B L O O D C O M P O N E N T T E S T I N G Experience of German Red Cross blood donor services with nucleic acid testing: results of screening more than 30 million blood donations for human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus Michael K. Hourfar, Christine Jork, Volkmar Schottstedt, Marijke Weber-Schehl, Veronika Brixner,* Michael P. Busch,* Geert Geusendam,* Knut Gubbe,* Christina Mahnhardt,* Uschi Mayr-Wohlfart,* Lutz Pichl,* W. Kurt Roth,* Michael Schmidt,* Erhard Seifried,* and David J. Wright* for the German Red Cross NAT Study Group BACKGROUND: The risk of transfusion-transmitted human immunodeficiency virus-1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections is predominantly attributable to donations given during the early stage of infection when diagnostic tests may fail. In 1997, nucleic acid amplification technique (NAT)- testing was introduced at the German Red Cross (GRC) blood donor services to reduce this diagnostic window period (WP). STUDY DESIGN AND METHODS: A total of 31,524,571 blood donations collected from 1997 through 2005 were screened by minipool NAT, predomi- nantly with pool sizes of 96 donations. These donations cover approximately 80 percent of all the blood col- lected in Germany during that period. Based on these data, the WP risk in the GRC blood donor population was estimated by using a state-of-the-art mathematic model. RESULTS: During the observation period, 23 HCV, 7 HIV-1, and 43 HBV NAT-only–positive donations were detected. On the basis of these data and estimated pre- NAT infectious WPs, the residual risk per unit trans- fused was estimated at 1 in 10.88 million for HCV (95% confidence interval [CI], 7.51-19.72 million), 1 in 4.30 million for HIV-1 (95% CI, 2.39-21.37 million), and 1 in 360,000 for HBV (95% CI, 0.19-3.36 million). Based on observed cases of breakthrough infections, the risk of transfusion-related infections may be even lower. CONCLUSION: The risk of a blood recipient becoming infected with HCV, HIV-1, or HBV has reached an extremely low level. Introduction of individual donation testing for HCV and HIV-1 would have a marginal effect on interception of WP donations. D uring the past two to three decades, there has been a substantial decrease in the risk of transfusion-transmitted blood-borne infec- tions. This has been a result of the deve- lopment of sensitive serologic methods and the recruitment of low-risk blood donors based on imple- menting progressively more restrictive donor eligibility criteria.1 Despite these measures, a residual risk of acquiring a transfusion-associated virus infection still exists. Fortunately, this residual risk has become ABBREVIATIONS: ChLIA = chemiluminescence assay; GRC = German Red Cross; ID = individual donation; IR-WP = incidence-rate window period; MP = minipool; WP(s) = window period(s). From the German Red Cross Baden-Württemberg–Hessen, Frankfurt, Germany; German Red Cross NSTOB, Springe, Germany; German Red Cross West, Hagen, Germany; Bavarian Red Cross, Wiesentheid, Germany; Blood Systems Research Institute, San Francisco, California; German Red Cross North, Lütjensee, Germany; German Red Cross Saxonia, Plauen, Germany; German Red Cross Mecklenburg-Western Pomerania, Neubrandenburg, Germany; German Red Cross Baden- Württemberg–Hessen, Ulm, Germany; and Westat, Rockville, Maryland. *Shared senior authorship; senior authors in alphabetical order. Address reprint requests to: M.K. Hourfar, Institute of Trans- fusion Medicine and Immunohematology, German Red Cross, Johann Wolfgang Goethe University, Sandhofstrasse 1, 60528 Frankfurt am Main, Germany; e-mail: k.hourfar@blutspende.de. Received for publication October 28, 2007; revision received January 28, 2008, and accepted January 28, 2008. doi: 10.1111/j.1537-2995.2008.01718.x TRANSFUSION **;**:**-**. Volume **, ** ** TRANSFUSION 1 extremely small, so that prospective studies measur- ing the rate of transmission in blood recipients are not feasible for accurate risk estimates. Nowadays mathematical risk models are necessary to determine the residual risk of viral transmission in countries with a low incidence of transfusion relevant viral infections.1,2 Today it is accepted that the residual risk is