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1 NAT Comparative analysis of triplex nucleic acid test assays in United States blood donors Susan L. Stramer, David E. Krysztof, Jaye P. Brodsky, Tracy A. Fickett, Benjamin Reynolds, Roger Y. Dodd, and Steven H. Kleinman BACKGROUND: This study assessed the clinical sensitivity of three fully automated, human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) triplex nucleic acid test (NAT) assays by individual donation (ID-NAT) and at operational minipool (MP-NAT) sizes used worldwide. STUDY DESIGN AND METHODS: MPX, Ultrio, and Ultrio Plus were used to test 2222 pedigreed, markerpositive samples with varying viral loads, each from a unique US blood donor. NAT-positive, seronegative yield samples (16 HBV, 156 HCV, and 23 HIV) were tested in replicates of three; undiluted; and in 1:6, 1:8, and 1:16 dilutions (MP6, MP8, and MP16), simulating various MP sizes. Seropositive samples (1276 HBV, 488 HCV, and 263 HIV) were tested by ID-NAT in singlet. RESULTS: MPX-MP6 and Ultrio Plus-MP16 had equivalent HCV sensitivity. Although Ultrio Plus-MP16 for HIV trended toward lesser sensitivity, this was not corroborated in a large substudy of low-viral-load samples in which Ultrio Plus-MP8/MP16 showed reactivity. MPX-ID and Ultrio Plus-ID HBV clinical sensitivity were identical, but MPX-MP6 was significantly more sensitive than Ultrio Plus-MP16; the differential yield projected to one HBV NAT yield per 4.72 million US donations. Ultrio Plus HBV sensitivity did not increase at MP8 versus MP16. Ultrio Plus versus Ultrio sensitivity was significantly increased in HBV-infected donors with early acute, late acute or chronic, and occult infections. No difference in sensitivity was noted for any virus for MPX-MP6 versus Ultrio Plus-ID. CONCLUSIONS: Our data support US donation screening with MPX-MP6 or Ultrio Plus-MP16 since the HBV DNA detection of Ultrio Plus was significantly enhanced (vs. Ultrio) without compromising HIV or HCV RNA detection. The Food and Drug Administration (FDA) has licensed several triplex (human immunodeficiency virus Type 1 [HIV-1], hepatitis C virus [HCV], and hepatitis B virus [HBV]) automated nucleic acid amplification (NAT) systems for blood donor screening. These include two on the TIGRIS System using transcription-mediated amplification (TMA; Ultrio and Ultrio Plus, Gen-Probe, San Diego, CA; and Novartis Vaccines and Diagnostics, Emeryville, CA) and one on the cobas s201 platform using polymerase chain reaction (PCR; MPX, Roche Molecular Systems, Pleasanton, CA). In addition, the MPX assay detects HIV-2 and HIV-1 non-m subtypes. 1 Ultrio Plus has been documented to have greater analytic sensitivity for HBV DNA than Ultrio. 2,3 Although Ultrio or Ultrio Plus has been broadly implemented in an individual-donation (ID) testing format, 4 US users have continued to screen minipools (MPs) of 16 samples (MP16). MPX is used worldwide in MPs of six samples (MP6). 1 Comparisons of Ultrio and MPX assays for one or more analytes have been conducted in Thailand, France, ABBREVIATIONS: ARC = American Red Cross; ChLIA = chemiluminescent immunoassay; ID = individual donation; LOD = limit of detection; MP(s) = minipool(s); NGI = National Genetics Institute; OBI = occult hepatitis B virus infection; S/CO = sample to cutoff; TMA = transcriptionmediated amplification. From the Scientific Support Office, American Red Cross, Gaithersburg, Maryland; Quality Analytics, Riverwoods, Illinois; Creative Testing Solutions, Tampa, Florida; Holland Laboratory, American Red Cross, Rockville, Maryland; and the University of British Columbia, Victoria, British Columbia, Canada. Address reprint requests to: Susan L. Stramer, PhD, Scientific Support Office, American Red Cross Biomedical Services, 9315 Gaither Road, Gaithersburg, MD 20877; susan.stramer@redcross.org. Received for publication December 17, 2012; revision received February 6, 2013, and accepted February 13, doi: /trf TRANSFUSION 2013;53: Volume 53, October 2013 TRANSFUSION 2525

2 STRAMER ET AL. Australia (using samples from Hong Kong blood donors), and the United States (using samples from Thai blood donors). 5-9 In three evaluations, Ultrio ID-NAT was compared to MPX-MP6 to mimic operational testing formats used in their respective countries. The US study focused only on HBV detection and compared MPX-ID to Ultrio- ID, MPX-MP6 to Ultrio-ID, and MPX-MP6 to Ultrio-MP16, and evaluated Ultrio at an intermediate pool size (MP6). Reference standards, seroconversion and/or routine donor samples were used in these studies. The present investigation compares ID-NAT and MP-NAT but test performance was assessed for all three viruses and comparisons with Ultrio Plus (in a pre FDAlicensed final configuration) were also performed. Specimens for this study were obtained from a frozen repository of NAT and/or seropositive samples accumulated since 1999 by the American Red Cross (ARC). This allowed a much larger number of blood donor samples to be evaluated from persons with newly acquired, persistent, or where appropriate resolved HIV, HCV, or HBV infection versus previous comparative studies. In addition, we analyzed donor NAT prevalence rates and frequencies of various combinations of marker-positive test results. MATERIALS AND METHODS Sample selection All samples in this study were selected from ARC s repository of frozen plasma units on the basis of predefined combinations of test-of-record NAT and serology results for HIV, HCV, and HBV. Table 1 describes the repository and selected sample subgroups. NAT-yield (seronegative) samples The test of record for HIV and HCV NAT-yield samples was the Procleix HIV-1 and HCV assay (duplex) MP16; for HBV NAT-yield samples, it was Ultrio-MP16. Reactive MPs resolved to the individual reactive donation were categorized by reactivity on discriminatory assays (dhiv, dhcv, dhbv). NAT-yield samples were confirmed through additional analyte-specific reactivity using alternate NAT reagents, and repeat NAT and antibody testing, on the retrieved plasma unit and on available follow-up samples. 10,11 HBV-yield samples The HBV-seronegative donations were sourced from a 2008 clinical trial and from operational use of Ultrio starting in April ,12 From March 2008 through June 2011, of 17 confirmed HBV-yield unique donors, 16 had a frozen repository plasma unit and were included. Results for five of these donations have been reported. 