papaya gave antibacterial activities
against various microorganisms including Bacillus
subtilis, Bacillus cereus, Staphylococcus aureus
Escherichia coli, Salmonella typhi, Pseudomonas
aeruginosa Enterobacter cloacae, Proteus vulgaris,
Klebsiella pneumoniae, and Shigella Flexner [12].
Papain which is the unique enzyme found in Carica
papaya is effective natural medicine in controling
both inflammation and edema associated with
surgical procedures [13]. It also produced therapeutic
beneficial effects in patients with inflammatory
disorders of liver, intestine and eye [14]. It was
reported that some diseases which have inflammatory
conditions such as rheumatism, asthma and arthritis
wound healing can be treated by using extracts from
leaves of Carica papaya [15]. An aqueous extract of
Carica papaya was also reported for its effect on
growth of various tumor cell lines and on human
lymphocytes and have shown positive significant
results [16]. The results also showed drastic growth
inhibitory activity of Carica papaya extract on tumor
cell lines [17]. Thus, literature search we did not found
any invivo study for its anticancer activity.
The present study was designed to study the
therapeutic efficacy of Carica papaya on DMBAinduced mammary carcinoma bearing rats. The status
of tumour marker enzymes such as cancer antigen 15
(CA-15) and LDH in the plasma, was evaluated in
control and experimental rats.
Study design: Experimental animal based study
Ethics approval: The study was approved by the

Institutional animal Ethics Committee, regulated by
the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA).
Collection of plant material: Plant The leaves of
Carica papaya were obtained from farms of Bijapur
district Karnataka India and the pharmacognostic
authentication was done by Department of Botany
KCP Sciences, College Bijapur. Aqueous leaf extract
of the Carica papaya was prepared by standard
extraction procedures[18].
Chemicals
and
reagents:
7,12Dimethylbenz(a)anthracene (DMBA), and other fine
chemicals were purchased from Sigma Chemical Co.
(St. Louis, MO,USA). All other chemicals and
solvents used were of analytical grade and highest
purity.
Animal: Adult female rats of Albino wistar strain
weighing 195-205g were procured from Central
Animal House, BLDEUs Sri BM Patil Medical
College Hospital & Research Center, Bijapur, India,
were used in the study. They were housed in the
quarantine room individually in polypropylene cages
for one week before the experiment was started. The
animals were maintained under standard conditions of
humidity, temperature (252 C) and light (12 h
light/dark). They were fed with standard rat pellet
diet and water ad libitum.
Experimental design
The rats were divided into five groups with six
animals in each group and were given the following
dose regimen.
Grouping of animals
Group-I: Normal control-vehicle treated (olive oil 1
ml) by gastric intubation.
Group-II: Tumor control-DMBA (65mg/kg orally
single dose in olive oil)
Group-III: Single dose DMBA and ALQECP
200mg/kg orally for 3 months
Group-IV: Single dose DMBA and ALQECP
200mg/kg orally for 21 days post 3 months of DMBA
Group-V: Single dose of DMBA and ALQECP
locally at tumor for 21 days post 3 months of DMBA
Mammary carcinoma was confirmed by palpation of
tumor and tumor volume was measured by calliper
once they have attained measurable size[19].At the end
of study period blood samples were collected from
retro orbital plexus by using heparinised capillary
tubes under ketamine anaesthesia for biochemical
668
Gurudatta et al.,
investigations (post 3 months+21 days treatment) and

the end animals were sacrificed by cervical
dislocation.
Estimation of tumor volume: Tumor volume from
all the rats was calculated using below formula
TV=(w(2)L/2 {TV=Tumor volume, W=Tumor
width, L=Tumor length}
Biochemical estimations: The levels of cancer
antigen (CA15-3) were estimated by ELISA kit
sandwich method and lactate dehydrogenase (LDH)
levels were estimated by colorimetric absorbance
method.
Statistical analysis: Values are given as the mean
S.D of six rats. The results were statistically
evaluated using Students t-test using SPSS 16
(Statistical Package for Social Sciences) software and
one-way analysis of variance (ANOVA). The
differences between the groups were considered as
significant at *p<0.05
RESULTS
Values of CA15-3 were increased in group-II rats
(tumor control) significantly, whereas in group-III
(prevention group) the levels of CA15-3 were found
to be reduced substantially and the P value < 0.001.
Similarlay, CA-15-3 levels were reduced significantly
in group-IV (treatment group) and P<0.005. The
levels of LDH were seen to be increased in group-II,
where as in group-III LDH levels were decreased and
P<0.001.similarly group-IV LDH levels also reduced
significantly but not to the level of group-III. There is
no significant change in group-V LDH levels when
compared to group-II.
Table 1: Levels of CA-15, LDH and tumor volume
Groups
CA15-3
LDH
Tumour
(IU/ml)
(mU/ml)
volume
(cm3)
Group-I
7.93(0.42)
4.67(0.65
Group-II 16.92(4.89)
8.87(1.98)
3.302.8
Group-III 13.43(1.66)
5.73(0.63)
2.6856
Group-IV 14.48(0.34) # 6.38(0.57) # 2.6840
Group-V 16.74(2.33)
7.58(0.80)
3.1042
Data expressed as (meanSD, n=6) p-values <0.001
when compared group-I. #P-Values <0.05
DISCUSSION
Approximately 13,000 plants have been screened in
last five years for the development of new drugs for
the treatment of cancer. This present study was

carried with an aim to evaluate the anticancer
potential of aqueous leaf extract of Carica papaya on
DMBA induced mammary tumors in rats. The result
showed that administration of aqueous leaf extract of
Carica papaya at a dosage of 200 mg/kg body wt
showed anticancer effect. CA15-3 is a tumor
marker used to monitor specifically, breast cancer,
along with other types of cancer. It is found on the
surface of many types of cancer cells and shed into
the blood stream. It is used to monitor advanced stage
cancer. According to Keshaviah et al Elevated CA153 was found to be associated with an increased
chance of early recurrence of breast cancer.(LDH)
enzyme is a tetramer recognized as a marker with
potential use in assessing the progression of the
proliferating malignant cells. In the present study,
increase in the activities of LDH in carcinoma
bearing animals, could be attributed to over
production of enzymes by proliferated cells and
further release of their isoenzyme from destructed
cells and it is a fairly sensitive marker for solid
neoplasm [17,20]. Numerous other reports also revealed
the elevated levels of LDH in various types of
cancers [21]. The rise in LDH may also be due to the
higher glycolysis in the cancerous condition, which is
the only energy-producing pathway for the
uncontrolled proliferating malignant cells. The
increased activity of LDH, seen in the present study
substantiates the over expression of these enzymes in
a wide range of malignancies including breast
tumours and may be responsible for neoplastic
transformation[22-23]. Oral administration of ALQECP
drug reduced the activity of LDH enzyme to nearnormal levels, which may indicate the anticancer
effects of the drug.
Tumor volume shows there is significant decrease in
the tumor volume in group-III animals indicating
once again the anticancer effect of ALQECP in
DMBA induced mammary tumors in rats. Aqueous
extract of papaya leaves in an unknown composition
shown to possess anticancer activity and inhibition of
cell proliferation in a various cancer cell lines, this
has been patented by Morimoto et al [25]. Similarly,
the ALQECP showed antitumor activity and
immunomodulatory activity in tumor cell lines and it
proved upregulation of immunomodulatory genes by
microarray studies [26].
669
Gurudatta et al.,
Mechanism of action
AtUniversity of Florida researchers Nam Dang and
his colleagues in Japan have documented papayas
powerful anticancer properties and impact various
lab-grown-tumor[26].
International team of doctors and researchers from
US and Japan have discovered that enzymes present
in papaya leaf tea have cancer-fighting properties
against a vast category of tumors[27].
Papaya is high Source of Enzyme Papain which
Effective against Cancer. Papain is an endolytic plant
cysteine protease enzyme isolated from papaya
(Carica papaya L.) latex.(Abu-Alruz et al., 2009)[28]It
preferentially cleaves peptide bonds involving basic
amino acids, particularly arginine, lysine and residues
following phenylalanine. (Menard et al., 1990)[29] The
unique structure of papain gives its functionality that
helps to understand how this Protieolytic enzyme
works and its useful for a variety of purposes.
(Carica papaya L.) Latex, (Mitchel, 1970)[30]. Many
cancer cells having a protective coating of fibrin.
That is why they go unnoticed for many years.
Papain breaks fibrin coat of cancer cell wall. So
ultimately it helps against the cancer. Papaya has
larger stores of Cancer Fighting Lycopene. According
to Stahl et al., 1992 and Knachik et al., 2002,
lycopone is a member which helps in overcoming the
toxic manifestations of cancel cells.
CONCLUSION
Among the various markers for the detection of
cancer CA15-3 and and LDH are important
biochemical parameters that give a clear
understanding of the progression and proliferation of
cancer cells. In this study it was found that there is
increase in the levels of markers such as CA15-3 and
LDH and also the tumor volume in tumor control,
these marker levels were decreased by the
administration of aqueous leaf extract of Carica
papaya in a dose of 200 mg/kg body wt. not only
prevented the progression of cancer growth but also
has significant effect in reducing the both CA15-3
and LDH levels in treatment group.
Limitation of study: However, further investigation
using cell culture studies, animal studies and clinical
trials
are
required
for
confirming
the
chemoprevention and therapeutic potential papaya
leaves and check the adverse affects if any.
Acknowledgement: My sincere thanks to BLDE

University, Bijapur for allowing me to use the animal
house. I am thankful to teaching and non teaching
staff of dept of pharmacology of Shri B.M Patil
medical college Bijapur, Karnataka. I am also
thankful to all the teaching staff of Dept of
Pharmacology, MGM Medical College, Kamothe,
Navi Mumbai for their timely support
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Leena Motivale, Rao
KVK: Anti-cancer activity of phenolic antioxidants
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Halmenschlager, Renato Moreira Rosa, Joao
Antonio Pegas Henriques, Ana Lgia Lia de Paula
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metabolites from plants of the
genus
Hibiscus. Food Chemistry. 2010; 118:1-10. 8
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papaya.
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Nutrition. Accessed on 5th December 2011.
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Parshad O, Weaver S. The use of papaya
on
pressure ulcers. Am J Nurs 2002; 102: 73 77.
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10. Wimalawansa S J. Papaya in the treatment of

chronic infected ulcers. Ceylon Med J 1981; 26:
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11. Gurung S, SkalkoBasnet N. Wound healing
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12. Yismaw G, Tessema B, Mulu A, Tiruneh M. The
invitro assessment of antibacterial effect
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13. Papaya's healing properties confirmed by
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14. Rakhimov M R. Pharmacological study of papain
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Soladoye A. Anti inflammatory activities
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17. Helmes MH, Modia A, El-Moneim A, Moustafe
MS, EI-Balc A, Safinoz MEL: Clinical value of
serum LDH, ceruloplasmin, cathepsin-D and lipid
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18. Mohamed Abd Elgadir, Mohamed Salama, Aishah
Adam: Carica Papaya As A Source Of Natural
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Bensalah, A. Gamouh1,R. Aboufatim,A. Benharref,
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Co Ltd. Cancer prevention and treating composition
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Ripoll D. A protein engineering study of the role of
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Gurudatta et al.,
DOI: 10.5958/2319-5886.2015.00128.9

&
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Received: 5 Jun 2015
Research article
Copyright @2015
ISSN: 2319-5886
ROLE OF INFLAMMATION IN PELLAGRA: AN OBSERVATIONAL STUDY

*Desireddy Neelima1, Bandi Hari Krishna2, Masthan saheb3, Natham Mallikarjuna Reddy4
1
Department of Dermatology, Velammal Medical College, Madurai, Tamil nadu, India

Department of Physiology, Narayana Medical College, Nellore, Andhra Pradesh, India
3
Department of Dermatology, Shanthiram Medical College, Nandyal, Andhra Pradesh, India
2,4
*Corresponding author e mail: neelimadermatlogist@gmail.com

ABSTRACT
Background and objectives: Recent studies in medical science have established a fundamental role for
inflammation in mediating all stages of this disease from initiation through progression and, ultimately, the
thrombotic complications of atherosclerosis. Earlier studies documented the role of inflammation in
pathophysiology of many diseases. But the information regarding the role of inflammation in pellagra is less.
Therefore we hypothesized that, high levels of inflammatory markers like C Reactive Proteins (CRP), Tumor
Necrosis Factor alpha (TNF alpha) and Interleukin 6 (IL6) will be present in pellagra patients. Materials and
methods: Clinically diagnosed pellagra patients aged between 18 to 40 years were recruited (n=63) from
department of dermatology. Age and gender matched controls were recruited among staff and residents.
Inflammation was assessed by using markers like C Reactive Proteins (CRP), Tumor Necrosis Factor alpha (TNF
alpha) and Interleukin 6 (IL6) Results: There was no significant difference in anthropometric parameters. But,
inflammatory markers hs CRP, TNF alpha and IL6 were significantly high in patients suffering from pellagra,
when compared to age and gender matched controls (p<0.001) Conclusion: From this study, we can conclude
that, the estimation of levels of inflammatory markers in pellagra patients at early stage will help to take measures
to prevent the progression of disease.
Keywords: C Reactive Proteins, Tumor Necrosis Factor alpha, Interleukin 6, Pellagra, Inflammation.
INTRODUCTION
Pellagra was first coined by Casal in 1735, it was
endemic in Asia and Africa where staple food is
nicotinic acid deficient corn-based diet and related to
poverty among refugees or displaced population [1].
Classically pellagra is characterized by combined
deficiency of the essential amino acid tryptophan
and the vitamin niacin [2]. Pellagra is characterized
by the four classic symptoms dermatitis, diarrhea,
dementia, and death. Other symptoms include
anxiety, anorexia, cheliosis, psychosis, delirium,
constipation, dermatitis occurring on sun exposed
areas, diminished strength, intermittent stupor,
glossitis, nausea, melancholia,
paralysis of
extremities, stomatitis, peripheral neuritis, vomiting

and weight loss[3,4].
Inflammation is the body's attempt at self-protection;
the aim is to eliminate harmful stimuli, including
irritants, damaged cells or pathogens - and start the
healing process. Chronic inflammation lasts for
several months and even years. It can result from
failure to eliminate whatever was causing an acute
inflammation, an autoimmune response to a self
antigen - the immune system attacks healthy tissue,
mistaking it for harmful pathogens, a chronic irritant
of low intensity that persists[5].
672
Neelima et al.,
Recent studies in medical science have established a

fundamental role for inflammation in mediating all
stages of this disease from initiation through
progression and, ultimately, the thrombotic
complications of atherosclerosis. Earlier studies
documented the role of inflammation in
pathophysiology of many diseases [6]. But the
information regarding the role of inflammation in
pellagra is less. Therefore we hypothesized that, high
levels of inflammatory markers like C Reactive
Proteins (CRP), Tumor Necrosis Factor alpha (TNF
alpha) and Interleukin 6 (IL6) will be present in
pellagra patients.
Hence, in this study we studied the levels of
inflammatory markers like hs CRP, TNF alpha and IL
6 in clinically diagnosed pellagra patients.
Study design: This is an observational study.
Ethical approval: The study was approved by
institute ethics committee and obtained written
informed consent.
Inclusion criteria: Clinically diagnosed pellagra
patients aged between 18 to 50 years of both gender
were recruited (n=63) from department of
dermatology.
Exclusion criteria: We excluded patients suffering
from chronic hypertension, diabetes mellitus,
coronary artery diseases and other diseases where
inflammatory markers were raised.
Age and gender matched controls were recruited
among staff and residents.
Sample size: Sixty three
Methodology: Age, gender, height, weight were
recorded for all the participants. The medical chart
was reviewed for clinical characteristics, such as
hypertension, diabetes, coronary artery disease etc.,
Blood was collected through vein puncture by
aseptically, allowed to clot and centrifuged at 3,000
RPM at 4oC for 10 min (Remi-refrigerated centrifuge)
and the serum was separated and stored in a frozen
state at - 20oC for analysis. Inflammatory markers i.e,
hsCRP, TNF alpha, IL 6 were assessed by using
commercially available kits [7].
Statistical analysis: Statistical analyses were
performed using Statistical Package for Social
Sciences 16. Data expressed as mean SD.
Independent students pairedt test was applied to
compare various parameters between groups. The

null hypothesis was rejected at p<0.05.
RESULTS
The baseline physiological characteristics of study
participants are Table 1.There were no significant
differences in baseline characteristics like age, gender
and other anthropometric parameters like height and
weight.
As shown in Table 2, the inflammatory markers hs
CRP, TNF alpha and IL6 were significantly high in
patients suffering from pellagra, when compared to
age and gender matched controls (p<0.001)
Table: 1 Physiological characteristics of study
participants.
Parameter
Pellagra patients
Controls
Age (Years)
47.54 5.70
48.02 5.43
Men/Women,
50/13
48/15
Height (cm)
161.547.34
162.148.66
Weight (Kg)
68.26 3.23
67.364.62
Table: 2. Between and within group difference of
Oxidative stress and inflammatory markers.
Parameter
Pellagra patients Controls
hs CRP
(ng/ml)
TNF alpha
(pg/ml)
IL 6 (pg/ml)
9192.26+ 2568.93 2655.21+1286.35*

200.64+ 81.45
128.74+ 43.59*
311.54 + 94.51
204.23+ 73.21*
* p<0.001.
hs CRP: high sensitive C-reactive protein, TNF alpha:
Tumor Necrosis Factor - alpha, IL6: Interleukin 6.
DISCUSSION
Despite of recent advances in the management and
pathophysiology of pellagra, the information about
the role of inflammation in the pathophysiology of
pellagra is less. Therefore in this study we assesses
the inflammation in pellagra patients by using hs
CRP, TNF alpha and IL 6.
TNF- initially defined in 1975, was eventually
named as cachectin because of its putative role in the
progression of cachexia. TNF- is released in
response to an array of inflammatory stimuli which
are associated with multiple cell signaling pathways
involved in the regulation of immune response. TNF exerts its biologic action by means of two TNF-
receptors, TNFR1 and TNFR2, which are depicted
673
Neelima et al.,
by all nucleated cells. These kinds of receptors

are entered into the cell membrane and
consequently may also be cleaved and unveiled
into the blood circulation. Lower quantities of these
soluble TNF- receptors could strengthen and
extend the biologic action of circulating TNF-,
however higher levels of receptors may buffer
the biologic outcomes of surplus circulating TNF[8]. Even though TNF- is the best inflammatory
marker in HF, some other cytokines may possibly
perform a role.
Interleukin 6 (IL-6) is a pro inflammatory
cytokine that offers a sophisticated role in the
control and propagation of the immune reaction and,
like TNF-, may collocate certain aspects of the HF
phenotype in animal models [9]. Generally, IL-6
seems to cause a hypertrophic reaction in myocytes,
possibly leads to unfavorable remodeling.
C-reactive protein (CRP) was found in 1930 and
named by its reaction with the C-polysaccharide of
Streptococcus pneumonia. It is produced from the
liver in response to inflammatory stimuli, and it
activates the conventional complement cascade. As
a biomarker of inflammation, this is routinely
available in laboratories because of its role in the risk
stratification of patients at risk for ischemic heart
disease (IHD) [10].
In this study the levels of TNF alpha was
significantly high in pellagra patients. This indicates
that, in pellagra patients, the high levels of
inflammatory markers may lead to other diseases like
hypertension, coronary heart
diseases
etc.
Limitations: Future studies should include more
sample size and more precise inflammatory markers.
CONCLUSION
In conclusion, the results of this study indicate that
estimation of levels of inflammatory markers in
pellagra patients at early stage will help to take
measures to prevent the progression of disease.
ACKNOWLEDGEMENT
We sincerely acknowledge all participants of the
study. We extend our sincere thanks to dean, director
and other staff who provided the laboratory facilities
to estimate the inflammatory markers.
REFERENCES
1. Mason JB. Lessons on nutrition of displaced
people. J Nutr. 2002; 132(7):2096 03.
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Nutrition.
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Reduces Oxidative Stress And Inflammation. JEP
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8. Von Haehling S, Jankowska EA, Anker SD.
Tumour necrosis factor-alpha and the failing
heart--pathophysiology
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9. Janssen SPM, Gayan-Ramirez G, Van den Bergh
A, Herijgers P, Maes K, Verbeken E, et al.
Interleukin-6 causes myocardial failure and
skeletal muscle atrophy in rats. Circulation. 2005
;111(8):99605.
10. Ridker PM. Role of inflammatory biomarkers in
prediction of coronary heart disease. Lancet.
2001; 358(9286):9468.

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Neelima et al.,
DOI: 10.5958/2319-5886.2015.00129.0

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Health Sciences
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Copyright @2015
th
th
Discussion
ISSN: 2319-5886
Accepted: 23rd Apr 2015
OPTIMAL eCRF DESIGN, USER FRIENDLY INTERFACE AND PROPER TRAINING:

QUINTESSENTIAL FOR HIGH QUALITY DATA IN REAL WORLD EVIDENCE (RWE) STUDIES
*RiturajMohanty1, ArunGowda2, AnupNair3, AnilSharma4, UttamBarick5
1
M.B.B.S, Associate Project Manager, 2M.B.B.S, Director, 3M.Sc, Clinical Research Associate,
4,5
M.Sc, Clinical Research Consultant, Focus scientific research center (FSRC), part of phamax AG, #19 KMJ
Ascend, 17th C Main, 1st Cross, 5th Block, Koramangala, Bengaluru, Karnataka, India 560095.
* Corresponding author email: rituraj.mohanty@fs-researchcenter.com
ABSTRACT
Background: The success of a Real World Evidence (RWE) study lies in collecting and processing high quality
data. Data in RWE study can be collected in a paper format as in a Case Report Form (CRF) or electronically in
the format of electronic Case Report Form (eCRF). For a multi-country/multi-centric study, eCRF can offer
advantages over the conventional paper CRF for collection of data. An approach which is a combination of
optimal eCRF design, user friendly interface and proper training can facilitate in collection of high quality data.
Aim: The aim of this article is to highlight the significance of optimal eCRF design, user friendly interface and
proper training in RWE studies. Conclusion: Implementation of EDC system with eCRF can be advantageous for
a multi-country/multi-centric RWE studies, as it facilities real time monitoring, which can yield adequate data of
high quality. Implementation of eCRF can be cost and time effective.
Keywords: Electronic Case Report Form (eCRF), Electronic Data Capture (EDC), Patient Registry, Real World
Evidence (RWE), Stakeholders
INTRODUCTION
A patient registry (an example of RWE study) is an
organized system that uses observational study
methods to collect uniform data to evaluate specified
outcomes for a population defined by a particular
disease, condition, or exposure, and that serves one or
more predetermined scientific, clinical, or policy
purposes. If designed and conducted properly it can
provide useful data on the real-world view of clinical
practice, patient outcomes, safety and comparative
effectiveness [1]. Apart from the study protocol the
most important element of a RWE study is the tool
designed and used to collect data. CRFs and
questionnaires have been traditionally used for
collecting data in clinical studies. Data collection in
CRF though simple is time-consuming, tedious and
error prone [2]. Moreover in cases where the study is
spread over a larger geographical area, the logistics

associated with paper based CRF is substantial and
cumbersome. An Electronic Data Capture (EDC)
system is a computerized system designed for the
collection of clinical data in electronic format.
EDC system typically has eCRF, which is basically
the CRF in electronic format into which data is
populated directly with the help of an appropriate
browser. The convenience of eCRF from data
collection point of view is that the data can be
monitored and evaluated in real time. The system
allows the study monitor to access anonymous data
from centers throughout the study duration and check
them on a real time basis. A well designed eCRF also
has numerous edit checks within itself, thereby
675
Rituraj et al.,
prompting the investigator/end user if the data

entered is incorrect or illogical.
Some of the core features of the EDC system are
ideally characterized by:
1. Different levels of user access so that data can be
monitored at multiple levels
2. Secured platform with password protection and
validated by electronic signatures
3. Facilitates remote data capture and real time
monitoring
4. Reduced data redundancy as data captured once
is populated in relevant fields
5. Facilitates real time query management thus
reducing total turnout time
6. Generation of reports which has data on
recruitment and other parameters
7. Central storage of anonymous data with back up
8. Automation leading to faster study start up and
closure
9. Early database lock leading to timely study
completion [3]
10. Cost effective as compared to paper based CRF
in certain scenarios [4]
eCRF Design; Designing the eCRF is a critical step
in conduct of a RWE study. Contrary to randomized
clinical trials, generally, in RWE studies or patient
registries, the protocol does not mandate the
investigator for any intervention. The investigator
collects and populates patient data as per the standard
clinical practice and local guidelines/regulations. A
particular RWE study conducted on a multi-country,
multi-centric platform involving data collection from
different geographical regions typically follows a
trend specific to local practices and requirements of
that particular region. Keeping the above in view,
optimal eCRF design becomes quintessential if the
right amount and right quality of data have to be
collected to yield meaningful analysis and results.
The fundamental basis of eCRF design is that it
should ideally be FDA 21 CFR Part 11 compliant
[5,6]
.It should also have appropriate internal and
external security safeguards [7].The operations team
ideally plays a crucial role in the design and
validation of the eCRF. The team responsible for
designing the database and eCRF should receive the
study protocol as soon as possible and the initiation
of eCRF design need not wait for the protocol to be
finalized, but can start in parallel to the review and
finalization of the protocol. The data parameters
derived ideally from the protocol are listed down,
logically segregated into sections/visits and

transcribed into paper CRF. The CRF should undergo
thorough review by clinical/scientific experts to make
sure that all the data parameters necessary to satisfy
the data requirements of the primary and secondary
objectives/outcomes of the protocol are considered
and included. An easy to use eCRF, both from an
investigator and a data analyst perspective is one that
has maximal drop down menus and radio
buttons/check boxes for collection of data and
minimal free text entry. End-to-end testing of the
eCRF portal is critical. The eCRF should be
rigorously tested by populating dummy data and all
types of user scenarios to ensure that all real life
events are considered and replicated without any
errors. The training eCRF portal should be fully
tested, validated and approved before release.
Training needs to be delivered to individual
investigators and all other end users identified at that
stage of the study. Indicative approach to eCRF
designing is outlined in Figure 1. To maintain the
sanctity of data and restrict the access to various
types of functionality on the eCRF portal, relevant
user specific access rights should be generated and
provided as agreed and approved between the sponsor
and other stakeholders. Despite best trainings, a user
guide should be made available to the respective sites
and other stakeholders.
User Interface: No matter how technically correct
the eCRF portal might be, it fails to serve its purpose
if the investigators/end users find it difficult to use.
The interface needs to have the following basic
components which would make it widely acceptable
in studies which are conducted across the world
within multi-cultural setups:
1. Compatibility with different browsers
2. Minimal hardware requirements
3. Minimal internet bandwidth requirement
4. Simple log in and log out procedures
5. Dedicated drop down menus or icons for different
functionalities
6. Ease of toggling from page to page
7. Secure and easily retrievable password protection
8. Help option next to login screen listing frequently
encountered eCRF issues with possible resolutions
9. Clear and simple user guide with local language
support
10. 24 hour helpdesk to solve any technical queries
676
Rituraj et al.,
Acceptability of the eCRF portal is enhanced if the

investigators/end users find it simple and uncluttered.
One of the important aspects of the interface is that it
should have provision for remote access by the
technical team in case the investigator/end user

encounters difficulties in populating data or using any
of the functionalities.
Fig 1: Schematic approach to eCRF design and validation (indicative only)

Training: A very important aspect of the entire
activity is ensuring that the investigators/end users
are properly trained and understand how to use the
eCRF portal.
It is quite common to encounter some level of
resistance and hesitancy from a handful of
investigators towards the eCRF approach of data
collection. It is always better to identify such
investigators prior to their enrolment on the study.
Some investigators express their resistance towards
eCRF once the study has been initiated and patient
enrolments are on-going. Hence, the eCRF training
sessions should also encompass a module which
clearly enumerates the reasons why this study is on
eCRF and the benefits it provides to all in terms of
time, efforts, money and the ease of conducting
studies. Wherever required and applicable, a case
study objectively portraying these variables in
tangible terms and/or numeric values should always
be targeted. Such portrayal has a tendency to simplify
the explanations and hence can have a higher
probability of connecting with the intended audience
who are mostly physicians.
Trainings, however good should never be a
standalone activity. Trainings for a longer duration
study or for those studies where numerous changes
are consistently incorporated in the protocol/eCRF
needs to be repetitive in nature. Such repeat sessions
may not necessarily be face to face but can be web
based interactive sessions with voice support and live
video transmission wherever applicable/possible.

