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Nucleic Acid Testing vs Infectivity

Nucleic Acid Testing vs Infectivity. Michael P. Busch, M.D., Ph.D. Blood Centers of the Pacific Blood Systems, Inc . EPFA, Lisbon Portugal May, 2001. NAT vs Infectivity.

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Nucleic Acid Testing vs Infectivity

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  1. Nucleic Acid Testing vs Infectivity Michael P. Busch, M.D., Ph.D. Blood Centers of the Pacific Blood Systems, Inc. EPFA, Lisbon Portugal May, 2001

  2. NAT vs Infectivity • Overview of stages of infections and importance of understanding stage-specific gEq:infectivity ratios (concentration vs volume infused) • Review of NA dynamics (pre/peri/post-SC) for HIV, HCV and HBV • Review infectivity data for each stage • Need for future studies • animal transmission models • lookback studies (NAT/SC donors; recipient cases)

  3. Stages of TTVIs and infectivity • Pre-ramp-up viremia • Ramp-up viremia • Plateau phase / peak viremia • Peri-SC viremia • Post-SC viremia • Persistent carrier (viral load set point) • “Resolved” infection? • Immunosilent carrier • Transient Viremia w/o SC

  4. Stages of TTVIs and infectivity • Pre-ramp-up Viremia • Low-level, intermittent “blips” of RNA/DNA detected prior to quantifiable ramp-up phase • Innoculum vs focal replication that seeds dissemination vs abortive replication • Can this occur transiently w/o subsequent ramp-up/SC? • Is this viremia infectious? • Kinetic, viral sequencing & infectivity studies in progress

  5. Testing Algorithm for HCV NAT-conversion Panels Alpha Therapeutic / NGI screening (Antibody & 512 Pool PCR) 63 HCV NAT-conversion Panels ( 774 Donations ) 5 Control Panels ( 54 Donations ) dHCV TMA Testing Intermittent Pre-Ramp-Up Viremia * in 25/41 (61%) panels >3 pre-r/u units Ramp-Up & Plateau Phase Quantitative Viral Load ( Roche Monitor ) Replicate dTMA ( x 3 ) & NGI UltraQual PCR Testing * Ramp-Up = consistent RNA detection with progressive increase in viral load.

  6. NGI 512 PCR x108 Neat PCR x107 HCV Monitor PCR (gEq/mL) % dTMA Positive (4 replicates) 100 x106 75 x105 50 x104 x103 25 x102 -60 -40 -20 0 10 20 Days Pre/Post 1st Quantitative RNA+ Donation Low-Level Intermittent HCV ViremiaPreceding Ramp-Up Phase Assay sensitivity (6 x 102) (BCP ID 10083)

  7. Representative HIV Conversion Panels with Pre-Ramp-Up “Blip” Viremia(RNA “blip” observed in 7/19 informative panels) Alpha / BCP Case 1012

  8. Representative HBV Panel with Pre-Ramp-Up Viremia (pre-ramp-up viremia observed in 12/23 informative panels) 140.00 120.00 1,000,000 100.00 Pre-Ramp-Up Ramp-Up 100,000 80.00 HBV DNA Load (gEq/mL) 10,000 HBsAg S/C 60.00 1,000 40.00 100 20.00 - + 10 0.00 -37 -35 -29 -26 -21 -18 -11 -8 0 3 8 Day from 1st + HBsAg test

  9. Stages of TTVIs and Infectivity • Ramp-up viremia • Persistent viremia with progressive increase in viral load leading to peak or plateau viremia • Doubling time used to project WP differences of ID-NAT, MP-NAT and Ag assays • Presumed infectious (chimp study in progress)

  10. 12 10 8 6 4 2 0 -2 -20 -15 -10 -5 0 5 10 15 20 HCV RNA Production Rate and Linear Regression Model during Ramp-up Viremia N = 101 samples from 37 donors Log HCV RNA [gEq/mL] DT = 14.9 hrs (95% CI: 12.9-17.8) Day from first HCV RNA positive sample

  11. Individual Rates and Linear Regression Model of HIV RNA Production during Early Infection. 9 N = 97 Samples from 44 Plasma donors DT: 21.5 hrs (95% CI: 19.2-24.6) 8 LOG HIV RNA [gEq/mL] 7 6 5 4 3 2 1 -10 -5 0 5 10 15 20 Day