mainly attributed to the transfusion of donations during the early, antibody-negative phase of infection. During this stage, the donor may have viremia without displaying any symptoms of infection and may therefore also not be deferred by the medical professional performing the examination before the donation. Further potential sources of risk are immunovariant viral strains not reli- ably detected by serologic assays, immunosilent carriers, procedural testing errors, and occult hepatitis B virus (HBV) carriers if anti-hepatitis B core antigen (HBc) testing is not performed. Although serologic assays have improved substan- tially over time and both human immunodeficiency virus (HIV) and hepatitis C virus (HCV) combined antibody and antigen assays are currently available, detection of patho- gens by means of nucleic acid amplification technology (NAT) still permits for the most efficient reduction of the window periods (WPs). At German Red Cross (GRC) blood donor services, the first studies to evaluate the feasibility of NAT to improve blood safety began in the mid-1990s. Subsequently, minipool (MP)-NAT screening for transfusion-transmissible viruses was implemented at all GRC blood donor services.3-5 Although controversial, testing for HBV DNA is also part of routine NAT screening in all GRC institutions. Ten years later a comprehensive set of NAT-screening data for HIV-1, HCV, and HBV is available, covering more than 30 million donations. This report reviews the evolv- ing method and results of NAT in Germany and considers the implications of these findings for future strategies for blood donor screening. Additionally, the residual WP risks for transfusion-transmitted HCV, HIV-1, and HBV in the GRC blood donor population are estimated for the first time using a state-of-the-art mathematic model that employs NAT yield rates and the relationship between the durations of the pre-NAT infectious and NAT detection WPs. MATERIALS AND METHODS Study population The data reported in this study were collected from January 1997 through December 2005 at the GRC blood donor services, which cover all geographic regions of Germany. A total of 31,524,571 allogeneic blood donations were routinely tested for anti-HCV (predominantly HCV enzyme-linked immunosorbent assay Enh. SAVe, Ortho Diagnostics, Raritan, NJ; and PRISM HCV, Abbott, Delken- heim, Germany), anti-HIV (predominantly Enzygnost HIV-1/2 Plus, Dade Behring, Marburg, Germany; and PRISM HIV-1/2 O, Abbott), hepatitis B surface antigen (HBsAg; predominantly Enzygnost HBsAg 5.0, Dade Behring; and PRISM HBsAg, Abbott), and Treponema pal- lidum antibodies. All reactive samples were retested in duplicate. Repeatedly reactive samples were further tested by confirmatory assays such as immunoblots (for HIV and HCV), neutralization (for HBsAg), and individual- donation (ID)-NAT (in particular for serologically reactive, MP-NAT nondetected donations). Additional testing for elevated alanine aminotransferase (ALT) levels was per- formed in all participating blood donor services during the study period. In general, donations were excluded from transfusion if ALT values were above twice the upper limit of normal. Testing for anti-HBc was not mandatory during the study period and was conducted only at some GRC blood donor services (total number of donations screened for anti-HBc, 3,769,766 [12%]). All donations included in this study were screened for HCV RNA, whereas 31,395,566 and 31,359,486 dona- tions were additionally tested for HIV-1 RNA and HBV DNA, respectively. Donations included in this study account for approximately 80 percent of the total dona- tions in Germany during the study period. NAT procedures At the GRC, NAT was implemented on a voluntary basis before commercial assays for blood donor screening were available. Therefore, in the first years of NAT, a variety of NAT approaches were used at the GRC institutions. These included pure in-house NAT methods,5 as well as adapta- tions of commercial NAT that were originally developed for clinical laboratories. Extraction and amplification methods applied were very heterogeneous in the first years of NAT, as were the NAT sensitivities. The introduction of mandatory HCV NAT in 1999, with