Repeat training for minor updates in protocol
affecting the eCRF data collections can also be
scheduled using the simple teleconference mode.
The trainer needs to be well prepared and equipped
with all the support documents and tools necessary to
impart such training sessions effectively. The list of
frequently asked questions (FAQs) should always be
shared well in advance with the sites/
investigators/end users along with the technical
requirements documents. The trainer should always
know the targeted audiences linguistic and cultural
gaps. Based on this assessment if the services of
interpreters are necessary then they should be
appointed for the training sessions and the relevant
audience informed of the same well before the start of
the session.
The training session needs to have inbuilt checks and
balances to periodically assess whether the training
serves its intended purpose and the audience is able to
well comprehend the contents. This can be done
through frequent questions on whatever has been
discussed till that point and getting an
online/telephonic feedback based responses. Based on
the responses gathered, the trainer should always
assess any gaps in the understanding of the audience
and modulate his/her further sessions/slides
accordingly. During remote trainings it is always
sought that the training platforms login credentials
have been first shared with all. It should be ensured
677
Rituraj et al.,
that all have successfully logged into it and proceed

on that platform with the new dummy data entries as
per the training document pre-shared with them. The
trainer should always ensure that the pace maintained
for the trainings never exceed beyond a point even if
audience seems very receptive. Neither should it be
too slow to keep pace with slow learners as this can
trigger negative interests among the bulk of those
who have been receptive and in sync with the trainer.
If required, the trainer can conduct an additional
session with the slow learners on need basis. The
option of submitting questions should always be
incorporated. All the questions collated by the end of
the session should be answered. If the time doesnt
permit to discuss all the questions, then the trainer
should request them to submit their remaining
questions and assure them of a time bound email
based response. The trainer should maintain a
question and answer log of that session and share it
within a specified time period post training with all
the audience via email. The trainer should also share
his/her contact details with them for any future
correspondence or queries related to the training
imparted and must ensure that prompt responses are
provided to all correspondences.
DISCUSSION
With ever increasing need of more data and better
data by pharmaceutical companies and other
stakeholders involved, the future of EDC system
seems promising. Technology has bridged the gap
between different stakeholders promoting their active
participation in any particular study. Newer devices
and interfaces are being developed at a rapid rate and
the EDC system will have to keep pace with them in
order to be compatible. As the acceptability of
technology increases, EDC systems will have to be
made device and platform agnostic. The EDC system
will need to evolve to provide interoperability
between differential clinical and e-health systems
among several standard consortiums [8,9]. In this
modern age of automation, large scale integration of
processes on a real time basis is desirable to obtain
credible results within justified and acceptable
timelines. Paper based CRF have been used
extensively in the past but as studies get expansive
and diversified, integration of EDC system for data
collection will be critical for study success. A study
evaluating EDC in developing country has
demonstrated that if EDC system is well designed and

introduced with care, and work processes are adjusted
to EDC, it will become a more time effective,
potentially more accurate and therefore cost effective
method than the standard paper-based data collection
method [10].
CONCLUSION
In
multi-country/multi-centric
RWE
studies,
implementation of EDC system with eCRF can be
advantageous as it facilities real time monitoring,
which can yield adequate data of high quality.
Collection of data in eCRF can be cost and time
effective. In order to encourage acceptance of
investigators/end
users
to
EDC
system,
implementation of an approach which combines
optimal eCRF design, user friendly interface and
proper training could be critical for success.
ACKNOWLEDGEMENT
The authors would like to acknowledge the efforts of
Mahafroz Khatib, Scientific Associate and Barani
Kumar, Graphic Designer at FSRC for their
invaluable support in the development of this article.
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DOI: 10.5958/2319-5886.2015.00130.7

&
Health Sciences
www.ijmrhs.com
Copyright @2015
th
th
Review article
ISSN: 2319-5886
BILATERALLY OCCURRING MUCOSAL ALTERATIONS OF THE ORAL CAVITY-A REVIEW

Ceena Denny1, *Junaid Ahmed2, Ravikiran Ongole3, Nandita Shenoy4, Almas Binnal5
1,4
Associate Professor, 2Professor and HOD, 3Professor, 4,5Reader, Department of Oral Medicine and Radiology,
Manipal College of Dental Sciences, Manipal University, Mangalore - 575001, India
*Corresponding author email: junaa@hotmail.com
ABSTRACT
Introduction: Lesions of the oral cavity could be unilateral / bilateral and could be the initial manifestation of
certain underlying pathology. Oral diagnosticians may be the ones who diagnose them in their initial stages.
Unilateral lesions have been well documented whereas bilateral soft tissue lesions have been rarely documented in
the literature. Hence we classified commonly occurring bilateral oro mucosal soft tissue lesions. Aim: To classify
bilaterally occurring oro-mucosal soft tissue lesions bilateral occurrence of lesions could be a normal variant or
indicative of pathology. Some of the lesions may or may not be symptomatic and some can even have a malignant
potential. It is imperative to know the different types of bilaterally occurring lesions as diagnosing such lesions of
the oral mucosa by the clinician is important through an adequate knowledge and thorough examination, followed
by investigation for the proper management and better prognosis for the patients.
Keywords: Bilaterally occurring, Intraoral lesions, Mucosal lesions
INTRODUCTION
A majority of oral lesions occurring in the oral
cavity are unilateral in nature. The anatomical
structures in the oral cavity appear bilaterally and
this feature usually provides the clinician a vital clue
to differentiate between a normal anatomy and a
clinical
pathology.
Bilaterally
occurring
maxillofacial pathologies though not very commonly
encountered in our daily practice, are important
since they may prove to be reliable indicators of
certain kind of lesions and hence aid in early
diagnosis thereby helping to reduce the morbidity
and mortality rates.
Bilaterally occurring oral lesions can occur both
intraorally and extraorally. The extraoral lesions that
can occur bilaterally include those affecting the
salivary glands, most commonly sjogrens syndrome,
mumps & sialadenosis. Muscular hypertrophy is
another commonly occurring extraoral bilateral
swelling in the orofacial region. The present review
paper will discuss exclusively about the intraoral

bilaterally occurring soft tissue lesions.
Intraoral bilaterally occurring mucosal alterations
can be broadly classified as:
Normal anatomic variants: Lingual tonsils,
Leukedema, Retrocuspid papillae, Palatal rugae
Lingual varices, Parotid papilla, Linea alba buccalis,
Racial pigmentation.
Developmental disturbances: Affecting the LipCongenital lip pits, Commissural lip pits, Cleft lip
Pigmented lesions: Physiologic pigmentation,Peutz
jeghers syndrome, Addisons disease, Heavy metal
pigmentation, Kaposis sarcoma, Drug induced
pigmentation, Post inflammatory pigmentation,
Smokers melanosis
White lesions of the oral cavity: White sponge
nevus, Benign hereditary intraepithelial dyskeratosis,
Traumatic keratosis, Lichen planus, Angular chelitis,
Oral hairy leukoplakia
Ahmed et al.,
680
Normal variants:
Lingual Tonsil: Referred to as the fourth tonsil in
the waldeyers ring of lymphoid tissue. Situated at
the root of the tongue behind the circumvallate
papilla in front of the epiglottis.[1]The lingual tonsils
form nodular bulges in the root of the tongue, and
their general structure is similar to that of the
palatine tonsil. Crypts are deep, may be branched,
and are lined by a wet stratied squamous
epithelium that invaginates from the surface.
Leukedema: Sandstead and Lowe in 1953 was the
first to describe leukedema.[2]It is a common
mucosal alteration than a pathologic change
characterized by a grayish-white lesion of the buccal
mucosa in humans. Although it can involve the
labial mucosa and the soft palate, it most commonly
affects the buccal mucosa bilaterally.[3] It can occur
in any age group, but more commonly seen in the
adults.[4] Although present in population of different
countries and ethnic groups, it is more profound
among the black Americans.[5]Stretching of the
buccal mucosa makes the lesion disappear and this
characteristic of leukedema differentiates it from
other white lesions.[6]
Retrocuspid papillae: First reported by Hirschfeld
in 1947, retrocuspid papilla is a circumscribed round
or dome-shaped sessile nodule found on the lingual
surface of the mandibular cuspids near the
mucogingival junction measuring about 2-4mm. It is
soft, homogenous and pink in colour.[7](Fig 1)
Fig 1: Retrocuspid papilla

Palatal rugae: Palatal rugae are bilateral, irregular,
asymmetric ridges of the mucous membrane
extending laterally from the incisive papilla and the
anterior part of the median palatal raphe. Winslow
was the first to describe them in 1732. Its size
increases due to normal growth, but remains in the
Ahmed et al.,
same position throughout the patients life and it is

unique in each person.[8]
Lingual varices: Varicosities are benign lesions of
blood which are commonly seen in the head and
neck region. In the oral cavity it is commonly seen
on the ventral surface of the tongue, other sites
involved are the lips and the cheeks.[9, 10]Varicosities
are more prominent in the elderly & are usually seen
occurring bilaterally on either sides of the midline on
the ventral surface of the tongue. Varicosity in the
oral cavity presents as purplish blue spots, nodules
or ridges and are usually asymptomatic.[11](Fig 2)
Fig 2: Lingual varices on the ventral surface of the

tongue
Parotid papilla: The parotid papilla is a small

elevation of tissue that marks the opening of the
parotid duct on the inner surface of the cheek usually
opposite the maxillary second molar.[12]It can
occasionally appear as an exaggerated growth giving
it an appearance of a pathological condition.
Linea alba buccalis: Its a Normal variation which
appears as a white line extending from the corners of
the mouth to the posterior region at the level of
occlusal plane bilaterally, usually associated with
frictional keratosis /trauma from the facial surface of
teeth.[13]
Racial pigmentation: Physiologic pigmentation,
which is common in African, Asian and
Mediterranean populations, occurs due to an
increase in the melanocyte activity rather than an
increase in their number. It varies from light to dark
brown. In the oral cavity, the attached gingiva is
most commonly affected and is seen bilaterally in a
ribbon like fashion. [14] The other sites affected are
the buccal mucosa, hard palate, lips and tongue.
Developmental disturbances:
Soft tissue disturbances affecting the lip
Congenital lip pits and commissural lip pits:
Developmental defects that usually occur on the
681
paramedian portion of the vermilion border of lower

lip and commissural lip pits are those seen at the
commissural areas.
It shows an autosomal
inheritance pattern. Presence of congenital lip pits,
with or without cleft lip and/or palate is termed as
Van Der Woude syndrome.[15]Other syndromes
associated with congenital lip pits are popliteal
pterygium syndrome, oral-facial-digital syndrome
and Marres and Cremers syndrome.[16] (Fig 3)
Fig 3: Commissural lip pits

Cleft lip: A cleft lip and palate is an abnormal gap
in the upper lip and the roof of the mouth. As the lip
and palate develop separately, it is possible for the
development of an infant to be born with only a cleft
lip, only a cleft palate, or a combination of both.
Cleft can be either unilateral / bilateral/can be either
only lip /palate or can be a combination.[17]
Pigmentation: Pigmentation other than due to race
can occur due to increased melanin deposition in
association with certain systemic diseases like Peutz
- Jeghers syndrome (mucocutaneous pigmentation
spots of mouth hand and feet and intestinal
polyps),[18]Addisons Disease ( homogenous or
blotchy pigmentation of skin, oral cavity,
conjunctiva and genitalia) [19,20] and can have both
oral and extraoral manifestations.
Heavy metal pigmentation: Increased levels of
heavy metals in the blood can cause discoloration in
the oral cavity. It appears as a blue black line along
the gingival margin extending bilaterally.[ 21]
Post inflammatory pigmentation: Pigmentation of
the oral mucosa which is usually seen associated
with healed chronic oro-mucosal lesions like lichen
planus, lichenoid reaction, pemphigus etc. appears as
brown to black discoloration in the oral cavity and is
usually seen as brownish black diffuse pigmentation
present bilaterally.[22] (Fig 4)
Fig 4: Post inflammatory pigmentation on the left

buccal mucosa
Smokers melanosis: It was Hedin in 1977 who
termed the brownish pigmentation present in the oral
cavity associated with use of tobacco. It can occur
anywhere in the oral cavity but more commonly it
affects the attached gingiva in the mandibular
anterior region and the interdental papilla.[23]
Kaposis sarcoma: Is a malignant, multifocal
systemic disease that originates in the vascular
endothelium.[24]It is the most common malignancy
seen associated with HIV. Orally it manifests as
single or multiple painless, brownish red to
violaceous macule or papule. The most commonly
affected sites are the hard palate and gingiva. [25](Fig
5)
Fig 5: Kaposis sarcoma affecting the palate

White lesions of the oral cavity:
White sponge nevus: An autosomal dominant
disorder which affects the skin and the oral mucosa.
In the oral mucosa it appears as bilateral,
asymptomatic white, diffuse corrugated plaques. It
usually affects the buccal mucosa, ventral surface of
the tongue, labial mucosa, alveolar ridge and floor of
mouth in the descending order.[26]
Hereditary benign intraepithelial dyskeratosis
(HBID): Rare autosomal dominant disorder
682
Ahmed et al.,
characterized by elevated epithelial plaques located

on the ocular and oral mucous membranes.It has a
seasonal variation with increase in the severity
during the spring and summer.[27] Oral lesions are
usually asymptomatic and appear as multiple white
plaques, bilaterally.[28]
Traumatic keratosis/ frictional keratosis: A
chronic white lesion/patch which is benign and selflimiting seen usually on the buccal mucosa as a
result of constant rubbing of two surfaces. Usually it
is seen bilaterally along the line of occlusion or
because of constant rubbing of the wisdom teeth in
buccal mucosa against the cheek. It disappears once
the causative agent is removed.[29]
Lichen planus: Is a chronic immune mediated
disease which has a mucocutaneous involvement
with varied presentations. In the oral cavity, its
usually seen bilaterally and presents as either
reticular, plaque, bullous, erosive, atrophic and/or
pigmented forms. More commonly it affects the
middle aged women. Lichen planus can involve any
site but more common sites are buccal mucosa,
gingiva, dorsum of the tongue, labial mucosa, and
lower vermilion lip in descending order.[30] (Fig 6)
nonscrapable, bilateral lesion on the lateral /ventral

surface of the tongue and it could be an early
indicator of undiagnosed HIV. (Fig 8)
Fig 7: Angular Chelitis
Fig 8: Hairy leukoplakia on the lateral border of

the tongue
DISCUSSION
Fig 6: Oral lichen planus on the buccal mucosa

Angular Chelitis: Also known as perleche which is
derived from French meaning to lick ones lip. Seen
in older people and manifests as inflammation of the
lips at the two corners of the lips, which starts as
erythema followed by ulceration and then
crustations. Patient complains of soreness, pain and
burning sensation. Predisposing factors leading to
angular
chelitis
include
malnourishment,
immunocompromised conditions, decreased vertical
dimension of mouth, superimposed candidal
infection, xerostomia etc.[31] (Fig 7)
Oral hairy leukoplakia: Caused by Epstein BarrVirus and is most commonly seen in patients
infected with HIV. It manifests as hairy white
Among the various lesions occurring in the oral

cavity few lesions could be normal variants, while
others could be with or without malignant potential
and most of them are not diagnosed at an early
stage.32 Some of these lesions occurring in the oral
cavity could heal spontaneously while other lesions
may need treatment to prevent any further
complications. Biopsy maybe considered for those
lesions that persist to rule out any dysplastic
changes.33 Hence, diagnosing such lesions in the
very early stage could in turn be beneficial for the
patient. We have attempted here to provide a concise
classification of the soft tissue lesions that most
commonly occur bilaterally in the oral cavity. We
also believe this should be useful for the dental
practitioners for easy identification of such unique
lesions.
CONCLUSION
683
Ahmed et al.,
The occurrence of a lesion can be a vital indicator to

the kind of disease group it belongs. To formulate
appropriate treatment strategies for the management
of oral disorders, it is imperative that one should be
trained to recognize and differentiate normal
morphological conditions that occur bilaterally from
Oro-mucosal pathologies.Furthermore, the early
clinical diagnosis of such bilaterally occurring
maxillofacial pathologies can result in better, faster
intervention and management of such unique
conditions.
The aim of this paper here is to create awareness and
provide a ready reckoner about these specific
conditions which can provide a useful guide for a
more efficient diagnosis by the clinician.
ACKNOWLEDGEMENT
I acknowledge all the staff of my department for
their valuable contribution
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DOI: 10.5958/2319-5886.2015.00131.9

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Health Sciences
www.ijmrhs.com
Volume 4 Issue 3

Review article
Coden: IJMRHS
Copyright @2015
ISSN: 2319-5886
SIMULATED ANIMAL EXPERIMENTS IN TEACHING AND RESEARCH

*Chirag B. Mistry1, Shreya M. Shah2, Jagatkumar D. Bhatt3
1
Tutor, 2Additional Professor, 3Past-Professor and Head, Department of Pharmacology, Medical College, Baroda,
Gujarat, India.
*Corresponding author email: drchiragm@gmail.com
ABSTRACT
Animal experiments are of paramount importance in the pre-clinical screening of new chemical entity. On
the other hand, various regulatory guidelines for animal experiments are becoming more stringent in the face
of worldwide protests by animal rights activists. Moreover, simulated animal experiments softwares are being
developed and they can be implemented in the postgraduate and graduate students' curriculum for demonstration
of standard physiological and pharmacological principles compared to real time animal experiments. In fact,
implementation of virtual experiment will decrease hand on experience of animal experiments among medical
students, but after medical graduation, animal experiment is lest utilized during their day to day clinical
practice. Similarly, in case of postgraduate pharmacology curriculum, computer based virtual animal experiments
can facilitate teaching and learning in a short span of time with various protocols, without sacrificing any animal
for already established experimental outcomes.
Keywords: Clinical Practice, Graduate Teaching, Simulated Animal Experiment, New Chemical Entity,
Postgraduate Study, Software.
Key message: Animal experiments are essential for confirmation of the efficacy of new chemical entity with
confirmation of safety before starting clinical trials on human. Similarly, for already established experimental
principles, simulated animal experiment can improve teaching and learning by demonstration of virtual
experiments in short span of time without sacrificing any animal. However, for research and development of new
chemical entity, currently there is no software or tool that can analyze like a live animal or human.
INTRODUCTION
Advances in medical research in the past 100 years
have led to the development of many novel drugs and
they have facilitated many new ways to diagnose,
treat and prevent diseases in humans as well as in
animals. On the other hand, many evolving diseases
like the mutant strains of flu, viral infections like
AIDS, multi drug resistant tuberculosis and various
cancers require new chemical entities for their cure
and prolongation of survival[1].
History of animal experiments:
Proponents of vivisection like tests, experiments, and
"educational" exercises involving harm to animals,
claim that they had played a crucial role in virtually

all medical advances[2]. On the other hand, several
medical historians argue that key discoveries in such
areas as heart disease, cancer, immunology,
anesthesia, and psychiatry were in fact achieved
through clinical research, observation of patients, and
human autopsy[3, 4, 5].
In biomedical research, specially bred and reared
laboratory animals like mice, rats, hamsters, guinea
pigs, rabbits, cats, dogs, monkeys and other lower
forms were studied as a sole experimental purpose for
development of new chemical entity after various
Chirag et al.,
686
high throughput screening and computational

chemistry methods[6, 7].
In the USA, total number of animals used in 2010
were more than 1 million, which include rats, mice,
birds and fish. Most procedures were carried out
predominantly on mice, rats followed by guinea pigs,
rabbits and hamsters[8].
In routine practice, researchers or clinicians cannot
start treatment with a new chemical entity from
humans directly, as it must be preceded by a good
efficacy and safety data of preclinical testing. On the
contrary, due to constant worldwide objection of
various scientific, legislative authorities, and animal
rights activists, currently many experienced
pharmacologists are now shifting towards alternatives
to animal experiments[1].
Guidelines for animal experimentation:
On the whole, there are restrictions on animal
experimentations due to consideration of vanishing
species of rare animals globally. In the developed
world like the United States of America, animal
testing is primarily regulated by the Animal Welfare
Act of 1966 (AWA), and the Animal Welfare
Regulations, which is enforced by the Animal Care
division of the Animal and Plant Health Inspection
Service (APHIS) of the United States Department of
Agriculture (USDA)[9]. The AWA contains
provisions to ensure that the animals of covered
species used in research receive a certain standard of
care and treatment[10, 11, 12].
Similarly, in the United Kingdom, the Animals
Scientific Procedures Act 1986 suggests experiments
to be regulated by three licenses, which details the
numbers and types of animals to be used, the
experiments to be performed, and the purpose of the
experiment[13].
In Australia, Animal Ethics Committees (AECs)
determine whether the use of an animal is valid or
not. AECs follow the code in order to ensure the well
being of the animals used for research and the Code
emphasizes
following
responsibilities
of
investigators, teachers and institutions for using
animals like: 1) To ensure that the use of animals is
justified, taking into consideration the scientific or
educational benefits and the potential effects on the
welfare of the animals; 2) To ensure that the welfare
of animals is always considered; 3) To promote the
development and use of techniques that replace the
use of animals in scientific and teaching activities; 4)
To minimize the number of animals used in
projects; and 5) To refine methods and procedures to

avoid pain or distress in animals used in scientific
and teaching activities[14].
The Canadian Council on Animal Care (CCAC) is set
up to act in the interests of the people of Canada to
ensure that the use of animals for research,
teaching and testing employs optimal physical and
psychological care according to acceptable scientific
standards. These experiments should increase the
level of knowledge, awareness and sensitivity to
relevant ethical principles[15].
In developing countries like India there is a similar
trend towards development of alternatives to animal
experiments because current guidelines given by the
Committee for the Purpose of
Control and
Supervision of Experiments on Animals (CPCSEA),
Indian National Science Academy (INSA) and the
principles of Good Laboratory Practice (GLP)
have contributed to the eventual framing of the
norms for conducting standard animal research that
restricts animal experiments. Similarly, as per the
Indian Council of Medical Research (ICMR)
guideline, research protocol on animals must serve
core purposes like acquisition of new knowledge, and
the testing of compounds, chemicals or devices for
evaluation of safety and effectiveness[8].
On the whole, toxicological studies are mandatory on
animal models to ensure the safety of new chemical
entity before starting a clinical trial on human. After
all, there is always a need of standard protocol,
justifying that research involving animals will
contribute significantly to present and future
knowledge, which can eventually lead to the
protection, improvement of the health of either
humans or animals. According to standard guidelines
for clinical and pre-clinical studies, new drug
research as well as tests meant for assuring the
quality and efficacy of pharmaceutical products,
vaccines or biological are based on structured
experiments on animals[8].
Restrictions on experiments on red listed and
threatened species:
In India, depending on the availability, types of
animals used for research are similar to
internationally, but the species may vary according to
geographical variation. On the contrary, there is a
stringent restriction for experiments on the
endangered species of animal as mentioned under the
red listed animals of The International Union for the
Conservation of Nature and Natural Resources[16].
Chirag et al.,
687
Similarly, according to the guideline of Medical