  12. Individual Rates and Linear Regression Model of HBV DNA Production during Early HBV Infection 8 N = 70 samples from 21donors DT: 2.6 days (95% CI: 2.6-3.2) Log HBV DNA [gEq/mL] 7 6 5 4 3 2 1 0 -60 -50 -40 -30 -20 -10 0 10 20 30 Day from first HBsAg positive sample

  13. Window Period Viremia Parameters Derived from NAT Analysis of Plasma Donor Panels * Based on follow-up of 30 plasma donors detected as MP-NAT-pos by Alpha and Bayer (95% CI 29-85 days)

  14. -20 -15 -10 -5 0 5 10 15 20 Projections of HCV Window Period Reductions by MP / ID NAT Using the Linear Regression Model of HCV Production 12 Log HCV RNA [gEq/mL] 10 HCV RNA 8 6 MP NAT [1,000 gEq/mL] a 4 c 2 ID NAT [50 gEq/mL] b 0 d -2 Day from first HCV RNA positive sample

  15. Incremental WP Closure and Projected Yield (U.S.) of ID over MP-NAT Screening Assumes sensitivity of ID NAT = 50 gEq / mL, and MP NAT = 1000 gEq / mL Does not include detection by ID-NAT of intermittent, low-level viremic episodes preceding ramp-up viremia

  16. geq/ml NucliSens-Ampliscreen 1:48 TMA 1:16 TMA single infectious window Days Detection of HIV in antibody negative window phase

  17. Relationship of Infectivity & Viral Load pre-SC Animal Models • Analyses of Plasma Stocks with Defined CID50. • Correlate limit of detection of NAT assays to 1 CID50. • Characterize viral load at several CID dilutions. Calculate viral load : CID50 ratio. • For HBV/HCV ~10 gEq/CID50. • Primary Infection Transmission Studies (Alter et al. Transfusion 1999) • Infect 1O animals, draw samples through SC. • Characterize serial samples by NAT, Ag, Ab assays. • Serially transfuse eclipse and NAT(+) blood into 2O animals. • Correlate detection of viremia with development of infectivity. • Transfuse Human WP Samples into Test Animal • Plasma donor SC panels. • Pre-SC units from blood donors with documented transmission.

  18. Infectivity Early in HCV Infection Assessed by Transmission to Chimpanzees D3 D2 B HCV RNA Level D1 A C Time

  19. Relationship of Infectivity & Viral Load pre-SC Human Transmission Data • Investigate Recipients of Prior Donations by NAT / Ab SC Donors (Donor directed lookback) • Model Duration of "Infectious WP" Based on Rate of Recipient Infection Relative to ID Intervals Petersen, et al. Transfusion 1993;33:552-7 • For Cases with Documented Transmission and Available Plasma, Characterize Viral Load and Detection by MP/SD NAT Tests Schttler, et al. Lancet 2000 Robbins, et al. JAMA 2000 Roth, et al. submitted • Transfuse human infectious plasma with low viral load into animals to validate sensitivity of the models

  20. HIV Test Results for Donor, Recipients in Singapore Transmission CaseRobbins, et al. JAMA, 2000

  21. Reactivity (+ / Total) of Donor Screening NAT assays on a Coded Plasma Panel of Singapore transmission case and controlsRobbins, et al. JAMA, 2000

  22. HCV Transmission by Blood Donation Negative by NATSchüttler CG, et al. Lancet 2000; 355:41-2 • Donation 8 weeks prior to SC donation • HCV transmission by platelet concentrate (~50mL plasma) but not RBC (~5mL plasma) • NAT studies of FFP, incl. “enhancedinput” PCR assays, negative for HCV RNA • Conclusion: “Even a negative NAT test cannot completely prevent transmission of HCV.”

