Group 3:

JIMENEZ, Jenny B.
PINEDA, John Patrick
LAXA, Kate Charossel
Tourniquet Test (Rumpel Leads Test)

 Inflate the blood pressure cuff on the upper arm

to a point midway between on the systolic and
diastolic pressure for 5 minutes.
 Release cuff and make an imaginary 2.5 cm2 or
1 inch just below the cuff, at the antecubital
fossa.
 Count the number of petechiae inside the box
 A test is positive (+) when 20 or more petechiae
per 2.5 cm2 or 1 inch per square are observed.
Confirmed
diagnosis
A. Culture of the virus

 Culture is positive within first 5 days

(sensitivity <50%); can also culture
from liver at autopsy.
B. Detection of the virus in tissue, serum or CSF by: 
 Haemeagglutination Inhibition test
> the gold standard for serological diagnosis.
However, because it is labour intensive and requires
paired samples for interpretation, this test is now
being used mainly for research purposes to
differentiate between primary and secondary
dengue infections.
 Immunofluorescence assay 
Procedures:
 Direct:
>Add fluorescein-labeled antibody to patient
tissue
>Wash and examine under fluorescent
microscope.
 Indirect:
>Patient serum is added to tissue containing
known antigen.
>Wash and examine under fluorescent
microscope.
 IgM capture Enzyme-Linked
Immunosorbent assay (ELISA)
 most widely used serological test. The antibody
titre is significantly higher in primary infections
compared to secondary infections. Once the IgM
is detectable, it rises quickly and peaks at about
2 weeks after the onset of symptoms and it
wanes to undetectable levels by 60 days.
 However in some patients, it may persist for
more than 90 days. A positive result thus has to
be interpreted and correlated cautiously with
the clinical picture. If the dengue IgM test is the
only available diagnostic test in the hospital,
then establishing a negative early in the illness,
and demonstrating a positive serology later will
be essential to exclude false negative results.
Procedures: “Sandwich Technique”

 Monoclonal or polyclonal antibody adsorbed

on solid surface (bead or microtiter plate)
 Patient serum added; if antigen is present in
the serum, it binds to antibody coated bead or
plate.
 Excess labeled antibody (antibody conjugate)
added; forms antigen-antibody labeled
antibody “sandwich” (conjugate directed to
another epitope of antigen being tested) 
Continuation of sandwich technique….

 Substrate added, incubate and

read absorbance.
 Washing required between each
step.
 Direct relationship between
absorbance and antigen
concentration.
 Indirect IgG ELISA test
 In primary and secondary dengue
infection, dengue IgG was detected
in 100% of patients after day 7 of
onset of fever.
 Therefore, dengue IgG is
recommended if dengue IgM is still
negative after 7 days in secondary
dengue.
Recommendation
 In order to establish serological
confirmation of dengue illness a
seroconversion of dengue IgM needs to be
demonstrated. Therefore, a dengue IgM
should be taken as soon as the disease is
suspected.
 Dengue IgM is usually positive after 5-7
days of illness. Therefore, a negative IgM
taken before 5-7 of illness does not
exclude current dengue infection.
 If the dengue IgM is negative before day 7,
a repeat sample must be taken in the
recovery phase to confirm the diagnosis.
 IgM Dot Enzyme Immunoassay
 A dengue rapid test that is a
commercially available kit for detection
of dengue antibody.

* The yield of rapid tests was shown to be higher when samples

were collected later in the convalescent phase of infection,
with good specificity and could be used when ELISA facilities
were not available. But the result had to be interpreted in the
clinical context because of false positive and negative results.
It is recommended that the dengue IgM Capture ELISA test
could be done after a rapid test, to confirm the status.
Serological tests for dengue have been shown to cross react
with other flavivirus, non-flavivirus, previous vaccinations and
connective tissue diseases.
C. Detection of genomic sequence of virus by
RT-PCR (sensitivity >90% early;<10% in 7
days) still a research tool.

 Reverse Transcriptase- Polymerase

Chain Reaction (RT-PCR)
 It is useful for the diagnosis of dengue
infection in the early phase (<5 days of
illness). It was shown to have a sensitivity of
100% in the first 5 days of disease, but
reduced to about 70% by day 6, following
the disappearance of the viraemia.
 An additional advantage of RT-PCR is the
ability to determine dengue serotypes.

Limitations of RT-PCR:
 It is only available in few centers with
facilities and trained personnel
 Expensive
 It requires special storage temperatures
and short transportation, time between
collection and extraction.
*In view of these limitations, the use of RT-PCR should only
be considered for in-patients who present with diagnostic
challenges in the early phase of illnesss.
D. WB and neutralization tests can be used to
confirm acute infection if necessary.

 Neutralization test
 the most sensitive and specific method
versus constant plaque reduction test.
 Similar in principle and result of
interpretation of Hemeagglutination
inhibition.
Procedures: “Neutralization Reactions”

 Antibody-binding > neutralization (antibody

neutralizes toxin)
 After binding, antibody is not available to react in
indicator system.
 Results:
>No agglutination or No hemolysis = positive
reaction
>Agglutination or hemolysis = negative reaction
(antibody not bound in original reaction and is
available to react
with indicator cells)
*Generally, positive control samples
used in inhibition or neutralization
tests show no reaction and negative
control samples show a reaction
(opposite of results in direct
agglutination testing).
 WBC decreased (2000-5000/uL) with
toxic granulation of leukocytes and
neutropenia; may have marked
atypical lymphocytes.
E. Virus isolation
 the most definitive test for dengue
infection. It can only be performed in the
lab equipped with tissue culture and other
virus isolation facilities. It is useful only at
the early phase of the illness. Generally,
blood should be collected before day 5 of
illness; e.g. before the formation of
neutralizing antibodies.
Virus isolation…
 During the febrile illness, dengue virus can
be isolated from serum, plasma and
leucocytes. It can also be isolated from post
mortem specimens.
 The monoclonal antibody
immunofluorescence test is the method of
choice for identification of dengue virus. It
may take up to two weeks to complete the
test and it is expensive.
F. Non-Structural Protein-1 (NS1 Antigen)
 a highly conserved glycoprotein that seems
to be essential for virus viability.
 Secretion of the NS1 protein is a hallmark of
flavivirus infecting mammalian cells and can
be found in dengue infection.
 This antigen is present in high concentrations
in the sera of dengue infected patients
during the early phase of the disease.
 The detection rate is much better in acute
sera of primary infection (75%-97.3%) when
compared to the acute sera of secondary
infection (60-70%). The sensitivity of NS1
antigen detection drops from day 4-5 of
illness onwards and usually becomes
undetectable in the convalescence phase.
Brazilian Journal of Infectious
Diseases
Dengue hemorrhagic fever and acute hepatitis: a case report
Dengue fever is the world's most important viral hemorrhagic fever
disease, the most geographically wide-spread of the arthropod-born
viruses, and it causes a wide clinical spectrum of disease. We report
a case of dengue hemorrhagic fever complicated by acute hepatitis.
The initial picture of classical dengue fever was followed by painful
liver enlargement, vomiting, hematemesis, epistaxis and diarrhea.
Severe liver injury was detected by laboratory investigation,
according to a syndromic surveillance protocol, expressed in a self-
limiting pattern and the patient had a complete recovery. The
serological tests for hepatitis and yellow fever viruses were
negative. MAC-ELISA for dengue was positive.