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International Journal of Recent Scientific Research
Vol. 2, Issue, 6, pp. XXX -XXX, June, 2011
ISSN: xxxxx

EMERGING TRENDS OF CANDIDEMIA AND ANTIFUNGAL SUSCEPTIBILITY IN A TERTIARY

NEONATAL INTENSIVE CARE UNITS
Vinodkumar C.S1, Umakanth Patil2, Kalapanavar N.K3, Basavarajappa K.G4,
1
Department of Microbiology, S. S. Institute of Medical Sciences and Research Centre, Davangere-577005, Karnataka, INDIA
2
Department of Pharmacology, S. S. Institute of Medical Sciences and Research Centre, Davangere-577005, Karnataka, INDIA
3
Department of Paediatrics, S. S. Institute of Medical Sciences and Research Centre, Davangere-577005, Karnataka, INDIA

ABSTRACT
Septicemia is the leading cause of morbidity and mortality in neonates. In this study, 1647 consecutive neonates suspected of having
septicemia from various neonatal intensive care unit hospitals in central Karnataka were investigated for isolation of microorganisms.
Two samples of blood were collected aseptically for isolating the etiology. The cultures were positive in 877 (53.2%) cases for aerobic
bacteria and 96 (10.9%) for Candida species. Among Candida species, C.tropicalis- 39(40.6%) was the predominant organism
followed by C.albicans 22(22.4%) and C.guillermondi 17(17.7%). The standard macrobroth dilution method was carried out to
determine the minimum inhibitory concentration (MIC); C.krusei ATCC 6258 standard strain was included for quality control
purpose. 4(25.0%) stains of C.albicans were resistant to amphotericin-B, 5-fluorocytosine respectively and 2(12.5%) to fluconazole.
High-level resistance to fluconazole, ketoconozole and 5-fluorocytosine was observed in C.krusei.

© 2010 IJRSR. All rights reserved.

