HCV Genotype 4 in EAST AFRICA

1. HCV Genotype 4 in EAST AFRICA Francis Ssali Site Investigator, Joint Clinical Research Centre, Kampala, Uganda

2. General population HCV prevalence by country and region in Sub-Sahara Africa This data was based on small studies, with mostly screening data of limited reliability ValsaMadhava et al, The Lancet Infectious Diseases 2002; 2:293-302

3. Global distribution of HCV Genotypes, WHO 2009

4. HCV Diagnostic pitfalls in East Africa • There a discordance or variability in the results of the available testing methods • Which tests to use (Kits) ? • In what Algorithm? • Limited generalizability of the current prevalence data.

5. HCV Prevalence Studies in East Africa

6. Screening of blood Bank Samples for HCV in Kampala, 1999 • 2592 blood donors (82% male) screened by EIA and and confirmed with RIBA HCV tests: • 107(4.1%) HCV EIA positive • 15(0.6%) RIBA positive (overall) • 47(44%) of 107 RIBA Indeterminate • 45(42%) RIBA Negative Hladik W. et al Tropical Medicine and International Health. 2006;11(6):951–954

7. VIRAL HEPATITIS IN THE DART STUDY COHORT HCV prevalence of 2.4% in the DART Study

8. HCV among pregnant women in Rwanda & Uganda,2001-2002 • Retrospective testing for HBV & HCV on Baseline samples from 247 HIV+ of 413 pregnant women in the SIMBA(Stopping Infection from Mother-to-child via Breastfeeding in Africa) • 165 from Kampala, Uganda and 82 from Kigali, Rwanda • HCV screening by 4th generation EIA (Innogenetics, Gent, Belgium) and positive samples were retested with a second EIA(Ortho-Clinical Diagnostics, Raritan,NJ) Pirillo Maria F. et al, Journal of Medical Virology,2007;79:1797–1801

9. HCV prevalence In The SIMBA Study • Comment: • The Initial HCV screening test kit is based antigens derived from genotype 1a, 1b,2 & 3a • Potential for underestimate Genotype 4 • Sera from 5 of 8 initially HCV positive were positive on the second EIA.

10. HCV & IVDU in Nairobi, Kenya, 2000 • 74 (22.2%) of 333 Samples were Anti-HCV positive. • 38 (52.2%) of 69 Anti-HCV+ samples were HCV RNA PCR+ • 7 (27%)were Genotype 4 • 19(73%) were Genotype 1a T. Muasya, East African Medical Journal, 2008;85(7):318-325.

11. HCV in Nairobi, UVDUs Kenya Genotype1a Isolates Kenya Genotype 4 Isolates T. Muasya, East African Medical Journal, 2008;85(7):318-325.

12. Viral Hepatitis Among 167 HIV Positive Children in Dar es Salaam, Tanzania Overall HCV prevalence = 13.6% Safila P Telatela et al, BMC Public Health. 2007; 7: 338.

13. HCV in Tanzania Children, Muhimbili Dar es Salaam • Post transfusion HCV in Tanzania children aged 15-59 months • Over all prevalence = 7.1% • No increase risk of Paediatric HCV due to blood transfusion Kitundu J. et al Annals of Tropical Paediatrics2001;21(4):343-348

14. HCV results from Rural SW Tanzania.(Lugalawa Study) ? Favorable outcome or variable diagnostic specificity ? Denaturation of Nucleic acids in Sample storage & Transportation Puato M. et al, Digestive and Liver Disease2007;39:891–892

15. P<0.001 L.G. Johnston et al. International Journal of Drug Policy 2010;21:485–492

16. Prevalence of HCV, HIV, HBV among 408 IVDUs in Zanzibar, 2012 Khatib A. et al. IAS, 2013 e-Poster TUPE339

17. HCV In Kampala, Uganda, 2008 • 500 hospitalized patients HIV positive and HIV- Negative (1:1) at Mulago Hospital • Anti-HCV + Samples re-tested with HCV PCR • Overall HCV prevalence(Anti-HCV) of 13/500 (2.6%) • Only 3(23%) of the 13 Anti-HCV+ samples were positive by RNA PCR. • No difference in HCV prevalence between HIV+ and HIV- groups. • Age > 50 was the only significant risk factor. O’Reill, J. I. et al Journal of Tropical Medicine, 2011. doi:10.1155/2011/598341

