Lab diagnosis of Dengue

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 : Spherical 40-50 nm Diameter Inner core ( C) Lipid envelope with glycoprotein polymers (E) Membrane (M) 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.

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

5. KINETICS OF DENGUE VIRUS REPLICATION AND HOST RESPONSE

6. 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)

7. 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

8. 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

9. 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 1 st week of illness , increasing slowly thereafter and is still detectable after several months and even for life.

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

11. 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.

12. 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.

13. 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.

14. 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.

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

16. 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

17. 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 Antigen detection by antibody staining Cytopathic effect Plaque formation Suckling mice (intracranial) S & S of encephalitis Antigen detection by antibody staining Culture.

18. 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. 

19. 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.

20. 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.

21.   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. 

22. 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%

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

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

25. 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.

26. 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

27. 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.

28. 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.

29. 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.

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

31. 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.

32. 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

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