a required minimum sensitivity of 5000 IU per mL for all known HCV genotypes, led to a decline in the variety of methods employed. Since 1999, the predominant methods used pools of 96 donor samples, enrichment of viruses by high-speed or ultracentrifugation (at 48,000 ¥ g or 402,000 ¥ g), and generic extraction of the viral nucleic acids, predominantly by use of chaotropic salts and silica surfaces. Extracted viral nucleic acids were amplified and detected by commercial (Roche Molecular Systems, Pleasanton, CA; and Artus Biotech, Hamburg, Germany),6,7 semicommercial,8 or in-house real-time polymerase chain reaction (PCR) methods.5 These methods provide almost similar results with 95 percent sensitivity levels of approximately 800 to 1500 IU HCV per mL, 1000 to 5000 IU HIV-1 per mL, and 100 to 700 IU HBV per mL for a single donation comprised in a MP. A NAT- HOURFAR ET AL. 2 TRANSFUSION Volume **, ** ** only WP donation was considered to be confirmed, if the donation was PCR-positive in the absence of serologic markers and if seroconversion was observed in a follow-up sample. Residual risk estimations Incidence rates and the duration of WPs of transfusion- transmitted infections were estimated according to the approach by Busch and coworkers.9 Analogous to the incidence-rate window period (IR-WP) model by Schreiber and colleagues,10 which has found widespread use in recent years,11-13 the residual risk attributed to WP donations is calculated by using the relationship Risk Window period Incidence rate = ( ) × ( ). By using alternative methods to calculate the WPs and the incidence rate, however, this model improves the esti- mates made in the IR-WP model. Wald-type confidence intervals (CIs) for the residual risk were computed. The standard error for the residual risk was estimated by a Taylor series expansion involving the standard error esti- mate of the incidence rate and standard error estimate of the WP. The incidence rate is derived from the yield of viremic, antibody-negative donations detected by routine MP NAT (assumed to be Poisson distributed) and an esti- mated person-time (number of donations times the NAT- only [viremic preseroconversion] WP). Thereby, the whole donor collective is considered for the calculation of the incidence rate, whereas in previous applications of the IR-WP model, viral incidence for all donors was projected from seroconversion rates derived only from repeat donors. The standard error for the incidence rate was esti- mated by a Taylor series expansion involving the standard error estimate of the yield and standard error estimate of the NAT only WP. The duration of the infectious WPs is estimated by back-extrapolation of acute viral replication dynamics during the ramp-up phase of primary viremia. In this study, the WP has been estimated on the basis of the sen- sitivities of the MP-NAT method that was used for screen- ing most of the donations (10,817,276 donations; 34.3%) during the observed period.5 The 50 percent sensitivity levels for this MP-NAT method are 293.6 IU per mL (95% CI, 93.3-398.3) for HCV, 222.3 IU per mL (95% CI, 43.2- 336.9) for HIV-1, and 82.7 IU per mL (95% CI, 44.9-107.3) for HBV. Calculation of WPs (t) were based on doubling times (l) of 10.8 hours for HCV, 20.5 hours for HIV-1, and 2.56 days for HBV, respectively,14-16 using the mathematical relation C C t = × 0 2 / . λ The standard error for the infectious WP was estimated by a Taylor series expansion involving the standard error esti- mate of the doubling time and standard error estimate of the sensitivity of MP-NAT. Calculation of the NAT-positive HCV and HIV-1 WPs were performed as described by Page-Shafer17 and O’Brien,18 except that different sensitivities of the NAT assays were considered. For calculation of incidence rates, we included WP cases, which failed to be detected due to technical shortcomings, but had a viral load above the 95 percent detection limit of the NAT method applied. For HBV, the period during which a donation can be MP-positive but antigen-negative was calculated on the basis of previously published detection sensitivities of the Abbott PRISM HBsAg test. Based on the data of Minegishi and colleagues,19 Kleinman and Busch20 calculated