18. HCV Testing among Hospitalized patients in Kampala, Uganda, 2006 • 380 (55.5% female) consecutive acute medical admissions(Median age 38yrs, Range 13-87yrs) screened for: • Anti HCV • Rapid Strip Assay (RSA) • Enzyme Immuno Assay(3rd Generation EIA) • Recombinant Immunoblot Assay(RIBA) • Positive samples were tested for serum RNA • Nucleic Acid testing (NAT) Seremba E. et al, Journal of Medical Virology,2010;82:1371-1378

19. Reduced reliability of the HCV Screening Tests in Kampala • 14 (29.2%) of 48 Positive Screen (Anti HCV) samples were positive by RNA PCR. • HIV positivity was associated with a higher rate of False positive HCV EIA. Seremba E. et al, Journal of Medical Virology,2010;82:1371-1378

20. Reliability of HCV Screening in Kampala… • 12 ( 25%) of 48 Positive Anti HCV but PCR negative possibly had past infection that was no longer active • RSA was falsely negative in 7 (50%) of 14 Positive samples by HCV-RNA • EIA falsely negative in 4 (28.6%) of 14 HCV-RNA Positive samples. • HCV genotyping done on 11 samples: • All were Genotype - 1a Seremba E. et al, Journal of Medical Virology,2010;82:1371-1378

21. Why is there a high rate of False HCV test results in the East African Studies ? • Background Flavivirus infections (e.g. West Nile Virus) , Recent Influenza vaccination & Autoimmune diseases? • Not evident in the Mulago Hospital, Kampala Samples. • HIV Infection ? • was associated with a higher rate of false positive HCV EIA, in Mulago Hospital Kampala • Insufficient Primer homology to adequately detect HCV-RNA in East African Isolates? • Suboptimal Storage and shipping conditions? Seremba E. et al, Journal of Medical Virology,2010;82:1371-1378

22. HCV genotype-4 in Uganda, Rwanda • HCV RNA testing and Genotyping done on previously stored HCV EIA positive (151 of 2900) samples at the UVRI, Entebbe, for an East African population Hematology -normal range study, 2008 (Stevens). Kato Kitondwe, Makerere University, (MSc Dissertation) 2011

23. HCV genotype-4 in Uganda, Rwanda (Samples from UVRI)… • Negative controls were from the same study population • Only 9 of 151 had positive PCR • Viral load range 19 – 1,058,000 U/ml. • All the 9 isolates were Genotype 4 Kato Kitondwe, Makerere University, (MSc Dissertation) 2011

24. Phylogenetic analysis of HCV Isolates from Rwanda and Uganda (n=9) Uganda Isolates Kato Kitondwe, Makerere University, (MSc Dissertation) 2011

25. HCV Clinical Cases. Two examples in Kampala, Uganda • There is low clinical awareness and the diagnosis is often missed CASE1 • 42yr Female seen at JCRC in June 2013 with Jaundice and a palpable spleen. • Labs: • ALT 13.2 u/L(NR <40), AST 15.5u/L(NR<40) T.Bili 2.38mg/dl(NR<1). Malaria Smear negative. Hb 7.1g/dl, Plts141x103/μL • HBSAg+, HBV-AnticoreIgG+, Anti HCV+, HIV-Negative • No further HCV tests done for reasons of cost. • Awaiting further HBV evaluation

26. CASE 2 • 39yr Male seen Nov.2012 with 4mths ofRUQ-Abdominal pain. No physical exam signs. BMI 25.4 • Labs: • ALT 26, AST 19, T. Bilirubin 17.8μmol/L(NR<17) • HBSAg-Negative, HCV Elisa Positive • HCV RNA 185,252 U/ml (Taqman) (Jan 2013) • Genotype 4 (Lancet Lab, SA) • Liver Biopsy: • Focal partial chronic inflammation limited to portal triads • No Steatosis /Necrosis/ Fibrosis.

27. Case 2 (cont) • CASE 2 Continued • March 2013. Pegasys 180μg SC. Weekly + Ribavirine 600mg p.o. BID • Missed Wk4 VL (for reasons of cost) • Wk12 VL =Undetectable (<15 U, Taqman). • Complete Early Virologic Response • Awaiting Wk 24 VL • Question: • Continue to 36 or 48 Wks of therapy?

28. Concluding Remarks • There is considerable variability in the overall prevalence of HCV in East Africa, with higher rates emerging in special populations. • There is need to have more reliable HCV testing methods. • Genotype 4 and perhaps genotype 1 appear to be the dominant HCV infections. • There is a paucity of HCV treatment outcome data in East Africa.