Key words: Neonatal septicemia, Candida species, Antifungal agents
INTRODUCTION
Neonatal mortality rate is one of the indicators measuring
the health status of a nation. There could be various
reasons for neonatal mortality but septicemia continues to
be a major cause of neonatal mortality and morbidity
worldwide1. Neonatal septicemia refers to infection of
infants during first month of life,2. According to National
Neonatal Perinatal Database (NNPD) 2002, the incidence
of neonatal septicemia has been reported to be 30/1000
live births in India2-4. A very wide spectrum of organisms
has been described to cause neonatal septicemia, and most
predominant organisms are Klebsiella pneumoniae,
Staphylococcus aureus, Group B and D Streptococci,
Escherichia coli, Pseudomonas aeruginosa, Coagulase
negative Staphylococci and Candida species2-5. Candida
species are the most common fungal pathogens isolated
from blood cultures of neonates5,6. Numbers of risk
factors are associated with the development of neonatal
candidaemia, such as very low birth weight, prematurity,
prolonged antibiotic therapy, prolonged use of fat
emulsion in total parenteral nutrition and use of artificial
ventilation7-9. Though majority of nosocomially acquired
Candidaemia is due to C.albicans10, the emergence of
non- albicans Candida species like C.tropicalis,
C.glabroata, C.krusei and C.parapsilosis as predominant
species causing blood stream infection in premature
newborns in neonatal intensive care units11-13 are also
reported.
* Corresponding author: +91 9964402525
E-mail address: vinodmicro@yahoo.com
Drug resistance is the major cause of increase in
morbidity and mortality in neonates. Among the different
antifungal agents, resistance to the polyenes compounds
has remained an uncommon problem, but resistance to
flucytosine and azoles now appears to be increasing in
importance, especially after the wide spread use of
fluconazole for extended period12,13. The aim of the study
is to determine the incidence and antifungal susceptibility
of Candida species in neonatal septicemia.
MATERIAL AND METHODS:
A total of 1647 clinically diagnosed cases of septicemia
were studied prospectively for over the period of 5 years
Neonatal sepsis were suspected when any of the signs and
systems or predisposing factors such as reduced activity,
fever, refusal of feed, seizures, prolonged jaundice, birth
asphyxia, umbilical sepsis, prematurity, abdominal
distensions and history of premature rupture of membrane
were noted in the newborns. Two samples of blood were
collected from each case using aseptic precautions. About
2 ml of blood was added immediately into 20 ml of brain
heart infusion broth with 0.025% sodium polyethol
sulphonate as anticoagulant. The bottles were incubated
for seven days and subcultures were done appropriately2.
Candida species were isolated from 96 cases, which were
further confirmed by germ-tube test, urease test, reduction
of tetrazolium media, different conidial arrangement on
corn meal agar, sugar assimilation and fermentation test14-
16
.
 International Journal of Recent Scientific Research, Vol. 2, Issue, 5, pp. XXX -XXX, June, 2011
The antifungal susceptibility for yeasts was tested using
broth macrodilution technique recommended by National
Committee for Clinical Laboratory Standards 16. Media
used was RPMI-1640 with glutamine, buffered to pH 7.0
at 250C with morpholinopropane sulphonic acid buffer
(final concentration 0.165 mol/1). Serial two-fold dilution
of antifungal agents was prepared (0.1ml of various
antifungal concentrations in 12 x 75 mm tubes). For
inoculum preparation, 5 colonies of  1mm diameter from
24 hours old cultures of Candida species were suspended
in 5ml of normal saline and mixed well for 15sec. The
cell density was spectrophotometrically adjusted to 0.5
McFarland standards.
This procedure yielded a stock suspension of 1.5 x 106
cells / ml. A working suspension was made by a 1:100
dilution followed by 1:20 dilution of the stock suspension
with RPMI 1640 broth medium which results in 5.0 x 102
to 2.5 x 103 cells /ml. A volume of 0.9ml of inoculum was
added to each tube. The tubes were incubated at 350C for
48 hours. Plate counts were performed on representative
inocula to check viability of Candida. The lowest
concentration of an antifungal that substantially inhibited
growth of the organism was detected visually. The end
point of amphotericin B was easily defined but in case of
5-fluorocytosine and azoles, minimum 80 percent
inhibition was taken as the end point standard (standard
was prepared by diluting 0.2ml of drug free control
growth with 0.8ml of media).C.krusei ATTC 6258
standard strain was included for quality control purpose.
The range of concentration tested for fluconazole was
0.125-64g/mL and for amphotericin B was 0.03-
16g/mL.
RESULT
Out of 1647 clinical suspected septicemia, 96 infants had
candidaemia. The predisposing factors that possibly led to
neonatal candidaemia are depicted in table-1. All the 96
infants received systemic broad spectrum antibiotic
therapy, 91(94.7%) were low birth weight and 72(75%)
were preterm babies [Table-1]. The percent prevalence of
Candida species in neonatal septicemia is 10.9%, with
Candida tropicalis 39(40.6%) being the predominant
isolate followed by C.albicans 22(22.4%), C.guillermondi
17(17.7%), C.krusei 14(14.5%) and C.parapsilosis
04(4%) [fig-1]
Table 1 Clinical features in neonates with systemic
candidiasis (n=96)
Clinical features Incidence
No. (%)
Antibiotic therapy 96(100)
Low birth weight 91(94.7)
Preterm babies 72(75.0)
Respiratory distress syndrome 62(64.5)
Bacterial sepsis 45(45.8)
Ventilator therapy 34(35.4)
Skin lesions 16(16.6)
Central line 08(8.3)
Neurological features 05(5.2)
45 39(40.6%)
40
se 35
t 30
al
22(22.4%)
os 25
I
f 20 17(17.7%)
o 14(14.5%)
re 15
b 10 4(4 .01%)
m
u 5
N
0
Candida species
FIGURE 1 Prevalence of Candida Species In Neonatal Septicemia
The test organisms were interpreted depending on
minimum inhibitory concentration (MIC) by broth
dilution against different antifungal agents. In case of 5-
Fluorocytosine,  4g/ml (Sensitive), 4 – 16 intermediate
and  32g/ml (Resistant); for amphotericin-B, 1g/ml
(Sensitive), and 1g/ml (Resistant); for Ketoconazole, 
0.125g/ml (Sensitive), and  1g/ml (Resistant) and for
Fluconazole, 8g/ml (Sensitive), 16-32g/ml
(intermediate), 64g/ml (Resistant) In-vitro antifungal
susceptibility of 96 Candida species for antifungal agents
like amphotericin-B, fluconazole, ketoconazole and 5-
fluorocytosine is depicted in table-2 & table-3. Among
Candida albicans, 4(18.2%) strains were resistant to
amphotericin-B, 4(18.2%) to 5-fluorocytosine, 2(9.7%) to
fluconazole and none of the isolates were resistant to
ketoconazole
High-level resistance was seen in Candida krusei to
fluconazole 6(42.9%), followed by 4(28.6%) to
amphotericin-B, 3(21.4%) to ketoconazole and 2 (14.3%)
to 5-fluorocytosine. Resistance to Candida tropicalis was
observed in 4(10.3%), 6(15.4%), 4(10.3%) and 4(10.3%)
to amphotericin-B, ketoconazole, 5-fluorocytosine and
fluconazole respectively. Among C.guillermondi,
3(17.6%) were resistant to amphotericin-B, 2(11.8%) to
fluconazole, 1(5.9%) to 5-FC and none of the isolates
were resistant to ketoconazole. In Candida parapsilosis, 2
isolates of each were resistant to amphotericin-B and
fluconazole.
DISCUSSION
Candida species are normally found on skin and mucous
membrane of healthy individuals and therefore
candidaemia is generally an endogenous infection 18-20.
Candidaemia is the most frequently encountered fungal
infection especially in those babies with the predisposing
factors like prolonged antibiotic therapy, intravascular
catheterization, endotracheal intubation, parenteral
nutrition and artificial ventilation7-9. In the present study,
the factors that possibly led to candidaemia were broad-
spectrum antibiotic therapy, low birth weight and
prematurity. Parenteral nutrition was not common as the
babies were breast-fed.
 International Journal of Recent Scientific Research, Vol. 2, Issue, 5, pp. XXX -XXX, June, 2011