an infectious WP of 38.4 days (95% CI, 33.0-43.7 days) for the Abbott PRISM. For donations tested with a traditional enzyme immunoassay (EIA) for HBsAg, we assumed a prolonged WP of 42.8 days. This assumption is based on the data of the Medicines and Healthcare Products Regu- latory Agency (MHRA) evaluation report, which describes an additional WP of 4.4 days for the Dade Behring Enzygnost HBsAg 5.0 compared to the Abbott PRISM chemiluminescence assay (ChLIA).21 The Enzygnost HBsAg 5.0 was the predominant test system in those GRC institutes not using the ChLIA technology. RESULTS Yield of WP donations A total of 73 NAT-only–positive donations were observed from the beginning of NAT at the GRC. Twenty-three WP donations for HCV and 7 for HIV-1 were confirmed, respectively (Table 1). Among the 43 NAT-only HBV-positive donations, 22 donations were WP donations. Anti-HBc testing could also have identified the remaining 21 donations, indicating that these were so-called “occult” HBV carriers rather than acute infections. Five of the HBV WP donations were found at GRC institutions that perform HBsAg screening with the Abbott PRISM (Table 2). Of the 23 HCV WP donations detected by MP-NAT, 8 donations were otherwise nontransfusable due to increased ALT values. In the case of HIV-1, 2 of the 7 WP donations would not have been released for transfusion due to confidential unit exclusion. Thus, the yield of WP donations, which would have been released for transfu- sion without NAT was 15 for HCV, 5 for HIV-1, and 22 for HBV. At the GRC, every allogeneic blood donation is con- verted on average into 1.4 transfused blood products (1 unit of red cells [RBCs], 0.1 unit of platelet [PLT] concen- trate, 0.3 units of fresh-frozen plasma [FFP]). Because plasma is quarantined until the donor returns to give his or her next donation, which must screen negative by all markers, FFP does not run the risk of containing infec- NAT yield in Germany Volume **, ** ** TRANSFUSION 3 tious HIV and HCV from WP donors. This does not apply for HBV, however, because of the transient nature of HBs antigenemia and lack of anti-HBc screening during the study period. Assuming that none of the products made from otherwise transfusable WP donations would have been discarded, for example, due to expired shelf-life, our data indicate that the introduction of MP testing likely pre- vented approximately 17 cases of transfusion-transmitted HCV infection and 6 posttransfusion HIV-1 infections. For HBV, approximately 60 transmissions (31 of those origi- nating from NAT-only WP donations) might have been prevented. A fraction of the recipients of these donations, however, might not have been at risk of being infected due to prior HBV vaccination. WP donations missed by NAT During the study period, nine HBV, one HIV-1, and one HCV WP donations missed by MP-NAT screening have been reported to the coordination office of the GRC NAT- study. In the case of HBV, six of these donations were derived from a single apheresis donor with a pure HBV genotype G infection.22 Detection of HBV DNA failed, although three donations showed a viral load above the 95 percent detection limit of the MP-NAT assay used. This could probably be attributed to the primer design with respect to sensitive detection of the rare genotype G. Addi- tionally, the antigen-negative WP was substantially prolonged, which was apparently caused by a decreased synthesis of incomplete viral forms in the early HBV geno- type G infection period. Three plasma units could be discarded before further processing, whereas six PLT concentrates, derived from three double-unit plateletpheresis collections, were trans- fused into four recipients. Two out of the four recipients were infected with the same rare genotype G. They did not show clinical symptoms of an acute hepatitis and died within 6 months after transfusion for reasons unrelated to the contaminated blood products. In one case, a PLT concentrate of a donor testing nega- tive for the presence of HBsAg with the Abbott PRISM was released before final NAT results were obtained. The delay in reporting test results was due to a laboratory contami- nation with HBV amplicons, leading to positive results of the majority