West Nile virus: the disease and Panbio product training

1. West Nile virus: the disease and Panbio product training

2. General information

3. WNV: the emerging epidemic • Previously unknown in the Western hemisphere, West Nile virus has spread through much of the United States since its first appearance in New York in 1999.

4. WNV: Europe • The incidence of West Nile fever in Europe is largely unknown (5) • 1960s - cases observed in southern France, southern Russia, Spain, southwestern Romania • 1970s, 1980s, and 1990s cases seen in Belarus, western Ukraine, southeastern Romania, and Czechland. • It has been hypothesized that there could be greatly increased WNV activity in Africa in the next few years. This in turn may be followed by an epidemic occurrence of West Nile fever in Europe (5)

5. European distribution of WNV* * based on the virus isolation from mosquitoes or vertebrates, including humans (black dots), laboratory-confirmed human or equine cases of West Nile fever (black squares), and presence of antibodies in vertebrates (circles and hatched areas).5

6. WNV: the emerging epidemic • West Nile virus infection peaks with mosquito activity in the summer months, July to September. • West Nile virus outbreaks in temperate areas over consecutive years indicate an efficientmosquito overwintering mechanism. • Year-round transmission of West Nile virus has been documented in subtropical regions of theUnited States.

7. Transmission cycle • In nature, West Nile virus is maintained in a mosquito-bird-mosquito transmission cycle, primarily involving Culex species mosquitoes.

8. Additional routes of transmission • Laboratory-acquired infections • Laboratory workers have been accidentally infected with West Nile virus (e.g. needlestick injury). • Intrauterine transmission (and possibly breast feeding). • Blood and blood products transfusion, organ transplantation.

9. Outcomes of WNV infection • Most human WNV infections are subclinical • Clinical infections rangefrom uncomplicated WN fever to fatal meningoencephalitis. All age groups and both genders appear equally susceptible to WNV infection, however the incidence of encephalitis and death increases with age. • Of those infected with West Nile virus: • 1 in 5 - experience mild, febrile illness lasting 3 - 6 days (symptoms include: malaise, headache, eye pain, gastrointestinal problems and rash). • 1 in 150 - develop meningitis or encephalitis (incubation period 2 - 14 days)Meningoencephalitis is rare in younger people, but incidence increases markedly in patients aged 50 and over.

10. Outcomes of WNV infection • Immunosuppression may increase risk of severe disease. • Severe muscle weakness is a diagnostic clue and an important predictor of death. • A few cases of a poliomyelitis-like syndrome involving anterior horn cells have been reported, characterised by flaccid paralysis without pain or paresthesia.

11. Serology of West Nile virus • Most patients have detectable levels of IgM antibodies 7-8 days after the onset of symptoms. • IgM antibody has been shown to persist for >500 days in approximately 60% of cases. • Most patients exhibit IgG antibodies 3-4 weeks post infection.

12. Panbio WNV ELISAs

13. PanbioELISA methodology • The Panbio West Nile Virus ELISAs are designed to assist in the clinical laboratory diagnosis of West Nile virus infection in patients with clinical symptoms consistent with encephalitis/meningitis. West Nile Virus IgM Capture ELISA (E-WNV02M) West Nile Virus IgG Indirect ELISA (E-WNV01G)

14. PanbioWNV IgM Capture ELISA • Superior specificity and is even simpler to use • Results in only 2.5 hours • No background subtraction necessary therefore only 1 well required per patient (unlike other manufacturer’s products)

16. Agreement with CDC MAC EIA • 98.6% overall agreement with CDC MAC EIA For further study details, please see Instructions for use

17. Clinical sensitivity • 100% sensitivity with serum from patients with WNV encephalitis/meningitis For further study details, please see Instructions for use

18. Cross-reactivity • Only 3.8% cross-reactivity when tested against a comprehensive panel of other disease state serum. For further study details, please see Instructions for use

19. PanbioWNV IgGIndirect ELISA • Results in only 1.5 hours • Simple procedure – no soaking step prior to starting the assay • Colour coded reagents

21. Negative presumptive agreement • Overall agreement of 91.4% obtained for endemic normal samples For further study details, please see Instructions for use

22. Positive presumptive agreement • IFA characterized • Good agreement obtained from 3 study sites For further study details, please see Instructions for use

23. Sensitivity For further study details, please see Instructions for use

24. Posters/Performance data

25. Publications/Posters Malan et al. (2004) Evaluations of Commercial West Nile virus Immunoglobulin G (IgG) and IgM enzyme immunoassays show the value of continuous validation. J. Clin. Microbiol. 42:727-733. Valks, A. et al. (2004) In-house development of a next generation West Nile virus IgM capture ELISA. Poster presented at the 2004 Clearwater Virology Meeting, USA. Fox J. et al. (2005) Immunoglobulin G Avidity in the Differentiation Between Early and Late Antibody Response to West Nile Virus. Poster presented at the 2005 Clearwater Virology Meeting, USA. McPherson, T.J., H.B. Lee, J.B. Johnson and G.G. Daves. Comparison of CDC and Panbio West Nile IgM Capture ELISA for human West Nile virus testing. Poster presented at the 6th National Conference on West Nile Virus. San Jose, California, February 8-9, 2005

26. Malan et al. (2004) • Panbio and Focus WNV IgM and IgG ELISAs were compared. With the first panel both performed similarly demonstrating high sensitivity and specificity. However when a less-biased panel was used the Panbio WNV IgM ELISA gave a false positivity rate of 6.3% compared to 2.5% for the Focus kit. Similarly the Panbio IgG ELISA demonstrated a false positivity rate of 27% while the Focus kit gave 12% false positives. • To correct the problem with false-positives during the 2003 season both Panbio and Focus made available supplementary procedures involving an antigen subtraction step. • Since this publication, Panbio has redesigned the WNV IgM ELISA (E-WNV02M – next generation) such that false-positivity is minimised. This data was presented at Clearwater Virology Meeting 2004 (Valks et al.). In the next generation assay only one well is required per patient rather than two as is the case for the Focus IgM ELISA.

