1. Instructions for users This slide presentation provides an overview of the diagnostic and laboratory testing aspects of JE. Below many of the slides, there are notes to explain the information in the slide. You should adapt the presentation so it is relevant to your audience in your country. This presentation is very long if used in total. Different sections could be presented by different presenters or in different sessions. Provide examples of the actual materials (e.g., specimen tubes) and include practical sessions when possible. Suggestions are provided.

2. Diagnostic and Laboratory Aspects of Japanese Encephalitis

3. Learning Objectives Participants will: Understand how diagnostics can enhance surveillance for JE. Review different tests that can be done to diagnose JE infection. Revise principles of MAC ELISA testing. Know types of specimens that can be tested. Be familiar with the collection, storage and transport requirements for specimens.

4. Why do we need diagnostics? JE surveillance is based on surveillance for cases of acute encephalitis syndrome (AES). AES has — Many potential agents (e.g., JE, herpes simplex, Ebstein Barr viruses). — Several epidemic agents (e.g., JE, Enterovirus 71, Nipah viruses). There are no specific signs or symptoms that distinguish JE from other causes of AES, so laboratory testing is needed to determine if JE is the cause of illness.

5. How can you use diagnostic information? Allows you to determine the proportion of all encephalitis (AES) cases that are due to JE. — e.g., to estimate the specific disease burden from JE Allows you to determine if any of the encephalitis (AES) cases are due to JE. — e.g., is an outbreak of encephalitis due to JE or another agent?

6. Different countries, different stages, different purposes (1) No previous JE control (e.g., Cambodia) Aim of surveillance — Determine disease burden from JE. Strategy — Monitor AES cases nationwide. — Use diagnostics in certain sentinel sites. — Estimate proportion of all AES cases due to JE.

7. Different countries, different stages, different purposes (2) Achieve control of JE (e.g., China) Aim of surveillance — Monitor progress towards control of disease. — Identify at-risk areas and populations that require further interventions. Strategy — Confirmatory test required for most cases.

8. Laboratory criteria for diagnosis: WHO surveillance guidelines for JE Laboratory confirmation of a JE virus infection includes: 1.Presence of JE virus-specific IgM antibody in a single sample of cerebrospinal fluid (CSF) or serum as detected by an IgM-capture ELISA specifically for JE virus; OR 2.Detection of JE virus antigens in tissue by immunohistochemistry; OR 3.Detection of JE virus genome in serum, plasma, blood, CSF, or tissue by reverse transcriptase PCR or an equally sensitive and specific nucleic acid amplification test; OR 4. Isolation of JE virus in serum, plasma, blood, CSF, or tissue; OR 5.A four-fold or greater rise in JE virus-specific antibody as measured by hemagglutination inhibition (HI) or plaque reduction neutralization assay (PRNT) in acute and convalescent- phase serum samples. The samples should be collected 14 days apart.

9. Options for diagnostic tests in humans Isolation, amplification, or antigen detection? – Viraemia in JE is low and short so the likelihood of a positive result is low. – High technology requirement and expensive. Antibody-based (serology) methods? – High sensitivity (if properly timed samples). – Options include PRNT, ELISA, and HI.

10. Antibody-based (serology) methods Plaque reduction neutralization assay (PRNT) — Least cross-reactivity; most specific. — Up to 14 days to complete test. — Costly — High biosafety level requirements. — Early acute phase specimen less likely to test positive with PRNT than ELISA — Requires acute- & convalescent-phase serum. Haemagglutination Inhibition (HI) — High levels of cross-reactivity with other flaviviruses, low specificity. — Anamnestic response with secondary flavivirus infection.

11. Antibody-based (serology) methods (2) JE-specific IgM capture ELISA — Advantages –Simple. –Sensitivity of IgM detection good as competition by IgG molecules eliminated. — Disadvantages –Some cross-reactivity with other flaviruses. Summary: ELISA is most feasible testing methodology

12. Equipment for performing the ELISA test Pipettes Incubator ELISA reader

13. How does the JE IgM capture enzyme linked immunosorbent assay (ELISA) work?

14. Y Y Y Y Y Y Anti-human IgM Sample containing human IgM * Japanese encephalitis E protein Y Anti-E JEV Mab Reporter molecule

15. Y Y Y Y Y Y * Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y * Y Y Y Y Y Y Y * Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y * Y

16. Type of specimens (1) CSF — Preferred sample whenever possible. SERUM — Most common sample for testing.

17. Type of specimens (2) Serum — Easy to collect. — A JE IgM positive result from the serum of a patient with encephalitis is a good indicator of acute infection (even though there is a problem of some cross- reactivity with other flaviviruses such as dengue virus).

18. Type of specimens (3) CSF — The finding of JE IgM in CSF confirms the diagnosis of JE. — IgM to JE virus rises earlier in CSF than in serum and rises to higher levels in CSF than in serum (2 to 4 times) — Whenever possible when CSF is collected for management purposes, an additional tube should be collected for ELISA testing.

19. Additional tests There is an ongoing dengue outbreak. There is a suspected case in an area not having data supportive of JE transmission. When JE vaccination coverage is very high. Further confirmatory tests (testing for other flaviviruses that circulate in the area) should be carried out if:

20. Timing of specimen collection First specimens (blood and CSF) should be collected on admission to hospital or when patient first seen. A follow up specimen (blood) should be collected at least 10 days after onset (or before discharge or death).