  23. HCV Transmission by Blood Donation Negative by NATSchüttler CG, et al. Lancet 2000; 355:41-2 Follow-up study in collaboration w/ Dr. Gerlich • Coded panel with 3 aliquots (2.5 mL) of implicated plasma and 2 positive (low-level HCV RNA ramp-up) and 1 negative controls. • GenProbe, NGI • Controls correctly identified by both assays • GenProbe HCV dTMA (+) in 2 of 3 replicates; NGI Ultraqual PCR (-) on all 3 replicates • Sequencing and chimp transmission studies in progress

  24. Donor directed lookback studies from NAT+/SC donorsRoth et al. Lancet (submitted)

  25. Relationship of Infectivity & Viral Load pre-SC Human Transmission Data 2. Investigate Donors to Recipients with reported post-transfusion infections (Recipient traceback) • Retest stored aliquots of donation plasma • Recall implicated donors • Perform sequencing/phylogenetic analysis to confirm linkage

  26. Analysis of Reported Post-Tx Cases in JapanMatsumoto et al. Transfusion 2001 • 141 “TA-HBV” cases reported ’97-99 • 103 fully investigated: donation samples & f/u available • 15 HBV DNA(+) donations linked to 14 cases • 9 WP units - donors SC to anti-HBc/anti-HBc on f/u • 5 tested HBsAg EIA+, all w/ >4,000 gEq/mL • 2 units anti-HBc+ at donation and f/u • 3 low-level HBV DNA(+) donations w/ HBV(-) donors on f/u • 1 case w/ 43 HBV DNA- donations, 1 donor SCd on f/u • 92 reported “TA-HCV” cases: all donations NAT(-) and all donors (-) on f/u • 0 cases of “TA-HIV” reported

  27. Stages of TTVIs and infectivity • Plateau phase (HCV) • Prolonged, stable high-titer viremia preceding SC • Minor fluctuations in viral load may reflect host cell capacity to support viral replication • Peak viremia (HIV & HBV) • Rapid rise then fall in viral load corresponding to immune control and clearance of virus • Results in clearance or post-SC steady state viremia

  28. Stages of TTVIs and infectivity • Peri-SC phase • cellular and humoral immune response results in down-modulation of viral production and, in some cases, clearance • Smooth decline in viral load to clearance or steady state viremia in most cases • Some cases evidence marked fluctuation in viremia including intermittent neg-pos ID NAT results

  29. S/CO 10 10,000,000 9 8 1,000,000 7 46 Days 6 100,000 HCV PCR Quantification 5 PCR 4 10,000 3 2 1,000 1 100 0 20 40 60 80 100 120 140 160 180 200 Virologic/Serologic Profile – HCV Panel 6211 Days

  30. Time to Detection of Transfusion-Transmitted HCV Infection by Test Time toMean95% CI RNA 12.6 1.5-23.7 ALT 51.5 18.5-84.5 EIA3.0 70.7 32.8-128.6

  31. HEPATOLOGY Vol. 29, No. 3, 1999

  32. 6 10 5 10 4 10 3 10 2 10 HCV NAT-Positive Donor with fluctuating viremia during plateau-SC phase RIBA – – – – – – Ind Ind + + ALT (S/CO) Viral load 30 1,500 25 1,250 20 1,000 15 EIA 750 Viral load TMA 10 500 ALT 5 250 0 0 -147 0 30 60 90 120 -365

  33. Stages of TTVIs and infectivity • Immunosilent carriers • Persistent viremia in absence of detectable SC • Case reports for HIV, HCV and HBV • Recipient infection documented for HCV • Durand et al. Ann Intern Med 2000 • Peoples et al. Transfusion 2000 • Transient Viremia without SC • Confirmed by alternate sample and f/u with serotyping • Frequency in HR populations detectable by high sensitivity NAT is now under study.

  34. ARC HCV NAT Reactive Donations Confirmed with Follow up Sample(s) N = 25 Seroreactive Seronegative Seroreactive, NAT Negative Seronegative, NAT Negative Number of Days

  35. Stages of TTVIs and infectivity • Post-SC • Chronic seropositive carrier state • Set-point viral load (NA vs Ag levels) • Variable infectivity to NA/Ag ratio • Defective viral particles • Excess Ag production • Neutralizing Ab • Impact of unit storage and recipient susceptibility • “Resolved” infection • Sensitivity of NAT vs infectivity in blood? • Persistence of infections virus in liver or other tissues • Waning Ab titers w/ persistent cellular immune responses