27. Valks et al. (2004): Summary • Valks, A., Hafner,G., Hazell, S., Xu, W. (2004) In-house development of a next generation West Nile virus IgM capture ELISA. Poster presented at the 2004 Clearwater Virology Meeting, USA. • 394 specimens were tested on the Panbio WNV IgM Capture ELISAs (Original and Next Generation kits). Of the 394 samples, 72 were classified as positive and 322 as negative for WNV infection according to MAC ELISA and/or IFA. Of the 322 negative samples, 71 were known non-specific reactors on the original WNV ELISA. The population was therefore biased specifically to challenge the robustness of the new assay. A cross reactive panel of specimens characterized as positive for a number of other disease states was also tested on the Original and Next Generation kits.

28. Valks et al. (2004): Results • False positive signals reduced • Negative population tightened • Improved positive to negative ratio • Assay run time only 2hr 10min • Incorporation of liquid antigen removesneed for Ag reconstitution • No subtraction method needed toobtain good specificity Next generation WNV IgM ELISA

29. Fox et al (2005): Summary • Fox, J.L. et al. (2005) Immunoglobulin G Avidity in the Differentiation Between Early and Late Antibody Response to West Nile Virus. Poster presented at the 2005 Clearwater Virology Meeting, USA. • Thirteen seroconversion panels, each consisting of between four and seven plasma samples collected from U.S. blood donors classified as WNV RNA positive by nucleic acid technology (NAT) screening, were obtained. The index sample (Day 0) was positive by a WNV Transcription Mediated Amplication (TMA) assay and non-reactive when tested for WNV IgM. Each panel contained at least two follow up specimens taken at various intervals ranging from 10 to 208 days post index. All samples were independently tested by the CDC using PRNT. • Samples were tested on the Panbio WNV IgM ELISA and the IgG ELISA with and without avidity reagent.

30. Fox et al (2005): Results • IgG avidity profiles of "primary" individuals had low Avidity Index (< 40%) for the first 20-30 days post index. • After ~ 40 days post index in these panels, the Avidity Index rose to > 40% (Fig 1). • For the "secondary" samples all positive IgG samples had an Avidity Index of > 55% regardless of the days since the index donation (Fig 1).

31. Fox et al (2005): Conclusion • When both IgM and IgG are present in a patient sample, IgG avidity testing in conjunction with the WNV ELISA appears to be useful in identifying recent, primary infections.

32. McPherson et al. (2005) • McPherson, T.J., H.B. Lee, J.B. Johnson and G.G. Daves. Comparison of CDC and Panbio West Nile IgM Capture ELISA for human West Nile virus testing. Poster presented at the 6th National Conference on West Nile Virus. San Jose, California, February 8-9, 2005 • Excellent poster to promote the next generation Panbio WNV IgM ELISA (E-WNV02M). • Independent study

33. McPherson et al. (2005) • First part of study compared the “old” and “new” Panbio WNV IgM Capture ELISAs using 83 sera, including a large number of samples that produced false-positives previously in the “old” generation of the Panbio kit. • Results of the first part showed that the old ELISA gave poor agreement with the CDC ELISA with 27/82 sera giving false positive results. The “new” (ie next generation) version of gave good agreement with the CDC ELISA (>97.5%) • Second part of the study compared the new Panbio kit (next generation) with a procedure relative to the CDC WNV IgM Capture ELISA. A total of 1050 sera were selected for the study.

34. McPherson et al. (2005) • There was 98.8% agreement between the Panbio Next generation WNV IgM ELISA and an ELISA based on the CDC WNV IgM ELISA (1050 samples were tested). • The study demonstrated a tremendous improvement in the performance of the Panbio next generation WNV IgM Capture ELISA. The new test is reliable and produces very few false positive values. • The sensitivity was 97.7% and specificity was 99.2% relative to the CDC protocol.

35. Ordering information Panbio WNV ELISA are FDA Cleared and CE marked

36. Also available

37. Panbio promotional material • WNV: The facts brochure • WNV: Now there’s a better way brochure • Scientific posters

38. www.panbio.com

39. References • 1. West Nile virus 2002 case count. http://www.cdc.gov/ncidod/dvbid/westnile/surv&controlCaseCount02.htm • 2. Morbidity and Mortality Weekly Report, December 20, 2002. Provisional surveillance summary of the West Nile virus epidemic – United States, January to November 2002. http://www.cdc.gov/mmwr/PDF/wk/mm5150.pdf. • 3. 2003 West Nile virus Activity in the United States. http://www.cdc.gov/ncidod/dvbid/westnile/surv&controlCaseCount03.htm (reported May 21 2004). • 4. West Nile virus 2004 case count. http://www.cdc.gov/ncidod/dvbid/westnile/surv&controlCaseCount04_detailed.htm • 5. Hubálek, J, & J. Halouzka (1999) West Nile Fever–a Reemerging Mosquito-Borne Viral Disease in Europe. Em. Infect. Dis. 5:643-50. http://www.cdc.gov/ncidod/eid/vol5no5/hubalek.htm • 6. Centers for Disease Control and Prevention. Epidemic/epizootic West Nile virus in the United States: Revised Guidelines for the Surveillance, Prevention and Control.www.cdc.gov • 7. Hazell S MLO 2004 June; 10-12, 16. • 8. Petersen LR et al. NEJM 2002; 347:1225-26.