of pools tested. Subsequent examinations revealed that the viral load of the donation was well above the 95 percent detection limit of the HBV MP-NAT assay. The seronegative recipient of the PLTs showed a serocon- version to HBsAg and anti-HBc without clinical symp- toms.23 This case dates back to the early phase of NAT where real-time PCR was not yet established. In 2001, two PRISM HBsAg-negative, HBV DNA– positive WP donations were identified after seroconver- sion of a multiple-time repeat donor. By analyzing TABLE1.RatesofpositivityforHIV-1,HCV,andHBV Period ResultsforHCVResultsforHIVResultsforHBV Numberof donations MP-NAT- only–positive Rateper 10 6 donations Number ofdonations MP-NAT- only–positive Rateper 10 6 donations Numberof donations MP-NAT- only–positive* Rateper 10 6 donations 1997-19999,211,14170.769,082,13610.119,046,05690.99 2000-200211,301,93360.5311,301,93340.3511,301,933272.39 2003-200511,011,497100.9111,011,49720.1811,011,49770.64 Total31,524,571230.7331,395,56670.2231,359,486431.37 *Testingforanti-HBcwasnotmandatoryduringthestudyperiodandwasconductedonlyatsomeGRCblooddonorservices(totalnumberofdonationsscreenedforanti-HBc:3,769,766 [12%]). HOURFAR ET AL. 4 TRANSFUSION Volume **, ** ** repository samples from the donor, it was shown that the two donations preceding the index donation were viremic with an HBV concentration of less than 100 IU per mL. The viral load in these donations was below the detection limit of the MP-NAT method applied in the reporting blood donor service. The only HIV-1 infection at the GRC, since introduc- tion of NAT screening, occurred in 1998, a case involving HIV-1 transmission to a recipient by contaminated RBCs. The donation showed a viral load of approximately 20,000 IU per mL, but tested negative by MP-NAT because HIV-1 subtype A, which is rare in Germany, was not covered by the PCR primers used23 at that time. The female donor of the respective donation likely acquired the infec- tion by sexual contact with a person from a country with high HIV-1 subtype A prevalence. The design of the HIV-1 PCR at the affected test site was changed after that break- through infection occurred. Very recently, an HCV-positive WP donation with a low virus burden was identified by donor directed look- back procedures after seroconversion. The donation before the index HCV-seropositive donation had a viral load of less than 100 IU per mL and was therefore not detected by MP-NAT. Atypically for the early phase of an HCV infection, the donor showed an increased ALT value of 294 U per L. Therefore, the donation was not trans- fused. In this case, there was no history of medications that affect liver enzymes and no proof of a coinfection with directly hepatocytopathic viruses, which could serve as an explanation for the increased ALT value. At the GRC there is a high percentage of multiple-time repeat donors (approx. 92%). Whenever an HIV, HCV, or HBV infection is diagnosed in a recipient of blood prod- ucts or a repeat donor is detected as infected, lookback investigations are initiated, including analysis of manda- tory repository samples from the donor. Owing to the high fraction of multiple-time repeat donors and extensive hemovigilance measures, it can be accepted that, at least for HIV and HCV, no infections in recipients have been overlooked. In the case of HBV, the situation might be different because of the transient antigenemia and the fact that anti-HBc testing was not performed for the majority of donations to detect seroconversions in repeat donors. Residual risk estimation As shown in Table 3, the incidence rates per 100,000 person-years, calculated for the entire period of observa- tion, were 0.53 for HCV, 0.87 for HIV-1, and 3.23 for HBV. The MP-NAT–negative infectious WPs were estimated at 9.7 days for HIV-1, 6.3 days for HCV, and 32.4 days for HBV. An additional 10.6, 50.0, and 7.0 or 11.4 days were esti- mated for the NAT yield phase preceding positive sero- logic results for HIV-1, HCV, and HBV (PRISM or traditional EIA), respectively. DISCUSSION From beginning of NAT in January 1997 to December 2005, a total of 73 MP-NAT-only–positive donations have been identified at the GRC blood donor services (Table 1). Our