  36. Rate of HCV Viremia (dHCV TMA) among Seropositive Subjects with Divergent Risk Factors and Immune Status

  37. Systematic Review of Role of Polymerase Chain Reaction in Defining Infectiousness among People Infected with Hepatitis C VirusDore GJ, Kaldor JM, McCaughan GWBr Med J 1997; 315:333-7 • 2022 people exposed to anti-HCV(+) sources • 1148 exposed to PCR(+) sources: 148 transmissions • 874 exposed to sources (-) for HCV: 0 transmissions • Transmission rates after: • Perinatal exposure 6.2% • Needlestick 6.1% • Solid organ / bone marrow transplantation 78% • Blood components 88%

  38. # (%) pos on SD-NAT (+) Original NAT Screening # RIBA+ / NAT- Specimens Tested 15 (25%) * Minipool 60 2 (13%) Single Donation 15 * Mean SD-NAT s/co = 5.0 (range 1.05 – 9.2) Results of SD-NAT Re-testing of HCV-RIBA-3-positive Specimens that Tested Negative by MP vs SD NAT

  39. HCV viral load in anti-HCV+ donors and outcome in recipientsOperskalski et al. AABB 2001

  40. Correlation of HIV NAT withSupplemental HIV Serological DataARC 09/08/99 – 08/31/00 Western Blot Result NATResult Pos Ind Neg Total Pos Neg Total 213(94%) 13 226(5.6%) 38 1,901 1,939 (1.5%) 21 1,856 1,877 272 3,770 4,042

  41. Characteristics of WB Pos/TMA Neg Samples identified by ARC NAT Screening 09/08/99 – 08/31/00 Sample Pool Neat HIV-1/HIV-2 WB HIV PCR 1 NR 1.43 41, 120, 160 Neg 2 NR 1.03 41, 160 Neg 3 NR 1.62 41, 160 Neg 4 NR 1.11 41, 55, 160 Neg 5 NR 2.50 24, 41, 51, 61, 160 Neg 6 NR 20.18 all bands Neg 7 NR 1.22 24, 41, 160 Neg 8 NR 1.11 17, 41, 120, 160 Neg 9 NR 1.84 41, 160 Neg 10 NR 1.40 24, 41, 160 Neg 11 NR 1.70 17, 24, 41, 51, 160 Neg 12 NR 20.00 all bands Pos (200 copies/mL) 13 NR 17.84 all bands Neg All samples p24 Ag negative

  42. HIV-1 Transmission vs RNA Load and Storage 6  RBC (+)  RBC (–)  Platelets (+)  FFP (+)  FFP (–) 5 4 HIV-1 RNA Load (Log10) Copies/mL 3 2 1 0 10 30 50 Days to Administration Busch et al. JID 1994

  43. HBV DNA Detection in anti-HBc+HBsAg- US blood donationsKleinman et al. AABB 2001 • 5121 anti-HBc-only donations from 5 REDS sites tested • 1231 anti-HBc+, anti-HBs <100 IU/mL by Prism • 395 rep samples screened by PCR (<10 IU/mL) w/ confirmation by 2 PCR assays on sep aliquot • 4 HBV DNA+ detected (4/107 anti-HBs-neg) • HBV DNA load 10-100 copies/mL • Project 1 HBV DNA+ per 48,955 Tx units, similar to projected yield of HBV DNA+ pre-HBsAgWP units

  44. Infectivity of HBV DNA PCR+, HBsAg-, anti-HBc+ bloodPrince et al. Transfusion 2001 • 3 patients sampled 25-30 years post acute HBV • HBsAg-/anti-HBc+/anti-HBs+ • HBV DNA+ w/ 200-1300 copies/mL • 1.35 mL serum + 2-4 x 107 PBMC inoculated • Chimps monitored 15 months • All chimps neg for HBsAg, HBV DNA, etc.

  45. Infectivity of HBsAg (-) Hepatic Allografts with Antibodies to HBVDodson, et al. Transplantation 1997;64:1582-4 * 4/18 transmitting donors had low-titer anti-HBs

  46. Conclusions • Relationship between infectious units and RNA/DNA levels varies by stage of infection • During primary (pre-SC) infection, 1-10 gEq/mL plasma appears able to transmit infection • Ab+/NAT- donations must be presumed infectious (can’t d/c serology or reenter donors) • Need for future studies at international level • animal transmission model systems • donor lookback studies (prior units from NAT+ and Ab SC donors) • investigation of donors in TA-recipient infections

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