Table 2 Minimum inhibitory concentration of candida species by broth dilution method for amphotericin- b and
fluconazole

Amphotericin- B 1g/mL (Sensitive), 1g/mL (Resistant)

Fluconazole  8g/mL (Sensitive), 16-32g/mL (Dose depedendent Susceptibility),
64g/mL (Resistant)

Table 3 Minimum inhibitory concentration of candida species by broth dilution method for
5-fluorocytosine and ketoconazole

The candidaemia in neonates is most commonly due to
C.albicans. The present study shows non- albicans
Candida has emerged as important nosocomial pathogens.
Over all isolation of non- albicans Candida species are
higher (76.5%) as compared to C.albicans (23.5%). Thus
it is interesting to speculate that increased use of
fluconazole in our hospitals may have contributed partly
to shift in the incidence of candidaemia due to non-
albicans Candida species.
Since the dawn of the antimicrobial drug era, resistance
has shadowed the success of infectious disease therapy. In
present decade, on one hand there are new, more
efficacious antifungal agents in the field, on other hand
there are increase in number of treatment failure cases.
While testing the susceptibility of Candida species to
fluconazole, it is observed that significantly higher
number of non- albicans Candida species (17.3%) were
resistant (MIC  64g/mL), as compared to 12.5% of
C.albicans (NCCLS MIC interpretive break point
concentration). Although resistance to amphotericin B is
considered rare in Candida species, increased in MIC had
been observed in our study. 4(25.0%) C.albicans were
resistant (MIC > 1g/mL) compared to 7.7% of non-
albicans Candida species to amphotericin-B. Increasing
incidence of Candida sps from neonatal septicemia and
higher MIC values of non- albicans Candida sps to
antifungal agents appear to be major challenge in NICU.
This resistance pattern and MIC values of Candida sps
observed in the present study are comparable with other
reports 12,13,18-20. In conclusion the present study highlights
Candida species as important pathogen in neonatal blood
stream infections. If our findings are confirmed in future
studies elsewhere, we predict that clinicians will
encounter increasing episodes of candidaemia due to non-
albicans Candida species with the risk of increased
antifungal resistance.
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