10 TABLE 1. Description of sample population Dilution number of replicates tested Number tested (% of available samples)* Total number of positive donors over study period (prevalence) Total number of donors presenting over study period Period of time samples acquired Sample type HBV yield 04/03/ /06/2011 9,061, (1:533,022) 16 (94) 1 3, 6 3, 8 3, 16 3 HCV yield 07/29/ /30/ ,924, (1:96,890) 156 (84) 1 3, 6 3, 16 3 HIV yield 02/29/ /13/ ,792, (1:544,566) 23 (79) 1 3, 6 3, 16 3 HBsAg Pos; anti-hbc NR 05/16/ /04/2009 6,706, (1:20,762) 15 (5) 1 1 HBsAg Pos; Anti-HBc RR 01/08/ /09/2009 7,346,020 1,933 (1:3,800) 511 (26) 1 1 Anti-HBc RR 10/07/2004-4/20/2009 9,698,682 77,076 (1:126) 750 (1) 1 1 Anti-HCV Pos 11/29/ /31/2009 4,818,066 3,578 (1:1,347) 488 (14) 1 1 Anti-HIV Pos 12/01/ /09/2009 6,629, (1:12,973) 263 (52) 1 1 * Each sample came from a unique donor; either all donors with an available plasma unit were used or a targeted number were used. 1 = neat, 6 = 1:6, 8 = 1:8, and 16 = 1:16. Includes both ARC and non-arc donors. A total of 205 samples were previously found to be HBV DNA reactive. 1:130,000 is the adjusted rate for 15 (16%) donors with HBV naturally occurring acute infection. NR = nonreactive; Pos = confirmed positive; RR = repeat reactive TRANSFUSION Volume 53, October 2013

3 NAT HBV, HCV, AND HIV SENSITIVITY COMPARISON HCV-yield samples From July 1999 through June 2008, of 185 confirmed HCVyield unique donors, 156 had a frozen repository plasma unit and were included. HIV-1 yield samples From February 2000 through August 2007, of 29 confirmed HIV-1 yield unique donors, 23 had a frozen repository plasma unit and were included. Seropositive samples Hepatitis B surface antigen confirmed-positive samples These donations were reactive by hepatitis B surface antigen (HBsAg) enzyme immunoassay (EIA; Auszyme monoclonal procedure C EIA, Abbott Laboratories, Abbott Park, IL) or, after November 2006, by chemiluminescent immunoassay (ChLIA; Abbott PRISM) and positive by the companion neutralization protocol. Of 2256 unique donors who met this criterion from January 2006 to January 2009, a total of 606 units were evaluated further consisting of two subgroups based on their anti-hbc results (Ortho anti-hbc enzyme-linked immunosorbent assay [ELISA], Ortho Clinical Diagnostics, Raritan, NJ; or Abbott PRISM HBcore after November 2006). The anti- HBc reactive subgroup consisted of 511 randomly selected donors. For anti-hbc nonreactive donors, 95 plasma units were available. Additional testing to confirm HBV infection from the 95 donors included repeating HBsAg testing and neutralization, as appropriate, and HBV DNA PCR (UltraQual 1000, National Genetics Institute [NGI], Los Angeles, CA). The exclusion of HBsAgpositive, anti-hbc nonreactive, HBV DNA negative donors with true infection was negligible. 12 Vaccination history was ascertained by follow-up interviews of donors by regional staff. 12,13 Samples were defined as false positive when signal levels were low and neutralization was observed only in a 1:500 sample dilution but not in the undiluted sample (and thus was due to processing contamination or variability in generated results that are close to the assay cutoff in the 1:500-diluted sample pair, occasionally resulting in the erroneous appearance of neutralization). 12,13 This further analysis demonstrated naturally acquired HBV infection in only 15 (16%) of 95 donors. Thus, the final HBsAg confirmed-positive sample set consisted of 526 samples (511 anti-hbc reactive and 15 anti- HBc nonreactive). Anti-HBc-reactive samples These donations were reactive by anti-hbc EIA or ChLIA (Ortho anti-hbc ELISA or Abbott PRISM HBcore after November 2006) but nonreactive for HBsAg. Of the 77,076 unique donors who met this criterion from October 2004 to April 2009, 750 units including 205 donors who were identified as HBV DNA positive by routine supplemental PCR under a standard ARC protocol for evaluation of anti- HBc reactivity were studied. This protocol used the Ampliscreen assay (Roche Molecular Systems), with the 1-mL multiprep sample preparation technique and triplicate testing having a 95% limit of detection (LOD) of approximately 1 to 2 IU/mL. 14 The remaining 545 samples were randomly selected from anti-hbc reactive donors on two separate occasions but HBV DNA negative in the supplemental testing algorithm. Anti-HCV confirmed-positive samples These donations were reactive by anti-hcv EIA (Ortho v3.0 ELISA, Ortho Diagnostics) and positive by recombinant immunoblot (RIBA 3.0, Novartis). Of 3578 unique donors who met these criteria from November 2007 to May 2009, a total of 488 randomly selected units were studied including those testing MP-NAT nonreactive but repeat antibody confirmed positive. Anti-HIV-1 confirmed-positive samples These donations were reactive by anti-hiv-1 and HIV-2 EIA (Abbott HIV-1/HIV-2 rdna EIA) and positive by either HIV-1 Western blot (Calypte Biomedical, Rockville, MD) or immunofluorescence assay (Sanochemia, Vienna, Austria). Of 511 unique donors who met these criteria from December 2006 through May 2009, a total of 263 units were studied including those testing MP-NAT nonreactive but repeat antibody confirmed positive. Sample preparation Plasma units were initially frozen according to standard operating procedures for frozen plasma component manufacture and shipped to the ARC Scientific Support Office for further processing. Samples were thawed and aliquots of 3 ml were prepared without dilution and, for NAT-yield donations, at dilutions of 1:6, 1:8, and 1:16 (MP6, MP8, and MP16), prepared using pedigreed HIV-, HCV-, and HBV-negative plasma. Samples were labeled under code and refrozen so that further testing was performed without knowledge of prior test results or sample dilution. Samples underwent no more than three freezethaw cycles, in compliance with test kit sample handling requirements. Initial study testing Frozen aliquots were shipped to Creative Testing Solutions in St Petersburg, Florida. A separate coded aliquot was used for each test performed. Two automated NAT assay systems were utilized: the Procleix Ultrio assay on the TIGRIS platform and the Roche MPX assay on the s201 platform. Commercially obtained FDA-licensed reagents were used; all tests were performed by personnel trained Volume 53, October 2013 TRANSFUSION 2527