data, however, indicate that 35 percent of the HCV- positive WP donations found since 1997 would not have been released for transfusion due to increased ALT values. These data are consistent with reports from the United States, where approximately one-third of the units detected by HCV-NAT would have been discarded owing to elevated ALT levels.24 Since January 2004, ALT reactivity is no longer a release criterion for blood products in Germany.25 Mainly due to requirements of plasma fractionating companies, however, testing has been continued throughout the study period and was discontinued at most GRC institutes in 2006 or 2007. In the case of HIV-1, two of the eight WP donations would not have been released because the donors confi- dentially excluded their donations from transfusion. The donors, both young male, reported previous homosexual contacts and can be classified as test seekers.26 The confi- dential unit exclusion option has since been criticized for its low positive predictive value, which results in the loss of many transfusable blood units.27 In these two cases, however, it has accomplished its given function as an extra layer safeguard to improve blood safety.28 In the mid-1990s, there was an intense discussion as to whether the novel NAT techniques, that is, PCR or transcription-mediated amplification, should be intro- duced to screen every blood donation for the presence of viral nucleic acids. Mostly it was argued that a marginal benefit could only be obtained at very high costs. Addi- tionally, there was the concern that NAT, from a technical point of view, was immature and not suited for routine testing of thousands of blood donations per day. There- fore, in most countries the decision to introduce the tech- nique was postponed until commercial platforms for NAT were available. TABLE 2. Yield of HBV NAT-only–positive samples in donations tested with Abbott PRISM and traditional EIA Tested with Number of donations MP-NAT only–positive WP donations Rate per 106 donations (WP-donations) Abbott PRISM 14,203,017 9 5 0.63 (0.35) EIA 17,156,469 34 17 1.98 (0.99) Total 31,359,486 43 22 1.37 (0.70) NAT yield in Germany Volume **, ** ** TRANSFUSION 5 In contrast, starting in 1996, at the GRC blood donor services, several studies were conducted to evaluate the feasibility and efficacy of NAT for routine screening. Owing to the economic and technical limitations at that time, a strategy of creating MPs, predominantly with pool sizes of 96 donations, was chosen to allow high- throughput screening and timely release of components at reasonable costs. Encouraged by the promising results of these studies, most GRC institutes started routine NAT on a voluntary basis in 1997 using methods which were, although to some extent comparable to the methods used today, on a rather experimental level. As described previously, during the first 2 years of routine NAT screening, one HIV-1 and at least one HBV infection occurred after transfusion despite NAT. Detailed examination of these events in fact suggested technical shortcomings as the most probable reason for the failure of the NAT assay to detect viremic donations. These cases, however, do not constitute an argument against early introduction on a voluntary basis, because seven HCV and seven HBV NAT-only–positive donations were identified during the same time span. For HIV-1, six NAT-only WP donations were identified before screening for HIV-1 RNA became mandatory in 2004. In contrast during the same period, three HIV-1 infections by NAT unscreened blood were reported from non-GRC institutes, which account for approximately 20 percent of the German blood supply.23,29 Based on the incidence of WP donations observed in the interval from January 1997 to December 2005, the residual risk of acquiring a transfusion-transmitted virus infection was calculated at 1 in 4,300,000 for HIV-1, 1 in 10,880,000 for HCV, and approximately 1 in 360,000 for HBV (Table 3). Although these data are striking evidence for the efficacy of MP-NAT, there is the discussion in the transfusion community that the test strategy should be modified to ID testing when fully automated test systems are available.30 Supporters of ID testing in this context refer to reports