DIAGNOSIS OF DENGUE

Dr.M.sravana durga ,
1st year junior resident
MD paediatrics
 DIAGNOSIS OF DENGUE

IN EARLY STAGE AT THE END OF

ACUTE PHASE
1.VIRUS ISOLATION

2. NUCLEIC ACID SEROLOGICAL METHODS

DETECTION

3.ANTIGEN DETECTION
 ANTIBODY RESPONSE
DURING PRIMARY DENGUE DURING SECONDARY DENGUE
INFECTION INFECTION

slow increase of specific antibodies raise rapidly and react broadly

IgM antibodies are 1st to appear after IgM are significantly lower and
3-5 days and declines over 2-3 undetectable in some cases
months

IgG detectable in low titers at end of Dominant is IgG detectable in high

1st week and may persist for life levels even in acute phase
IgG primary
 Specimen collection

• Acute phase specimen s1

specimen as soon as possible after onset of illness

• Convalescent phase specimen s2

specimen shortly before discharge-

• Late convalescent phase specimen s3

7-21 days after the acute serum was drawn
 Collection, storage and shipment requirements of specimens

Specimens Time of Clot Storage Shipment

type collection retraction

Acute phase 0-5 days after 2-6 hours, 4 Serum-70 Dry ice
blood onset

Recovery 14-21 days 2-24 hours, Serum-20 Frozen or

(convalescent) after onset ambient ambient
phase blood
(S2+S3)

Tissue As soon as 70 C or in Dry ice or

possible formalin ambient
Diagnostic methods Specimen Time of collection Time to
after onset of results
symptoms

Viral isolation and Whole blood, serum, 1-5 days 1-2 wks
serotype tissues
identification

Nucleic acid Tissues, whole 1-5 days 1 or 2 days

detection blood, serum
,plasma

Antigen detection Serum 1-6 days 1 day

Tissue for immuno- NA >1 day
chemistry

IgM ELISA Serum ,plasma, After 5 days 1-2 days

whole blood
IgM rapid test 30 min

IgG(paired sera) by Serum, plasma, Acute sera, 1-5 days, 7 days or more
ELISA ,HI or whole blood convalscent after 15
neutralization test days
 CURRENT DENGUE DIAGNOSTIC
METHODS
1. Virus Isolation
2. Nucleic acid detection
3. Detection of antigens
4. Serological tests
5. Haematological tests
 Virus isolation
• Specimens should be collected early in course of
illness from – serum
plasma
peripheral blood mononuclear cells
tissues collected at autopsy
• Because dengue virus is heat labile, for storage upto
24 hrs - +4 to +8 degree C
• longer storage - -70 degree C
• Cell culture is the most widely used method
• It generally takes 1-2 weeks
 Nucleic acid detection
• They offer better sensitivity compared to virus isolation

• All nucleic acid amplification tests involve

1.nucleic acid extraction and purification
2.amplification
3.detection

• Different methods – RT PCR

real time RT PCR(quantification)
isothermal amplification methods (NASBA)
 DETECTION OF ANTIGENS
• By using ELISA and dot blot essays directed to

1. envelope \membrane (E\M) antigen

2. NS1 antigen

• High concentrations of these antigens detected in

both primary and secondary infections upto 9 days
 Serological tests
• Includes
1.MAC ELISA
2.IgG ELISA
3.IgM\ IgG ratio
4.IgA
5.Haemagglutination inhibition test .
MAC ELISA
• Total IgM in patients sera is captured by anti micro chain
specific antibodies coated onto a microplate

dengue specific antigens (1 to 4) are bound to captured anti

dengue IgM antibodies

detected by monoclonal and polyclonal dengue antibodies

directly or indirectly conjugated with an enzyme

Non colored substance to colored .

 Ig G ELISA
• Used for detection of recent or past dengue
infection.

• IgG antibodies are lifelong but a fourfold or greater

increase in IgG antibodies in acute and convalscent
paired sera – recent infection.

• Uses the same antigen as like MAC ELISA.

• Lacks specificity within the flavi virus.

 IgM \IgG ratio
• Can distinguish between primary and secondary
infections .
• If Ig M OD ratio > 1.2 – PRIMARY
Ig G
<1.2 – SECONDARY
 IgA
• Positive detection for serum anti dengue IgA occurs
one day after that for IgM
• Peaks around day 8 after onset of fever and
decreases rapidly
• Cannot differentiate between primary and secondary
infections
 Haemagglutination inhibition test
• Principle:
ability of dengue antigens to agglutinate red
blood cells of ganders or trypsinised human O rbc

Anti dengue antibodies in sera can inhibit this

agglutination and the potency of inhibition –
HI(haemagglutination inhibition) TEST
• The essay doesn’t discriminate between infections by
closely related flavi virus nor between
immunoglobulin isotypes
• During secondary dengue infections HI antibody
titres raise rapidly usually exceeding 1:1280
Interpretation of Dengue diagnostic tests (adopted from Dengue and
Control [DENCO] Study)

Highly Suggestive Confirmed

One of the following One of the following

1. IgM + in a single serum sample 1. PCR +
2. IgG + in a single sample with a 2. Virus culture +
Hl titre of 1280 or greater 3. IgM seroconversion in paired
sera
4. IgG seroconversion in paired
sera or IgG titre increase in
paired sera
 Haematological tests
• A drop in the platelet count < 100000 per microlitre
– usually observed between day 3-8 following the
onset of illness .

• Hematocrit >20% or more

• Additional laboratory investigations :
cbc
blood glucose
blood gas analysis lactate if available
serum electrolytes and BUN ,creatinine
serum calcium
liver function tests
Coagulation profile
Group and match for fresh whole blood or fresh packed
Cardiac enzymes or ECG
serum amylase and ultrasound
any other test if indicated