4 STRAMER ET AL. in the use of each system according to the manufacturer s instructions. All seropositive samples were submitted undiluted; that is, they were tested by ID-NAT in singlet on both testing platforms. For NAT-yield samples, three replicates were tested undiluted, and in MP6, by each system, as well as by Ultrio-MP16. In addition, HBV-yield samples were tested by Ultrio-MP8. Early in the study, a research version of the Procleix Ultrio Plus assay became available. Since this test had not yet been FDA licensed, testing could not be conducted by Creative Testing Solutions, but instead was performed under code using final reagent formulations at Gen-Probe. NAT-yield and seropositive samples were tested consistent with the remainder of the protocol. Quantitative viral load determinations HIV, HCV, and HBV viral loads were obtained using commercial PCR assays at NGI (SuperQuant, 100 copies/ml limit of quantitation; or UltraQual-1000, 5 copies/ml LOD). SuperQuant nonreactive samples were further tested by UltraQual HCV RNA confirmed-positive, antibody-negative samples were genotyped at NGI using assays manufactured by InnoLIA (InnoLIPA, InnoLIA, Ghent, Belgium). Further study testing: HIV-yield sample substudy Of the 23 HIV-yield samples, 17 with low viral loads (< copies/ml) were selected for additional testing to further evaluate HIV detection by Ultrio versus Ultrio Plus. ARC prepared multiple sample aliquots at 1:8 and 1:16 dilutions. Twelve 3-mL aliquots per donor were shipped to Gen-Probe: three each for testing by Ultrio- MP8 and MP16 and three each for testing by Ultrio Plus- MP8 and MP16. Each aliquot was pipetted into five separate test wells by the TIGRIS, thereby producing 15 results at each dilution for each assay. The rationale was that detection in at least 14 of the 15 replicates indicated an observed detection rate of approximately 95% with a lower 95% confidence interval (CI) of greater than 68%. Statistical analysis NAT-yield samples were tested in triplicate on each of three test platforms at multiple pool sizes as described above. The results were analyzed using a modified approach to McNemar s test 15 for analyzing clustered matched-pair data as described by Durkalski and colleagues. 16 This method produces a chi-square statistic that is evaluated with one degree of freedom. Given the number of sample configurations and test methods included, we limited the total number of pairwise comparisons to only those of key interest: MPX-ID versus Ultrio Plus-ID, MPX-MP6 versus Ultrio Plus-ID or Ultrio Plus-MP16, and Ultrio-MP16 versus Ultrio Plus-MP16. Further analyses included only Ultrio Plus comparisons of: ID, MP6 or MP8 to MP16. For seropositive samples, tested undiluted in singlet on the same three test platforms, each test method was compared to the other two methods using the traditional McNemar s test with continuity correction for matched (paired) data (QuickCalcs, GraphPad Software, Inc., online tool graphpad.com/quickcalcs/mcnemar2.cfm). RESULTS Published analytic sensitivities for the viral targets Table 2A provides a summary of HBV DNA analytic sensitivities of the commercially available triplex NAT assays. These data are limited to those studies that used wellcharacterized WHO stock preparations. HBV DNA analytic sensitivity at a 95% LOD is 10 to 15 IU/mL (95% range, 8-40 IU/mL) for Ultrio, 2 to 4 IU/mL (95% range, 2-10 IU/ ml) for Ultrio Plus, and approximately 4 IU/mL (95% range, 3-8 IU/mL) for MPX. Thus, Ultrio Plus is more sensitive for HBV DNA detection than Ultrio, and Ultrio Plus and MPX HBV DNA sensitivity is comparable when testing by ID-NAT. As seen in Table 2B, the published HCV analytic sensitivities (95% LOD with 95% CI) for Ultrio and Ultrio Plus are equivalent (2-6 IU/mL), whereas that of MPX is approximately two- to threefold lower (7-22 IU/ ml). Similarly, for HIV-1, the analytic sensitivity for Ultrio and Ultrio Plus, according to the package insert studies, is also equivalent (16-40 IU/mL); however, that of MPX is approximately two- to threefold lower (42-58 IU/mL). Overall sample design and criteria for evaluation Table 1 summarizes the ARC repository and relevant samples. More than 2200 samples were included with more than 10,300 test results generated in eight different result categories. Donor prevalence rates were the lowest for confirmed NAT-yield (seronegative) donors ranging from approximately 1:500,000 for HIV and HBV to 1:100,000 for HCV (Table 1, Fig. 1). This is followed by a rate of 1:20,000 for HBsAg-positive, anti-hbc nonreactive donors (a mixture of acute infections, recent HBV vaccinees, and donors testing falsely positive). However, only 16% of such samples had verified acute infection; thus the adjusted rate for HBsAg-positive donor prevalence due to naturally occurring acute infection is 1:130,000 (see Materials and Methods). The frequency of HBsAg-positive, anti-hbc reactive donors was 1:3800 (reactivity to two or more HBV markers is considered indicative of active HBV infection). Antibody-positive donors had the highest prevalence rates, with 1:13,000 for anti-hiv positive donors, 1:1300 for anti-hcv positive donors, and 1:130 for anti-hbc reactive donors having reactivity on more than one occasion. These donor groups include those with 2528 TRANSFUSION Volume 53, October 2013

5 NAT HBV, HCV, AND HIV SENSITIVITY COMPARISON Evaluation, assay, and source* Gen-Probe Ultrio (US); WHO Gen-Probe Ultrio (ex-us); WHO Gen-Probe Ultrio Plus (US); WHO Gen-Probe Ultrio Plus (ex-us); WHO Ultrio Plus combined studies (ex-us) WHO Roche TaqScreen MPX (US); WHO TaqScreen MPX combined studies* (ex-us) WHO TABLE 2A. Analytic sensitivity by probit analysis of HBV NAT methods in current use Number of System replicates studied 50% IU/mL LOD (95% CI) 95% IU/mL LOD (95% CI) TIGRIS ( ) 10.4 ( ) TIGRIS ( ) 14.7 ( ) TIGRIS ( ) 3.4 ( ) TIGRIS ( ) 2.1 ( ) TIGRIS ( ) 4.4 ( ) Cobas s NA 3.8 ( ) Cobas s ( ) 4.6 ( ) * Two different WHO standards were used in these evaluations; both are derived from the same standard diluted in cryoprecipitate-removed, supernatant plasma. The only difference between the two is the freeze drying-run. Probit analysis on combined data using dilution panels prepared by BioQControl/DDL, Rijswijk, the Netherlands, and Hong Kong Red Cross Blood Transfusion Service; analysis performed by Van Drimmelen, BioQControl/DDL. Probit analysis on combined data using the Roche package insert according to Assal and colleagues. 21 NA = not available. The 50 and 95% IU/mL lower LOD and associated CI for each appears in bold font. TABLE 2B. Analytic sensitivity by probit analysis of HCV and HIV-1 NAT methods in current use Number of Evaluation, assay, or source* System replicates studied 50% IU/mL LOD (95% CI) 95% IU/mL LOD (95% CI) TIGRIS ( ) 3.0 ( ) Gen-Probe Ultrio (US); HCV WHO TIGRIS ( ) 3.9 ( ) Gen-Probe Ultrio (ex-us); HCV WHO TIGRIS ( ) 4.4 ( ) Gen-Probe Ultrio Plus (US); HCV WHO TIGRIS ( ) 3.1 ( ) Gen-Probe Ultrio Plus (ex-us); HCV WHO Roche Cobas s NA 11.0 ( ) TaqScreen MPX (US); HCV WHO TIGRIS ( ) 20.3 ( ) Gen-Probe Ultrio (US); HIV WHO TIGRIS ( ) 25.8 ( ) Gen-Probe Ultrio (ex-us); HIV WHO TIGRIS ( ) 18.9 ( ) Gen-Probe Ultrio Plus (US); HIV WHO TIGRIS ( ) 27.6 ( ) Gen-Probe Ultrio Plus (ex-us); HIV WHO Roche TaqScreen MPX (US); HIV WHO Cobas s NA 49 ( ) * Two different WHO standards were used in these evaluations; both are derived from the same standard diluted in cryoprecipitate-removed, supernatant plasma. The only difference between the two is the freeze-drying run. NA = not available. The 50 and 95% IU/mL lower LOD and associated CI for each appears in bold font. Volume 53, October 2013 TRANSFUSION 2529