of HIV-1 and HCV transmissions via RNA- screened blood donations.31,32 The data presented in this report, however, do not lend support to the conclusion that ID testing would add significantly to blood safety. By testing more than 30 million blood donations for HCV and HIV-1, only a single WP donation was missed by MP-NAT due to insufficient sensitivity. The respective HCV-positive donation had a viral load of less than 100 IU per mL. As described previously, a release for transfusion was pre- vented by a nonspecific increase in ALT levels that coin- cided with the early phase of the infection. By examining the donation using a sensitive ID-NAT method, it was found to be positive in only two of four replicates. This case likely represents a virus infection at the very begin- ning of the ramp-up phase or intermittent low-level viremia during the pre–ramp-up phase.14 Glynn and col- leagues33 showed that those donations might not be reli- ably detected by an ID-testing strategy. Very recently, the TABLE3.TheresidualriskofHCV,HIV-1,andHBVinfectionderivedfromtheincidenceofNAT-reactive,serology-negativedonations Numberof incidentcases NAT-only WPindays Person-years atrisk Incidencerateper 100,000person-years (95%CI) InfectiousWP indays Residualrisk(numberper milliondonations)(95%CI) Residualrisk(1pernumber ofmilliondonations)(95%CI) HIV-18*10.6915,3050.87(0.21-1.54)9.70.23(0.05-0.42)1:4.30(2.39-21.37) HCV2350.04,316,8200.53(0.31-0.76)6.30.09(0.05-0.13)1:10.88(7.51-19.72) HBV†9*7.0271,2883.32(0.22-6.42)31.42.85(0.14-5.56)1:0.35(0.18-7.06) HBV‡1711.4534,5193.18(1.17-5.19)31.42.73(0.94-4.52)1:0.37(0.22-1.06) HBV†‡26805,8073.23(0.40-6.05)31.42.77(0.30-5.24)1:0.36(0.19-3.36) *IncludesoneHIVWPdonationmissedbyNATandfourHBVWPdonationsmissedbyNATduetoproceduraltestingerrors. †AbbottPRISMusedforHBsAgscreening. ‡EIA(predominantlyEnzygnostHBsAg5.0)usedforHBsAgscreening. HOURFAR ET AL. 6 TRANSFUSION Volume **, ** ** first case of HCV transmission in Germany after introduc- tion of mandatory NAT has been reported by a non-GRC blood donor service.34 As was the case with the HCV- positive donation at the GRC, the respective donation was detected erratically even when tested by ID-NAT assays. The estimated prevalence of anti-HCV and anti-HIV-1 in Germany is 0.63 and 0.06 percent, respectively.35,36 The prevalence of these serologic markers among first-time donors is 0.1 and 0.005 percent, respectively, and thus approximately 1 order of magnitude lower. In repeat donors, approximately 2.4 anti-HCV–positive and 0.62 anti-HIV–positive donations were found among 105 blood donations. Taking into account the higher frequency of HCV infections in the donor population and the compa- rable length of the MP-NAT–negative WP, it can be expected that residual risk calculations yield a higher risk for HCV than for HIV-1. Hence, the risk of an undetected infection despite MP-NAT was previously estimated at 1 in 5,540,000 for HIV-1 and 1 in 4,400,000 for HCV, respec- tively.37 These calculations for the German blood supply for the years 2000 to 2002 were based on the traditional IR-WP model and incidence rates per 100,000 person- years of 1.09 and 0.66 for HCV and HIV-1, respectively. Of note, using the novel mathematic model by Busch et al., we found a reversed relationship (Table 3). The inci- dence rates per 100,000 person-years were 0.53 and 0.87 for HCV and HIV-1, respectively. This observation is con- sistent with the report of O’Brien for the Canadian Blood supply.18 This group found by direct comparison of both methods that the estimate for HCV incidence and residual risk with the classical IR-WP model was significantly higher than that derived based on the new method. O’Brien and colleagues suggested that this discrepancy could be due to the inclusion of false-positive samples and/or chronically infected donors as seroconverting donors in the calculations used in the classical IR-WP model. The discordant results, however, could also be an indication that the novel strategy by Busch and coworkers more accurately reflects the risk structure of the blood donor collective by deriving the risk estimates from the observed rate of very recent (NAT yield) infections, thus accounting for certain behaviors