various methods of diagnosing dengue.

1. Introduction
• Dengue is the most rapidly spreading
mosquito borne viral disease in the
world.
• An estimated 50 million dengue
infections occur annually.
• Approximately 2.5 billion people live in
dengue endemic countries.

2. DENGUE VIRUS
• Arthropod – borne: Arbovirus
• Genus: Flavivirus.
• Family: Flavivirdae.

3. Virions:
1. Spherical
2. 40-50 nm Diameter
3. Inner core ( C)
4. Lipid envelope with glycoprotein polymers (E)
5. Membrane (M)
6. Seven Non Structural Proteins : NS1, NS2a, NS2b,
NS3, NS4a, NS4b, NS5.
• Genome: infectious, 11kb ssRNA, +ve polarity
• Based on the analysis of E protein : 4 Serotypes –
DENV1, DENV2, DENV3, DEV4.
• Virus is inactivated by heat and disinfectant, containing
detergent or lipid solvent.

5. Laboratory diagnosis
• Two Basic Methods :
1. Detection Of Dengue Virus
• Isolation Of Virus By Culture
• Detection Of Viral Nucleic Acid
• Detection Of Viral Antigens
2. Detection of Anti Dengue Antibodies

7. KINETICS OF DENGUE VIRUS
REPLICATION AND HOST
RESPONSE

8. ONSET OF ILLNESS
4 to 5 days
Virus detected in serum, plasma,
Circulating blood cells and tissues
Virus isolation
Nucleic acid detection
Antigen detection (NS1 Ag)

9. After the acute phase…..?
• Antibody detection
• Antibody response to infection differs
according to the immune status of the host.
• First antibody to appear – IgM
• Secondary antibody – IgG

10. Primary Dengue
Dengue infection in a person not previously infected
with flavivirus nor immunized with a flavivirus vaccine.
primary antibody response
(IgM – slow increase)
Detectable in
• 50% of patients – Day 3 to 5
• 80% of patients – Day 5
• 99% of patients – Day 10

11. • IgM levels peak at 2 weeks after the onset of
symptoms
• Declines to undetectable levels over 2 to 3
months.
• Anti dengue serum IgG is detectable at low
titres at the end of 1st week of illness ,
increasing slowly thereafter and is still
detectable after several months and even for
life.

12. • The physiological definition of a primary
infection is a higher molar fraction of IgM and
a lower fraction of IgG.

13. Secondary Dengue
• Previous infection with a dengue virus/
vaccination with non dengue flavivirus vaccine.
Antibody titres rise rapidly ( IgG)
• IgG detectable at high levels in the acute stage
appearing before or simultaneously with IgM,
peaks at 2 weeks after the onset of symptoms
and declines over 3-6 months.

14. • Early convalescent stage IgM levels are
significantly lower in secondary infections
than in primary.
• The physiological definition of a secondary
case is characterized by a lower molar fraction
of IgM and higher molar fraction of IgG.
• To distinguish primary and secondary –
IgM/IgG antibody ratios are commonly used.

16. Collection And Handling Of Specimen
• Acute Serum S1 – collection of specimen as soon
as possible after onset of illness, hospital
admission or attendance at a clinic.
• Convalescent Seum S2 – shortly before discharge
or in the event of fatality, at the time of death.
• Late Convalescent Serum S3 – 7-21 days after
collection of acute seum.
• Optimal interval between S1 and S2/S3 – 10 days
• Blood collection – Tubes/ vials/ high quality
absorbent paper.

17. Specimen Collection Procedures
• 2-5 ml of aseptically collected venous blood
• Use tubes or vials with screw-caps.
• If a specimen cannot be analyzed or shipped
within 24 hours , serum should be seperated and
stored frozen.
• Ship specimens for serology or culture on ice.
• Minimum Volume – 0.1 ml of undiluted
specimen.
• Adequate specimen- 0.3 to 2 ml.

18. • Gold Standard for most viral infections.
• Sample:
– Serum
– Plasma
– Anticoagulated whole blood (leukocyte culture)
– CSF
– Tissues from dead patients (autopsy)
– Mosquito
Virus Isolation

19. Limitations:
•During Viraemia only (early 5 days)
•Slow results (Days) vs. PCR (Hours)
•Expensive to maintain the technique
•Strict storage and transportation needs
Heat-labile.
Refrigeration(4-8 degree C) or wet ice for 24 hours
Frozen at - 70 deg C, or more, for longer storage
Never ever freeze in ordinary Freezer (-20C).
Virus Isolation

20. • Inoculation of samples into:
– Mosquitoes ( Adults/ Larvae)
• Antigen detection in head squash by immunofluoresence.
– Cell culture lines
• Vertebrate cell-line: Vero, LLC-MK2
• Insect cell-line: C6/36,AP61
1. Antigen detection by antibody staining
2. Cytopathic effect
3. Plaque formation
– Suckling mice (intracranial)
• S & S of encephalitis
• Antigen detection by antibody staining
• Culture.

21. GENOME DETECTION
RT-PCR :
• They offer better sensitivity compared to virus
isolation with a much more rapid turnaround
time. In situ RT-PCR offers the ability to detect
dengue RNA in paraffin embedded tissues.
• Three basic steps:
nucleic acid extraction and purification
 amplification of the nucleic acid
 detection and characterization of the amplified
product.