Council of India, for undergraduate teaching,
demonstration of the effects of various drugs but this
has been phased out in most institutes[17, 18].
Current requirements of animal research:
According to the European Research Commissioner
Philippe Busquin, use of animals to test drugs is a
necessary evil to safeguard human health[7]. In other
words, the underlying principle for carrying out
research on specific models of animals with similar
human biology can help to predict probable drug
outcome of animal experiments towards human.
Moreover, no new chemical entity can be introduced
into clinical practice or clinical research, unless it has
undergone all phases of toxicity tests in animals[8].
Basically, there are seven major areas of medicine,
and biology where there is a need for using animals
for experiments: (1) Fundamental biological, and
medical research (2) Developing new treatment for
diseases (3) Preparations of natural/herbal products
used in medical research, and treatment (4) Safety
testing of chemicals, and drugs (5) Study of genetic
disorders (6) Development of new diagnostic tests for
diseases (7) Biology, and medical education[8].
Role of animal activists in control animal
experiment: In spite of the mounting need for
safer and newer drugs that can treat currently
incurable diseases, the opposition to animal
experiments has always existed. Worldwide, there are
various factors that affect the research, and
development of drugs by means of conducting animal
experiments as a prerequisite to human research. In
fact, all the currently available effective drugs used
for human or animals, had undergone the same
process of animal experimentation in the past[8].
All over the world, various movements like the
animal rights movement which originated in
Europe, America and the anti - vivisection movement
laid down by Regan, have been carried out by these
activists to express their dissatisfaction on animal
experiments via news, blogs, and tweets over the
internet[8].
In other words, animal experiments cannot be done
without justifiable future benefits to animal or
human, appropriate indication, protocol and approval
of legal authorities of the nation. Moreover, there are
various responsibilities of a research scientist
starting from breeding and rearing of animals for
carrying out the experiment as per animal ethics
guidelines of the respective country. In the
absence of genuine reason to carry out research,

just repeatedly performing same animal experiments
of well established principle like Vasomotor
Reversal of Dale does not make any sense for future
clinicians[8].
Overall, the media has a major role in the decision
making of lay people and by adopting an extreme
moralistic stance, the animal activists look onto
animal researchers as cruel and corrupt, consumed by
desire for ever more research papers publications or
grants, which may not be the reality of many ethical
researchers. The fundamental pragmatic value of
biomedical study on both humans and animals is the
relief of suffering with the enhancement of well
being. Consequently, all biomedical research is
structured in a well designed manner to relieve
suffering of humans, without belittling the animals
used in the process[8, 19].
Alternative to animal experiments for academic
purpose: Considering the real life scenario of
requirements of novel drugs, need of animal
experiment cannot be nullified at any level just due to
activists who are opposing animal experiments, but
none can answer name of newer drug for
unpredictable newer disease in the absence of
experienced research scientists working with animal
experiments.
In history, Russell and Burch in 1959 developed the
concept of alternative to animal experiments, which
includes refinement, reduction, and replacement.
Similarly, the guideline of CPCSEA also supports
their concept with the addition of the fourth R that is
their rehabilitation, as an added measure for their care
[20, 21, 22]
.
In routine practice, implementation virtual
experiments can minimize to a great extent the use of
animals for drug development and testing[7, 17, 21].
Eventually, various professional and non professional
bodies had developed software and program for
virtual animal experiments. As shown in Figure 1,
one such computer assisted learning tool can reduce
and replace the animal experiments and to refine the
demonstrations as well the teaching process by a
software[23], which displays complete video
demonstrations of different procedures like isolation
and mounting of animal tissues followed by on screen
interactive interface to study the effects of various
drugs on the isolated tissues. As shown in Figure 2,
this software simulates experiments without causing
Chirag et al.,
688
undue pain to animals and the presentations were

made user friendly and self explanatory[23].
On the other hand, a similar effort was done in Ex
Pharm Pro software that is available over the internet
and free trial version can be utilized for
undergraduate and postgraduate teaching and
learning. This software can make quick
understanding of the already established experiment
with saving time without sacrificing any animal. As
shown in Figure 3, students can perform experiments
through the computer, like effect of drugs on rabbit
eyes, bioassay of histamine on the ileum of guinea
pig, ciliary motility of esophagus, isolated frogs
heart, blood pressure and heart rate of dog[24].
Fig 3: Determination of PA2 values of Atropine on

rat ileum preparationby Schhild splot method
through simulated animal experiment software- XCology Pro[25].
In short, Ex Pharm Pro software is well structured
virtual animal experiment software in a manner of
question answer mode followed by tutorial and
examination modes with ability to control the level of
examination by subscriber moderator or examiner. In
addition to that, as shown in Figure 4 it shows almost
identical graphs as seen in the recording of
physiograph showing the effects of various standard
panels of drugs as well as blockers as observed in real
animal experiments[24].
Fig 1: An interface showing video demonstration and

links for the navigation of simulated experimental
pharmacolgogy software.-X- Cology[23]
Fig 2: An interface showing interactive interface or

performing
bioassay
through
simulated
experimental pharmacology software-X-cology[23]
Abbreviations: Sal; Saline, Nopi: Norepinephrine,

Phen: Phenotolamine, bpm : beats per minute, BP:
Blood pressure, mmHg: milli meters of mercury.
Fig 4: Study of the effect of drugs or unknown
compound on anaesthetized dogs blood pressure by
stimulated animal experiment software. Ex Pharma
Pro[24]
Overall ExPharmPro is a useful educational software
which simulates animal experiments
to help
students for learning containing five experimental
programs which simulate animal experiments in
Pharmacology. In short, the current version of
689
Chirag et al.,
ExPharm demonstrates five programs named 1.

Effects of drugs on the rabbits eye, 2. Effects of
drugs on the frogs heart, 3. Effects of drugs on the
frogs oesophagus, 4. Bioassay of histamine on the
ileum of guinea pig, 5. Effects of drugs on the blood
pressure and the heart rate of dog[24].
However, the total numbers of virtual experiments of
the Ex Pharm Pro are less in number like the tip of
the iceberg compared to overall undergraduate and
postgraduate medical curriculum. On the other hand,
experts of experiments can develop almost all types
of basic experiments at reasonable cost that medical
or pharmacy institutes can afford. Moreover,
additions in such software can be carried out through
inviting software from junior researchers with the
help of network of Indian Pharmacologist[25].
Advantages of simulated animal experiment in
medical institutes:
Computer aided learning in Pharmacology can do
replacement,
reduction
and refinement
by
avoidance of repetition of experiments. Moreover,
it saves the anguish of analysis of unambiguous and
incomplete data due to variations in animal limited
survival time. If junior researchers can submit
software of virtual experiments free of cost and after
their incorporation, they can be provided free of cost
or at subsidized rates, which can save time and
money of the medical institutes and government,
which can be utilized for other essential programs
like
Pharmacovigilance,
Haemovigilance,
Prescription audit or Generic drugs prescribing[24, 25].
Simulated experiments can avoid negative student
perceptions of unsuccessful experiments and
successful experiment depending upon various
factors including the experience of junior researcher.
It can benefit undergraduate students by decreasing
the risk of exposure to various chemicals, zoonotic
disease and addresses the concern regarding
discarding of sacrificed animals[24, 25].
Limitations of existing simulated animal
experiment: Even though virtual experiments can
simplify teaching and learning, there are factual
limitations of these simulated software like: (1) It
cannot substitute preclinical research over animals for
the purpose of clinical trials, (2) New chemical entity
cannot be studied over it, (3) Research for any newer
experimental hypothesis cannot be done over it, (4)
In real time experiment, the effect of blocker cannot
be removed with so much rapidity, which is available
in exam mode only[24].
Opinion of experienced pharmacologist: As per

current development, animal laboratories are not
necessary for teaching skills of biological and
medical principles for medical students, and 85 % of
U.S. and Canadian medical schools have eliminated
animal labs from their educational curricula.
Conversely, for all practical purposes, the existence
of animal experiments and animal house is only for
namesake in some medical schools[26].
Current MCI guidelines for animal experiments:
According to the MCI guideline For teaching
Physiology and Pharmacology in Undergraduate
curriculum, the required knowledge and skills should
be impacted by using computer assisted modules. As
per the CPCSEA guidelines, only an animal hold area
is required. On the other hand, the proposed new
curriculum of MCI "Vision-2015" for undergraduates
considers simulation lab as an alternative to
experiments for medical students [17, 27]. In view of
this initiative, there can be a reduction or replacement
in future use of animals by the MCI amendment of
the year 2009, which had recommended all medical
colleges to use alternatives to animal experiments in
the undergraduate medical course[17, 20].
The MCI guideline also advices postgraduate
students to demonstrate the effects of various drugs,
to determine the nature of an unknown drug by bioassay, drug screening methods and to learn various
animal experiment skills as a part of the syllabus of
M.D. Pharmacology, which considers animal
experiments as one of the components of practical
examination[17, 28].
Chirag et al.,
CONCLUSIONS
In short, there is a need to put greater emphasis on
clinical teaching that can be more useful in clinical
practice after medical graduation. On the other hand,
implementation of the virtual animal experiment
might decreases expertise of animal experiments
among undergraduate medical students, but
experiments are lest utilized during their day to day
clinical practice.
Similarly, for postgraduate study, computer operated
software of virtual animal experiment can enhance
teaching and learning. Moreover, it can help junior
residents, to carry out experiments for undergraduate
practical demonstration in short span of time with
different protocols without sacrificing any animal for
the already established experimental outcome.
690
However, for research and development of new

chemical entity, currently there is no software or tool
that can analyze like a live animal or human.
Conflict of interest: There is no conflict of interest.
Acknowledgement: Authors are thankful to Elsevier
publishers for providing a demo version of simulated
animal experiment software Ex Pharm Pro.
Moreover, we are also grateful to the administrative
staff of Medical College, Baroda for providing
subscription of simulated animal experiments
software.
9.
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DOI: 10.5958/2319-5886.2015.00132.0

&
Health Sciences
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st
Volume 4 Issue 3
Coden: IJMRHS
th
Copyright @2015
ISSN: 2319-5886
Review article
DECIPHERING LEPTOSPIROSIS-A DIAGNOSTIC MYSTERY: AN INSIGHT
*Mohit Bhatia1, B L Umapathy2
1
Senior Resident, Department of Microbiology, Govind Ballabh Pant Institute of Post Graduate Medical
Education and Research, New Delhi
2
Professor, Department of Microbiology, Sree Mookambika Institute of Medical Sciences, Kulasekharam,
Kanyakumari District, Tamil Nadu
*Corresponding author email: docmb1984@gmail.com
ABSTRACT
Leptospirosis is an emerging infectious disease which has been recognized as the most common zoonotic
infection in the world. It affects human beings and many other species of vertebrates . Most commonly, the
infection is acquired by direct or indirect exposure to urine of reservoir animals through contaminated
soil, mud & water entering via small abrasions or breaches in the skin & mucous membranes during
occupational, recreational or vocational activities. The signs & symptoms resemble a wide range of
bacterial & viral diseases & sometimes can present as food poisoning, chemical poisoning & snake bite
also due to which the diagnosis is often missed. This review article aims to focus on the role of Dark Field
Microscopy (DFM), culture, Enzyme Linked Immuno Sorbent Assay (ELISA), Macroscopic Slide Agglutination
test (MSAT), Microscopic Agglutination Test (MAT) and Faines criteria in the diagnosis of leptospirosis.
Keywords: Dark Field Microscopy, Enzyme Linked Immuno Sorbent Assay, Macroscopic Slide Agglutination
test, Microscopic Agglutination Test, Faines criteria
INTRODUCTION
Leptospirosis also known by various names like
Weils disease, Pretibial fever, Fort Bragg
fever, Peapickers fever in different parts of
the world is an acute bacterial infection caused
by spirochetes belonging to the genus Leptospira
that can lead to multiple organ involvement and
fatal complications.[1] It has been recognized as the
most common zoonotic infection in the world.[2]
Leptospirosis has a wide geographical distribution
and occurs in tropical, subtropical and temperate
climatic zones. The incidence is higher in the
tropics than in temperate regions.[3] Most countries
in the South East Asia region are endemic to
leptospirosis.[2]
Leptospirosis affects human beings and many
other species of vertebrates .[2] Most commonly,
the infection is acquired by direct or indirect

exposure to urine of reservoir animals through
contaminated soil, mud & water entering via
small abrasions or breaches in the skin & mucous
membranes during occupational, recreational or
vocational activities.[4] A small number of organisms
can cause infection. The incubation period usually
ranges from 7-10 days. Leptospirosis may follow a
biphasic course. During the rst 10 days, there is a
phase of leptospiraemia when the leptospires multiply
in blood and spread to different organs. The chances
of recovery of leptospires from blood or other tissues
or body uids is usually high during this stage. This
phase is followed by immune phase or leptospirurea
phase when the organisms are excreted in the urine.
In this phase, the chance of recovery of organisms
693
Mohit et al.,
from the blood is low. The ideal specimen for

isolation or demonstration of leptosires during
immune phase is urine.[5] The signs & symptoms
resemble a wide range of bacterial & viral
diseases & sometimes can present as food
poisoning, chemical poisoning & snake bite also
due to which the diagnosis is often missed.[6] The
most
common clinical syndrome is anicteric
leptospirosis which is a self limiting illness.[4]
Weils disease or icteric leptospirosis, is generally
the most severe illness, with symptoms caused by
liver, kidney & vascular dysfunction (jaundice,
oliguria/anuria, bleeding & lethal pulmonary
haemorrages).[4] The case fatality rate of leptospirosis
is upto 10%.[4]
The clinical diagnosis of leptospirosis may be
difficult to arrive at. A high index of suspicion is
required in patients with Pyrexia of Unknown
Origin (PUO) with or without jaundice, especially in
presence of history of animal contact, diagnosis
may be established by laboratory investigations.[7]
The commonly followed case definition, which is
also recommended by the WHO and International
Leptospirosis Society prescribes that any person
presenting with acute onset of fever, headache
and body aches associated with severe muscle
tenderness particularly in
calf
muscles,
haemorrhages
including
sub-conjunctival
haemorrhage, jaundice, cough, breathlessness and
haemoptysis, oliguria, signs of meningeal irritation
should be suspected as a case of leptospirosis and
investigated[8]. A suspect, who tests positive in any
of the screening tests such as dipstick, lateral flow
or latex agglutination test should be considered as a
probable case. Successful isolation of Leptospires
from clinical specimens, a four-fold or higher rise
in titre or seroconversion in paired Microscopic
Agglutination Test (MAT) or a positive Polymerase
Chain Reaction (PCR)
is
considered
as
confirmatory evidence of current leptospiral
infection [8]. Owing to shortcomings of laboratory
tests in establishing early diagnosis of leptospirosis,
the World Health Organization (WHO), introduced
Faines criteria which includes the scoring of clinical,
epidemiological and laboratory parameters of patients
(Parts A, B and C respectively) [9].This criteria has
been simultaneously modified and validated by Brato
et al and Shivkumar et al, who recommended addition
of abdominal symptoms, local factors like rainfall and
newer investigations in the total scoring respectively

[10,11]
. Microbiological diagnosis of leptospirosis
aims at demonstrating the leptospires, by culturing
them or by demonstrating an appreciable antibody
response to them.[12]
Laboratory diagnostic tests are broadly divided
into two categories viz., Direct evidence isolation of organism or demonstration of
leptospires by dark field microscopy or
amplification of specific fragment of leptospiral
DNA; and Indirect evidence- detection of
antibodies to leptospires.[13]
This review article aims to focus on the role of Dark
Field Microscopy (DFM), culture, Enzyme Linked
Immuno Sorbent Assay (ELISA), Macroscopic Slide
Agglutination
test
(MSAT),
Microscopic
Agglutination Test (MAT) and Faines criteria in the
diagnosis of leptospirosis.
REVIEW
Dark-Field Microscopy (DFM)
DFM has often been employed for examination of
body fluids such as blood, urine, CSF and
dialysate fluid.[14] The specimens should be taken
aseptically and sent to laboratory without delay.
They must not be frozen.[5] Oxalate, citrate,
heparin or EDTA may be used as anticoagulant
for blood or pleural fluid.[5,15] Approximately 104
leptospires/ml of sample are necessary for one
organism per field to be visible by DFM.[14]
DFM is the procedure of choice for the
demonstration of the organisms in tissue fluids.[16]
It is particularly useful for observing leptospires
in culture, particularly when they are present in
large numbers. It can be used for observing
agglutination in the MAT.[17] However, it is
technically demanding. Recognizing leptospires is
difficult, particularly when only small numbers
are present.[17] Reading the results is always
subjective as in the majority of the samples the
number of organism per field ranges from 0-2
and there is always doubt about typical
motility.[5] Double centrifugation of the sample at
low speed to separate the cellular elements, and
then at high speed, help concentrate the
leptospires.[12] The usefulness of differential
centrifugation is limited and the motility of the
organism further reduces after centrifugation at
higher g.
694
Mohit et al.,
The advantage of this technique is that laboratories,

where the facilities for the other tests are not
available, can undertake this technique. But the
results should be conrmed with other standard tests.
The various limitations of this technique include the
following: Low sensitivity (40.2%) and specificity
(61.5%). Serum proteins and brin strands in blood
resemble leptospires thereby making it extremely
difficult to diagnose the disease. The concentration of
organism is frequently too low in the specimens. This
technique requires expertise. [5]
Culture: Leptospires are obligate aerobes with an
optimum growth temperature of 28 C to 30C.[14]
They can be grown in liquid, semi-solid
(containing 0.2-0.5% agar) or solid media.[18] The
only organic compounds required for their growth
are Vitamins B1 and B12, and long chain fatty
acids . Fatty acids are the main source of carbon
and energy for leptospires. These are also required
as a source of cellular lipids, as leptospires
cannot synthesize fatty acids de novo. Liquid
medium is used for the cultivation of leptospires
which can later be used for harvesting antigens to be
used in various serological tests. Liquid medium can
be converted into semisolid and solid by the
addition of agar or agarose. Semisolid media
contain 0.2-0.5% agar whereas solid medium
contain 0.8-1% agar.[18,19] Semisolid medium is
commonly used for the isolation of leptospires
and for maintaining the cultures. In semisolid
media, growth reaches a maximum density in the
form of a discrete zone under the surface of the
medium usually within 7-21 days. This growth is
known as Dingers ring and is related to the
optimum oxygen tension.[14] Solid medium is not
ideal for isolation or maintenance of leptospires
and mainly used for the research purpose to
clone the leptospires from mixed letospira
cultures.[19]
A wide variety of culture media can be used for
the cultivation of leptospires.[19] The routinely
used culture media are described briefly here:
a) Media that contain rabbit serum: These include
Korthofs medium, Fletchers medium and Stuarts
medium. Rabbit serum contains nutrients including
high concentrations of bound vitamin B12 which
helps in the growth of leptospires. All these
media can be used for the isolation of leptospires
from the clinical specimens and for the
maintenance of leptospires but not for the

preparation of antigens for MAT.[19]
b) Fatty acid albumin medium: In this medium long
chain fatty acid is used as a nutritional source
and serum albumin as detoxicant. This medium is
popularly known as Ellinghausen-McCulloughJohnson-Harris (EMJH) medium and widely used
for isolation, maintenance and preparation of
antigens for MAT and for growing leptospires in
bulk.[19]
c) Protein-free medium: In this medium long chain
fatty acids are treated with charcoal to detoxify
the free fatty acids which are highly toxic to
leptospires.[19]
Culture media can be enriched by addition of 1%
fetal calf serum or rabbit serum to cultivate the
fastidious leptospiral serovars.[19] Due to the ability
of leptospires of incorporating purine and not
pyrimidine bases into their nucleic acids, they are
resistant to the antibacterial activity of the
pyrimidine
analogue,5-Fluorouracil.[12] Selective
culture media, containing 5-FU 50-1000 g/ml or
a combination of nalidixic acid 50 g/ml,
vancomycin 10 g/ml and polymixin B sulphate 5
units/ml or a combination of actidione 100 g/ml,
bacitracin 40 g/ml, 5 FU 250 g/ ml, neomycin
sulphate 2 g/ml, polymixin B sulphate 0.2 g/ml
and rifampicin 10 g/ml can be used to avoid the
contamination.[19] Contaminated cultures may be
passed through a 0.2m or 0.45m filter before
subculture into fresh medium.[20]
The infecting strains can often be isolated in
culture, provided that suitable material is obtained
before antibiotics have been administered. Early in
the course of illness during the leptospiraemic
phase the samples of preference are blood or
cerebrospinal fluid; later during the phase of
leptospiruria it is urine.[12]
Blood: Leptospires have rarely been isolated from
blood weeks after the onset of symptoms.[14] Blood
should be cultured in the first ten days of the
illness and before antibiotics are given. Venous
blood is collected with aseptic precautions and
ideally inoculated at the bedside into blood
culture bottles containing culture medium for
Leptospira. Only few drops of blood are inoculated
into several tubes, each containing 5 ml of a
suitable medium as large inocula will inhibit the
growth of leptospires.[17] Cultures are incubated
695
Mohit et al.,
between 28 to 30C for 4 to 6 weeks or

longer.[5,14] Culture are examined using dark field
illumination initially on first, third and fifth days
followed by 7-10 days interval up to 6 weeks.[5]
Blood culture is particularly valuable in man, as
the serological response can be slow and may be
absent altogether if antibiotics are given early.
Urine: During the leptospiruria phase which is
characterized by increasing concentrations of
antibodies after about 1 week from onset of clinical
illness, the urine and the renal cortex post mortem
are the most suitable inocula for the isolation of
leptospires from humans. Fresh mid-stream urine is
collected and inoculated immediately. One drop of
undiluted urine is inoculated into the first tube
containing 5 ml of culture medium. Alternatively,
urine samples may be centrifuged for 30 minutes at
1600 g or 1 min at 10,000 g and the pellet resuspended in medium, after which ten-fold serial
dilutions are made immediately in 1 or 2
additional tubes. Since urine is acidic and
decreases the viability of leptospires, it should be
inoculated into the medium within 2 hours after
voiding. Viability is reported to be increased in
urine samples neutralized with sodium bicarbonate
and by using phosphate-buffered bovine serum
albumin solution. Media containing 5-fluorouracil
or appropriate antibiotics that suppress the growth
of bacterial contaminants and leave leptospires
unaffected may be beneficial in reducing the
contamination of media inoculated with urine
samples obtained from various patients clinically
suspected to be suffering from leptospirosis.[17]
Cerebrospinal Fluid (CSF): Leptospires may be
observed in CSF by DFM and can be isolated
by inoculating 0.5ml CSF into 5 ml semi-solid
culture medium during the first week of illness
(usually upto 10 days of onset of clinical signs &
symptoms).[17,21]
The various advantages of this technique include the
following: Isolation in culture is a denite proof of
infection. Circulating serovars can be identied.
Local isolates can be used as antigens in MAT and
vaccine development. [5]
The various disadvantages of this technique include
the following: Leptospires are fastidious organisms
which require special medium for isolation.
Leptospires grow slowly and therefore, isolation of
leptospires from clinical specimens takes several days
to several weeks. The technique is laborious, time

consuming and is not possible in small laboratories.
Contamination of culture media by other microorganisms or by saprophytic leptospires is common in
routine practice. The successful isolation rate is less
due to prior use of antibiotics, imperfectly cleaned
glass ware or wrong incubation temperature and pH.
[5]
The various serological tests used for the diagnosis

of leptospirosis can be divided into two groups,
those, which are Genus specific, and those,
which are Serogroup specific.(13) The various Genus
specific tests are Macroscopic Slide Agglutination
test (MSAT), Indirect Fluorescent Antibody Test
(IFAT),
Indirect Hemagglutination Test (IHA),
Counter Immuno Electrophoresis
(CIEP),
Complement Fixation Test (CFT), Enzyme Linked
Immuno Sorbent Assay (ELISA), Microcapsule
Agglutination Test (MCAT) and Lepto-Dipstick. The
Serogroup/serovar specific tests are Microscopic
Agglutination Test (MAT) and Serovar specific
ELISA.[12]
Leptospires have a complex antigenic structure.[12]
Within the two species L.interrogans and L.biflexa,
there are many serotypes (now referred to as
sero-varieties or serovars) that are distinguished
by
cross-agglutinin-absorption
tests
or
by
antigenic factor analysis. Some of the serovars are
closely related because of common antigens and
form clearly defined serogroups.[18] The somatic
antigen is genus specific. The surface antigen which
is a polysaccharide is serovar specific.[12] The outer
membrane is lipopolysaccharide (LPS) in nature
and is a potent immunogen. It is the major antigen
and the target of antibody and complement-mediated
bactericidal activity. Antibodies directed against this
antigen are protective in nature. Flagellar antigen is
composed of both genus and serotype specific
antigens. Some serovars, e.g. L. icterohaemorrhagie,
have an additional Vi antigen associated with
virulence.[12] The leptospiral outer membrane
contains both transmembrane proteins, such as the
porin OmpL1 and lipoproteins such as LipL41 and
LipL36 .[22] Outer membrane proteins (OMPs) that
are exposed on the leptospiral surface are potentially
relevant in pathogenesis because of their location at
the interface between leptospires and the mammalian
host. Results of surface immunoprecipitation studies,
immunoelectron
microscopy
&
topological
696
Mohit et al.,
considerations suggest that OmpL1 and LipL41 are

