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Dengue fever (DF) is an acute febrile viral disease presenting with headache,
bone or joint and muscular pains, rash and leukopenia.
Dengue haemorrhagic fever (DHF) is characterized by
high fever, haemorrhagic phenomenon often with hepatomegaly and in severe cases signs of circulatory
failure. Such patients may develop hypovolaemic
shock resulting from plasma leakage which is then called as dengue shock
syndrome (DSS).
Epidemics
of dengue fever or dengue-like disease are reported from many countries of
the South-East Asia Region. The peripheral/intermediate laboratories play a
vital role in confirming the diagnosis of the disease by performing some of
the tests at their own level and by sending the clinical specimens to the
reference laboratories for confirmation of the diagnosis.
 Laboratory
diagnosis
Laboratory diagnosis is based upon two main methods.
1. Detection
of virus
This is done by isolation of virus or demonstration
of antigen. These are definitive diagnostic tests. However, only a few
laboratories perform these tests. This requires specific infrastructure and
skilled manpower.
2. Serodiagnosis
This is performed by detection of dengue specific
antibodies. The serological tests are simple, rapid and most laboratories can
perform these. However, accurate diagnosis cannot be made with these as cross
reactions with other flaviviruses occur.
 Collection
and transportation of specimens for serodiagnosis
Usually three blood specimens are collected which help
in proper interpretation of results
a) S1
: At admission
b) S2
: 10-14 days after S1
c) S3
: 14-21 days after onset of disease
Blood specimens should be collected aseptically in
tubes/vials. Filter paper method can also be used for collection and transportation
of the specimen (see Chapter 2).
Transportation
If delay is anticipated, separate the serum and
transport it to the laboratory on wet-ice at the earliest. Do not send frozen
whole blood. When the specimen is collected on filter paper the same may be
sent wrapped in a polythene bag, at room temperature, preventing it from
exposure to moisture during transportation.
Specimen
suitable for culture
In outbreak situations, the intermediate laboratories
may be required to collect and transport specimens to referral laboratories
for culture or demonstration of viral antigen. The important points in this
exercise are:
 Collection of specimen early in the course of
disease usually within 5 days of onset of fever.
Prompt delivery to the laboratory.
Dengue virus is heat labile,
specimens awaiting transportation should be kept in refrigerator or packed in
wet ice.
The specimens used for isolation of the virus from the
suspected patient are:
Serum/plasma
Washed leucocytes
Cerebrospinal fluid
From
autopsy
Homogenized liver, lung, spleen, lymph node,
CSF, pleural fluid.
The virus can also be isolated from vector
mosquito.
Information accompanying the specimen
Following information must accompany the specimens:
Name, age, sex of the patient
Name of father/mother
Complete address
Name of the hospital sending the specimen
Registration number of the patient
Date of onset of illness
Date of hospitalization
Date of collection of specimen and number of
specimen
Brief clinical history
Provisional diagnosis
Recognizing
cases of DF/DHF/DSS
An outbreak of DF/DHF in the community should be
suspected when:
Children are suffering from undiagnosed fever
of 2-7 days duration.
Patients have petechiae,
bleeding from nose or gums, melaena or haematemesis.
Patients remain ill despite a drop in
temperature and develop signs of shock.
Unexplained death due to shock, with or without haemorrhage.
Rapid Immunochromatographic test for detection of IgM and IgG antibody
This test is commercially available and can detect both IgG and IgM antibodies against
dengue with the same kit. Perform the test following the instructions given
by the manufacturer along with the diagnostic kit.
Serological
tests done in reference laboratory
Following serological tests are done in reference laboratories:
MAC-ELISA
This measures IgM antibodies
in the serum. It is very efficient test method and is valuable for
laboratories that perform a high volume of testing.
Haemagglutination-Inhibition
test
This is a simple, sensitive and reproducible test which
can be easily done and is also not very expensive. It, however, requires
pretreatment to remove nonspecific inhibitors and paired sera are required
for interpretation.
Neutralization
test
The most sensitive and specific method is the serum
dilution versus constant plaque reduction test.
Dot-blot
immunoassay
This is a commercially available kit for detection of
dengue antibody.
Reporting
of results
Detection of IgM
antibody Positive
A four fold rise in paired sera for HI
antibody Positive
collected 10 days apart
A very high titre
(>1280) in single serum sampleStrongly
suggestive
No change in serum HI antibody in paired
sera Negative
collected 7-10 days apart
No IgM antibody in
paired serum samples Negative
Positive culture/antigen detection Positive
Quality assurance
Observe internal quality control methods
covering collection, transportation, equipment, reagents and training of
technical staff.
Laboratory should participate in a quality
assurance programme for dengue serology.
Biosafety
Safe laboratory techniques as outlined in
Chapter 8 must be practised.
Use of personal protective equipment,
appropriate methods for collection and transportation, proper procedures for
serum separation and decontamination and disposal of potentially infectious
materials be strictly adhered to.
Referral
The specimens are to be sent to the reference
laboratories for:
Confirmation of diagnosis
Isolation and characterization of virus
In outbreak situations
Part of Quality Assurance Programme
Further
reading
1. Dengue haemorrhagic
fever: Diagnosis, treatment, prevention and control. WHO, Geneva,
2nd Ed, 1997.
2. Viral haemorrhagic
fevers. Report of a WHO Expert Committee, WHO Technical Report Series No 721,
1985.
3. Lam SK Dengue haemorrhagic
fever 6:39,1995.
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