22. • Many laboratories utilize a nested RT-PCR assay, using
universal dengue primers targeting the C/prM region
of the genome for an initial reverse transcription and
amplification step, followed by a nested PCR
amplification that is serotype-specific.
• A combination of the four serotype-specific
oligonucleotide primers in a single reaction tube (one-
step multiplex RT-PCR) is an interesting alternative to
the nested RT-PCR.
• The products of these reactions are separated by
electrophoresis on an agarose gel, and the
amplification products are visualized as bands of
different molecular weights in the agarose gel using
ethidium bromide dye, and compared with standard
molecular weight markers. In this assay design, dengue
serotypes are identified by the size of their bands.

23. REAL TIME RT-PCR
• The real-time RT-PCR assay is a one step assay
system used to quantitate viral RNA and using
primer pairs and probes that are specific to
each dengue serotype. The use of a
fluorescent probe enables the detection of the
reaction products in real time, in a specialized
PCR machine, without the need for
electrophoresis.

24. Isothermal amplification methods
• NASBA (nucleic acid sequence based
amplification) assay is an isothermal RNA-specific
amplification assay that does not require thermal
cycling instrumentation. The initial stage is a
reverse transcription in which the single-stranded
RNA target is copied into a double-stranded DNA
molecule that serves as a template for RNA
transcription. Detection of the amplified RNA is
accomplished either by
electrochemiluminescence or in real-time with
fluorescent-labelled molecular beacon probes.

25. Antigen detection
• Sample – Serum
• NS1 and E/M antigens- ELISA and Dot Blot Assay
• Upto 9 days after onset of illness in both primary
and secondary infections.
• NS1 antigen is produced by all flaviviruses and
secreted from mammalian cells.
• Commercial kits gives rapid results, Helpful in
field settings but do not differentiate between
dengue serotypes.
• NS1 antigen ELISA – sensitivity 86% and
specificity 100%

26. Antigen Detection
1. Methods -
• Fluorescent antibody
• Immunoperoxidase
• Avin-biotin enzyme assay
2. Tissues -
• Acetone Fixed Leucocytes
• Snap-Frozen tissues
• Formalin Fixed tissues

27. SEROLOGICAL DIAGNOSIS
MAC – ELISA (IgM antibody-capture Enzyme-
Linked Immunosorbent Assay)
a) in primary and secondary dengue can measure
dengue specific IgM. IgM elicited by dengue can
be differentiated from other flavivirus elicited
IgMs.
b) MAC-ELISA has good sensitivity and specificity
but only when used five or more days after the
onset of fever.
c) Cross reactivity occurs with some flaviviruses.

28. • Total IgM in patients' sera is captured by anti-μ
chain specific antibodies (specific to human IgM)
coated onto a microplate. Dengue-specific
antigens, from one to four serotypes (DEN-1, -2, -
3, and -4), are bound to the captured anti-dengue
IgM antibodies and are detected by monoclonal
or polyclonal dengue antibodies directly or
indirectly conjugated with an enzyme that will
transform a non-coloured substrate into coloured
products. The optical density is measured by
spectrophotometer.

30.  The IgG ELISA
• is used for the detection of recent or past dengue infections (if paired
sera are collected within the correct time frame).
 IgM/ IgG ratio
• A dengue virus E/M protein-specific IgM/IgG ratio can be used
to distinguish primary from secondary dengue virus infections.
• Primary infection – greater than 1.2(1/100) or 1.4(1/20)
• Secondary infection – lesser than 1.2 or 1.4

31. HEMAGGLUTINATION- INHIBITION
TEST
• The haemagglutination-inhibition (HI) test is based on the
ability of dengue antigens to agglutinate red blood cells (RBC),
Anti-dengue antibodies in sera can inhibit this agglutination
and the potency of this inhibition is measured in an HI test.
• It has the ability to differentiate between Primary and
Secondary infections.
• Optimally the HI test requires paired sera obtained upon
hospital admission (acute) and discharge (convalescent) or
paired sera with an interval of more than seven days.

33. • In positive tests there are fourfold or greater increases in
titre between acute and convalescent sera, with peak titres
always exceeding 1: 2560 in secondary responses, and
generally falling below this ratio in primary responses.
•The assay does not discriminate between infections by
closely related flaviviruses (e.g. between dengue virus and
Japanese encephalitis virus or West Nile virus) nor between
immunoglobulin isotypes.
•Less sensitive than ELISA.

35. RAPID IMMUNOCHROMATOGRAPHY
• Test Kits are available commercially
• Quick/easy/early detection of Dengue ( 5-
30mins).
• Good as a screening test but should not be the
only test used.
• Both IgM and IgG
• Claims to differentiate between primary and
secondary infections.
• Accuracy in testing for IgG is greater than ELISA
whereas accuracy is lower for IgM than ELISA.

36. Other Serological Tests
• Neutralization test
• Complement fixation test
• Dot-blot immunoassay

37. Haemotological tests
• WBC count
• Platelet count
• Haematocrit value
• Platelets and haematocrit values are commonly measured during
the acute stages of dengue infection.
• A drop of the platelet count below 100 000 per μL may be
observed in dengue fever but it is a constant feature of dengue
haemorrhagic fever.
• Thrombocytopaenia is usually observed in the period between day
3 and day 8 following the onset of illness.
• Haemoconcentration, as estimated by an increase in haematocrit of
20% or more compared with convalescent values, is suggestive of
hypovolaemia due to vascular permeability and plasma leakage.

38. Summary
• Upto five days of illness (acute stage) – NS1 antigen
detection
• From three days to 2 months (Depends upon the
immunity) – IgM detection
• After 3 weeks upto life sometimes – IgG detection
• To differentiate primary and secondary infection –
IgM/IgG ratio
• Patient monitoring – Platelet count and haemotocrit
values

39. THANK YOU