6 STRAMER ET AL. Fig. 1. Comparison of HIV-1, HCV, and HBV donor prevalence rates. RNA or DNA positive in the absence of serologic marker reactivity. Pos = confirmed positive; RR = repeat reactive. active infection (all categories); resolved infection (anti- HCV); true infection but MP-NAT nonreactive at the level of sensitivity provided by MP-NAT (anti-hbc, anti-hcv, and anti-hiv); and those testing falsely positive (HBsAg and anti-hbc). False positivity for HBsAg was addressed above, but also occurs for anti-hbc reactives since no antibody confirmatory test exists; this rate has been estimated at approximately 40% before the introduction of ChLIA. 14 NAT-yield (seronegative) sample comparisons HBV-yield samples (Table 3A) Of the 16 samples, 15 were initially identified by MP-NAT while one was identified by ID-NAT (100 copies/ml); this donor also had low-level anti-hbs reactivity (15.42 IU/L). Median viral loads were 200 copies/ml (range, <100 to 7900 copies/ml); only four samples had viral loads greater than 500 copies/ml ( copies/ml). Low viral loads were also evident by variable sample-to-cutoff (S/CO) values generated by test-of-record MP-NAT (Ultrio-MP16): S/CO values for samples with 300 copies/ml or fewer ranged from 4 to 18 by MP16 versus consistently higher for the Ultrio ID-dHBV discriminatory assay. Seven of 16 HBV-yield donors had detectable anti-hbs (7-97 IU/L). Of the 16 yield donors, five were previously described, four of which were HBV vaccine breakthrough infections, 10 similar to those described in subsequent studies. 7,8 Assay sensitivity decreased with increasing dilution from a maximum of detection for all sample replicates when tested ID (n = 48 for MPX) to a minimum of 56% with Ultrio-MP16. MPX-MP6 and Ultrio Plus-ID each detected 94% of sample replicates. HBV DNA detection was higher for Ultrio Plus compared to Ultrio, especially at MP8 and MP16. Ultrio Plus-MP8 detected 83% of replicates with four of 16 unique donor samples having one or more nonreactive replicates. Ultrio Plus-MP16 detected 75% of replicates with six samples having one or more nonreactive replicates; however, Ultrio- MP16 detected only 56% of replicates with 10 samples having variable detection. One donor with HBV vaccine breakthrough infection (<100 copies/ ml) and low-level anti-hbs reactivity (15.42 IU/L) showed limited detection by both Ultrio and Ultrio Plus and variable detection by MPX-MP6 including when 10 additional replicates of this sample were retested (Table 3A). This variability in detection likely reflects the low viral load of this sample and variations inherent in sample preparation and testing. There was no significant difference between MPX and Ultrio Plus when samples were tested undiluted (p = 0.179) or when MPX-MP6 was compared to Ultrio Plus-ID (p = 1.000); however, MPX-MP6 was more sensitive than Ultrio Plus-MP16, with six samples more frequently reactive by MPX-MP6 (p = 0.028). There was no significant difference in sensitivity for Ultrio Plus-MP8 versus MP16 (p = 0.157), although four additional replicates were detected at MP8. Significantly higher detection rates were observed for Ultrio Plus-MP16 versus Ultrio- MP16 (p = 0.029) and for Ultrio Plus-ID versus Ultrio Plus-MP16 (p = 0.020); Ultrio Plus-ID was the most sensitive assay or format among the Ultrio Plus and Ultrio comparisons. HCV-yield samples (Table 3B) Viral loads for 156 donors ranged from fewer than 100 to 380 million copies/ml, with a median of 2.8 million copies/ml. Subgenotypes were available for 152 samples: 88 (58%) were Subgenotype 1A or 1B, 28 (18%) were 2A or 2B, and 36 (24%) were 3A. No significant difference in sensitivity was observed among all assays and at all dilutions tested (99%- detection). Four donors had variable detection by all three assay systems when diluted (including the one with <100 copies/ml). HIV-yield samples (Table 3C) Viral loads for 23 donors ranged from 200 to 7 million copies/ml, with a median of 3800 copies/ml. No signifi TRANSFUSION Volume 53, October 2013

7 NAT HBV, HCV, AND HIV SENSITIVITY COMPARISON TABLE 3A. Detection of 16 HBV-yield samples* (number reactive replicates/number tested) Dilution MPX Ultrio Ultrio Plus ID 48/48 44/48 92% 45/48 94% MP6 45/48 94% NT 43/48 90% MP8 NT 33/48 69% 40/48 83% MP16 NT 27/48 56% 36/48 75% * Of the 16 yield donors, five were previously described, four of which were HBV vaccine breakthrough cases. 10 For Ultrio Plus, two of three nonreactive replicates came from the same donor sample; similar results occurred with Ultrio; however, three of three replicates of this sample were reactive by MPX-MP6. MPX-MP6 retesting of 10 additional replicates from the same sample yielded only one reactive replicate (data not shown). For MPX-MP6, nonreactives included one replicate from three different samples. MP6 = 1:6 dilution, MP8 = 1:8 dilution, and MP16 = 1:16 dilution; NT = not tested. TABLE 3B. Detection of 156 HCV-yield samples (number reactive replicates/number tested) Dilution MPX Ultrio Ultrio Plus ID 460/460* 468/ /468 99% MP6 464/467* 99% NT 464/46 99% MP16 NT 464/468 99% 463/468 99% * Of 468 replicates, eight were invalid by MPX-ID and one by MPX-MP6; there was no sample where all three replicates were invalid. Most of the nonreactive replicates came from a single yield donor. This sample accounted for two nonreactive replicates on Ultrio Plus-ID and MPX-MP6 and three nonreactive replicates on Ultrio-MP16 and Ultrio Plus-MP6 and MP16. MP6 = 1:6 dilution, MP8 = 1:8 dilution, and MP16 = 1:16 dilution; NT = not tested. TABLE 3C. Detection of 23 HIV-1 yield samples (number reactive replicates/number tested) Dilution MPX Ultrio Ultrio Plus ID 69/69 69/69 69/69 MP6 68/69 99% NT 69/69 MP16 NT 64/69 93% 61/69 88% MP6 = 1:6 dilution, MP8 = 1:8 dilution, and MP16 = 1:16 dilution; NT = not tested. cant difference in sensitivity was observed among all assays when samples were tested undiluted or at MP6 (99%- detection). Although lower HIV sensitivity was observed at MP16 (88%-93% sensitivity for Ultrio Plus and Ultrio), the difference in detection between MPX-MP6 and TABLE 4. Detection of 526 HBsAg confirmed-positive and 750 anti-hbc reactive samples (number reactive/number tested) Sample type MPX Ultrio Ultrio Plus HBsAg positive Anti-HBc-NR 15/15 15/15 15/15 HBsAg positive Anti-HBc RR 495/510* 97% 463/511 90% 502/511 98% HBsAg NR anti-hbc RR 55/750 