present on the surface. Unlike leptospiral LPS,
OmpL1 and LipL41 are antigenically conserved
among pathogenic Leptospira species. OmpL1 and
LipL41 are expressed during infection of the
mammalian
host.[22]
LipL32,
the
32-kDa
lipoprotein is also a prominent immunogen during
human leptospirosis. The sequence and expression
of LipL32 is highly conserved among pathogenic
Leptospira species. LipL32 may be important in
the pathogenesis, diagnosis, and prevention of
leptospirosis.[23]
Enzyme linked Immuno Sorbent Assay (ELISA)
This is now widely used as a genus specific
screening test in man.[12] It is a very sensitive and
specific test for the biological diagnosis of
leptospirosis.[17] It is of particular value as a
serological screening test because of its relative
simplicity in comparison with the MAT.[17] Both
peroxidase and urease labelled conjugates have
satisfactorily been used. Stable reagents are
available and form the basis of bedside tests,
which are read visually. The use of computer
assisted automated readers and the appropriate
controls has improved the reproducibility and
predictive value of this test. They can be
performed with commercial kits or with antigen
produced "in house".[12] Antigen preparation for
ELISA is done cultivating Leptospira interrogans
serovar Copenhageni strain Wijnberg or L. biexa
serovar Patoc strain Patoc I in EMJH medium for 1012 days at 30C in shaking incubator. Abundant
growth is necessary to produce a good antigen.[5]
Several attempts have been made to develop
serotype specific ELISA tests with a variety of
extracted antigens. Tests based on boiled whole
cell antigens tend to be genus specific but those
based on ultrasound-disintegrated or phenolextracted preparations show considerable serotype
specificity.[12]
The ELISA test gives a positive response in the
diagnostic evaluation of leptospirosis a little earlier
than the MAT because it is more sensitive to
IgM antibodies. A response 68 days following
the appearance of the first clinical signs is
generally observed. On the other hand, the test
may become negative more quickly than MAT,
although low levels of specific IgM may persist.
A potential advantage of the ELISA test is that
it may help to differentiate between current

leptospirosis and previous leptospirosis since
antibodies from past infection or immunization
may not be detectable. However, if a total human
anti-Ig or IgG conjugate is used instead, the
positivity of the test may be extended, allowing
the detection of residual antibodies in recovered
or immunized patients. The level of positivity
observed with a total anti-Ig conjugate is then
always equal to or higher than the maximum
observed with anti-IgG or anti-IgM antibodies.[17]
Other advantages include the following: Single
antigenic preparation can be used for ELISA; Heat
stable antigens which are stable at room
temperature for long periods are generally used;
ELISA allows rapid processing of large number of
samples.
A limitation to the use of single serum samples
for serodiagnosisis the persistence of antibodies.
Anti-leptospiral IgM antibodies are also persistent,
but the rate of decline shows marked variation.
Thus, a single IgM positive sample taken during
an acute febrile illness with symptoms suggestive
of leptospirosis is presumptive evidence of
infection, but this finding requires confirmation by
testing a convalescent sample, preferably by the
use of an alternative method.[24] Infecting serovar
cannot be assessed which makes it comparatively
less specific.[12] Another disadvantage of this
procedure is that it usually requires calibration of
cut-off values and significant titres.[5]
Macroscopic Slide Agglutination Test (MSAT)
A rapid macroscopic slide agglutination test can
be used to screen human and animal serum
samples. This test is carried out with a dense
suspension of leptospires, which agglutinate into
clumps visible to the naked eye. It can be
performed on slides or plates. There can be different
ways to prepare the antigens. One may either use a
single serovar or multiple serovars to prepare the
antigens. When multiple serovars are used as
antigens, antigens can be pooled. Since the technique
will require lot of laboratory work, generally a single
non-pathogenic serovar patoc (strain Patoc 1) is
used.[12] The basic principle of the test is similar to
other slide agglutination tests used in other infectious
diseases such as enteric fever or brucellosis. The
reaction is recorded as ++ when the clumps are large
and denite, + when the smaller clumps are well
697
Mohit et al.,
dened but the suspension is not clear, +/- when ne

clumps are visible but the suspension is not clear and
negative when the mixture in the drop is unchanged.
Agglutination of + and ++ is considered as positive.[5]
It is a simple, rapid, and sensitive diagnostic test
for active leptospirosis and allows a provisional
diagnosis of acute leptospirosis to be made
within a few minutes.[12] The antigen is broadly
reactive and stable for six months at 4C to 8C.[5] It
is more sensitive than Microscopic Agglutination
Test (MAT) in the early stage of the disease. The
sensitivity of this test can be enhanced by adding
the locally-prevalent serovars. MSAT has shown
good correlation with both IgM ELISA and MAT
in various studies, and therefore can be used as a
valuable and simple screening test. It is not
however suitable for retrospective or survey work.[12]
A high percentage of false positive reactions are
observed, probably due to lack of standardization and
quality control of the antigen preparation. The
number of false negative reactions are comparatively
low.[5] Positive reactions should therefore be
confirmed by complement fixation or microscopic
agglutination tests.[12]
Microscopic Agglutination Test (MAT) : This test
is considered the gold standard for serodiagnosis
of Leptospirosis.[25] MAT is carried out with
suspensions of live cultures or with cultures
killed by
the
addition
of
neutralized
formaldehyde.[12] There is a difference in appearance
between the clumps of agglutinated living
leptospires and those of killed cultures. Living
leptospires are agglutinated into highly refractile
spheroids of various sizes which may join to
produce elongated masses of confluent spheroids.
By contrast, the agglutinated killed leptospires
form looser masses with an irregular often
angular,
outline;
these
appear
flattened,
resembling small piles of threads, or snowflakes,
or pieces of cotton wool.[12]
Agglutinating antibodies can be of both IgM and
IgG classes.[17] The degree of agglutination can
only be assessed in terms of the proportion of
free leptospires. The accepted endpoint of an
agglutination reaction is the final dilution of
serum at which 50% or more of the leptospires
are agglutinated. Preparations for MAT require
meticulous culture of a collection of the live
strains used as antigen suspensions, their regular
subculture and quality control for authenticity and

purity and also skilled educated personnel.[11]
A recent advance is the use of standardized
preparations of dried leptospires available to
accredited diagnostic laboratories from a central
reference laboratory. Multi-antigen MAT uses a
battery of strains giving comprehensive coverage
of all serogroups and provides an alternative to the
so-called genus-specific tests as a means of
diagnosing leptospirosis. However, the necessity to
maintain large number of live strains of L.
interrogans limits its use to reference laboratories.
Wherever possible, local isolates of known
identity should be included in the battery of
strains in order to increase both the sensitivity and
the specificity of this widely accepted gold standard
test.[12]
Interpretation of diagnostic MAT: The MAT is
usually positive 1012 days after the appearance
of the first clinical symptoms and signs but
seroconversion may sometimes occur as early as
57 days after the onset of the disease.[17] The
antibody response may be delayed if antibiotic
therapy was given before the test. A patient with a
compatible history may be considered to have current
leptospirosis if he has IgM antibodies to leptospira
and a MAT titre of >/=80.[26]
The optimal cut-off titre is assessed by carrying
out a baseline study on distribution of titres in
the community as well as among confirmed
patients. Using this data it is possible to estimate
the sensitivities and false positivity rates at
different cut-off titres. The titre that gives the
lowest number of false results is then chosen as
the optimal cut-off titre.[5]
In a non-endemic area, even a low level of antibodies
may signify leptospirosis in the first week of a
clinically compatible illness. The titre will rise in
a second specimen taken after 3 to 7 days. If the
titre remains below 100, even on repeated testing
then it could probably be due to previous
leptospirosis, and not current illness. In endemic
areas, the diagnosis will be confirmed if the titre
rises on retesting, but will be negated if it is
unchanged, assuming that the infecting serovar
was included among the antigens used for MAT.[12]
MAT is the serological test used in reference
laboratories, because of its high degree of
sensitivity and specificity.[27] MAT remains very
698
Mohit et al.,
useful for epidemiologic studies, identification of

strains, assessment of the probable infecting
serogroup and confirmation of illness for public
health surveillance.[28]
The disadvantages of MAT are that 1421 strains
have to be maintained in culture, which is often
very difficult. Procedure is complex and time
consuming. Reading results requires experienced
personnel.[5] It cannot be standardized as live
antigens are often used and various factors, such
as the age and density of the antigen culture, can
influence the agglutination titre.[17] Co-agglutinins
(cross-reactions) are frequently present in the sera
of patients with leptospirosis. Antibodies which
cause cross-reactions are often the first to appear
but they disappear rapidly. Homologous antibodies,
although they appear slightly later, persist much
longer, thus
allowing
the
presumptive
identification of the serogroup responsible for the
infection and also the detection of traces
indicating previous infections.[17] Some patients
have serological evidence of previous infection
with a different leptospiral serogroup. In these
patients, serological diagnosis is complicated
further by the anamnestic response, in which the
first rise in antibody titre is usually directed
against the infecting serovar from the previous
exposure. Only later does it become possible to
identify the serovar or serogroup responsible for
the current infection, as the titre of specific
antibody rises. Paradoxical reactions also occur in
patients
who have
such
infections, and
interpretation
of
serology
is
further
[14]
complicated.
The MAT cannot differentiate
between agglutinating antibodies due to current,
recent or past infections.[17] Therefore, the use of
MAT is only restricted to some of the specialised
laboratories.[29]
Faines criteria for diagnosis of leptospirosis
Faine had formulated a criteria for diagnosis of
leptospirosis
on
the
basis
of
clinical,
epidemiological and laboratory data (Parts A, B and
C respectively). A presumptive diagnosis of
leptospirosis may be made if:
(i) Parts A and B score = 26 or more (Part C
laboratory report is usually not available before
fifth day of illness; thus it is mainly a clinical and
epidemiologic diagnosis during early part of
disease).
(ii) Part A+B+C = 25 or more A score between 20

and 25: Suggests a possible but unconfirmed
diagnosis of leptospirosis.[9]
Shivakumar et al from Chennai have suggested
modification on Faines criteria to include local
factors like rainfall and newer investigations in the
total scoring.[11] As per this, epidemiological and
laboratory criteria (Parts B and C) are modified
only; no modification is made in the clinical criteria
(Part A).
The reasons for modification suggested are as
follows:
(i) Most of the leptospirosis is reported in
monsoon and post-monsoon period. Therefore they
have suggested rainfall separately to be adjusted
in the scoring criteria of Part B.
(ii) Microscopic agglutination test (MAT) is the
Gold Standard test, but is complicated and less
sensitive compared to newer tests like IgM
ELISA and Slide Agglutination Test (SAT). IgM
ELISA and SAT are simple, sensitive tests and
can be used to diagnose current leptospirosis.
Thus, they have been included with appropriate
score being assigned to each one of these.
The difficulties in utilizing MAT are due to the
following reasons:[30]
(a) The antibody titres rise and peak only in
second or third week. Thus, paired sera are required
to demonstrate four-fold rise of titre.
(b) The test is complicated requiring dark-field
microscopy and cultures of various serovars, which
may not be available in smaller laboratories.
The advantage of including simple diagnostic
tests (IgM ELISA or SAT ) in modified Faines
criteria is that it helps in diagnosing milder
forms of leptospirosis which are associated with
low clinical score (Part A). Suggestion of
modification of existing Faines criteria appears
justified; however further evaluation is required.[30]
Table 1shows comparison between Faines &
Modified Faines criteria.
699
Mohit et al.,
Table: 1. Table showing comparison between Faines & modified Faines criteria [9,11]
Faines criteria
Modified Faines criteria
PART A: Clinical data
PART
A:
Clinical data
Score
Score
Headache
2
Headache
2
Fever
2
Fever
2
If fever, temperature 39C or more
2
If fever, temperature 39C or more
2
Conjunctival suffusion (bilateral)
4
Conjunctival suffusion (bilateral)
4
Meningism
4
Meningism
4
Muscle pain (especially calf muscle)
4
Muscle pain (especially calf muscle)
4
Conjunctival suffusion + meningism +
10
Conjunctival suffusion + meningism +
10
muscle pain
muscle pain
Jaundice
Jaundice
1
1
Albuminuria or nitrogen retention
Albuminuria or nitrogen retention
2
2
PART B: Epidemiological factors
PART B: Epidemiological factors
Contact with animals or contact with
Rainfall
5
known contaminated water
Contact
with
contaminated
environment
4
10
Animal contact
1
PART C: Bacteriological and laboratory
PART
C:
Bacteriological
and
findings
laboratory findings
Isolation of leptospires in culture:
Isolation of leptospires in culture:
Diagnosis
Diagnosis
certain
certain
Positive serology (MAT)
Positive serology
Leptospirosis (endemic)
ELISA IgM positive*
15
2
Single positive low titre
MSAT positive*
15
10
Single positive high titre
MAT single high titre*
15
MAT rising titres (paired sera)*
25
*Only one of these tests to be scored
Leptospirosis (non-endemic)
5
Single positive low titres
15
Single positive high titres
25
Rising titres (paired sera)
CONCLUSION
Leptospirosis is probably an under diagnosed
infectious disease which mysteriously mimics several
clinical conditions. It is an emerging infectious
disease, the current incidence of which only
represents tip of an iceberg. Although, several
modalities are currently available for diagnosing this
deadly infectious disease, but each one of these has
certain limitations. Newer diagnostic tests for
leptospirosis are need of the hour which would aid
clinical diagnosis during the initial phase of the
disease and rapid case confirmation during outbreak
surveillance.
Acknowledgment : Dr Bibhabati Mishra, Director,
Professor and Head, Department of Microbiology,
Govind Ballabh Pant Institute of Post Graduate
Medical Education and Research, New Delhi.

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Medical
Research centre, Port Blair, WHO Country office
for India, WHO, 2007, pp.1-3
3. Narita M, Fujitani S, Haake DA, Paterson DL.
Leptospirosis
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exposure to water in the Yaeyama islands, Japan.
Am J Trop Med Hyg. 2005;73(4):652-56
4. The Spirochetes. In: Betty AF, Daniel FS, Alice
SW, (eds.), Bailey & Scott's.Diagnostic
Microbiology, 12th ed, St.Louis, Missouri,
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700
Mohit et al.,
5. Vijayachari
P,
Sameer
Sharma
&
Natarajaseenivasan K. Laboratory Diagnosis. In:
Leptospirosis Laboratory Manual, Regional
Medical Research centre, Port Blair ,WHO
Country office for India, WHO, 2007, pp.27-45
6. Sharma KK, Kalawat U. Early diagnosis of
leptospirosis
by
conventional
methods: One year prospective study. Indian J
Pathol Microbiol 2008; 5:209-11
7. Angnani R, Pathak AA, Mishra M. Prevalence of
leptospirosis in various risk groups. Indian J Med
Microbiol 2003 Oct;21(4):271-73
8. Singh SS. Clinical Manifestations. In:
Leptospirosis Laboratory Manual, Regional
Medical Research centre, Port Blair, WHO
Country office for India, WHO, 2007, pp.22-26
9. Faine.S. Guidelines for the control of
Leptospirosis. WHO offset publication 1982:67
10. Brato DG, Mendoza MT, Coredero CP,
Leptospirosis Study Group. Validation of the
WHO criteria using the MAT as the gold
standard in the diagnosis of leptospirosis. PJMID
1998;27:125-128
11. Shivakumar S, Shareek PS. Diagnosis of
leptospirosis utilizing modified Faines criteria.
JAPI 2004 Aug;52:678-79
12. Sambasiva RR, Naveen G, Bhalla P, Agarwal
SK.
Leptospirosis
in
India
and
the
rest of the world. Braz J Infect Dis
2003;7(3):178-93
13. Vijayachari P, Sehgal SC. Recent advances in the
laboratory
diagnosis
of
leptospirosis and characterization of leptospires.
Indian
J
Med
Microbiol.
2006
Oct;24(4):320-22
14. Levett PN. Leptospirosis. Clin Microbiol Rev
2001 April;14(2):296-326
15. Peter S. Leptospirosis. In: Joseph L, Kasper DL,
Braunwald E, Fauci AS, Hauser SL, Longo DL,
Jameson JL, (eds.), Harrisons Principles of
Internal Medicine, 17th ed, New York: McGraw
Hill, 2005, pp.988-95
16. Gangadhar N L, Prabhudas K , Sashi B ,
Munasira S , Barbuddhe SB, Rehaman H.
Leptospira infection in animals and humans:a
potential public health risk in India. Rev. sci.
tech. Off. int. Epiz. 2008;27(3):885-92
17. World Health Organization. Human
Leptospirosis: Guidance for diagnosis,
surveillance and control [Book on the internet].
World Health Organization;2003
http://whqlibdoc.who.int./hq/2003/2003/WHO_C
DS_CSR_EPH_2002.23.pdf
18. Coghlan JD. Leptospira, Borrelia, Spirillum. In:
Collee JG, Fraser AG, Marimon BP, Simmons A,
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Laboratory Manual, Regional Medical Research
centre , Port Blair ,WHO Country office for
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Ahmad SN, Shah S, Ahmad FMH. Laboratory
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Joseph M.V. Leptospirosis. In: Kasper DL, Fauci
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David AH, Mary KM, Adam MM, Frank M,
Garlo C, James M et al. Leptospiral Outer
Membrane Proteins OmpL1 and LipL41 Exhibit
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David AH, Garlo C, Richard LZ, Jeanne KB,
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Levett PN, Branch SL. Evaluation of two
enzyme-linked Immunosorbent assay methods for
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Hyg.2002;66(6):745-48
Sofia AS, Ahmed AS, Shaila M. Laboratory
Methods for Diagnosing Leptospirosis: A
Review. Bangladesh J Med Microbiol
2009;03(01):39-43
Prasad SR, Rajini M. Leptospirosis: An
Overview. The Journal of the Academy of
Clinical Microbiologists 2008;10(2):89-97
Mary DB, David AA, Sandra LB, Christopher
WW, Tin A, Richard AS et al. Evaluation of Four
Commercially Available Rapid Serological Tests
for Diagnosis of Leptospirosis. J Clin Microbiol
2003 Feb;41(2):803-09
Brandao AP, Camargo ED, Silva ED, Silva MV,
Abrao
RV.
Macroscopic
Agglutination Test for rapid diagnosis of human
Leptospirosis.
J
Clin
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1998
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Smits HL, Van der Hoorn MAWG, Goris MGA,
Gussenhoven
GC,
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Sasaki DM et al. Simple Latex agglutination
assay
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J Clin Microbiol
2000 Mar;38(3):1272-75
Dutta TK, Christopher M. Leptospirosis-An
Overview. JAPI 2005 June;53:545-51
701
Mohit et al.,
DOI: 10.5958/2319-5886.2015.00133.2

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LYMPHOEPITHELIOMA LIKE CARCINOMA OF ORBITAL ADNEXA: A RARE CASE REPORT

*Lohit Kumar Kalita1, Chayanika Kalita2, Ahmed Ali3, Umesh Chandra Sharma4
1
Assistant Professor, Department of Oncology, 2Assistant Professor, Department of Dermatology, 3Assistant

Professor, Department of Pathology, Guwahati Medical College & Hospital,4Vice Chancellor, Srimanta
Sankadeva University of Health sciences, Narakasur HillTop Guwahati, Assam, India.
*Corresponding author email:lkkalita2013@gmail.com
ABSTRACT
Lymphoepithelioma like squamous cell carcinoma is an extremely rare malignancy in ocular adnexa. Here we
have reported a case presenting with a growth arising from the adnexa of the right orbit with gradual loss of vision
followed by enlargement of the growth with ulceration and sudden loss of vision. Biopsy was taken from the
adnexa of the eye which revealed it to be Lymphoepithelioma type of squamous cell carcinoma.
Keywords: Carcinoma, Malignant, Vision
INTRODUCTION
Lymphoepithelioma like carcinoma is a histological
variant of malignant tumor arising from the
uncontrolled mitosis of transformed cells originating
in epithelial tissue or in cells that display epithelial
characteristics that have morphologic features similar
to those of undifferentiated nasopharyngeal
carcinoma(UDNPC).Histologically,Lymphoepithelial
carcinoma is characterized by nests, sheets, or
individual undifferentiated or poorly differentiated
malignant epithelial cells surrounded and infiltrated
by prominent components of small, mature
lymphocytes and plasma cells.[1,2] There is
considerable variation in the classification of
Lymphoepithelioma like carcinoma-while Lympho
epithelioma like carcinoma is perhaps most
commonly considered a subtype of squamous cell
carcinoma. It can also be classified as a form of large
cell carcinoma (i.e. while occurring in lung [3], and
can be regarded as a separate, unique entity [4]. In
most
anatomical
sites,
many
cases
of
Lymphoepithelioma like carcinoma are associated
with the Epstein-Barr virus. [5].
CASE REPORT
Lohit et al.,
The patient came to outpatient department with a

growth of the right eye (Fig-1) for last months that
suddenly became enlarged since last one week with
sudden loss of vision. He also had associated
weaknees, palpitation on working, and loss of
appetite. There is no history of fever. On examination
he had pallor and mild hepatosplenomegaly. The
growth of the right orbit is non tender, hard to soft in
consistency, bleeds on touch, adherent to underlying
structures. Blood routine examination report was
normal
except
low
haemoglobin.
Routine
biochemical parameters were normal except high
LDH level. Biopsy was taken from the growth and
sent for histopathological examination. The
histopathology revealed nest of poorly differentiated
to undifferentiated malignant cells surrounded by
sheets
of
lymphocytes
suggestive
of
Lymphoepithelioma type squamous cell carcinoma
(Fig-2).
702
Fig 1: Patient with growth at the right ocular

adnexa
reported in literature and comprehensive descriptions

of this entitys biologic behaviour and optimal
treatment are lacking. In 2013, B. Qiu et al of
department of radiation oncology, State key
Laboratory of Oncology in South China, Sun YatSen University Cancer center, Guangzhou, PR China,
reported 4 cases of Lymphoepithelioma like
carcinoma of ocular adnexa[10]. As Lympho
epithelioma like carcinoma involving ocular adnexa
is extremely rare. We have reported the case of
Lymphoepithelioma like carcinoma involving ocular
adnexa.
CONCLUSION
Lymphoepithelioma-like carcinoma is a hetrogenous
entity among ocular adnexal malignancies. Lacrimal
glands, lacrimal drainage system, eyelid and
conjunctiva are potential primary sites for the
development of the disease. Because of the low
incidence of this entity and limited studies, further
efforts with a large number of cases are required to
know more details about of the disease and to
determine optimal treatment and prognosis of the rare
disease.
Fig 2: Histopathological shows nest of poorly

differentiated to undifferentiated malignant cells
surrounded by sheets of lymphocytes suggestive of
Lymphoepithelioma
type
squamous
cell
carcinoma
DISCUSSION
Lymphoepithelioma like carcinoma itself is a very
disease where the more frequently involved
anatomical sites of is the nasopharynx, salivary
glands and the larynx [6]. More rarely, it affects organs
like lungs, esophagus, stomach, pancreas, skin,
cervix, endometrium, vulva, kidney, bladder, and
central nervous system [7]. In vast majority of cases,
patients are asymptomatic, and the lesion is
discovered incidentally by imaging [8]. Moreover,
Lymphoepithelioma like carcinoma have been
reported in several other sites in the head and neck
region, including the floor of the mouth, tonsil,
thymus,
larynx,
and
sinonasal
tract[9].
Lymphoepithelial carcinoma of ocular adnexa is an
extremely rare malignant tumor. To our knowledge so
far about 14 cases of Lymphoepitheliomal like
carcinoma involving at ocular adnexa have been
Lohit et al.,

REFERENCES
1. Lanier AP, Bornkamm GW, Henle W, et al.
Association of Epstein-Barr virus with
nasopharyngeal carcinoma in Alaskan native
patients: serum antibodies and tissue EBNA and
DNA. Int J Cancer. 1981; 28(3):3015.
2. Hyun J, Seong Kook P, Kyung Wook H, Mi Seon
K: Lymphoepithelial carcinoma of the maxillary
sinus with orbital invasion. Auris Nasus Larynx
2009, 36:487-90.
3. Travis, William D, Brambilla, Elisabeth, MullerHermelink, H Konrad et al. Eds. Pathology and
genetics of tumors of the lung, pleura, thymus
and heart. World Health Organization
classification of tumors. Lyon: IARC Press:
2014; 92:832
4. Skinner NE, Horowitz RI, Majmudar, B.
Lymphoepithelioma like carcinoma of the uterine
cervix cervix. South Med J. 2000; 93(10):1024-7.
5. Mayer EK, Beckley I, Winkler MH.
Lymphoepithelioma- like carcinoma of the
urinary bladder diagnostic and clinical
703
6.
7.
8.
9.
10.
implications. Nat Clin Pract Urol. 2007; 4 (3):

176-71.
Coskun BU, Cinar U, Sener BM, Dadas B:
Lymphoepithelial carcinoma of the larynx. Auris
Nasus Larynx 2005;32:189-93
Ewing J: Lymphoepithelioma. Am J Pathol
1929;5:99-07
Sone M, Nakahima T, Nagasaka T, Itoh A,
Yanagita N. Lymphoepithelioma-like carcinoma
of the larynx associated with an Epstein-Barr
viral infection. Otolaryngol Head Neck Surg
1998; 119:134-37.
Tsai CC, Chen CL, Hsu HC. Expression of
Epstein-Barr virus in carcinomas of major
salivary glands: a strong association with
lymphoepithelioma-like carcinoma. Hum Pathol.
1996; 27(3):25862.
Qiu B. Primary Lymphoepithelioma like
carcinoma of ocular adnexa: Clinicopathologic
features and treatment. Oncologic Pathology
Home, 2013; 20(2): 177
704
Lohit et al.,
DOI: 10.5958/2319-5886.2015.00134.4

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Case report
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Accepted: 2nd May 2015
OSSIFYING RETROPERITONEAL CYSTIC LYMPHANGIOMA IN A PREGNANT WOMAN

*TengliMandakiniB1, Ahmed Mateen M2
1
Associate Professor, 2Assistant Professor, Department of Pathology, Khajabandanawaz, Institute of Medical

