HCV Genotype 4 in EAST AFRICA

Francis SsaliSite Investigator, Joint Clinical Research Centre, Kampala,

Uganda

Valsa Madhava et al, The Lancet Infectious Diseases 2002; 2:293-302

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

Global distribution of HCV Genotypes, WHO 2009

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.

HCV Prevalence Studies in East Africa

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

VIRAL HEPATITIS IN THE DART STUDY COHORT

HCV prevalence of 2.4% in the DART Study

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

HCV prevalence In The SIMBA StudyHCV (n=247) HBV (n=266)

Kigali 4.9% 4.1% Kampala 0.6% 4.9%Overall prevalence

2.1%(95%CI=0.3-3.9)

4.1% (95%CI=1.7-6.8)

Viral hepatitis present in 6%

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.

HCV & IVDU in Nairobi, Kenya, 2000Anti HCV Positive Anti HIV 1

n (%) P Value n (%) P Value

Male (n=314)

61 19.4 0.0352 63 20 0.0005

Female (n=19)

8 42.1 11 57.9

• 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.

T. Muasya, East African Medical Journal, 2008;85(7):318-325.

Kenya Genotype1a Isolates

Kenya Genotype 4 Isolates

HCV in Nairobi, UVDUs

Viral Hepatitis Among 167 HIV Positive Children in Dar es Salaam, Tanzania

Safila P Telatela et al, BMC Public Health. 2007; 7: 338.

Overall HCV prevalence = 13.6%

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

HCV Serology Transfused No past Transfusion

Positive 5 (5.4%) 8 (8.6%)Negative 87 84

Total (n) 92 92

Kitundu J. et al Annals of Tropical Paediatrics2001;21(4):343-348

HCV results from Rural SW Tanzania.(Lugalawa Study)

General Population screened

• n=750

Number testing Positive by Elisa • 78 (10.4%)

Positive by RIBA

• 15 (2%)

Positive by RNA-PCR • 2 (0.2%)

Puato M. et al, Digestive and Liver Disease2007;39:891–892

? Favorable outcome or variable diagnostic specificity? Denaturation of Nucleic acids in Sample storage & Transportation

HIV Pos HCV Pos HCV+HIV Pos HBV Pos0

5

10

15

20

25

30

Prevalence of HCV, HIV & HBV Among MSMs in Zanzibar , 2007

% MSM IVDU(n=66)

% MSM Non IVDU (n=443)

% P

reva

lenc

e

P<0.05

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

P<0.001

Prevalence of HCV, HIV, HBV among 408 IVDUs in Zanzibar, 2012

Series10

5

10

15

20

25

30

11.3 25.4 5.9

Prevalence of HCV, HIV & HBV

HIV HCV HBV

%

Khatib A. et al. IAS, 2013 e-Poster TUPE339

All HIV All HCV All HBV0

5

10

15

20

25

30Hepatitis & HIV Co-infection

HBV & HCV only

HBV & HIV Only

HBV Only

HBV & HCV & HIV

HIV Only

HCV & HIV Only

HCV Only

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

HCV Testing among Hospitalized patients in Kampala, Uganda, 2006

Seremba E. et al, Journal of Medical Virology,2010;82:1371-1378

• 380 (55.5% female) consecutive acute medical admissions(Median age 38yrs, Range 13-87yrs) screened for:

1. 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)

Reduced reliability of the HCV Screening Tests in Kampala

Seremba E. et al, Journal of Medical Virology,2010;82:1371-1378

• 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.

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

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

HCV genotype-4 in Uganda, Rwanda

Kato Kitondwe, Makerere University, (MSc Dissertation) 2011

• 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).

Site EIA Used Number of Positive Number of Negative TotalKigali, Rwanda

Murex anti HCV Version 4.0 (Abbot) 13 2 15

Entebbe, Uganda

Innotest HCV Ab IV (Innogenetics) 10 1 11

Masaka, Uganda

Innotest HCV Ab IV (Innogenetics) 98 10 108

Kilifi, Kenya Innotest HCV Ab IV (Innogenetics) 30 3 33

Total 151 16 167

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

Phylogenetic analysis of HCV Isolates from Rwanda and Uganda (n=9)

Kato Kitondwe, Makerere University, (MSc Dissertation) 2011

Uganda Isolates

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-Anticore IgG+, Anti HCV+, HIV-Negative

– No further HCV tests done for reasons of cost.– Awaiting further HBV evaluation

CASE 2• 39yr Male seen Nov.2012 with 4mths of RUQ-

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.

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?

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.