7% Ultrio Plus-MP16 was nonsignificant (p = 0.052). These two assay configurations gave discordant results on five samples, including one MPX-MP6 nonreactive replicate and eight Ultrio Plus-MP16 nonreactive replicates (one or two nonreactive replicates per sample). Similarly, Ultrio- MP16 showed nonreactivity on at least one replicate for three of the same five samples with an additional inconsistently reactive sample (p = vs. Ultrio Plus-MP16). In contrast, all sample replicates were reactive by Ultrio Plus-MP6, representing a significant improvement in detection versus Ultrio Plus-MP16 (p = 0.033). Thus, in total, six samples (with viral loads of copies/ml) had at least one discordant replicate at MP6 or MP16. One sample having 260 copies/ml had one nonreactive replicate by MPX-MP6 and by Ultrio and Ultrio Plus-MP16. Ultrio Plus-MP16 had the lowest sensitivity with borderline significance in some comparisons; thus additional testing was performed to assess the reproducibility of this finding, using 17 of the 23 HIV-yield samples (viral loads <2 million copies/ml) including the six discordant samples mentioned above. Each was tested in replicates of 15 by Ultrio and Ultrio Plus-MP8/MP16. Ultrio Plus-MP8/ MP16 detected all replicates of all samples as reactive. Ultrio-MP8/MP16 each generated one nonreactive replicate for one sample and a nonreactive replicate for a second sample at MP8 (S/CO values, 0.03, 0.08, and 0.99, respectively); viral loads were 200 and 790 copies/ml, respectively. Thus, all 17 samples met the minimum detection criteria of 95% detection by both assays at both dilutions (see Materials and Methods). Seropositive samples 27/750 4% 46/750 6% * One invalid sample replicate. A total of 205 (27%) samples were previously found to be HBV DNA reactive by the routine supplemental ARC testing algorithm. NR = nonreactive; RR = repeat reactive. HBsAg and anti-hbc Three categories of HBV-seropositive donors were evaluated (Tables 1 and 4). The 15 HBsAg confirmed-positive, acutely infected donors had viral loads ranging from 1500 to 46 billion copies/ml and a median of 480,000 copies/ml, the highest viral loads of all sample categories. All 15 samples Volume 53, October 2013 TRANSFUSION 2531

8 STRAMER ET AL. TABLE 5. Detection of 488 anti-hcv and 263 anti-hiv confirmed-positive samples (number reactive/number tested) Sample type MPX Ultrio Ultrio Plus Anti-HCV 375/488 77% 380/488 78% 383/488 78% Anti-HIV 243/254* 95% * Nine invalid sample replicates. 253/263 96% 257/263 98% were reactive by all assays. Viral loads correlated well with HBsAg S/CO results (data not shown). For the 511 HBsAg-positive, anti-hbc reactive donors, MPX detected 495 (97% sensitivity with one invalid sample); similarly, Ultrio Plus detected 502 (98% sensitivity; p = vs. MPX), but Ultrio detected only 463 (90% sensitivity; p < vs. MPX and vs. Ultrio Plus). Of the 53 samples (10%) with discordant or concordantly nonreactive results between assays, nine were nonreactive by Ultrio Plus, 15 were nonreactive by MPX, and 48 were nonreactive by Ultrio (including five concordantly nonreactive by all three assays). Viral loads for the 53 discordant samples ranged from fewer than 100 to 1100 copies/ml with 16 having quantifiable viral loads of 100 or greater and a median of 200 copies/ml (PRISM HBsAg S/CO values for these 16 ranged from 28 to >600). All five concordantly nonreactive samples had viral loads of fewer than 100 copies/ml (PRISM HBsAg S/CO values, 1.27, 1.58, 10.52, , and ). Overall, 37 of the 53 discordants had fewer than 100 copies/ml (all with PRISM HBsAg S/COs of <17). Of the 750 anti-hbc reactive donors, MPX detected 55 (7%), Ultrio 27 (4%), and Ultrio Plus 46 (6%). Stated differently, MPX detected 28 additional samples compared to Ultrio (p < ), and Ultrio Plus detected 19 additional samples compared to Ultrio (p = ). The difference in MPX compared to Ultrio Plus was not significant (p = ). Of the 73 donor samples reactive by any method, 20 were concordantly reactive by all methods; among the 53 discordantly reactive samples, 35 were reactive by MPX, 26 by Ultrio Plus, and seven by Ultrio. The NAT test of record (i.e., the Ampliscreen multiprep method) used for routine confirmation of anti-hbc reactivity detected 38 of the 53 as reactive. Viral loads could be obtained for 17 samples ranging from 100 to 4700 copies/ ml, with a median of 447 copies/ml. Anti-HCV Of the 488 samples, 365 (75%) were reactive by test-ofrecord MP-NAT. ID-NAT performance was comparable among all three assays with 77% detection for MPX (375 reactive samples) and 78% for both Ultrio and Ultrio Plus ( reactive samples, respectively; Table 5); no significant differences were noted between assays. Of the 488 samples tested, 117 (24%) were nonreactive by one or multiple assays; 25 were reactive by at least one assay. All 117 had viral loads less than 100 copies/ml (anti-hcv S/COs ranged from 3.74 to >16). Anti-HIV Of the 263 available samples, nine gave invalid results on MPX. Thus, of 254 samples tested by MPX, 243 (95%) were reactive; reactivities of the 263 were 253 (96%) and 257 (98%) for Ultrio and Ultrio Plus, respectively (Table 5). No significant difference in detection between assays was observed. A total of 13 samples had discordant or concordant nonreactive ID-NAT results (Table 6). Ten of these donors had viral load determinations at index and follow-up and all were less than the LOD, either 5 or 100 copies/ml depending on the assay used (see Materials and Methods). Anti-HIV S/CO values were uniformly high, at or above the upper limit of the assay s dynamic range. Nine of the 13 donors were also HIV RNA nonreactive by routine screening; of the other four who were reactive with TMA- MP16, one was dhiv nonreactive. Ten donors (the nine who were MP16 nonreactive and the one who was dhiv nonreactive) were studied as part of an ARC follow-up study of HIV discordant donors. Their age range was 17 to 56 years, nine were first-time donors, four were male, and all were persistently anti-hiv positive and RNA negative when followed. Three of the 10 had low-level RNA reactivity (10%-50%) observed when the plasma unit was retested by Ultrio-ID in replicates of 10 (data not shown). HIV-infected persons who suppress their viral infection without receiving antiretroviral drugs are referred to as elite controllers. 24,25 Many, if not all, of our donors in this study would be similarly classified. Based on TMA-MP16 test-of-record results, the RNA-negative rate among anti- HIV confirmed-positive donors was 3.8% (10 of 263) or a rate within the overall ARC population of 1:342,000. When considering the three ID-NAT assays, the rate of RNA negatives among anti-hiv positives was 4.2% with MPX, 3.8% with Ultrio, and 2.3% with Ultrio Plus. Only five donors were nonreactive by all three assays for a rate of 1.9%, projecting to an RNA-negative rate of 1:684,000. A summary of significance testing for all assay comparisons is provided in Table 7. DISCUSSION Fully automated triplex (HIV, HCV, and HBV) NAT assays have been available in the United States since Guidance requiring HIV-1 and HCV NAT was issued in In November 2012, FDA issued guidance adding HBV DNA detection by NAT at a minimum sensitivity of 100 IU/mL (approx. 500 copies/ml), 18 and after the availability of MPX-MP6 and Ultrio Plus-MP16. While these two assays will meet the FDA requirement, the published 2532 TRANSFUSION Volume 53, October 2013