Sciences, Rouzabuzurg, Gulbarga, Karnataka, India.
*Corresponding author email: mandakinibt@gmail.com
ABSTRACT
Retroperitoneal cystic lymphangiomas are very rare lesions and may be misdiagnosed. Longstanding
lymphangiomas may show secondary changes like inflammation, hemorrhage, fibrosis and rarely ossification.
Treatment is complete surgical excision. We are reporting a rare case of ossifying retroperitoneal lymphangioma
in a pregnant woman which was misdiagnosed clinically as ovarian tumor. Our diagnosis was confirmed by IHCCD-31 and D2-40 positivity. Postoperative follow up for 3 years, patient is fine and she is full term pregnant now.
Keywords: Retroperitoneum, Cystic lymphangiomas, Ossification, Pregnant woman, IHC D2-40 and CD-31.
INTRODUCTION
Lymphangiomas are rare benign cystic neoplasms of
the lymphatic system, common in children but rare in
adults [1] . They are thought to occur due to
obstruction of the local lymph flow. Approximately
95% of the lymphangiomas are found in head neck
and axilla, and 5% appear in other regions like the
lungs, pleura, pericardium, esophagus, stomach,
jejunum, colon, pancreas, liver, gallbladder, kidney
and mesentery [2]. 1% of all lymphangiomas are
located in retroperitoneum and nearly 186 cases have
been reported [3]. The most common presentation of
lymphangioma is that of a soft, fluctuant mass that
enlarges, remains static, or waxes and wanes during
the period of clinical observation [4]. We report a case
of ossifying retroperitoneal cystic lymphangioma in a
pregnant woman.
CASE REPORT
A 28 years old woman, gravida 2, para 1, presented
with history of 3 months amenorrhea and mass in the
left side of the lower abdomen since two years, but
painful since three months. Clinically it was thought
to be an ovarian tumor. USG showed a
retroperitoneal cystic mass. The mass was enucleated

and pregnancy was terminated. The mass was sent for
histopathological examination to the department of
pathology KBNIMS, Gulbarga. Grossly it was a large
globular mass, grey white to grey brown in colour,
measuring 15x12x10 cms, well circumscribed. Cut
surface showed multiple cysts of varying sizes (0.3 to
2.5cms), filled with cloudy fluid and showed
hemorrhagic areas (Fig-1).
Microscopy showed multiple cystic spaces, these
cystic spaces were lined by flattened endothelial cells
(Fig-2). Most of the cystic spaces were filled with
eosinophilic material mixed with lymphocytes (Fig3). Stroma showed dense lymphocytic infiltration,
areas of hemorrhage, fibrosis and ossification (Fig-4).
Immuno- histochemistry was done showed positive
for D2-40 (Fig-5) and CD-31 (Fig-6). Our final
diagnosis was ossifying retroperitoneal cystic
lymphangioma. Postoperatively patient is fine. After
one year she conceived and at present she is full term
and healthy.
705
Tengli et al.,
Fig 4: Microscopy showing cystic spaces, areas of

fibrosis and inset showing focus of ossification.
10x10
Fig 1: Cut surface of large grey-white mass

showing multiple cystic spaces and grey-brown
hemorrhagic areas.
Fig 5: Microscopy showing IHC D2-40 Positivity
in the exfoliated endothelial cells. 40x10
Fig 2: Microscopy showing multiple large cystic

spaces filled with eosinophilic fluid, separated by
areas of fibrosis. 5x10
Fig 6: Microscopy showing IHC CD-31 Positivity
in the lining endothelial cells. 10x10
DISCUSSION
Fig 3: Microscopy showing a large cystic space

lined by endothelial cells and filled with
eosinophilic fluid containing lymphocytes. 40x10
It is often difficult to state whether Lymphangiomas

are
true
neoplasms,
hamartomas
or
lymphangiectasias. They are all benign lesions and
symptoms depend on their location and extent [1].
Cystic lymphangiomas more commonly occur in the
head and neck region and axillary region and very
rarely in the retroperitoneum. The most characteristic
radiologic finding is a large mass containing fluid
with or without septa [5]. Lymphangiomas are thought
to occur due to obstruction to the local lymph flow [2].
Several forms of lymphangiomas including simple
capillary, cavernous and cystic have been described
[2]
. The common presentations of intra-abdominal
cystic lymphangiomas are abdominal mass (as in our
case) and distension [6]. Complications of
retroperitoneal lymphangiomas are bleeding, rupture,
ascitis, infections, tortion and rarely obstruction of
ureter and hematuria [3]. Jeong HK, et al, reported a
706
Tengli et al.,
case of giant retroperitoneal lymphangioma which

showed focal bony ossification [7]. A M Buccoliero
and. V. Maio reported a case of calcified cystic
lymphangioma of the mesentry [8]. R J I Boskar and E
H Eddes reported a case of mesenteric lymphangioma
showing calcification and ossification [9]. Prognosis of
lymphangiomas is good because they rarely become
malignant [1]. Complete surgical enucleation is the
treatment of choice [10]. Rarely post-operative
recurrence may occur [11]. Differential diagnosis of a
retroperitoneal lymphangioma includes teratoma,
ovarian cystadenoma, retroperitoneal hematomas,
metastatic
lymphadenopathy,
fibrosarcoma,
[12]
leiomyosarcoma and liposarcoma . A higher index
of suspicion and a simple ultrasonography may lead
to an earlier correct diagnosis in many of the
patients[13].
CONCLUSION
We report this case of ossifying retroperitoneal cystic
lymphangioma in a 28 years old pregnant woman. It
has not yet been reported in a pregnant woman.
ACKNOWLEDGMENT
We are very thankful to our beloved-President(Khajabandanawaz Institute of Medical Sciences)-Dr.
Syed Shah KhusroHussaini for constant strategic
support and encouragement.
REFERENCES
1. Sook HC, Young P, Yun JJ, Seong HK, Dae WJ,
Byoung KS, et al. Asymptomatic lymphangioma
involving the spleen and retroperitoneum in
adults. World J Gastroenterol 2009;15(44):
5620-23.
2. Caglar Y, Kursat K, Cihat S, Ali C. Huge cystic
lymphangioma mimicking ovarian malignancy: A
case report.
Turk J Gastroenterol 2011;
22(3):344-46.
3. Tapan B, Daryoush S, Sean H, Susan I.
Retroperitoneal cystic lymphangioma in an adult:
A case report and review of the literature. World
J of GastrointestPathophysiol 2010; 1(5):171-76.
4. Sharon WW, John R. Goldblum, Enzinger&
Weisss Soft tissue tumors. 4th ed. Philadelphia:
Mosby Elsevier; 2004;955-83.
5. Yusuke Y, Keiji K, Kanji N, Masakazu Y,

Hiroshi K. A case of Laparoscopic Excision of a
Huge Retroperitoneal Cystic Lymphangioma.
Case report in urology : 2011; 1-3
6. Yavuz A, Fatih A, Serdar A, Ilknur C.
Mesenteric calcified cystic lymphangioma in an
adult patient. Turk J Gastroenterol 2011;
22(3):341-43.
7. Jeong HK, Min-Ok K, Young JC, Hyun YH,
Kang SP, Byung SC, et al. A giant retro
peritoneal lymphangioma in a patient with
neurofibromatosis type 1. Journal of the Korean
Surgical Society 2010; 7:543-46.
8. Buccoliero AM, Castiglione F, Maio V.
Calacified cystic lymphangioma of the
mesentery- A case report. Fetal and Pediatric
2009:infomahealth care.com
9. Basker RJI, Eddes EH. A mesenteric lymphangioma showing calcification of ossification.
Digestive surgery 2004; 21(3):182-83.
10. Anand SK, Rajendra KG, Budhwani KS, Roshan
C, Monika N. Giant retroperitoneal cystic
lymphangioma in a seven-months-old girl. J
Indian Assoc. Pediatrsurg 2007;12(3):161-62
11. Huseyin O, Ercan K, Zulkif B, and Bengu C.
Recurrent Retroperitoneal cystic Lymphangioma.
Yonsei Medical Journal 2005; 46(5):715-18.
12. Chan IYF, Khoo J.
Retroperitoneal
Lymphangioma in an Adult. J HK Coll. Radiol
2003; 6:94-96.
13. Chih-Cheng L, Chen-Sheng H, Hsun-Chin, ShihMing C, Chuen H. Intra-Abdominal Cystic
Lymphangiomas in Infancy and Childhood.
Chang Gung Med J 2004; 27(7):509-13.
707
Tengli et al.,
DOI: 10.5958/2319-5886.2015.00135.6

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Case report
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PROSTHETIC MANAGEMENT OF PATIENT WITH PAPILLON-LEFEVRE SYNDROME: A

CLINICAL REPORT
Sushen Sharma1, *Akhil Mittal1, TK giri2, Bhaskar Sengupta3, Vishal Jain4
1
PG student, Department of Prosthodontics, 2Professor, 3Professor & HOD, Department of Prosthodontics,

Principal,4PG student, Department of Paedodontics & Preventive Dentistry, DR R Ahmed Dental College &
Hospital, Kolkata, India.
*Corresponding author email: akhilmittal1988@gmail.com
ABSTRACT
Papillon Lefevre syndrome is a rare autosomal recessive genetic disorder, which is characterized by palmarplantar hyperkeratosis, with rapidly progressive periodontitis and premature loss of both deciduous and permanent
teeth. The etiology of PLS is multifactorial with genetic, immunological, and microbial factors playing a role in
etiopathogenesis. This is a case report of prosthodontic rehabilitation of a 14-year-old boy with Papillon-Lefevre
syndrome.
Keywords: Papillon Lefevre syndrome, Palmoplantar hyperkeratosis, Periodontitis, Overlay prosthesis
INTRODUCTION
Papillone Lefevre syndrome (PLS) is a very rare
autosomal recessive disorder characterized by
palmoplantar hyperkeratosis and severe early onset
periodontitis with premature loss of the primary and
secondary dentitions [1]. This syndrome was first
reported and described by two French physicians,
Papillon and Lefevre in 1924[1].
The prevalence of PLS has been reported as 1 to 4 per
million [2]. Parental consanguinity is demonstrated in
between 20% and 40% of the cases [1].
From a dental standpoint, young patients with PLS
have juvenile periodontitis, severe destruction of the
alveolar bone as early as within two years of eruption
in both primary and permanent dentitions. Associated
features may include calcification of falx cerebri and
choroid plexus, and retardation of somatic
development[3-5]. Primary dentition is usually
exfoliated prematurely by the age of 4 to 5 years [1,5].
After exfoliation, the inflammation subsides and the
gingiva appears healthy. With the eruption of
permanent dentition, the whole process of gingivitis
and periodontitis is repeated and there is subsequent

premature exfoliation of the permanent teeth by the
age of 14-15 years [1,5,6]. Later, the third molars also
undergo the same fate. Severe resorption of alveolar
bone gives the teeth a floating-in-air appearance on
dental radiographs.
Conventional removable prosthodontics with or
without osseointegrated dental implants can help to
restore
an
efficient
functional
dentition.
Psychological reassurance and regular follow-up will
be the key to success in these complicated
scenarios[7].
Akhil et al.,
CASE REPORT
A 14year old male patient reported to Dept. of
Prosthodontics of Dr. R Ahmed Dental College &
Hospital, Kolkata with chief complaint of unpleasant
appearance, difficulty in chewing (Fig.1). Family
history of patient revealed consanguineous marriage
708
of his parents. Patient gave a dental history of

premature exfoliation of his teeth from childhood.
Examination of patient showed Palmoplantar
keratosis, with dry scaly keratotic plaques over the
skin of his legs (Fig.2) along with multiple missing
teeth and only 3rd molars remaining, associated with
aggressive periodontitis (Fig.3). Radiographic
examination revealed the classic presentation of
floating in air appearance of remaining teeth
(Fig.4).
Considering Muller De Van statement8 The
preservation of that which remains is of utmost
importance and not the meticulous replacement of
that which has been lost, to simplify the treatment
plan and considering the age of patient and ongoing
growth any extensive surgical options were avoided.
To restore function and esthetics, compete denture
prosthesis overlaying the erupting 3rd molar was
considered as the treatment option. Impression of
maxillary arch was made with help of elastomeric
impressions due to presence of tuberosity undercut
and with zinc oxide eugenol impression paste for
mandibular arch (Fig.5).
This was followed by taking Jaw relation of patient to
determine
vertical
dimension.
After
teeth
arrangement try-in was done (Fig.6). And phonetics,
aesthetics were determined at this stage. Denture was
delivered and patient was put on follow up at
bimonthly interval. Patient was completely satisfied
with aesthetics and function of denture (Fig.7). After
4 month of denture wearing tooth started to erupt in
maxillary arch which was relieved adequately. As the
patient is still in growing stages, Regular follow up of
the patient need to be done to adjust and reline the
denture in case need arises.
Fig 2: Hand and feet showing Palmoplantar

keratosis
Fig 3: Intra-oral view of maxilla and mandible
Fig 4: OPG showing floating in air appearance
Fig 1: Pre-operative appearance
709
Akhil et al.,
Fig 5: Final impression of maxilla and mandible
Fig 6: Try-in denture
Fig 7: Final denture

DISCUSSION
Dental surgeon is the first to diagnose Papillonlefevre because of severe periodontitis affecting the
patient. This syndrome is inherited as an autosomal
recessive trait with a prevalence of 1 to 4 cases per
million[2]. PLS is characterized by marked destruction
of the periodontium (periodontoclasia) of both
dentitions with premature loss of teeth, marked
palmar and plantar hyperkeratosis. The gene has been
mapped to the long arm of chromosome 11. These
patients are usually normal at birth with only
reddening of palms and soles. Teeth erupt usually in
normal sequence, position and time. At around one
and an half to two years, marked gingivo-periodontal
process develops with edema, bleeding, alveolar bone
resorption and teeth mobility with consequent
exfoliation [1,5,6,7,8]. The pathogenesis of this syndrome
is still not understandable.
Etiology can be of Immunologic, genetic, and
microbiologic factors. As a genetic factor cathepsin C
gene is associated with Papillon-Lefevre syndrome
[9,10,11]
. The cathepsin C gene is seen in the epithelial
regions commonly affected by Papillon-Lefevre

syndrome, such as the palms, soles, knees, and
keratinized oral gingiva[12]. In addition, it is expressed
in various immune cells including polymorpho
nuclear leukocytes and macrophages, as well as their
precursors[13]. After exfoliation of all teeth, the soft
tissues usually acquire the normal healthy
appearance. The permanent dentitions usually start to
appear at normal time, but just after 2 - 3 years, the
gingivo-periodontal condition starts to deteriorate
again. All permanent teeth usually exfoliate within a
few years except for third molars which usually stay
longer[1,5,6]. Peripheral blood neutrophil chemotaxis
have been reported to be depressed. This decreased
chemotaxis suggests that neutrophils may be
important factor in periodontal destruction[14-18].
No definitive treatment is available for prevention
and management of periodontal destruction, although
strict oral hygiene maintenance, scaling and root
planning along with suitable antibiotic regimen may
improve the situation The treatment should be
planned with a multidisciplinary team approach
involving
paediatricians,
periodontists,
dermatologists, prosthodontists and psychologists[19].
However, edentulous patients can adapt to removable
prosthesis very quickly because of young age, better
oral stereognostic and oral motor abilities[20]. The
osseointegrated dental implants have revolutionized
the possible treatment options, but long-term effects
in these syndromic cases are still pending[19]. The
Papillon lefevre syndrome can adversely affect
growing children psychologically, socially and
aesthetically. A multi-disciplinary approach may
improve the prognosis and quality of life of these
children. Thus, oral rehabilitation in such patients is a
must.
Thus, prosthetic replacement in such patients is an
age specific, speciality treatment involving initial
replacement with complete or partial dentures and
future consideration for an implant-supported
prosthesis[21]. In the present case, prosthetic
rehabilitation with an overlay prosthesis was
considered as it would provide immediate satisfaction
to the patient in terms of aesthetics and function.
CONCLUSION
Tooth supported over denture in addition to
preserving the underlying tooth structure helps to
restore form and function. Rehabilitating such a
710
Akhil et al.,
patient at a young age is important to help them

adapt, maintain muscle tonicity and in overall
development of stomatognathic system, so as to
prepare them for more extensive treatment which
may be desired in future. Follow-up at regular
interval is important to see for any changes due to
erupting teeth and modify the denture accordingly.
10.
11.

REFERENCES
12.
1. Papillon MM, Lefvre P. Deux cas de
Keratodermie Palmaire Et Plantaire Symtrique
Familiale (maladie de Meleda) Chez Le Frere Et
La Soeur. Coexistence dans les deus cas
alterations dentaires grabes. Bulletin de la Soceite
Francaise de Dermatologie et de Syphiligraphie
1924; 31, 8287.
2. French D, Scott H, Overall CM. Papillone
Lefevre syndrome associated early onset
periodontitis: a review and case study. J Can Dent
Assoc. 1995; 61:432-38.
3. Gorlin RJ, Cohen MM, Levin LS. Syndromes of
the Head and Neck, 3rd edn.Oxford:Oxford
University Pres; 1990: 85355.
4. Hall RK (ed.). Paediatric Orofacial Medicine and
Pathology, 1st edn. London: Chapman &
HallMedical; 1994;Vol/issue: page no
5. Kressin S, Herforth A, Preis S, Wahn V, Lenard
HG. Papillon Lefvre syndromesuccessful
treatment with a combination of retinoid and
concurrent systematic periodontal therapy: case
reports. Quintessence International 1995; 26:795
03.
6. Wara-Aswapati N, Lertsirivorakul J, Nagasawa
T, Kawashima Y, Ishikawa I. PapillonLefevre
syndrome: serum immunoglobulins G (IgG)
subclass antibody response to periodontopathic
bacteria. A case report. Journal of Periodontology
2001; 72: 174754.
7. Subramanium P, Mathew S, Gupta KK. Papillon
Lefevre syndrome: a case report. J Indian Soc
Paedo Prev Dent 2008; 26:171-4.
8. DeVan M. The nature of the partial denture
foundation: suggestions for its preservation. J
Prosthet Dent. 1952;2:210-18.
9. Hart T C, Hart PS, Bowden DW, et al. Mutation
of the cathepsin C gene are responsible for
13.
14.
15.
16.
17.
18.
19.
20.
PapillonLefvre syndrome. Journal of Medical

Genetics 1999; 36: 881 87.
Toomes C, James J, Wood AJ et al. Loss-offunction mutations in the cathepsin C gene result
in periodontal disease and palmoplantar keratosis.
Nature Genetics 1999; 23: 42124.
Hart PS, Zhang Y, Firatli E, et al. Identification
of cathepsin C mutations in ethnically diverse
Papillon Lefvre syndrome patients. Journal of
Medical Genetics 2000; 37: 92732.
Cagli NA, Hakki SS, Dursun R, et al. Clinical,
genetic, and biochemical findings in two siblings
with PapilloneLefe`vre syndrome. J Periodontol.
2005; 76: 2322.
Sollecito TP, Sullivan KE, Pinto A, et al.
Systemic conditions associated with periodontitis
in childhood and adolescence (A review of
diagnostic possibilities). Med Oral Patol Oral Cir
Bucal. 2005; 10:142.
D'Angelo A, Margiotta V, Ammatuma P,
Sammartano F. Treatment of prepubertal
periodontitis. A case report and discussion.
Journal of Clinical Periodontology 1992; 19:
21419.
Brown RS, Hays G, Flaitz CM, O'Neill PA,
Abramovitch K, White RR. A possible late onset
variation of PapillonLefvre syndrome: report of
3 cases. Journal of Periodontology 1993; 64:
37986.
Tinanoff N, Tempro P, Maderazo EG. Dental
treatment of PapillonLefvre syndrome: 15 year
followup. Journal of Clinical Periodontology
1995; 22: 60912.
FQratlQ E, Gurel N, Efeoglu A, Badur S. Clinical
and immunological findings in 2 siblings with
PapillonLefvre
syndrome.
Journal
of
Periodontology 1996; 67: 1210 15.
Ghaffer KA, Zahran FM, Fahmy HM, Brown RS.
PapillonLefvre syndrome. Neutrophil function
in 15 cases from 4 families in Egypt. Oral
Surgery, Oral Medicine, Oral Pathology, Oral
Radiology and Endodontics 1999; 88: 320 25.
Etoz OA, Ulu M, Kesim B. Treatment of patient
with Papillon Lefevre syndrome with short dental
implants: a case report. Implant Dent 2010;
19:394-9.
Ahmed B, Mirza KM, Mehmood A, Hussain M,
Yazdanie N. Oral stereognostic ability in
711
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hypodontia patients. Pak J Med Research 2010;

49:14-7
21. Varri S, Chukka RS, Tadepalli A, Kiran R
Prosthetic Rehabilitation For A Case Of Papillonlefevre Syndrome: Indian Journal of Dental
Sciences. March 2014, 1(6):88-89
712
Akhil et al.,
DOI: 10.5958/2319-5886.2015.00136.8

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th
th
Case report
ROSAI-DORFMAN DISEASE WITH
INVOLVEMENT: A CASE REPORT
CERVICAL
ISSN: 2319-5886
LYMPHADENOPATHY
AND
ORBITAL
*Sameer Saleem 1, Sundas Younas 2, Kamran Qayyum 3

1, 2, 3
MBBS, Department of Internal Medicine, Khyber Medical College Peshawar, Pakistan
*Corresponding author email: sameersaleems@gmail.com

ABSTRACT
Rosai-Dorfman disease (RDD), which is also called as sinus histiocytosis with massive lymphadenopathy
(SHML), is a rare histiocytic disorder which occurs due to the over-production of non Langerhans sinus
histiocytes. It is a nonmalignant disorder that most frequently affects children and young adults and typically
presents with fever, night sweats, nonpainful cervical lymphadenopathy, leukocytosis and an elevated ESR.
Extranodal involvement may also occur, thus a variety of organs in the body can be affected. Although some viral
etiology has been implicated, the disease generally is considered to have an unknown cause. RDD can often be
misdiagnosed as lymphoma, leukemia or tuberculosis, so it is imperative to distinguish it from these conditions as
well as other forms of histiocytosis because of difference in the modes of treatment. Diagnosis of Rosai-Dorfman
disease is based on biopsy of affected tissue. Biopsy showing the presence of emperipolesis, or the engulfment of
lymphocytes and other immune cells by histiocytes that express S-100 antigen is diagnostic of Rosai-Dorfman
disease. Once diagnosed, further workup including imaging studies are undertaken in order to determine the
extent of the disease. In majority of cases, the disease resolves on its own however, treatments including
corticosteroids, chemotherapy, surgical treatment or radiotherapy are carried out in severe or persistent disease or
when organ function is at stake (e.g. breathing obstruction, kidney failure, visual problems). The case we report is
that of a 16 year old girl who presented with a 6 month history of gradual onset drooping of left upper eyelid with
mild proptosis of the left eye alongwith mild drooping of right upper eyelid, low grade fever, night sweats and
cervical lymphadenopathy. Blood workup showed increased ESR, CT scan of orbits showed superior orbital
masses and diagnostic biopsy revealed Rosai-Dorfman disease.
Keywords: Rosai Dorfman disease, Cervical lymphadenopathy, Emperipolesis, Histiocytes, Proptosis
INTRODUCTION
Rosai-Dorfman disease (RDD) or sinus histiocytosis
with massive lymphadenopathy (SHML), is a rare
disease resulting from increased production of non
Langerhans sinus histiocytes. [1, 2] Rosai and Dorfman
first described this disease in 1969. [3] Lymph nodes
are commonly involved but it can involve any organ.
[1]
Its main features are painless cervical
lymphadenopathy, fever and elevated ESR.
[4]
Different sites of extranodal involvement have
been reported, particularly the skin, orbit, and upper
respiratory tract. [5, 6] Central nervous system

involvement with no nodal disease has also been
reported. [7] RDD mostly occurs in children and
young adults with a slight male predominance and an
increased incidence in African descent. [8] There are
some viruses thought to be involved in the
pathogenesis of RDD however, the disease is
considered to have an unknown cause [9, 10, 11] The
presentation of RDD resembles a number of different
conditions including leukemia, lymphoma, TB and
713
Saleem et al.,
other causes of histiocytosis. The definitive way to

diagnose this disease is via a biopsy of the affected
tissue. The disease is usually a self-limiting one
however, on case to case basis depending upon the
organ involvement and severity of the disease,
different treatment modalities ranging from medical
to surgical or radiotherapy can be instituted to fight
this disease. [12, 14, 15]
The purpose of this case report is to highlight this rare
disease that we have encountered and to educate
clinicians about the fact that the disease can present
with a variety of symptoms and can involve different
organs that may often distract or misguide clinicians
into making a wrong diagnosis.
of the orbits showed bilateral superior orbital masses

(Fig 1) and mild proptosis of the left eye (Fig 2).
Incisional biopsy of the left Superior orbital mass was
performed that revealed the diagnosis of RosaiDorfman disease.
CASE REPORT
A 16 year old girl presented to the outpatient
department of Khyber Teaching Hospital, Peshawar,
Pakistan in February 2015 with a six month history of
gradual onset, painless left upper eyelid drooping
along with low-grade fever and night sweats plus a 20
day history of gradual onset right upper eyelid
drooping. She also noticed a few lumps in her
cervical region. She had no significant past medical
history of any major illness and no family history of
tuberculosis or blood disorders was found. Her vitals
were as follows, BP 120/80 mm Hg, pulse 90/min,
respiratory rate 15/min and temperature 100.2 F. On
examination she had bilateral painless cervical
lymphadenopathy and bilateral superior orbital
masses on palpation. Her vision was 6/6 in both eyes.
Ptosis was seen in both right (3mm) and left (5mm)
eyes. Mild left eye proptosis was also seen.
Extraocular movements were restricted in upper gaze
of both eyes, more so of the left eye. There was no
evidence of any visceromegaly and the rest of the
general physical and systemic examinations were
unremarkable. Lab investigations: Hb 11.1 g/dl, RBC
4.27 million/cmm, Hct 32.4 %, MCV 76 fl, MCH
25.9 pg, MCHC 34.2 g/dl, Platelet count
328000/cmm,
TLC
11000/cmm,
normocytic
normochromic picture with DLC showing 80%
neutrophils, 15% lymphocytes and 5% monocytes on
peripheral smear, ESR 70 mm/1st hour, negative HbS
and HCV screening, negative PPD and sputum AFB,
normal Liver function tests and normal Renal
function tests. Chest X-ray was normal, U/S and CT
scan of the abdomen and pelvis was normal. CT scans
Fig 1: CT scan orbit (Coronal View): Bilateral