21. Rationale for timing of specimen collection IgM antibody levels rise steadily after onset of encephalitis. The percentage of patients with IgM detectable in serum increases with days after onset.

22. Study: the kinetics of IgM and IgG On admission, only 53% of serum specimens and 68% CSF specimens were positive. Kinetics of IgM and IgG responses to JE virus in human serum and cerebrospinal fluid. Burke DS, Nisalak A, Ussery MA et al Journal of Infectious Diseases 1985. 151: 1093-1099

23. Therefore, it is important to collect a follow up blood specimen at least 10 days after onset (or before discharge or death).

24. Typical course of acute JE infection: presence of virus and viral markers

25. Encephalitis onset Concentration Concentration

26. Infection Encephalitis onset Concentration Concentration Incubation: 5 - 15days JE virus in blood Day 4 - 6 illness - 6 illness

27. Infection Encephalitis onset Concentration Concentration Incubation: 5 - 15d JEV - Blood D4– D6 illness D14– D21 illness JEV - CNS & tissue

28. Infection Encephalitis onset Concentration Concentration Incubation: 5 - 15d JEV - Blood D4– D6 illness D14– D21 illness JEV - CNS & tissue 1Y after illness Serum & CSF IgM Ab

29. Infection Encephalitis onset Concentration Concentration Incubation: 5 - 15d JEV - Blood D4– D6 illness D14– D21 illness JEV - CNS & tissue 1Y after illness Serum & CSF IgM Ab IgG, HI & Neut Ab

30. Collection and processing of specimens

31. Step 1: Complete forms and specimen identification Fill in the information on tube (identifying information, date of collection, and other information as required). Fill in the laboratory form that will accompany specimens.

32. Collection and processing of serum Collect blood in a tube that does not contain any chemicals or anticoagulants. Collect 5mL of whole blood (for very small children collect 1mL). Place tube upright for 30-60 minutes then when firm clot has formed, centrifuge tube for 20 minutes at 2500rpm. Remove serum with a pipette and place in a plastic storage tube (2-3mL microtube or cryovial). If 5mL of blood was collected it will result in about 2mL of serum.

33. Collection of CSF for JE ELISA testing Collect 2mL of cerebrospinal fluid into a sterile, plastic microtube or cryovial. Place in the refrigerator or, if ELISA testing will not occur within 7 days, place immediately in the freezer.

34. Storage of specimens

35. Principles of storage of serum and CSF (1) Both serum and CSF samples should be stored in microtubes or cryovials. If a serum or CSF sample can be tested within 7 days, it should be refrigerated until tested (4 ° C). If it will be tested after more than 7 days, it should be frozen (-20 ° C) (-70 ° C also acceptable for serum)

36. Principles of storage of serum and CSF (2) Periodically check the temperature of the refrigerator. There should be a constant electricity supply to the refrigerator and freezer; if possible, have generator back up. If electricity fails for longer than 2 to 3 hours during the storage of samples, the date and length of time of the power failure should be recorded. Do not use the refrigerator/freezer for storage of other goods such as food or drink.

37. Transport of specimens

38. Principles of transport of serum and CSF specimens The sample should be kept cold during transport and arrive at the testing lab cold (or frozen if it has been previously frozen). The transport time should be kept to a minimum. Ensure samples will arrive at the testing laboratory when a staff member can receive them and immediately put them into cold storage. Ensure all forms with patient information are complete and sent with the specimen.

39. Options for transport Cold box with dry ice. Container filled with liquid nitrogen. Cold box with wet ice.

40. Transport of serum and CSF specimens Tubes (with lids tightly closed) should be packed with absorbent material between tubes (e.g., cotton wool) in a sealed container (e.g., 250-500mL screw top plastic container or can) These containers and the dry ice (or wet ice) are then packed in outer packaging. If ice is used, the outer package must be leak-proof. If dry ice is used, the outer package must permit the release of carbon dioxide gas.

41. Rules to follow to avoid problems with specimen quality If a serum or cerebrospinal fluid sample is being stored frozen, keep it frozen until it is thawed for immediate testing. Repeated freezing and thawing will damage the antibody. Do not keep serum and cerebrospinal fluid samples for JE testing in warm conditions (such as a warm room) for more than 2 to 3 hours.

42. Summary of requirements SpecimenTiming of collection ProcessingStorageTransport SerumAdmission and 10 days after onset Collect blood, allow clot to form, centrifuge, collect serum Refrigerator or freezer (depending on when testing will occur) Cold or frozen (dry or wet ice) CSFAdmission-Same as serum (above)

43. Commercial JE diagnostics: Where are we now? PATH’s JE Project is working with several companies to accelerate development and increase accessibility of ELISA kits. The advantages of these kits include — No long blocking or incubation steps. — Shorter time to run tests (4 to 8 hours).

44. Acknowledgements Please include the following acknowledgement if you use this slide set: This slide set was adapted from a slide set prepared by PATH’s Japanese Encephalitis Project. For information: www.JEproject.org