9 NAT HBV, HCV, AND HIV SENSITIVITY COMPARISON Sample TABLE 6. Detection of 13 anti-hiv confirmed-positive samples with negative or discordant NAT results* Test-of-record anti-hiv-1/2 (S/CO) Test-of-record MP-NAT S/CO; if reactive (S/CO > 1.00), ID-NAT and dhiv-nat S/COs are listed MPX Ultrio (S/CO) Ultrio Plus (S/CO) (-) > (-) > (-) > (-) > (-) (-) > /2.55/0.29 (-) > (-) > (+) > /10.41/15.58 (-) > /9.19/12.89 (-) > /12.33/24.93 (-) > (+) * Of 263 anti-hiv confirmed-positive samples tested, 13 (4.9%) were RNA equivocal or negative initially as part of this study. Samples were considered antibody confirmed positive at index if repeat anti-hiv confirmed positive in a sample from the retrieved plasma unit; of 10 donors with follow-up, all were anti-hiv confirmed positive. The total number reactive samples of the 13 by NAT assay were two (MPX), three (Ultrio), and seven (Ultrio Plus). Five of the 13 samples remained RNA negative upon a single ID-NAT retest in each of the three assay systems. Ten samples had viral load determinations; all 10 had viral loads of fewer than 5 or fewer than 100 copies/ml (assay dependent). All reactive/positive values are in bold font. 95% LOD of Ultrio at 10.4 IU/mL (Table 2A) indicates that this assay will not meet this new requirement if used as MP16. Based on the goal of maximizing the detection of units with low-level HBV DNA, most TIGRIS users outside the United States have transitioned to Ultrio and Ultrio Plus-ID. 4 However, MP-NAT remains in use in the United States since HBV is not endemic, anti-hbc testing is in place, and HBV vaccine is universally recommended. To date, assessments have failed to justify the logistic changes and costs associated with changing to a smaller pool size or ID-NAT. 9,19,20 The goal of this study was to assess the sensitivity of the available triplex NAT assays at the operational pool sizes used worldwide to inform appropriate policy decisions. The comparisons included HBV, HIV, and HCV to ensure that the different assay configurations had adequate sensitivity for all analytes. This robust study used over 2200 routine donor samples covering a wide range of viral loads and serologic profiles. Although one limitation of the study was the sourcing of NAT-yield samples by MP16, there was a large representation of low viral load samples in both the NAT-yield and the seropositive categories. Within the United States, the most relevant comparisons are MPX-MP6 versus Ultrio and Ultrio Plus-MP16. A related issue is whether it is necessary for TIGRIS users to decrease their MP size from 16 to 8 or 6 to achieve increased sensitivity. Given an anticipated conversion from Ultrio to Ultrio Plus in the United States, we focused our analyses on Ultrio Plus. The comparison with regard to NAT-yield samples showed different results for each analyte. For HCV, MPX-MP6 and Ultrio Plus-MP16 showed equivalent sensitivity. For HIV, the decreased detection by Ultrio Plus-MP16 trended toward significance (p = 0.052; Tables 3C and 7) and an intermediate pool size compared to MP16 showed a significant increase in detection (p = for Ultrio Plus-MP6 vs. Ultrio Plus-MP16). However, these observations were not corroborated in a robust and statistically well-powered substudy that tested a large number of replicates focusing on HIV NAT-yield samples having the lowest viral loads, including six discordant samples. In the substudy, Ultrio Plus-MP8 and MP16 detected of all replicates, equivalent to the performance of each assay performed ID or at MP6 in the initial study. The substudy conclusion of comparable HIV assay sensitivity between MPX-MP6 and Ultrio Plus-MP16 is also supported by analytic sensitivity data (MPX with twoto threefold lower 95% analytic sensitivity but used in a pool size that is 2.67-fold smaller). These findings demonstrate that a robust comparison of assay performance requires the testing of large numbers of replicates, as shown in previous studies comparing HIV and HCV-NAT assays. 9,20,21 Significantly greater detection of HBV-yield samples was observed for MPX-MP6 versus Ultrio Plus-MP16; this is supported by analytic sensitivity data showing equivalent ID-NAT 95% LOD between assays; enhanced sensitivity of MPX-MP6 is attributable to the 2.67-fold smaller pool size. Although this difference in assay sensitivity at operational pool sizes could have an impact on HBV transmission, this effect is expected to be minimal given the very low incidence of acute, HBV NAT-yield infections in US blood donors. 10 Previously, we calculated the difference in yield between MPX-MP6 and Ultrio-MP16 as 1:1.43 million donations using a 30-day HBV-infectious window period, ARC donor incidence of 3.43 per 100,000 person-years, and window period reduction of 12.8 days for MPX-MP6 and 5.3 days for Ultrio-MP16. 9 Similarly, we Volume 53, October 2013 TRANSFUSION 2533

10 STRAMER ET AL. TABLE 7. Summary of significance testing by p values reported in the text* ID comparisons Dilutions Ultrio Plus-MP16 vs. Ultrio-MP16 Ultrio Plus-MP6 vs. Ultrio Plus-MP16 Ultrio Plus-MP8 vs. Ultrio Plus-MP16 Ultrio Plus-ID vs. Ultrio Plus-MP16 MPX-MP6 vs. Ultrio Plus-MP16 MPX-MP6 vs. Ultrio Plus-ID Ultrio Plus vs. Ultrio MPX vs. Ultrio MPX vs. Ultrio Plus Marker HBV yield Not done Not done Not done HCV yield Not done Not done Not done HIV yield Not done HBV acute HBsAg/anti-HBc <0.001 <0.001 Anti-HBc < Anti-HCV Anti-HIV * The comparisons of key interest for the NAT yield samples discussed in the text are MPX-ID versus Ultrio Plus-ID, MPX-MP6 versus Ultrio Plus-ID, MPX-MP6 versus Ultrio Plus-MP16, and Ultrio-MP16 versus Ultrio Plus-MP16. Results for given comparisons were identical. p < 0.05; p values demonstrating significant differences are in bold font. can project the difference in yield between MPX-MP6 and Ultrio Plus-MP16 as 1:4.72 million donations using an increase in window period reduction of 2.7 days for Ultrio Plus versus Ultrio 26 or 8-day total window period