Superior Orbital Masses
Fig 2: CT scan Orbit (Axial View): Mild Proptosis

of the Left Eye
Treatment: The patient was counseled by the
attending physician about the nature of the disease
and administered Inj. Methylprednisolone 1gm x OD
for 3 days followed by Tab Prednisolone 1mg/Kg
body weight x OD and advised follow up after 4
weeks. On follow up visit, examination showed that
her ptosis and cervical lymphadenopathy had
improved. She was also assessed for side effects of
steroid therapy. No side effects were noted. She was
advised follow up after 8 weeks.
DISCUSSION
Rosai-Dorfman disease (RDD), which is also called
as sinus histiocytosis with massive lymphadenopathy
(SHML), is a rare histiocytic disorder which occurs
due to the over-production of non Langerhans sinus
histiocytes. [1, 2] Rosai and Dorfman were the first
714
Saleem et al.,
people to identify this disease as a unique

clinicopathologic condition in 1969. [3] RDD
commonly affects lymph nodes however, it has the
propensity to involve any organ. [1] Cardinal features
include painless cervical lymphadenopathy, fever and
elevated ESR. [4] Extranodal involvement has been
reported in diverse anatomic sites, particularly the
skin, orbit, and upper respiratory tract. [5, 6] Central
nervous system involvement without nodal disease
has also been reported. [7] Rosai-Dorfman disease
though quite rare, is distributed worldwide with 80%
cases occurring in children and young adults with a
slight male predominance (58%) and has a general
predilection for individuals with African descent. [8]
The etiology of RDD is unknown, however certain
viruses like Human Herpes virus 6 and Epstein-Bar
virus via causing immune dysregulation have been
implicated in the pathogenesis of this disease. [9, 10, 11]
The diagnosis of Rosai-Dorfman disease is not easy
since its presentation can mimic a number of other
non-malignant as well as malignant conditions
ranging from bacterial or viral infections to
malignancies including leukemia and lymphoma.
Biopsy of the lymph node or affected tissue is
required for the diagnosis of this disease.
Proliferating S100 and CD68 antigens positive
histiocytes exhibiting emperipolesis i.e. phagocytosis
of intact lymphocytes and other immune cells, is the
classical histologic finding on biopsy in Rosai
Dorfman disease.
No specific treatment protocol is established for
Rosai Dorfman disease because the disease is rare
and its course is mostly self limiting. [14] However,
patients with severe, persistent disease or in cases
where organ function is compromised steroid therapy,
chemotherapy, surgical resection or radiotherapy can
be instituted with varying success rates. [12,13, 14, 15]
CONCLUSION
Rosai-Dorfman disease shares many of its presenting
features with leukemia, lymphoma, tuberculous
lymphadenitis and other causes of histiocytosis, so it
should be considered in the differentials of patients,
especially children and young adults who present
with painless cervical lymphadenopathy. It is also
important for physicians to recognize that the disease
can have a myriad of clinical manifestations
depending upon the tissue involved, as was the case
in our patient we presented in this case report having
cervical lymphadenopathy with orbital involvement

in whom lymphoma, leukemic deposits, orbital
pseudo tumor, Langerhans cell histiocytosis and
hemangioma were also amongst the list of
differentials until biopsy confirmed the diagnosis of
RDD. It is essential for pathologists as well to look
for the histopathologic features of this disease in
biopsy specimens, since if promptly diagnosed and
managed, can reduce unnecessary diagnostic workups
and mismanagement due to misdiagnosis of this
disease.
REFERENCES
1. Riyaz N, Khader A, Sarita S. Rosai-Dorfman
syndrome. Indian
J
Dermatol
Venereol
Leprol. 2005; 71:3424.
2. James, William D.; Berger, Timothy G.; et al.
Andrews' Diseases of the Skin: clinical
Dermatology. Saunders Elsevier.2006; ISBN 07216-2921-0.
3. Kong Y, Kong J, Shi D, Lu H, Zhu X, Wang J,
Chen Z: Cutaneous RosaiDorfman Disease: a
clinical and histopathologic study of 25 cases in
China. Am J Surg Pathol 2007; 21:341-50.
4. Foucar E, Rosai J, Dorfman R: Sinus
histiocytosis with massive lymphadenopathy
(Rosai-Dorfman disease): a review of the entity.
Semin Diagn Pathol 1990; 7:19-73
5. Puppin D Jr, Chavaz P, Harms M: Histiocytic
lymphophagocytic panniculitis (Rosai-Dorfman
disease): a case report. Dermatology 1992;
184:317-20
6. Andriko JW, Morrison A, Colegial CH, et al:
Rosai-Dorfman disease isolated to the central
nervous system. A report of 11 cases. Mod Pathol
2001; 14:172-78
7. Woodcock RJ, Mandell JW, Lipper MH: Sinus
histiocytosis (Rosai-Dorfman disease) of the
suprasellar region: MR imaging findings -- a case
report. Radiology 1999; 213:808-10
8. Sodhi KS, Suri S, Nijhawan R, Kang M, Gautam
V: RosaiDorfman disease: unusual cause of
diffuse
and
massive
retroperitoneal
lymphadenopathy. Br J Radiol 2005; 25:845-47.
9. Ensari S, Selcuk A, Dere H, Perez N, Dizbay Sak
S: RosaiDorfman
disease
presenting
as
715
Saleem et al.,
10.
11.
12.
13.
14.
15.
laryngeal masses. Kulak Burun Bogaz Ihtis

Derg 2008; 18:110-14.
Pinto DCG, Vidigal TA, Castro B, Santos BH,
DeSousa NJA: RosaiDorfman disease in the
differential
diagnosis
of
cervical
lymphadenopathy. Bras J Otorrinolaringol
2008; 74:632-35.
Levine PH, Jahan N, Murari P, Manak M, Jaffe
ES: Detection of human herpesvirus 6 in tissues
involved by sinus histiocytosis with massive
lymphadenopathy (RosaiDorfman disease). J
Infect Dis 1992; 166:291-95.
Yoon A, Parisien M, Feldman F, Young-In Lee
F: Extranodal RosaiDorfman disease of bone,
subcutaneous tissue and paranasal sinus mucosa
with a review of its pathogenesis. Skeletal
Radiol 2005; 34:653-57.
Montgomery EA, Meis JM: RosaiDorfman
disease of soft tissue. Am J Surg Pathol
1992; 16:122-29.
Pinto DCG, Vidigal TA, Castro B, Santos BH,
DeSousa NJA: RosaiDorfman disease in the
differential diagnosis of cervical lymphadenopathy. Bras J Otorrinolaringol 2008; 74:632-35.
Moore J, Zhao X, Nelson E: Concomitant sinus
histiocytosis with massive lymphadenopathy
(RosaiDorfman disease) and diffuse large B-cell
lymphoma: a case report. J Med Case
Reports 2008; 2:70
716
Saleem et al.,
DOI: 10.5958/2319-5886.2015.00137.X

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Volume 4 Issue 3
Coden: IJMRHS
th
Case report
Copyright @2015
ISSN: 2319-5886
PERSISTENT ARRHYTHMIAS AFTER ELECTROCUTION IN A PATIENT SCHEDULED FOR

GRAFT SURGERY
RekhiBalwinder K.1, RekhiHarnam S2, *Singh Praneet3, Singh Harcharan4
1
Professor,3Resident, Dept. of Anaesthesia, 2Professor, Dept. of Surgery, 4Professor, Dept. of Cardiology

Government Medical College and Hospital, Patiala, Punjab, India
*Corresponding author email:singhpraneet87@gmail.com
ABSTRACT
PVC may occur due to numerous etiologies. Manifestations may range from an asymptomatic presentation to
ventricular fibrillation and death. The incidence of PVC in healthy population may range from 0.5% in those
aged below 20 years old to 2.2% in those older than 50. Here we present a case of persistent arrhythmias after six
months of electrocution. Patient was admitted in our hospital for graft surgery post burns.
Keywords: Arrhythmias, Electrocution, PVC (Premature Ventricular Contractions), Holter Monitoring.
INTRODUCTION
Heart is most susceptible organ to electric injury with
wide
range
of
abnormalities.
Cardiac
arrhythmias can occur due to various etiological
factors. Manifestations may range from an
asymptomatic presentation to ventricular fibrillations
and death. 3-4% of admissions in the burn units are
accounted by electrical burn injuries.[1]Approximately
20% of all electrical injuries occur in children (with a
bimodal peak incidence in toddlers and adolescents),
usually via cable extensions or wall outlets.[2]Death
most often occurs in young males (male:female =
9:1).[2]
CASE REPORT
We present a case of high voltage electrocution 0f
11,000kV ac and supraventricular and ventricular
arrhythmias after six month of incidence. A 23 year
old male patient electrician by occupation weighing
50 kg was scheduled for post burn graft surgery lower
limb. About 5 months back patient got electrocuted
with 11,000kV tension wire while doing some repair
work. Baseline investigation of the patient were Hb
Singh et al.,
11.0 g% ,BT 25,CT 610, blood urea 34 mg%,

serum creatinine 1.2 mg%, serum Na 140 meq/l
,serum K 4.2meq/l , ECG attached, breath holding
more than 25 seconds.
In the operation theatre patient was found to be
having irregular pulse, ECG showed multiple PVC,
more than 13-14/min. without any symptom. Surgery
was deferred and patient was sent to cardiology for
expert opinion. Echocardiography was done, no
major abnormality was observed. No regional wall
motion abnormality was seen. Ejection fraction was
50%. After getting fitness patient was again
scheduled for graft surgery. Irregular pulse persisted
with heart rate 98/min BP 124/80 mm hg n right
upper limb. On auscultation lung fields were clear. 12
lead ECG (Fig. 1) showed 12-15 PVC/min.
Lignocaine 1.5% was given to abort ongoing
arrhythmias, but there was no response. Defibrillator
and anti-arrhythmic drugs were kept ready in case of
progression of multiple PVC to ventricular fibrillation
and unstable hemodynamic
Since the patient was hemodynamically stable
subarachnoid block was performed at L3-4 level
717
using bupivacaine 10mg plus 25g fentanyl. Patient

remained hemodynamically stable throughout the
procedure and shifted to recovery in satisfactory
conditions.
Postoperatively Holter ECG study (Fig. 2) was done
where the ventricular, supraventricular arrhythmia
was observed. The report showed ventricular ectopics
totaled 1254 averaging 59/hr. with 1247 single and

four paired and here trigemni and zero R on T.
Supraventricular ectopics totaled 713 averaging
33.6/hr- 663 single and 18 paired beats.
Supraventricular tachycardia occurred four times.
Fig 1: 12 Lead E.C.G.
Fig 2: Post-operative Holter Study

DISCUSSION
Cardiac arrhythmias are usually explained on the
basis of abnormalities of cardiac impulse conduction
(reentry) or impulse formation (automaticity).
Reentry excitation is the mechanism for most cardiac
arrhythmias and reflex excitation of tissue by return
Singh et al.,
of the same cardiac impulse in circuitous fashion. In

automaticity cardiac impulses are generated each time
to excite the heart. Enhanced automaticity leads to
cardiac arrhythmia by facilitating repetitive firing
from single focus. [3] The various events that may be
associated with cardiac arrhythmias encountered by
an anesthetist are
718
1) Use of Volatile anesthetics [4]

2) Arterial hypoxemia
3) Hypercarbia
4) Systemic hypertension
5) Endogenous or exogenous catecholamines
6) Electrolyte imbalance
7) Coexisting cardiac diseases.
In our patient no other disease was present so the
changes observed could be exclusively connected
with the electric accident. The mechanism of
electrical induced arrhythmias is not much clear. Due
to the differences in electrical resistance, current
travels more preferentially along blood vessels and
nerves, that makes the heart as an organ more
susceptible to injury by electrical currents.[5]Patchy
necrosis may be seen in heart muscle biopsy
specimens after injury by electrical currents, the
fibrotic tissue can act as a potential chronic
arrythmogenic
focus.[6]
Increased
cardiac
sodium/potassium pump activities and an increase in
potassium concentration have also been described as
causes.[7] Cardiac arrhythmias may occur at the time
of electrical shock or later, but mostly within the first
day
after
an
injury.[7,8]Arrowsmith
et
[9]
al. retrospectively evaluated 145 patients with
electrical injury in the same centre over a five year
period; 128 (88%) had suffered low voltage injury
and 17 (12%) had suffered high voltage injury
(>1000 V). The frequency of cardiac complications
was 3% (four patients). Atrial Fibrillation was
detected in only one patient in their study, in this case
normal sinus rhythm was restored after i.v. digoxin
infusion. Purdue et al. [10] recommended cardiac
monitoring of patients with electrical injury if there
was loss of consciousness, recorded arrhythmia in the
field, abnormal ECG on admission and rhythm
disturbance during monitoring in the emergency
room.
CONCLUSION
Since in our study no other concomitant disease
history was present, so the PVCs observed could be
exclusively related to the electrical incident that
happened approximately 5 months ago.
Of General Medicine & Cardiology of Govt. Medical

College, Patiala and associated Rajindra Hospital for
their support and interest.
REFERENCES
1. Cameron P,Jelinek G, Kelly A-M, Murray L,
Brown AFT, Heyworth J. Textbook of Adult
Emergency Medicine (2nd ed.), chapter 27.6:
Electric shock and lightning injury, 2004.
Churchill Livingstone; Postgraduate textbook.
2. Hettiaratchy S, Dziewulski P; ABC of burns:
Pathophysiology and types of burns. BMJ 2004;
328: 1427-29.
3. Stoelting RK, Dierdorf Sf. Anesthesia and
coexisting disease , 5th edition. Philadelphia:
Churchill His in dogs. Anesthesiology 1972;
336:112-8
4. Akhtar M. Management of tachyarrythmias
JAMA 1982; 247:671-4
5. Atlee JL, Rusy BF. Halothane depression of A-V
conduction studied by electrograms of Bundle of
His in dogs. Anesthesiology 1972; 336:112-8
6. Johnson RR, Eger EI, Wilson C. A comparative
interaction of epinephrine with enflurane,
isoflurane and halothane in man. AnesthAnalg
1976; 55:709-12.
7. Jensen PJ, Thompson PE, Bagger JP, Norgaard
A, Baandrup U. Electrical injury causing
ventricular arrhythmias. Br Heart J. 1987 Mar;
57(3):279-83
8. Ku CS, Lin SL, Hsu TL, Wang SP, Chang MS.
Myocardial damage associated with electrical
injury. Am Heart J. 1989 Sep; 118(3):621-24.
9. ArrowsmithJ ,Usgaocar RP, Dickson WA.
Electrical injury and the frequency of cardiac
complications.Burns.1997; 23:5768.
10. Purdue GF, Hunt JL. Electrocardiographic
monitoring after electrical injury: necessity or
luxury? JTrauma. 1986; 26:1667.
ACKNOWLEDGMENT
We are thankful to our colleagues in the Department
of Anaesthesia & Intensive Care and the Department
719
Singh et al.,
DOI: 10.5958/2319-5886.2015.00138.1

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RETAINED LARGE METALLIC SCREW
PENETRATING MAXILLOFACIAL TRAUMA
WITH
Copyright @2015
ISSN: 2319-5886
MANDIBULAR
FRACTURE
AFTER
Ramneesh Garg1, *Sheerin Shah2, Sanjeev Uppal3, Rajinder Mittal4, Sundeep Kaur5
1
Associate Professor, 2Assistant Professor, 3Professor and Head, 4Professor, 5MCh Resident, Department of
Plastic Surgery Dayanand Medical College and Hospital Ludhiana Punjab
*Corresponding author email: sheerinkathpal@gmail.com
ABSTRACT
Accidental penetrating injury to neck is uncommon. Because of location of important vital structures, any injury
in this area should be timely diagnosed and managed. Over the years a remarkable number of changes have
occurred in the treatment paradigm of such injuries. An evolution from no treatment, to routine exploration and
now to selective exploration has occurred because of better diagnostic and surgical skills. We report an interesting
unusual case of removal of 10cm metallic screw from the mandibular area and neck area. Close proximity of such
a rigid and sharp metallic body to neurovascular structures, airway and esophagus, posed a unique management
challenge.
Keywords: Retained metallic screw, Penetrating mandible injury, Traumatic neck injury
INTRODUCTION
In developing nations, like India, facial fractures
(open and closed) are most commonly caused by road
traffic accident [1]. These facial open wounds are
usually contaminated with dust, cement or glass
particles. Large foreign bodies like metallic screw are
rare to encounter. Such retained foreign bodies which
track down to neck, from gingiva buccal sulcus, are
difficult to diagnose and manage. Most of the
penetrating wounds to neck are caused by gun shot or
stab by sharp object [2]. Its important to understand
the mechanism of penetration for delineating the
extent of injury. The mortality in such cases is higher
because of major arterial or laryngeal injury [3].
This is a case of 46-year-old male who presented to

emergency department with complaints of pain and
bleeding from the oral cavity following a roadside
accident. He was riding a two-wheeler and had a head
on collision with a standing trolley. There was no
history of loss of consciousness, convulsions or

incontinence. On examination, he had multiple full
thickness lacerations over upper lip, anterior tongue
and lower gingivobuccal sulcus. The patient was
unable to occlude his teeth properly. There was
extensive bleeding from the oral cavity and facial
lacerations. There was loss of left side lower canine,
premolar and first molar. He had fracture of left
parasymphyseal area of mandible and a round foreign
body was visible in lower buccoalveolar sulcus. After
stabilization, CT Face with multiplanar images and
3D reconstruction was done. CT was suggestive of
segmental fracture of left parasymphyseal area with
alveolar ridge loss with foreign body measuring 10 x
1.5cm in the left buccoalveolar sulcus till the
supraclavicular area(Fig 1).On coronal and axial
images it was found that trachea and esophagus were
not pierced and the metallic body was lateral to
midline(Fig -2). X ray neck lateral view showed the
length and direction of foreign body (Fig -3).After
Sheerin et al.,
CASE REPORT
720
ENT consultation, the patient was taken up for

removal of foreign body, and fracture fixation.
Fibreoptic nasal intubation was done. Torn tongue
was repaired in layers. The metallic screw was visible
in left buccoalveolar groove and was extracted with
the help of forceps. The tract in the neck was
selectively
explored
through
buccoalveolar
laceration. The tract was confirmed to be in
subcutaneous plane and no vital structures were
damaged. The tract was washed with betadine and
saline.
The mandibular fracture was exposed and rigidly
fixed with mini plates (Fig 4).The tract was kept open
for drainage and secondary healing. Postoperative
period was uneventful. All wounds healed well and
patient was discharged on 5th day.
Fig 4: OPG showing mandibular fracture fixed

with Plates
DISCUSSION
Fig 3: soft tissue X ray neck (lateral view)
Worldwide, 5-10 % of all trauma patients have facial

fractures [4]. Life- threatening injuries have been
reported in 6.2% of facial fractures patients in a
Taiwanese study, with mortality causes including
hemorrhagic shock and compromised airway [5]. In
our part of country, most commonly hurt
maxillofacial patients are the ones on motorbike [1], as
is the present case also. Facial fractures with large
retained metallic screw in neck are rare to encounter.
Though it might eventually turn out to be uneventful
but because of vital structures like aero digestive
tract, vessels and nerves in the surrounding area, this
site is to be dealt with a great caution[6]. The sharp
threads of the screw may pose danger during and
after removal. Anatomically, neck is divided into
three zones. Zone I is the horizontal area between the
clavicle/suprasternal notch and the cricoid cartilage.
The proximal common carotid, vertebral and
subclavian arteries and the trachea, esophagus,
thoracic duct and thymus are located in Zone I. Zone
II is the area between the cricoid cartilage and the
angle of the mandible. It contains the internal and
external carotid arteries, jugular veins, pharynx,
larynx, esophagus, recurrent laryngeal nerve, spinal
cord, trachea, thyroid and parathyroids. Zone III is
the area that lies between the angle of the mandible
and the base of the skull. It has the distal extracranial
carotid and the vertebral arteries and the uppermost
segments of the jugular veins. In the present case, the
injuries were confined to the lower face, Zone I and
Zone II, without damaging major vessels, nerves as
well as the aero digestive tract. The amount of tissue
damage is depended on axis of weight transmission
and direction of kinetic force of the foreign body
Sheerin et al.,
Fig 1: Preoperative picture showing lip laceration
Fig 2: coronal and axial CT
721
impaction[7]. The foreign body in neck can easily

cause vascular injury giving rise to absent pulses,
absent arterial bleed and expanding hematoma.
Previous literature suggests that an urgent CT scan
with arteriography is must in such a case[8,9].Munera
et al [10] and Matsuyama et al[11]suggested that in
absence of angiography, suspected patients should
undergo color Doppler or MRI
In the present case, vitals were stable and there was
no active bleed from neck, buccoalveolar sulcus or
tongue and hard signs were not present, so only CT
face with 3D reconstruction was done to see the
extent of mandibular fracture and foreign body.
In the present case, we did early removal of the
foreign body by extraction and then performed
selective exploration of foreign body tract through the
buccoalveolar incision and already lacerated chin
wound. The protocol for addressing neck injuries has
changed in the recent time. Till 1990s, mandatory
neck exploration was practiced in all penetrating neck
injuries. It was reported by one series that it has
negative findings in 57% cases [12]. Sekharan et al [13]
and Mc Connell et al [14] states that for penetrating
neck trauma, there are various treatment option like
conservative treatment, routine neck exploration,
selective exploration and adjuvant invasive and non
invasive assessment. The decision for operative
choice depends on preoperative clinical presentation
(hard signs), vitals, and amount of blood loss and CT
/ angiography findings. A patient in shock and
bleeding neck wound should be managed with
priority with blood transfusion, tracheostomy and
ligation of vessels during surgery. Internal jugular
vein injury, laryngeal, tracheal and esophageal tear
should be repaired within 24 hours. Nason et al[15]
advocated the practice of early surgical treatment and
intravenous antibiotic to these patients for preventing
postoperative infection, necrosis and later on
contracture development.
In our case, the mandibular fracture was internal
reduced and rigidly fixed with plates after achieving
class 1 dental occlusion. The teeth gap can be
corrected on later sitting with dental implants.
approach for neck exploration can avoid surgical

trauma and scarring of neck
ACKNOWLEDGMENT: Nil
REFERENCES
1. Gupta AK, Garg R, Gupta A, Bajaj K. A
Foreign bodies to neck through oral vestibule are rare

and demand appropriate early diagnostic and
management strategy. Selective conservative
retrospective analysis of 189 patients of

maxillofacial injuries presenting to a tertiary care
hospital in Punjab, India. J Maxillofac Oral
Surg.2009;8(3):241-5
2. KH Kamil, KK Arzu, UzutMahmut, et al. T
imely management of penetrating neck trauma:
Report of three cases. J Emerg Trauma
Shock.2013;6(4).289-92
3. Nason RW, Assuras GN, Gray PR, Lipschitz J,
Burns CM. Penetrating neck injuries: Analysis of
experience from a Canadian trauma centre. Can J
Surg. 2001;44:1224
4. Chandran A, Hyder AA, PeekAsa C. The global
burden of unintentional injuries and an agenda
for progress.Epidemiol Rev.2010;32(1):110-20
5. Tung TC, Tseng WS, Chen CT. Acute life
threatening injuries in facial fracture patients: a
review
of
1025
patients.
J
Trauma.2000;49(3):420-4
6. Hunter TB, Taljanovic MS. Foreign Bodies.
Radiographics. 2003;23:73157
7. Singh RK, Bhandary S, Sinha BK, Karki P.
Penetrating injury of parotid gland and external
auditory canal: A unique combination. J Laryngol
Otol. 2004;12:9779.
8. Weaver FA, Yellin AE, Bauer M, Oberg J,
Ghalamber N, Emmanuel RP. Is arterial
proximity a valid indication for arteriography in
penetrating extremity trauma? A prospective
analysis.Arch Surg. 1900;125:125660
9. Johansen K, Lynch K, Paun M, Copass M. Noninvasive vascular tests reliably exclude occult
arterial trauma in injured extremities. J Trauma.
1991;31:51522
10. Mnera F, Soto JA, Palacio D, Velez SM,
Medina E. Diagnosis of arterial injuries caused
by penetrating trauma to the neck: Comparison of
helical CT angiography and conventional
angiography. Radiology. 2000;216:35662.
Sheerin et al.,
CONCLUSION
722
11. Matsuyama T, Okuchi K, Nogami K, Hata M,

Murao Y. Transorbital penetrating injury by a
Chopstick-case report. Neurol Med Chir.
2001;41:3458
12. Apffelstaedt JP, Mller R. Results of mandatory
exploration for penetrating neck trauma. World J
Surg 1994; 18:917.
13. Sekharan J, Dennis JW, Velder HC, Miranda F,
Frykberg ER. Continued experiences with
physical examination alone for evaluation and
management o penetrating zone II neck injuries:
Results of 145 cases. J Vas Surg. 2000; 32:483
9.
14. McConnell DB, Trunkey DD. Management of
penetrating trauma to the neck. Adv Surg. 1994;
27:9727.
15. Nason RW, Assuras GN, Gray PR, Lipschitz J,
Burns CM. Penetrating neck injuries: Analysis of
experiences from a Canadian trauma centre.
Canadian J Surg. 2001;44:1224
723
Sheerin et al.,
DOI: 10.5958/2319-5886.2015.00139.3

