1. Impact of Testing Strategies to Reduce Transmission Risk for HBV Ravi Reddy, M Vermeulen South African National Blood Service (SANBS) 29 July 2013

2. Overview of SANBS SANBS is a private not for profit company operating on a fee for service basis Provides a vein to vein blood transfusion service in 8 of the 9 provinces in SA. – WPBTS in Western Cape 807,000 units of blood collected annually (100% voluntary) – 2 800 units bled daily 2 testing centres - Johannesburg and Durban – Also test for Namibia BTS (ID-NAT as well) 7 blood processing centres 82 blood banks serving > 600 hospitals and clinics. SANBS is accredited with the South African National Accreditation System (SANAS) – Specific laboratories are ISO 17025 accredited HQ in Johannesburg

3. Donation Testing Strategy Quality assured testing of each donation – EQAS and internal QC systems in place Serology is mandatory – Anti-HIV-1,2, anti-HCV, HBsAg – Quality assured testing with sensitive serology assays leads to detection of most of the viral positive donations cost effectively Individual Donation Nucleic Acid Testing (ID-NAT) – Implemented in October 2005 – Primarily to reduce risk of HIV window period transmission but also HBV transmission as well

4. Rationale for ID-NAT in South Africa HIV prevalence in blood donors 0.21% HBV prevalence in Blood Donors is 0.1% Implemented ID-NAT testing in October 2005 – ULTRIO ® assay on the TIGRIS ® platform for: – HIV-1 RNA – HCV RNA – HBV DNA – Serology testing for anti-HIV, anti-HCV and HBsAg done concurrently Evaluated and implemented Ultrio Plus assay in May 2011 because of increased sensitivity for HBV detection in window period

5. Geographic Distribution of Chronic HBV Infection HBsAg Prevalence  8% - High 2-7% - Intermediate <2% - Low WHO

6. HBV 5 Year data CodeActive%New%Rejoined% Grand Total% Donations2967492 432064 399863 3799509 Concordant (NAT and HBsAg Pos) 2320.00830500.7061370.03434190.090 ID-NAT only pos 2170.00731940.0449650.01634760.0125 HBsAg only pos 20.0001124 0.028710.00031270.0033 Grand Total4510.01533680.7802030.05140220.106

7. Hepatitis B Summary 476 NAT+, HBsAg negative donations detected in 5 years – Hundreds of cases of transmission via transfusion prevented ID NAT testing has had a significant positive impact on reducing risk of HBV transmission via blood transfusion However still a risk of non detection of very low viral load donations One confirmed HBV transmission on a donation that tested negative with routine serology and ID-NAT – Donor triggered look back as subsequent donation positive Ultrio Plus Assay evaluated and in 2010/11

8. Confirmed pre-ID-NAT WP transmission case Donor triggered look back – RBC Platelet concentrate – Patient unable to be traced Donor details – 47 year old white male (low risk, regular donor) – 54 previous donations – Negative donation – 26/11/2008 – Date of Transfusion – 10/12/2008 – Positive donation – 26/01/2009

9. ID-NAT WP transmission case DonorRBC recipient 26-Jan-099-Apr-0930-Mar-09* S/CO HBsAg 1st screen3520.27743 S/CO HBsAg 2nd screen385 721 S/CO HBsAg 3rd screen395 760 S/CO Ultrio 1st screen15.313.314.3 S/CO Ultrio 2nd screen15.612.814.2 S/CO Ultrio 3rd screen14.9 0.12 15.7 S/CO dHBV23.1invalid25.2 anti-HBc IgM Pos anti-HBc total Pos anti-HBs Neg * 3.5 months after transfusion

10. Additional tests to Determine if Recipient infection caused by Transfusion Donor plasma index donation (26/11/08) – SANBS 3 out of 30 replicates positive – Gen Probe 7 out of 30 replicates positive with Ultrio Plus Phylogenetic analysis/sequencing – Donor and recipient – genotype A – Intra group nucleotide divergence Donor/recipient 0.31% (99.7% Shared Identity) Transmission by Blood Tranfusion confirmed

11. Ultrio, Ultrio Plus, TaqScreen Study performed Compare Sensitivity of NAT assays with a range of samples – 10 000 random samples – 40 HIV ID-NAT yield samples – 107 HBV ID NAT positive samples on Ultrio – HBsAg positive, HBV NAT negative samples – Other samples (HIV Window period transmission sample and HBV window period transmission sample)

12. Proportion reactive per NAT option on 107 Ultrio HBV ID-NAT yield samples NAT option % reactive p< 0.00001 P= 0.00015 tested in 6 replicates in each test option

13. Proportion HBV NAT reactive on WP* and OBI yield samples NAT option % reactive

14. Percent ID-NAT reactive on HBsAg+/Ultrio- yield samples copies/ml* *determined by probit analysis in Ultrio Plus against Eurohep standard % reactive (12 reps )