reduction for Ultrio Plus-MP16 and updated ARC donor incidence of 1.62 per 100,000 person-years (ARC, unpublished). The increased sensitivity of Ultrio Plus versus Ultrio was demonstrated with three of the four HBV sample sets (Tables 3A, 4, and 7) representing donors with early acute infections including those with vaccine breakthrough (p = 0.029), late acute or chronic infections in which both HBsAg and anti-hbc were present (p < 0.001), and from donors with occult HBV infection (OBI 22,23,27 ) having a median viral load of fewer than 500 copies/ml (p = ). Although HBV sensitivity may be enhanced by decreasing pool sizes for Ultrio Plus, in this study, Ultrio Plus-MP8 versus MP16 showed no significant difference for detection of HBV-yield donors (p = 0.157; Tables 3A and 7). The data also show that Ultrio-MP8 has equivalent sensitivity to Ultrio Plus-MP16 (detection of 69 and 75%, respectively). Thus, consistent with the results of an earlier study, 9 we are not advocating the use of Ultrio-MP6 or MP8. Comparisons of Ultrio Plus-ID versus MPX-MP6 demonstrated no difference in detection of NAT-yield samples for any of the three viruses studied. These results agree with previous MPX-MP6 versus Ultrio-ID studies, 5-9 three of which tested samples from HBV-endemic areas. 6-9 Although analytic sensitivity data suggests a sixfold enhancement for Ultrio Plus-ID due to the dilution factor for MPX-MP6, no such differences were observed. This may be because such differential detection would only occur if donors were to present in a very small window or that our initial MP16 sample selection strategy was not sensitive enough versus other studies sourcing samples via ID-NAT. 26,28 Despite differences in analytic sensitivity, we found no differences among the assays in the ID format when testing HIV and HCV clinical samples. Consistent with data generated from HBV analytic sensitivity panels, MPX-ID versus Ultrio Plus-ID demonstrated no significant difference in HBV DNA detection for acute-phase yield samples and HBV-seropositive samples. In contrast, Ultrio Plus versus Ultrio demonstrated significant improvements in HBV DNA detection in yield donors, HBsAg-positive and anti-hbc reactive donors, and OBI donors. As shown in this and other studies, ID-NAT was the most sensitive configuration for the detection of donors with low viral loads for HBV and HIV. 26,28 In contrast, for HCV, our study showed no increased detection by ID-NAT versus MP-NAT; this is due to the long RNApositive, seronegative window and the establishment of high viral loads early in the window period given the very short (10.8-hour) HCV doubling time. 11,19, TRANSFUSION Volume 53, October 2013

11 NAT HBV, HCV, AND HIV SENSITIVITY COMPARISON In addition to evaluating comparative assay sensitivity, our data also define viral marker prevalence rates in US blood donors and NAT positivity in seropositive donors. In this study, versus prior studies, 30,31 we report prevalence rates on a per-donor rather than a per-donation basis (Fig. 1). We found that detection of HCV acutely infected donors was fivefold greater than that of HIV or HBV; similarly, anti-hcv in donors was fourfold more prevalent than anti-hiv. The frequency of HBV infection varied depending on the stage of infection. As has been previously reported, caution must be taken when calculating HBV prevalence rates based on HBsAg-positive donors lacking other HBV screening markers since vaccine-derived HBsAg, false positivity due to crosscontamination during assay processing, and erroneous neutralization interpretations inherent in the procedure (as described in Materials and Methods) are common. 12,13 Thus, verified HBV infection was observed in less than 20% of HBsAg-confirmed-positive donors lacking anti- HBc. Overall, the trends in these data are consistent with those previously described for donations in the United States, in that HCV is the most prevalent followed by HBV and finally HIV. 13,30,31 We observed that 20% to 30% of anti-hcv positive donors lacked detectable HCV RNA by ID-NAT. We interpret this as an indication of viral clearance rather than of chronic persistent infection Anti-HBc reactive donors previously testing HBV DNA positive (i.e., OBI donors) were included in this study at a disproportionately high rate to assess the ability of the various ID-NAT assays to detect samples with low HBV viral loads. Ultrio Plus and MPX detected HBV DNA at a rate of 6% to 7% in our OBI-enriched, anti-hbc sample set; this was significantly higher (p < 0.001) than the 4% observed with Ultrio. As reported previously, OBI prevalence in anti-hbc reactive ARC donors is 0.95%. 12 Finally, we report the occurrence of HIV antibody positive donors who suppress their HIV infection (i.e., elite controllers); these HIV-infected individuals are able to achieve long-term control of viremia and avoid immunodeficiency without the need for antiviral therapy. 24,25 The frequency of this finding is dependent on the sensitivity of the RNA assay used and the number of replicates tested. In a French study examining the limitations of MP-NAT (MP8), 35 samples from six HIV antibody positive blood donors were selected for further testing based on RNA levels of fewer than 400 copies/ml; 11 (31%) were initially and repeat negative by MP8-NAT and three (9%) were initially and repeat negative by ID-NAT. 37 Of the HIV antibody positive donors in this study sourced using Ultrio-MP16, HIV RNA could not be detected by ID-NAT from 2.3% (Ultrio Plus) to 4.2% (MPX). Additionally, 1.9% of HIV antibody positive donors lacked detectable RNA by all three assays, a rate of 1:684,000 presenting donors. These rates have not been previously published for US blood donors, but are consistent with the 2.2% rate reported in France during the first 15 months after MP8- NAT implementation. 