&
Health Sciences
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Volume 4 Issue 3
Coden: IJMRHS
th
Case report
Copyright @2015
ISSN: 2319-5886
Accepted: 2nd Jun 2015
ENDOMETRIOSIS OF APPENDIX IN WOMEN PRESENTING WITH RIGHT LOWER ABDOMINAL

PAIN
*Radha Bai Prabhu T1, Velayudam DA2, Jayalakshmi M3
1, 2, 3
Department of Obstetrics & Gynaecology, Meenakshi Medical College and Research Institute,
Kancheepuram, Tamilnadu, India
*Corresponding author email: radhaprabhu54@ymail.com
ABSTRACT
Endometriosis is a well known gynaecological condition associated with infertility and chronic pelvic pain.
Review of literature shows that endometriosis can affect any tissue in the body, including the appendix. Here we
report a case of pelvic endometriosis involving the vermiform appendix in a 45 years old multiparous woman.
When women of the reproductive age present with recurrent lower abdominal pain on the right side,
endometriosis of the appendix should also be considered. At the time of surgery appendix should be inspected and
removed; especially in the presence of pelvic endometriosis.
Keywords: Extra genital endometriosis, Appendix, Right lower quadrant pain
INTRODUCTION
Endometriosis is a well known gynaecological
condition associated with infertility and chronic
pelvic pain. Review of literature shows that
endometriosis can affect any tissue in the body,
including the appendix.[1] Recurrent chronic right
lower abdominal pain is the most common
presentation. Endometriosis of the appendix should
be suspected and should be included in the
differential diagnosis in young women presenting
with right lower quadrant pain or symptoms of acute
appendicitis. This case describes endometriosis of the
appendix as a possible cause of chronic right lower
quadrant pain in a multiparous woman. .
45 years old, para 2 presented with lower abdominal

pain of 3 years duration and was severe in the
previous 6 months. She also complained of
congestive dysmenorrhoea and scanty periods for the
past 3 months. The pain was intermittent with a dull
ache and was more on the right side. Initially, the
pain was not related to her periods. However, in the
last 3 months the pain on the right side was very

severe, more so in the pre-menstrual and menstrual
phase. She also suffered from non-specific symptoms
such as dyspepsia and abdominal bloating. She was
seen by the medical Gastro-Enterologist and GI tract
problems were ruled out. She also suffered from on
and off painful micturition. There was no history of
fever or discharge per vaginum. She was married for
25 years, has delivered two children vaginally and the
last childbirth was 18 years ago. She has used IUCD
for contraception for nearly 10 years and got it
removed two years ago.
On examination, there was mild bloating of lower
abdomen with vague fullness and tenderness in the
right iliac fossa. On speculum examination, the cervix
was congested and there was a small endocervical
polyp measuring 1 cm. in size. On bimanual pelvic
examination, the uterus was retroverted, bulky, and
the left fornix was free. Through the right fornix,in
close association with the uterus, a tender cystic mass
measuring 10 cms. in size was palpable. Per rectal
examination, the tender mass was felt on the right
Radha et al.,
CASE REPORT
724
side and there was no nodularity in the pouch of

douglas (POD). A provisional diagnosis of right
adnexal mass probably due to pelvic inflammatory
disease/appendicitis/ ovarian mass was made.
Investigations showed that all her biochemical and
haematological parameters were within normal
except the leukocyte count, which was 14,000/cmm.
The CA 125 level was 10 miu. An ultrasound
examination showed a complex mass with internal
echoes measuring 6 cms. in diameter in the right
adnexal region. There was minimal free fluid in the
abdomen. The CT imaging also showed a right
adnexal mass with loculated fluid collection in the
POD. With the above findings she was taken up for
laparotomy with adequate bowel preparation. On
opening the abdomen there were dense omental
adhesions between the parietal wall, uterus and the
anterior surface of the bladder. There was
haemorrhagic fluid collection anterior to the bladder
and in POD. There were plenty of small bowel
adhesions and were released. The uterus was normal
in size, the tubes were congested and the ovaries were
enlarged and were fixed in the POD. On releasing,
the ovaries discharged chocolate coloured material
and a diagnosis of endometriosis was made. Total
hysterectomy with bilateral salpingo oophorectomy
was carried out. As there was extensive bowel
dissection Surgeon was called in to check the
integrity of the bowel. During the systematic
inspection of the bowel, the appendix was found to be
enlarged with a cystic mass of 5 cms in size at the tip.
Therefore, appendicectomy was also carried out. Her
post-operative period was uneventful. Histopathology
of the specimen was reported as endometriosis of the
tubes and ovaries. Excised appendix was reported as
sub acute appendicitis with features of endometriosis
with mesoappendix also showing evidence of
endometriosis. [Fig. 1]
Fig1: Picture showing endometrial gland within the

muscularis
DISCUSSION
Endometriosis of the gastrointestinal tract is rare, and
can present with a wide spectrum of symptoms. The
Gastrointestinal tract is affected in nearly 12% of
patients with pelvic endometriosis, of which 72% are
in the recto-sigmoid region, followed by rectovaginal
septum in 13%, small bowel in 7%, caecum in 4%
and appendix in 3% of cases. [2] The occurrence of
appendiceal endometriosis without evidence of pelvic
endometriosis is rare and is reported to be between
0.05% and 0.8%. However, in women with pelvic
endometriosis, the prevalence of appendiceal
endometriosis is estimated to be approximately
between 0.8% - 2.8%. [3, 4] When endometriosis
involves the appendix, there is acute inflammation of
the appendix due to partial or complete occlusion of
the lumen with endometriosis. As a result, chronic
right lower abdominal pain is the most common
symptom and nearly one third of them eventually
present with symptoms of acute appendicitis. [5,6]
Endometriosis can also present as mucocele
formation or appendicular mass. Other unusual
presentations are intussception of the appendix and
the perforation. [7, 8]
Appendiceal endometriosis patients can be
categorised into four groups in terms of
symptomatology [9]
1. Patients who present with acute appendicitis
2. Patients who present with appendix invagination.
3. Patients manifesting atypical symptoms such as
abdominal colic, nausea and melena.
4. Patients who are asymptomatic.
Based on the history it is difficult to diagnose
endometriosis of the appendix. In our patient though
the possibility of appendicitis was thought of, because
of her pelvic symptoms, Gynaecological condition
was a primary diagnosis. Leucocytosis with
predominance of polymorphonuclear leukocytes
accompanies acute appendicitis in most cases. In our
case, though it was not an acute manifestation, the
leukocyte count was elevated. There are no specific
features on USG and CT to diagnose endometriosis of
the appendix. Findings are similar to that of acute
appendicitis with dilated fluid filled appendix.[1,10]
Appendiceal endometriosis is often seen in patients
with ovarian endometriosis. Our patient also had
concomitant pelvic endometriosis with concomitant
ovarian involvement. Muscular and seromuscular
725
Radha et al.,
involvement is seen in two-thirds of cases, while the

mucosal surface is involved in one-third of patients.
Our case presented with a nodule at the tip of the
appendix and the sero-muscular layer was involved.
Though, our patient presented with recurrent attacks
of incapacitating lower abdominal pain, the pain was
not related to her menstrual cycle initially, therefore,
endometriosis of the appendix was not suspected preoperatively. As in other reports involvement of the
appendix with endometriosis was made only on histopathological examination. However, the patients
persistent right lower quadrant pain made us inspect
the appendix for possible concomitant pathology and
appendicectomy was proceeded with because of the
nodule. Incidental appendicectomy during surgical
treatment of pelvic endometriosis is controversial. In
one study, routine appendicectomy during
laparoscopic treatment of ovarian endometriosis
showed microscopic evidence of endometriosis in
13.2% of patients. [11] Though routine removal is
controversial, pre-operative counselling and obtaining
consent for appendicectomy is important. As well as
appendicectomy should be carried out while treating
patients with recto-sigmoid endometriosis, as the
involvement of appendix in these cases is high.[12] In
our case , as one of the differential diagnosis was
appendicitis, pre-operative consenting was taken for
appendicectomy. In cases where, there was incidental
diagnosis of endometriosis of the appendix, further
gynaecological assessment and post-operative follow
up are important.
CONCLUSION
Endometriosis of the appendix is rare. Pre-operative
diagnosis is difficult, and the definitive diagnosis is
usually established following histopathological
examination of the excised tissue. Recurrent chronic
right lower abdominal pain is the most common
presentation and pain may be occasionally cyclical.
Endometriosis of the appendix should be suspected
and should be included in the differential diagnosis in
young women presenting with right lower quadrant
pain or symptoms of acute appendicitis. While
treating cases of pelvic endometriosis surgically,
appendix should always be inspected and incidental
appendicectomy should be considered.
REFERENCES
1. Douglas C, Rotimi O. Extragenital endometriosis
a clinicopathological review of a Glasgow
hospital experience with case illustrations. J
Obstet Gynaecol. 2004; 24: 804-08.
2. Khairy GA. Endometriosis of the appendix: a trap
for the unwary. Saudi J Gastroenterol 2005; 11:
45-7.
3. Berker B, Lashay N, Davarpanah R, Marziali M,
Nezhat
CH,
Nezhat
C.
Laparoscopic
appendectomy in patients with endometriosis. J
Minim Invasive Gynecol. 2005; 12: 206-09
4. Gustofson RL, Kim N, Liu S, Stratton P.
Endometriosis and the appendix- a case series
and comprehensive review of the literature. Fertil
Steril. 2006; 86: 298-03.
5. Harris RS, Foster WG, Surrey MW, Agarwal SK.
Appendiceal
disease
in
women
with
endometriosis and right quadrant pain. J Am
Assoc Gynecol Laparosc. 2001; 8: 536-41.
6. Nasser S AI Oulaqi, Ashraf F Hefny, Sandyia
Joshi, Khalid Salim, Fikri M Abu-Zidan.
Endometriosis of the appendix. Afr Health Sci.
2008; 8: 196-98.
7. Uncu H, Taner D. Appendiceal endometriosis:
two case reports. Archives of Gynecology and
Obstetrics. 2008; 278: 273-75.
8. Samia Ijaz, Surjit Lidder, Waria Mohamid,
Martyn
Carter
and
Hilary
Thompson.
Intussusception of the appendix secondary to
endometriosis: a case report. Journal of Medical
Case Reports 2008, 2:12
9. Styliani Laskow, Theodossis S Papavramidis,
Angeliki Cheva, Nick Michalopoulos, Charilaos
Koulouris, Isaak Kesisoglou and Spiros
Papavramidis. Acute appendicitis caused by
endometriosis: a case report: Jl of Med case
reports. 2011; 5: 144
10. Genevieve L. Bennett, Chrystia M. Slywotzky,
Mariela Cantera and Elizabeth M. Hecht.
Unusual manifestations and complications of
endometriosis spectrum of imaging findings:
Pictorial Review. Am J Roentgenlogy. 2010; 194:
34- 46.
11. Wie HJ, Lee JH, Kyung MS, Jung US, Choi JS.
Is incidental appendectomy necessary in women
with ovarian endometrioma? Aust NJZ. 2008; 48:
10711
12. Harper AJ, Soules MR. Appendectomy as a
consideration in operation for endometriosis. Int J
Gynaecol Obstet. 2002; 79: 53-54

726
Radha et al.,
DOI: 10.5958/2319-5886.2015.00140.X

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Volume 4 Issue 3
Coden: IJMRHS
th
Case report
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ISSN: 2319-5886
POST VARICELLA ANGIOPATHY- A CASE REPORT

Nita R Sutay1, Md Ashfaque Tinmaswala2, Shilpa Hegde3
1
Professor and Head, 2,3Senior Resident, Department of Pediatrics, Grant Government Medical College and JJ
Hospital Mumbai, Maharashtra, India
*Corresponding author email: dr.ashfaq.memon@gmail.com
ABSTRACT
Chickenpox is a common viral illness in children. In most of the immunocompitent children its a self-limiting
disease and seldom causes complications. Neurological complications are one of the rare complications of
chickenpox. These complications may present as hemiparesis, focal deficits and arterial ischemic strokes (AIS).
These Ischemic strokes may be a manifestation of post varicella angiopathy. Here we present a case of 11 year old
girl who presented with left hemiparesis with left sided facial nerve palsy 15 days after chickenpox. An MRI was
done which was suggestive of multiple infarcts in cortical and subcortical regions and MR angiography was
suggestive of narrowing of right middle cerebral artery. Patient was treated with aspirin and LMW heparin in
addition to supportive measures.
Keywords: Post varicella angiopathy, arterial Ischemic stroke, Hemiparesis.
INTRODUCTION
Varicella in childhood is usually a self-limiting
disease. Although mortality and morbidity may be
more in immunocompromised individuals, in
immunocompitent individuals it usually doesnt lead
to any complications [1]. The complications which
may be seen in varicella infections are secondary
bacterial
infections,
bacterial
pneumonias,
thrombocytopenia, glomerulonephritis, myocarditis,
arthritis, orchitis and hepatitis [2]. Neurological
complications may include cerebellar ataxia,
encephalitis and stroke [3]. Post Varicella angiopathy
as a cause of arterial ischemic stroke is an unusual
occurance. Here we present a case post varicella
angiopathy in 11 year old girl who presented with left
hemiparesis and left sided facial nerve palsy 15 days
after chicken pox.
CASE REPORT
11 year old girl 5th by order of birth presented with
complaints of weakness of left upper and lower limbs
with deviation of angle of mouth towards right side,

difficulty in eating and drooling of saliva since 5
days. There was no history of convulsions, fever or
head trauma. There was a history of chicken pox 15
days back for which she was treated by a local
pediatrician on OPD basis. On admission the patient
was vitally stable with post inflammatory
hyperpigmented spots all over the body. On
neurological examination positive findings were left
upper motor neuron type of facial nerve palsy in the
form of deviation of angle of mouth to right side.
Power in left upper and lower limbs was 3/5 with
increased tone in left upper and lower limbs. Deep
tendon reflexes were exaggerated on left side along
with upgoing plantar on left side. Patient was
admitted in view of left hemiparesis and left facial
nerve palsy. Her routine investigations CBC,
coagulation profile and Hb electrophoresis was
normal. Screening for tuberculosis was also negative.
2D echo was done to rule out cardiac cause of stroke
727
Nita et al.,
but it was also normal. MRI Brain was done which

was suggestive of multiple infarcts in right cortical
and subcortical regions of right frontoparietal lobe
involving right centrum semiovale right corona
radiate, right internal capsule, right cerebral peduncle,
right insular cortex, right temporal lobe, right
lentiform nucleus. MR angiography was suggestive
of narrowing of distal half of M1 segment of right
middle cerebral artery with paucity of cortical
branches.
Fig 1: Narrowing of Right Middle Cerebral Artery

is seen on MR angiography
Fig 2: Right cortical and subcortical infarcts seen

on MRI
In view of history of chicken pox 15 days prior to
stroke and MRI findings patient was diagnosed to be
having post varicella angiopathy. Patient was started
on aspirin and LMW heparin in view of stroke. Oral
feeding was continued. Patient started showing
improvement and by 5-6 days was able to walk with
little support. Patient was adviced regular
physiotherapy and was discharged with an advice to
follow up with repeat MR angiography after 3
months.
DISCUSSION
The etiology of stroke in pediatric age group is quite
different from that of adults. Types of stroke in
pediatric age group are arterial and venous
thrombosis, intracranial bleeds, embolism and various
other conditions. Predisposing conditions for stroke
in children include cardiac diseases like congenital
heart diseases, arrhythmias, structural valvular heart
diseases, bacterial endocarditis causing mycotic
aneurysms, sickle cell disease and occlusive vascular
diseases like moya moya disease etc[4]. Varicella
angiopathy is one of less common causes of stroke in
childhood. The underlying mechanism of varicella
causing AIS is not clearly understood. Various
mechanisms have been suggested. One of the possible
explanation is intraneuronal migration of VZ virus
from trigeminal ganglion along the trigeminal nerve
to cerebral arteries [5]. VZ virus was present in the
media of the large cerebral arteries in adult patients
presenting with herpes zoster opthalmicus [6]. The
fact that the distribution of vasculitic lesions in
varicella infection associated arterial ischaemic
strokes corresponds to and matches the anatomical
location and density of trigeminal nerve innervations
at circle of willis further potentiates this theory.
Recurrence of stroke is more common in the varicella
associated AIS than nonvaricella AIS [7].
Radiological finding in AIS associated with varicella
is more likely to have infarcts in basal ganglia,
multiple infarcts and large vessel stenosis. In
pediatric age group basal ganglia infarcts may be
associated with the history of varicella ranging from
10-50% [8].
Primary prevention of post varicella AIS by varicella
vaccine is important but given the rarity of
complication of varicella infection in pediatric age
group and excellent prognosis of AIS in pediatric
patient means only modest impact is expected.
Treatment of varicella associated AIS is mainly
supportive. Antiviral therapy and anticoagulants can
also be given.Varicella associated AIS has a higher
mortality and morbidity in adults [9].As the survival
rate is excellent in children as compared to adults,
antiviral therapy and anti-inflammatory therapy may
not be given in pediatric patients presenting with AIS
associated with varicella infection[10]. Anticoagulant
therapy in initial phase of stroke should be considered
as this will prevent local extension of the thrombus
728
Nita et al.,
and chances of embolization. Long term therapy with

aspirin should be prescribed to all children having
suffered AIS due to varicella infection.
CONCLUSION
Strokes in children have distinct etiology. Post
varicella angiopthy is an important cause of AIS and
should be considered in differential diagnosis
especially when radiological findings of basal ganglia
infarcts, multiple infarcts and stenosis of large vessels
are found. As post varicella angiopathy may cause
AIS several months after initial infection its
important to carefully elicit past history of chicken
pox in pediatric patients presenting with AIS.
8. Inagaki M, Koeda T, Takeshita K, Prognosis, and

MRI after ischemic stroke of the basal ganglia.
Pediatr Neurol. 1992;8:104108.
9. Gilden DH, Kleinschmidt-DeMasters BK,
LaGuardia JJ, Mahalingam R, Cohrs RJ.
Neurological complications of the reactivation of
the varicella-zoster virus. N Engl J Med.
2000;342:635645.
10. Rand Askalan, Suzanne Laughlin, Supriya
Mayank, Anthony Chan, Daune MacGregor,
Maureen Andrew, et al. Chickenpox and Stroke
in Childhood- A Study of Frequency and
Causation. Stroke 2001;32:1257-62.

REFERENCES
1. Martin G.Myers, Lawrence R Stanberry and Jane
F Seward Varicella Zoster virus In: Kliegman
RM, Behrman RE, Jenson HB, editors.Nelson
Textbook of Paediatrics. 17th ed. Philadelphia:
Saunders; 2004. pp. 1057-1062
2. Rivest P, Bdard L, Valiquette L, et al. Severe
complications associated with varicella: Province
of Quebec, April 1994 to March 1996. The
Canadian Journal of Infectious Diseases.
2001;12(1):21-26.
3. Hayes B, Baker L, Alhajeri A, Ryan S, Lynch B.
Ischaemic stroke in children
secondary to post varicella angiopathy. Ir Med J.
2007 Jan;100(1):332-3. PubMed
PMID: 17380923.
4. Michael V. Jhonston Acute Stroke Syndromes
In: Kliegman RM, Behrman RE, Jenson HB,
editors.Nelson Textbook of Paediatrics. 17th ed.
Philadelphia: Saunders; 2004. pp. 2035-2038
5. Mayberg MR, Zervas NT, Moskowitz MA.
Trigeminal projections to supratentorial pial and
dural blood vessels in cats demonstrated by
horseradish peroxidase histochemistry. J Comp
Neurol 223:46-56, 1984.
6. Eidelberg D, Sotrel A, Horoupian DS, Neumann
PE, Pumarola-Sune T, Price RW. Thrombotic
cerebral vasculopathy associated with herpes
zoster. Ann Neurol. 1986;19:714.
7. Hattori H, Higuchi Y, Tsuji M. Recurrent strokes
after varicella. Ann Neurol.
2000 Jan;47(1):136. PubMed PMID: 10632115.
729
Nita et al.,
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PRIMARY CONJUNCTIVAL TUBERCULOSIS - A RARE CASE REPORT

Kulkarni Dinesh R1, Sulegaon Ritesh V2, Chulki Shashidhar F3,
1
Associate Professor, 2Assistant Professor, 3Professor & Head, Department of Pathology, Bidar Institute of
Medical Sciences, Bidar, Karnataka, India
*Corresponding author email: docritz@gmail.com
ABSTRACT
Tuberculosis is an endemic disease in India. Primary conjunctival tuberculosis is an uncommon condition and
with better treatment of pulmonary tuberculosis it is now becoming extremely rare. Primary conjunctival
Tuberculosis can present as unilateral conjunctivitis, hence laterality, chronicity and non-resolution of symptoms
on treatment are indications for biopsy. In our patient conjunctival Tuberculosis was diagnosed on histopathology,
which resulted in early implementation of antikochs treatment and complete resolution of the disease condition.
Keywords: Conjunctiva, Tuberculosis, Histopathology.
INTRODUCTION
Tuberculosis is a major public health problem and is
the second leading cause of death from infectious
disease worldwide. [1] The recognized association of
Tuberculosis with ocular disease dates to 1700s,
when iris lesions in tuberculous patients were
described. Recognition of choroidal tubercles was
first noted between 1830 and 1844. The current
incidence of ocular Tuberculosis is uncertain, about
1.4% of patients with Pulmonary Tuberculosis have
ocular manifestations, but many patients with Ocular
Tuberculosis have no evidence of Pulmonary
Tuberculosis. [2,3] Ocular Tuberculosis occurs via
hematogenous spread during pulmonary or
extrapulmonary lesion and/or via local spread from
an active sinus or meningeal infection.[3,4] In children,
Primary ocular infection occurs when the bacilli enter
the body through the conjunctiva. Most commonly
reactivation of dormant lesions in ocular tissue leads
to symptomatic disease. Additionally immune
mediated Ocular Tuberculosis can occur due to
hypersensitivity to Mycobacterium Tuberculosis
antigens from a distant focus.[3] Depending on the
immuno-allergic state of the patient, clinical picture is
variable.[5] Ocular Tuberculosis is often unilateral and

asymmetric and spreads to the central nervous system
via optic nerve.[3] In areas endemic for tuberculosis,
considering it in the diagnostic algorithm of nonresolving unilateral conjunctivitis would be
worthwhile.[2]
CASE REPORT
A 45 year male from a middle class family presented
with persistent redness and itching in left eye for 15
days. His ophthalmic examination was within normal
limits. He was treated for allergic conjunctivitis.
Subsequent follow up after 15 days did not show any
improvement. He was asked to continue same
treatment. He came after three months with persistent
redness and formation of a small nodular firm mass
on bulbar conjunctiva. His systemic examination did
not reveal any abnormality. Laboratory investigations
showed lymphocytic leukocytosis with markedly
raised ESR (37 mm at one hour). He was advised to
get the mass excised. He was physically fit for
surgery and under local anaesthesia the mass was
730
Ritesh et al.,
excised and sent for histopathological examination.