15. UltrioUltrio Plusincreasep value Donations775444789948 pre-HBsAg WP47 (1:16 499)80 (1:9 874)170%0.005 post-HBsAg WP10 (1:77 544)17 (1:46 468)167%0.19 OBI94 (1:8249)162 (1:46 468)172%0.00004 HBV-NAT yield151 (1:5204)259 (1:3 050)172%<0.00001 HBsAg positive820 (1:946)841 (1:939)103%0.89 HBsAg+/DNA-38 (1:20 406)16 (1: 49 372)0.002 All HBV infections971 (1:799)1100 (1:718)111%0.016 HBV ID-NAT Yields – One year Data Ultrio (2010 vs. Ultrio Plus 2011)

16. HBV-DNA cps/mL Viral load distribution in all HBV NAT yields detected by Ultrio versus Ultrio Plus

17. Estimated WP reduction with Ultrio and Ultrio Plus (14.5 vs 24.7 days; 1.7 fold) days cps/ml Ultrio HBsAg WP eclipse Ultrio PlusHBsAg Ultrio Plus Start WP ID 50 3.16 cps/ 20 ml Ultrio Plus 50% LOD 4.1 cps/ml Ultrio 50% LOD 63 cps/ml HBsAg S/CO=1 3000 cps/ml 14.5 days 24.7 days 12.9 days 22.9 days screening period WP transmission risk Ultrio1:27 000 Ultrio PLus1:43 000 WP Ultrio

18. Probable OBI transmission case Donor Recipient Drawing date25/1/1211/4/12¥11/5/1211/6/124/7/12¥31/1/13 StatusRBC Issued Look back Follow up Ultrio Plus S/CONeg 15.3/15.5/15.115.6/14.9 NegPos dHBV S/CO 22.7neg HBsAgNeg PosNeg Anti-HBc IgMNeg Pos Anti-HBc totalPos Anti-HBs titre<22.6<10284 IU/L Viral load1.6 cps/mL*43 cps/mL ¥ 100 % homology of whole HBV genotype D genome sequence of donor sample 11/4/12 and recipient sample 4/7/12 (analysis kindly performed by Dr Marco Koppelman, Sanquin, Amsterdam) * Estimated from 5/20 reps Ultrio Plus reactive by probit analysis ( 5.1% probability of transmission estimated with ID 50 of 316 virions)

19. Testing Strategy for Hepatitis B Countries with high incidence/prevalence of HBV need to carefully review algorithms for HBV testing and assay selection – Maximise safety and minimise product discard High prevalence countries – HBsAg and ID-NAT but not anti-HBc No discard of anti-HBc positive units (6-8% of products usable) Majority of early and occult infections detected Minimal risk of not detecting post HBV DNA window period donations and some OBI’s Ultrio Plus in ID-NAT format has significantly improved sensitivity and reduced risk

20. Course of HBV markers and residual transmission risk with ID-NAT Vermeulen et al, Transfusion 2012;52:880-892. 1 st WP ~10 days (Ultrio Plus) 2 nd WP < 1day (Ultrio Plus) OBI transmission risk unknown

21. SANBS Algorithm for HBV Testing ID-NAT and HBsAg testing Concordant positive – confirmed ID-NAT only (2 x U+ and dHxV) Non repeat reactive – donation discarded, anti- HBc and anti-HBs marker added – 0.12% of donations discarded (mostly false positive) – Donor not notified – When donor returns Additional tests performed Blood can be used if all routine tests and anti-HBc negative

22. Algorithm for HBV Testing (cont’d) ID-NAT repeat reactive (2 out of 4), serology negative – Additional tests on plasma bag – Donor recalled and additional tests done – If confirmed positive defer donor – If deferral, medical division contacts and counsels donor – If all tests on follow up donation negative (review need for additional tests or re-instate)

23. HIV infections in six years of ID-NAT screening of 4,520,230 donations 7993 (96.5%) HIV RNA +, anti-HIV– window period 228 (2.8%) HIV RNA +, anti-HIV + concordant HIV RNA –, anti-HIV + elite controller 64 (0.77%) HIV-RNA anti-HIV 82/228 (38.4%) HIV-Ag + 59 in first time donors (0.71%) 4 in lapsed donors (0.05%) 1 in repeat donor (0.01%)* 136 HIV-Ag-, RNA+ infections avoided (1:33,237)

24. Impact of ID NAT on Blood Safety - HIV Significant positive impact on blood safety contributing to major public health success story – 136 ID- NAT positive, anti-HIV negative, p24 antigen negative donations detected in 6 years of testing. – Without ID-NAT there would have been numerous HIV transmissions via blood transfusion – Major public health success story – Increased collections by 95 000 units over 5 years and increased confidence in the blood supply – Could increase Black donors from 7% to 31% of donor base over 5 years Since ID NAT implementation, no reported case of HIV transmission since October 2005 – HIV prevalence in blood donors has increased (0.07% to 0.23%) – Highlights impact of ID-NAT on improving blood safety

25. Summary Implementation of ID-NAT testing has resulted in a significant increase in safety of the South African blood supply compared to previous testing strategy ID-NAT continues to interdict infectious donations that are missed in mini pool format SANBS evaluation and one year data confirmed that Ultrio Plus is more sensitive than Ultrio for HBV

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