37 In summary, this study demonstrates that MPX-MP6 and Ultrio Plus-MP16 produce comparable findings in US blood donors and that Ultrio Plus improves HBV DNA detection compared to Ultrio without compromising the detection of HIV or HCV RNA. The observed HBV DNA sensitivity improvement has important policy implications for blood donation screening. ACKNOWLEDGMENTS The authors acknowledge Gen-Probe staff for performing the Ultrio Plus testing and specifically Dr Jeffrey M. Linnen for his useful discussion of the manuscript content. CONFLICT OF INTEREST None of the authors had a conflict of interest. REFERENCES 1. Roche Molecular Systems, Inc. cobas TaqScreen MPX Test for use on the cobas s 201 System product insert: 1636, Rev Indianapolis, IN, USA. 2. Gen-Probe, Incorporated. Procleix Ultrio Assay product insert: , Rev. A San Diego, CA, USA. 3. Gen-Probe, Incorporated. Procleix Ultrio Plus Assay product insert: , Rev San Diego, CA, USA. 4. Roth WR, Busch MP, Schuller A, Ismay S, Cheng A, Seed CR, Jungbauer C, Minsk PM, Sondag-Thull D, Wendel S, Levi JE, Fearon M, Delage G, Xie Y, Jukic' I, Turek P, Ullum H, Tefanova V, Tilk M, Reimal R, Castren J, Naukkarinen M, Assal A, Jork C, Reesink HW et al. International survey on NAT testing of blood donations: expanding implementation and yield from 1999 to Vox Sang 2012;102: Assal A, Barlet V, Deschaseaux M, Dupont I, Gallian P, Guitton C, Morel P, van Drimmelen H, David B, Lelie N, DeMicco P. Sensitivity of two hepatitis B virus, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) nucleic acid test systems relative to hepatitis B surface antigen, anti-hcv, anti-hiv, and p24/anti-hiv combination assays in seroconversion panels. Transfusion 2009;49: Margaritis AR, Brown SM, Seed CR, Kiely P, D Agostino B, Keller AJ. Comparison of two automated nucleic acid testing systems for simultaneous detection of human immunodeficiency virus and hepatitis C virus RNA and hepatitis B virus DNA. Transfusion 2007;47: Phikulsod S, Oota S, Tirawatnapong T, Sakuldamrongpanich T, Chalermchan W, Louisirirotchanakul S, Tanprasert S, Chongkolwatana V, Kitpoka P, Praphan Phanuphak P, Wasi C, Nuchprayoon C; The Working Group for NAT Study in Thai Blood Donations. One-year experience of nucleic acid Volume 53, October 2013 TRANSFUSION 2535

12 STRAMER ET AL. technology testing for human immunodeficiency virus type 1, hepatitis C virus, and hepatitis B virus in Thai blood donations. Transfusion 2009;49: Louisirirotchanakul S, Oota S, Khuponsarb K, Chalernchan W, Phikulsod S, Chongkolwatana V, Sakuldamrongpanish T, Kitpoka P, Chielsilp P, Tirawatnapong T, Wasi C; the Working Group for NAT Study in Thai Blood Donations. Occult hepatitis B virus infection in Thai blood donors. Transfusion 2011;51: Stramer SL, Krysztof DE, Brodsky JP, Fickett TA, Reynolds B, Phikulsod S, Oota S, Lin M, Saldanha J, Kleinman SH. Sensitivity comparison of two Food and Drug Administration licensed, triplex nucleic acid test automated assays for hepatitis B virus DNA detection and associated projections of United States yield. Transfusion 2011; 51: Stramer SL, Wend U, Candotti D, Foster GA, Hollinger FB, Dodd RY, Allain JP, Gerlich W. Nucleic acid testing to detect HBV infection in blood donors. N Engl J Med 2011; 364: Stramer SL, Glynn SA, Kleinman SH, Wright DJ, Dodd RY, Busch MP; National Heart, Lung, and Blood Institute Nucleic Acid Test Study Group. Detection of HIV-1 and HCV infections among antibody-negative blood donors by nucleic acid amplification testing. N Engl J Med 2004;351: Stramer SL, Zou D, Notari EP, Foster GA, Krysztof DE, Musavi F, Dodd RY. Blood donation screening for hepatitis B virus markers in the era of nucleic acid testing: are all tests of value? Transfusion 2012;52: Zou S, Stramer SL, Notari EP, Kuhns MC, Krysztof D, Musavi F, Fang CT, Dodd RY. Current incidence and residual risk of hepatitis B infection among blood donors in the United States. Transfusion 2009;49: Katz L, Strong DM, Tegtmeier G, Stramer S. Performance of an algorithm for the reentry of volunteer blood donors deferred due to false-positive test results for antibody to hepatitis B core antigen. Transfusion 2008;48: McNemar Q. Note on the sampling error of the difference between correlated proportions or percentages. Psychometrika 1947;12: Durkalski VL, Palesch YY, Lipsitz SR, Rust PF. Analysis of clustered matched-pair data. Stat Med 2003;22: US Food and Drug Administration. Guidance for industry: nucleic acid testing (NAT) for human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV): testing, product disposition, and donor deferral and reentry. May 2010 [cited 2012 Dec 2]. Available from: URL: GuidanceComplianceRegulatoryInformation/Guidances/ default.htm 18. US Food and Drug Administration. Guidance for industry: use of nucleic acid tests on pooled and individual samples from donors of whole blood and blood components, including source plasma, to reduce the risk of transmission of hepatitis B virus. Oct [cited 2012 Dec 2]. Available from: URL: GuidanceComplianceRegulatoryInformation/Guidances/ default.htm 19. Busch MP, Glynn SA, Stramer SL, Strong DM, Caglioti S, Wright DJ, Pappalardo B, Kleinman SH; NHLBI-REDS NAT Study Group. A new strategy for estimating risks of transfusion-transmitted viral infections based on rates of detection of recently infected donors. Transfusion 2005;45: Busch MP, Glynn SA, Wright DJ, Hirschkorn D, Laycock ME, McAuley J, Tu Y, Giachetti C, Gallarda J, Heitman J, Kleinman SH for the National Heart, Lung, and Blood Institute Nucleic Acid Test Study Group. Relative sensitivities of licensed nucleic acid amplification tests for detection of viremia in early human immunodeficiency virus and hepatitis C virus infection. Transfusion 2005;45: Assal A, Barlet V, Deschaseaux M, Dupont I, Gallian P, Guitton C, Morel P, David B, De Micco P. Comparison of the analytical and operational performance of two viral nucleic acid test blood screening systems: Procleix Tigris and cobas s 201. Transfusion 2009;49: Allain JP. Occult hepatitis B virus infection: implications in transfusion. Vox Sang 2004;86: Raimondo G, Allain JP, Brunetto MR, Buendia MA, Chen DC, Colombo M, Carxi A, Donato F, Ferrari C, Gaeta GB, Gerlich WH, Levrero M, Locarnini S, Michalak T, Mondell MU, Pawlotsky JM, Pollicino T, Prati D, Puoti M, Samuel D, Shouval D, Smedile A, Squadrito G, Trepo C, Zoulim F et al. Statements from the Taormina expert meeting on occult hepatitis B virus infection. J Hepatol 2008;49: Deeks SG, Walker BD. Human immunodeficiency virus controllers: mechanisms of durable viral control in the absence of antiretroviral therapy. Immunity 2007;27: Hatano H, Delwart EL, Norris PJ, Lee TH, Dunn-Williams J, Hunt PW, Hoh R, Stramer SL, Linnen JM, McCune JM, Martin JN, Busch MP, Deeks SG. Evidence for persistent low-level viremia in individuals who control human immunodeficiency virus in the absence of antiretroviral therapy. J Virol 2009;83: Vermeulen M, Dickens C, Lelie N, Walker E, Coleman C, Keyter M, Reddy R, Crookes R, Kramvis A. Hepatitis B virus transmission by blood transfusion during 4 years of individual-donation nucleic acid testing in South Africa: estimated and observed window period risk. Transfusion 2012;52: Hollinger FB. Hepatitis B virus infection and transfusion medicine: science and the occult. Transfusion 2008;48: Vermeulen M, Lelie N, Sykes W, Crookes R, Swanevelder J, Gaggia L, Le Roux M, Kuun E, Gulube S, Reddy R. Impact of individual-donation nucleic acid testing on risk of human immunodeficiency virus, hepatitis B virus, and 2536 TRANSFUSION Volume 53, October 2013