The postoperative period was uneventful.. The biopsy
report suggested tuberculous granulomatous lesion.
Clinical examination, lab investigations, x-ray chest
and USG abdomen ruled out any evidence of primary
tuberculosis. He was treated with standard four drugs
anti tubercular therapy for six month. Morphology:
Received single small grayish soft tissue bit,
processed as such. Microscopic features: Section
stained with H & E showed stratified squamous
epithelium of conjunctiva and beneath fibro
collagenous stroma along with many granulomas
composed of central caseous necrosis, surrounded by
epithelioid cells, lymphocytes and Langhans type
giant cells with areas of fibrosis (Fig. 1). Langhans
giant cells are more suggestive of tuberculous
granulomas. Ziehl Neelsen stain did not reveal acid
fast bacilli, so the diagnosis of tuberculous granuloma
was made.
exogenous infection. There are five clinical types ulcerative, nodular, hypertrophic, granulomatous, and
pedunculated. It may occur more commonly in young
than old patients, and may be associated with regional
lymphadenopathy. It runs a chronic course, and may
heal with scarring or may spread to involve adjacent
tissues and structures.[2,4]
Spread of infection from a contiguous focus leads to
secondary form of tuberculous conjunctivitis. There
are six clinical types - ulcerative, nodular,
hypertrophic, granulomatous, pedunculated, and
lupoid. It may occur more commonly in adults than
children and may not be associated with regional
lymphadenopathy. In both instances, the diagnosis is
confirmed on histological examination.[4]
In our patient despite the use of broad spectrum
antibiotics, steroids and anti allergic drugs, the
redness still persisted with formation of conjunctival
mass and was eventually diagnosed as of tuberculous
origin only after histopathological examination of the
excised conjunctival mass. Criteria for diagnosis of
ocular tuberculosis are not well established and the
same may also explain the variation of reported
incidence and epidemiology of ocular tuberculosis
over time and geography.[3] Treatment of tuberculosis
is curative regardless of site, if it is instituted early
and if the organism remains sensitive to all first-line
antituberculous drugs.[6] There was proper response to
four drug anti-tubercular regime in our patient.
CONCLUSION
Fig. 1 shows lining epithelium and beneath

granulomas composed of epithelioid cells,
lymphocytes, Langhans giant cell and caseous
necrosis. (H & E: 4x X 10x).
DISCUSSION
Despite all advancement made in treatment and
management, tuberculosis still remains one of the
major public health problems, mainly in developing
countries. The incidence of drug sensitive and multi
drug resistant tuberculosis is very high in India.[1] The
usual presentation is pulmonary tuberculosis, but
extrapulmonary tuberculosis is also important clinical
problem, which is difficult to diagnose.[1,6] In current
clinical practice, the incidence of active tuberculous
lesions of the conjunctiva are so rare that the
physicians index of suspicion is very low.[2] Primary
tuberculous conjunctivitis occurs as a result of an
Ritesh et al.,
We would like to conclude that although ocular

tuberculosis is rare, it is therefore necessary to
consider it in any unusual chronic conjunctivitis, with
or without regional lymphadenopathy, particularly in
endemic areas where the incidence of tuberculosis is
still high.
REFERENCES
1. WHO Global Tuberculosis report 2013.
www.who.int/tb/publications/global_report
2. Rose JS, Anupriya A, Renu R, Meera T. Primary
conjunctival tuberculosis in a 14 year old girl.
Indian J Tuberc 2011; 58:32-34.
3. Bramante CT, Talbot EA, Rathinam SR, Stevens
R, Zegans ME. Diagnosis of Ocular
Tuberculosis: A Role for New Testing
731
Modalities?
Int
Ophthalmol
Clin. 2007
Summer;47(3):45-62
4. Cook CD, Hainsworth M. Tuberculosis of the
conjunctiva occurring in association with a
neighbouring lupus vulgaris lesion. Brit J Ophthal
1990; 74:315-16.
5. Sollom AW. Primary conjunctival tuberculosis.
Brit J Ophthal 1967; 51:685-87.
6. Fanning A. Tuberculosis: Extrapulmonary
disease. CMAJ 1999; 160:1597-03.
732
Ritesh et al.,
DOI: 10.5958/2319-5886.2015.00142.3

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Coden: IJMRHS
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ISSN: 2319-5886
Case report
CONCURRENT ORIGIN OF RIGHT GASTROEPIPLOIC AND LEFT COLIC ARTERIES FROM

INFERIOR PANCREATICODUODENAL ARTERY: RARE VARIATION OF SPLANCHNIC
ANASTOMOSIS
*Mutalik Maitreyee M
Assistant Professor, Department of Anatomy, MIMER Medical College, Talegaon Dabhade, Pune, India
*Corresponding author email: maitreyeemadhav@gmail.com
ABSTRACT
In the present case, inferior pancreaticoduodenal artery, the first branch of superior mesenteric artery, was
exceptionally giving rise to right gastroepiploic artery and left colic artery simultaneously. Right gastroepiploic
artery is a branch of foregut artery, while left colic artery is a branch of hindgut artery. Concurrent origin of
branches of foregut as well as hindgut arteries from a midgut artery i.e. superior mesenteric artery is very rare.
Usual left colic artery from inferior mesenteric artery was also present but was supplying smaller area than usual.
It can be explained as persistence of unusual channels and obliteration of usual ones along the dorsal splanchnic
anastomosis during the embryonic development. The field of vascularization of superior mesenteric artery was
extended beyond its usual boundaries both proximally as well as distally, which is clinically important as
unawareness of the variations may lead to significant morbidity and mortality.
Keywords: Bypass graft, Colic artery, Gastroepiploic artery, Pancreaticoduodenal artery, Splanchnic
anastomosis, Mesenteric artery
INTRODUCTION
Fields of vascularization of celiac trunk (CT),
superior mesenteric artery (SMA), and inferior
mesenteric artery (IMA), which are ventral branches
of abdominal aorta, constitute the basis for dividing
the abdominal gastrointestinal tract into three
respective embryological regions - foregut, midgut,
and hindgut[1]. The midgut forms the third and fourth
parts of the duodenum, jejunum, ileum, and twothirds of the way along the transverse colon[2]. These
parts of the gut are usually supplied by SMA. The
part of the abdominal gut proximal to it is supplied by
CT and distal to it is supplied by IMA. Inferior
pancreaticoduodenal artery (IPDA) arises as a first
branch of SMA. It usually divides directly into
anterior and posterior branches which anastomose
with
the
similar
branches
of
superior
pancreaticoduodenal artery which is a branch of
gastroduodenal artery (GDA), arising from hepatic

branch of CT (foregut artery). It is a site of
anastomosis between SMA and CT. Both the
branches of IPDA supply the pancreatic head, its
uncinate process and the second and third parts of the
duodenum[2]. GDA (foregut artery) also gives rise to
right gastroepiploic artery (RGEPA), which supplies
the stomach by running along its greater curvature.
Left colic artery (LCA) is a first branch of IMA
(hindgut artery), which ascends towards left side and
immediately divides into ascending and descending
braches. Ascending branch anastomoses with left
branch of middle colic, while descending one with
the highest sigmoid artery[2]. In the present case as
both RGEPA and LCA were arising from IPDA
simultaneously, this branch of SMA was unusually
supplying two distinct parts of the gut and hence field
733
Maitreyee
of vascularization of SMA was extended beyond its

usual boundaries both proximally as well as distally.
Such variations related to both foregut and hindgut
arteries together on a same branch of midgut artery
are very rare.
CASE REPORT
The observations were done during routine
anatomical dissection for undergraduate students on a
formalin-fixed 70-year-old male cadaver while
dissecting the abdominal region. During careful
dissection of arteries of the gut it was seen that the
inferior pancreaticoduodenal artery (IPDA), a first
branch of superior mesenteric artery (SMA) was
showing an unusual branch near its root. The IPDA
then continued further and gave its usual anterior and
posterior branches and further continued as right
gastroepiploic artery (RGEPA), running along the
greater curvature of the stomach [Figure 1].
Fig 1: Origin of Right gastroepiploic and accessory

left colic arteries from inferior pancreaticoduodenal
artery [L: liver; A: aorta; S: stomach; P(R): reflected
pancreas; CT: celiac trunk; SMA: superior mesenteric
artery; IPDA: inferior pancreaticoduodenal artery;
RGEPA: right gastroepiploic artery; LCA(S): accessory
left colic artery from SMA; MC: middle colic artery;
RC: right colic artery; IC: ileocolic artery; IMA:
inferior mesenteric artery; TC(R): reflected transverse
colon]
The RGEPA was not originating from its usual artery
i.e. gastroduodenal artery (GDA). The unusual branch
arising near the root of IPDA crossed SMA
anteriorly, approached splenic flexure, and terminated
into two branches, ascending and descending [Fig 2].
The ascending branch was running along splenic
flexure and the adjacent transverse colon and the

descending branch along the upper part of descending
colon and continued further by forming marginal
artery [Figure 3]. We found that it was an extra left
colic artery as we also found the usual LCA arising
from IMA, hence we called this branch of IPDA as an
accessory LCA.
The inferior mesenteric artery (IMA) was giving its
usual three branches i.e. left colic, sigmoid, and
superior rectal arteries. Here the left colic artery
(LCA) was directed downwards towards lower part of
descending colon [Figure 2]. In the present case, the
LCA from IMA was not branched, was running
downwards, and near the lower part of the descending
colon it terminated by joining with the lower part of
the marginal artery [Figure 3]. The above
observations showed that the colon on the left side
was supplied by two left colic arteries having
different origins. One LCA was arising from IMA as
usual, but with smaller area of distribution. However,
another artery originated from IPDA near its root was
supplying distal part of transverse colon, splenic
flexure, and upper part of descending colon, which
we called as (was) an accessory LCA.
Fig 2. Accessory left colic artery supplying splenic

flexure and upper left colon [TC: transverse colon;
SF: splenic flexure; A: aorta; P(R): reflected pancreas;
CT: celiac trunk; SMA: superior mesenteric artery;
IPDA: inferior pancreaticoduodenal artery; LCA(S):
accessory left colic artery from SMA; a: ascending
branch of LCA (S); d: descending branch of LCA (S);
MC: middle colic artery; RC: right colic artery; IC:
ileocolic artery; IMA: inferior mesenteric artery]
734
Maitreyee
Fig 3: Accessory left colic artery and usual left colic

artery [TC: transverse colon; SF: splenic flexure; A:
aorta; P: pancreas; SMA: superior mesenteric artery;
IPDA: inferior pancreaticoduodenal artery; LCA(S):
left colic artery from SMA; a: ascending branch of LCA
(S); d: descending branch of LCA (S); IMA: inferior
mesenteric artery; MA: marginal artery; DC:
descending colon; LCA(I): usual left colic artery from
IMA]
The peculiarity of the case was one artery i.e. the

IPDA (midgut artery) was giving rise to two unusual
arteries concomitantly and those arteries were
supplying to two completely different parts of the gut
- RGEPA proximally, supplying greater curvature of
stomach (a foregut derivative) and an accessory LCA
distally, supplying distal part of transverse colon,
splenic flexure, and upper part of the descending
colon (hindgut derivatives).
DISCUSSION
Variations in the branching pattern of superior
mesenteric artery are commonly seen and well
documented. It may be a source of the common
hepatic, gastroduodenal, accessory right hepatic, and
accessory pancreatic or splenic arteries[2]. Bergman et
al mentioned that SMA may give rise to branches
which are usually derived from other sources, like
hepatic or its branches, cystic, gastroduodenal or its
right gastroepiploic, left gastric, and accessory left
colic arteries. Inconstant branches also have been
noted. These are the dorsal pancreatic (21% of cases
studied), inferior pancreatic, right hepatic (accessory
or replacing, 14%), common hepatic, and accessory

middle colic. In some cases, the inferior mesenteric,
splenic, gastroduodenal, right gastroepiploic, or even
the cystic artery arise from the superior mesenteric[3].
Origin of right gastroepiploic artery (RGEPA) from
SMA[4,5]or accessory left colic artery from SMA[6,7]
have been observed and mentioned in literature, but
they always occurred as separate events. To the best
of our knowledge, the concurrent origin of both the
arteries from SMA has not been reported.
The embryological events occurring during the
establishment of the vasculature of gut may form the
basis of this variation. Each primitive dorsal aorta on
each side gives off many paired segmental branches
to the digestive tube. After fusion of the dorsal aortae,
they merge as unpaired trunks that are distributed to
the
primitive
digestive
tube.
Longitudinal
anastomotic channels connect these branches along
the dorsal and ventral aspects of the tube, forming
dorsal and ventral splanchnic anastomoses. These
vessels obviate the need for so many arteries and are
reduced to three CT, SMA, and IMA. Some of the
channels in the anastomosis persist according to the
blood supply of the region while remaining channels
disappear. The dorsal splanchnic anastomosis persists
in the gastroepiploic, pancreaticoduodenal, and
primary branches of the colic arteries, whereas the
ventral splanchnic anastomosis forms the right and
left gastric and the hepatic arteries. The variations are
due to modifications of the usual processes by which
the vessels are developed[8]. The uncommon origin of
RGEPA and LCA from IPDA found in the present
case can be explained as persistence of unusual
channels and obliteration of usual ones along the
dorsal splanchnic anastomosis.
Prior knowledge of branching pattern of these arteries
is essential to successfully accomplish surgical and
other interventional procedures. RGEPA is one of the
arteries used for coronary artery bypass graft (CABG)
[9,10]
. The RGEPA is also used as an alternative inflow
source in acute mesenteric ischemia11. This suggests
the significance of verification of origin of RGEPA
before it could be used in any procedures. Yoshihara
et al have mentioned an anomalous right
gastroepiploic artery graft arising from SMA5.
Similarly the knowledge of variations in the origin of
left colic artery will be useful in procedures or
surgeries related to colon. In a situation like a present
case, where a large territory is supplied by SMA,
735
Maitreyee
injury or thrombosis of the artery may lead to

significant morbidity or mortality.
CONCLUSION
In the present case, the SMA, a midgut artery was
crossing its usual boundaries both proximally as well
as distally to supply parts of foregut and hindgut i.e.
a large visceral territory was supplied by a single
vessel. Such occurrence can be explained as the
variation in the formation of dorsal splanchnic
anastomosis during embryonic development. The
dominance of the SMA found in the present case was
remarkable. Though most of the times such variations
remain asymptomatic, in cases like injury or
thrombosis of SMA, surgical procedures on the gut or
intended use of RGEPA in coronary artery bypass
graft (CABG) unawareness of the variations may lead
to significant morbidity and mortality.
Conflict of interests: The author declares that there
is no conflict of interests.
Acknowledgments: The author is thankful to Dr
Garud, Dr Reddy, Dr Shinde, and Mrs Anandi of
Anatomy Department, Bharati Vidyapeeth Medical
College, Pune, India.
REFERENCES
1. Schoenwolf, Bleyl SB, Brauer PR, Francis-West
PH. Editors. Larsens Human Embryology. 4th
Ed., pp. 408. Philadelphia: Elsevier/Churchill
Livingstone; 2009.
2. Standring S. Editor. Grays Anatomy. 40th Ed.,
Section 8. Abdomen and Pelvis. pp. 1039-1325.
New York: Elsevier/Churchill Livingstone; 2010.
3. Bergman RA, Afifi AK, Miyauchi R. Superior
Mesenteric Artery. Illustrated Encyclopedia of
Human
Anatomic
Variation:
Opus
II:
Cardiovascular System: Arteries: Abdomen:
Variations in Branches of Celiac Trunk. A digital
library of Anatomy information. Anatomy
Atlases.
Available
at
http://www.anatomyatlases.org/AnatomicVariant
s/Cardiovascular/Text/Arteries/MesentericSuperi
or.shtml (Accessed 31st December, 2013).
4. Sakamato H, Akita K, Sato T. An anomalous
right gastroepiploic artery arising from the
superior mesenteric artery. Surg Radiol Anat
1999;21(4):283-6.
5. Yoshihara S, Yaegashi T, Naito M, Kutsukake Y,

Kamiya M. An anomalous right gastroepiploic
artery graft arising from the superior mesenteric
artery. Cardiol J 2011;18(2):202-3.
6. Kim DH, Jin C, Jung Y, Lee JH, Choi IJ, Kim
DK. The accessory left colic artery arising from
the superior mesenteric artery: A case report.
Korean J of Anat 2009;42(3):209-12.
7. Rusu MC, Vlad M, Voinea LM, Curca GC, Sisu
AM. Detailed anatomy of a left accessory
aberrant colic artery. Surg Radiol Anat
2008;30(7):595-9.
8. Standring S. Editor. Grays Anatomy. 40th Ed.,
Chapter 13. Early embryonic circulation. pp. 207209. New York: Elsevier/Churchill Livingstone;
2010.
9. Grandjean JG, Boonstra PW, den Heyer PD,
Ebels T. Arterial revascularization with the right
gastroepiploic artery and internal mammary
arteries in 300 patients. J Thorac Cardiovasc Surg
1994;107:1309-16.
10. Ishida T, Kurosawa H, Nishida H, Aomi S, Endo
M. Sequential bypass using the right
gastroepiploic artery for coronary artery bypass
grafting. Japanese J of Thorac and Cardiovasc
Surg 2003;51(7):277-81.
11. Oda T, Ono H, Muranaka H, Takai F. The right
gastroepiploic artery as an alternative inflow
source in acute mesenteric ischemia. J Vasc Surg
2005;41(6):1061-4.
736
Maitreyee
DOI: 10.5958/2319-5886.2015.00143.5

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Case report
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TYPHOID AFTERMATH: PRESENTING AS VASCULITIS, NEURORETINITIS AND MACULAR

NEUROSENSORY DETACHMENT
*Rohit Laul1, Atif Ali MIR2, Shazia Shafi3
1
MS FERC, 2MS FVRS, DNB MNAMS, Practitioners, Dr. Agarwal's Eye Hospital, Chennai, Tamil Nadu, India
*Corresponding author email: drlaulrs@rediffmail.com

ABSTRACT
Systemic immune-mediated reactions are known to occur following typhoid illness. Vasculitis, neuroretinitis and
macular neurosensory detachment are amongst the rarely documented aftermath of typhoid fever. A 32-year-old
male came with complaints of decreased vision in both eyes with history of typhoid fever (treated adequately 4
weeks prior and declared cured 2 weeks prior to ocular manifestations) who was found to have vasculitis,
neuroretinitis and neurosensory detachment at macula. His BCVA was 1/60 in right eye and 6/12 in left eye. The
inflammation completely resolved and there was marked improvement in the visual acuity, 6/12 in right eye and
6/6 in left eye after treatment with oral steroids. Immune-mediated vasculitis and neuroretinitis, the ocular
aftermath of typhoid fever responded well to oral steroids
Keywords: Neuroretinitis, Immune-mediated neuroretinitis
INTRODUCTION
Salmonella typhi causes typhoid infection. It can give
rise
to
fever,
gastroenteritis
and
septicemia. Salmonella rarely affects ocular tissue[1].
This may be direct infection or due to an immunemediated mechanism. Ocular complications of
typhoid fever include iritis, choroiditis, retinal
hemorrhage, panophthalmitis and endophthalmitis as
reported by Hersing and Duke-Elders. After complete
resolution of typhoid fever, there are documented
evidences of endophthalmitis[2,3]. Here we are
reporting a
patient who presented with
neuroretinitis and had typhoid fever 4weeks prior to
presentation
CASE REPORT
A 32-year-old Indian male presented to Dr Agarwal's
Eye Hospital, Salem with sudden, painless diminution
of vision in both the eyes (right eye > left eye) for 2
weeks. He gave a past history of typhoid fever 4
Rohit et al.,
weeks ago. Lab investigations during the fever

showed positive Widal test with high `O' antigen
(1:160) and `H' antigen (1:40) titres, while `AH' and
`BH' antigens were non-reactive. At the onset fever,
treatment was initiated with oral ofloxacin 400 mg
twice daily for 14 days; following which, the fever
resolved. He started experiencing diminution of
vision 2 weeks after the completion of treatment. At
presentation, his best corrected visual acuity was
1/60,in the right eye and 6/12 in the left eye. On
examination with slit lamp, anterior segments of both
eyes were within normal limits except for relative
afferent pupillary defect in the right eye. Colour
vision was defective in right eye. On fundus
examination media was clear, mild disc pallor was
noted. Vasculitis with multiple areas of deep
neuroretinitis was seen. Macular neurosensory
detachment was evident on OCT scans (Figure 1).
The left eye (Figure 1) had clear media, normal disc
737
and macular oedema, and one patch of neuroretinitis.

Lab tests for HIV.
TB, syphilis, connective tissue disorders, rheumatoid
arthritis and SLE were negative. Diagnoses of
immune mediated vasculitis, neuroretinitis and
macular neurosensory detachment secondary to
typhoid infection were made. After consulting with a
physician
he
was started
on
prednisolone
60mg/kg/day. Steroids were tapered over 2 months
with regular monitoring of health status and ocular
response. He had markedly recovered after 2month of
treatment & his visual acuity was 6/12 in right eye
and 6/6 in left eye. At 6 months follow-up (Figure 2)
all the retinal lesions had completely resolved with
pigmentary changes in the macula and mild pallor of
the optic disc in the right eye. OCT at 6
months revealed retinal pigment epithelium changes
and thinning of inner retinal layers over the lesions, in
addition to complete resolution of macular
neurosensory detachment (Figure2).
RIGHT EYE
Fig 1 : Both eyes at presentation
Fig 2 : Both eyes after treatment
LEFT EYE
This is a case of immune-mediated retinitis secondary

to typhoid fever. There can be a viral etiology which
cannot be neglected. The lesions in this patient were
posterior to the equator, did not show centrifugal or
circumferential extension, and did not affect the
arterioles, not associated with aqueous flare or cells
or vitritis. Taking into consideration the time of onset
of ocular complaints and onset of fever, clinical
improvement with oral steroids without the use of
antiviral medications, the most likely diagnosis was
immune mediated vasculitis, neuroretinitis and
macular neurosensory detachment as a aftermath of
typhoid infection. There is not much regarding this
type of pathology secondary to typhoid fever in the
literature [4-6]. Reports of similar cases[5] assume that
the origin of this kind of pathology is due to retinal
infiltration. Pathogenesis of immune-mediated
vasculitis secondary to typhoid infection could be
because of immunologic effects which can give rise
to an immune response, affecting self-antigens by
reacting with them due to homology or molecular
mimicry giving rise to autoimmunity[7]. Immunemediated vasculitis is a clinical diagnosis most often
when there is past history of infection few weeks or
days prior to the onset of ocular manifestations. Lab
work up can help to find out the exact etiology many
a times. Malaria, dengue fever, Chickungunya fever
and other viral infections may lead to immunemediated vasculitis. Some non-infectious etiologies
can be Behcet's syndrome and intraocular lymphoma
may also manifest as immune-mediated vasculitis[8].
Due to paucity of published literature on such rare
clinical scenarios, management remains controversial.
Mild cases may resolve spontaneously without
treatment, but severe cases may be treated with oral
corticosteroids, if these are not contraindicated. In
this case, treatment with oral steroids was initiated
due to inflammation of the vessels, macula and disc
which had caused profound diminution of vision.
CONCLUSION
Though these cases are rare, an ophthalmologist may
encounter such cases which are immune-mediated
neuoretinitis, non-infectious neuroretinitis, vasculitis
with or without macular neurosensory detachment
after complete resolution of typhoid fever that may be
effectively treated with a course of oral steroids
DISCUSSION
Rohit et al.,
738
Conflict of interest: The authors declare that they

have no competing interests (financial or nonfinancial).
REFERENCES
1. Duke-Elder S, Perkins ES. Diseases of the Uveal
Tract. Kimpton, London; 1968.
2. Rachitskaya AV, Flynn HW, Davis JL.
Endogenous
endophthalmitis
caused
by
salmonella serotype B in an immunocompetent
12-year-old
child. Arch
Ophthalmol. 2012;130:802804.
3. Sinha MK, Jalali S, Nalamada S. Review of
endogenous
endophthalmitis
caused
by
Salmonella species including delayed onset
Salmonella
typhi
endophthalmitis. Semin
Ophthalmol. 2012;27:9498
4. Thapar S. Characteristic OCT patterns of
posterior uveitis. Science City, Kolkata; 2010.
5. Vishwanath S, Badami K, Sriprakash KS, Sujatha
BL, Shashidhar SD, Shilpa YD. Post-fever
retinitis: a single center experience from south
India. Int Ophthalmol. 2013;17.
6. Fusco R, Magli A, Guacci P. Stellate
maculopathy
due
to
Salmonella
typhi. Ophthalmologica.1986;192:15458
7. Hughes EH, Dick AD. The pathology and
pathogenesis of retinal vasculitis. Neuropathol
Appl Neurobiol. 2003;29:32540
8. Balansard B, Bodaghi B, Cassoux N, Fardeau C,
Romand S, Rozenberg F, Rao NA, Lehoang P.
Necrotising retinopathies simulating acute retinal
necrosis
syndrome. Br
J.Ophthalmol.
2005;89:96101.
Rohit et al.,
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International Journal of Medical Research

A COMPARATIVE STUDY OF ORAL OLANZAPINE AND ORAL HALOPERIDOL ON GLUCOSE

Cause: Genetic: Some researchers estimate

receptor: Interest has also focused on the

Int J Med Res Health Sci. 2015;4(3):477-482

function of the NMDA glutamate receptor in the

Neuroleptic malignant syndrome has been reported

Int J Med Res Health Sci. 2015;4(3):477-482

Olanzapine and controls took oral Haloperidol along

Diabetes Mellitus and Haloperidol group had 5

Int J Med Res Health Sci. 2015;4(3):477-482

1st and 6th week 10.2 7.6

1st and 6th week

3rd and 6th week

1st and 3rd week

1st and 6th week

3rd and 6th week

3rd and 6th week

1st and 3rd week

1st and 6th week 12.6 7.4

3rd and 6th week

1st and 3rd week

glucose there was no significant increase for the 1st

Int J Med Res Health Sci. 2015;4(3):477-482

Fig1: Mean change in Fasting Blood Glucose

Fig2: Mean change in Post Prandial Blood

persons age 45 to 64 years. In patients with

Int J Med Res Health Sci. 2015;4(3):477-482

Acknowledgement: I thank the Director of Institute

Conflict of interest: Nil

Int J Med Res Health Sci. 2015;4(3):477-482

International Journal of Medical Research

SEROPREVALENCE OF HEPATITIS B IN A TERTIARY CARE CENTRE IN BIJAPUR,

More than two billion people worldwide have

Int J Med Res Health Sci. 2015;4(3):483-485

Several surveys for HbsAg screening have been

2013, 9428 samples were screened out of which 156

WHO definition one could categorize Karnataka into

1. Brian WJ. Hepatitis B. In: Topley and

4. Sayed A. Quadri, H.J. Dadapeer, K.

Int J Med Res Health Sci. 2015;4(3):483-485

International Journal of Medical Research

HISTOPATHOLOGIC AND CYTOMORPHOLOGIC CORRELATION IN LEPROSY

HOD, 1Department of Pathology, Panjabrao Deshmukh Medical College, Maharashtra, India

*Corresponding author email: sharayukalmegh@gmail.com

compare the histological and cytological procedure

Int J Med Res Health Sci. 2015;4(3):486-489

MATERIAL AND METHODS

sample was submitted for routine processing,

All cases (16/16) of histologically confirmed

Int J Med Res Health Sci. 2015;4(3):486-489

Table 2: cytohistological correlation along RJ spectrum

leprosy. In TT leprosy 62.5% (10/16) cases were

Int J Med Res Health Sci. 2015;4(3):486-489

in present study this is lower than 76.6% as reported

Int J Med Res Health Sci. 2015;4(3):486-489

International Journal of Medical Research

Department of Genetics, Osmania University, Hyderabad-500 007, Telangana, India

*Corresponding author email: ishaq_50@rediffmail.com

estimated to be around 5% [2, 3] and that of

Medicine, Princess ESRA Hospital, Hyderabad for

Int J Med Res Heath Sci. 2015;4(3):490-495

Subjects who were already diagnosed as suffering

The prevalence of hypothyroidism was highest in the