of blood donors after a recent exposure (e.g., self deferral or test seeking). Results from previous studies have implied that donors might delay their return around the time of infec- tion.38,39 This “deliberate delay” in donating by donors engaging in risk behavior could also serve to explain why in many countries the yield of NAT, particularly for HCV, was significantly lower than estimated by use of the IR-WP model.30 Risk factors for HIV and HCV are to a large extent distinct, and for HIV there is no such discrepancy between the rates of MP-NAT yield cases predicted by the IR-WP model and observed MP-NAT yield. By examining the respective HIV-positive donors, we found another poten- tial explanation for the discrepancy between HIV and HCV. For the majority of the donors of HIV WP donations, there was some suspicion that they were homosexual men. If this particularly high-risk population, although not allowed to donate in Germany, uses NAT of blood donations to get knowledge about their health status very early after expo- sure, this might serve as an unwelcome explanation for the higher risk for HIV compared to HCV. If this assumption turns out to be true, there should be a discussion if the offer of free and anonymous HIV tests (for example, at public health services) could reduce the risk for the blood supply. Discussion about MP-NAT screening for HBV has been very controversial,40-43 and NAT screening for HBV is not mandatory in Germany and many other countries. The slow doubling time of HBV and the resulting low con- centrations of HBV DNA in blood during the WP suggested that the yield of MP-NAT screening (in particular in pools of 24 samples per pool) would be extraordinary small in countries with a low incidence for HBV. Additionally, the medical consequences of transmission are not severe, when compared to HIV and HCV. HBV infection in immu- nocompetent adults very infrequently results in recog- nized acute or chronic disease. Owing to these reasons a poor cost-effectiveness was assumed for HBV MP-NAT.43 The most common argument against introduction of MP-NAT in the United States, however, was that HBsAg immunoassays with improved sensitivity were awaiting licensure by the FDA. Biswas and colleagues16 suggested that some formerly investigational HBsAg assays, that is, the Abbott PRISM might be even more sensitive than MP-NAT. In Germany, the Abbott PRISM HBsAg test has been used since 1997 in some GRC blood donor services. During the whole study period, 14,203,017 donations were screened for HBsAg using the PRISM ChLIA technology, whereas the remaining 17,156,469 donations were screened using traditional EIAs (Table 2). Disregarding slight differences in the sensitivities of the MP-NAT assays used at the different GRC institutions and slight differences in the epidemiology of transfusion- transmissible infections, there is a considerable difference in the yield rate of NAT-only–positive WP donations between institutes using the Abbott PRISM and those using traditional EIAs. Nonetheless, several NAT-only WP dona- tions have been identified in institutions screening with the Abbott PRISM, although NAT assays with pool sizes of 96 donations and a moderate sensitivity were applied (see Table 2). The reason for the failure of the Abbott PRISM to identify these donations has not been elucidated in detail. The observation that the antigen-negative WP, however, can be substantially prolonged in certain HBV genotypes may speak in favor of continuation of HBV MP-NAT, even at moderate sensitivities. Reduction of the pool size to allow higher sensitivities in the detection of HBV DNA is cur- rently not considered in Germany for economic reasons. To summarize, early introduction of MP-NAT at the GRC prevented numerous transfusion-associated HIV, NAT yield in Germany Volume **, ** ** TRANSFUSION 7 HCV, and HBV infections. Current risks of acquiring transfusion-transmitted infections with these viruses are extremely low. Additionally testing for anti-HBc, which became mandatory in Germany since October 2006, will reduce the risk for HBV to the WP risk. Data presented in this study do not lend support to the conclusion that ID testing would add significantly to blood safety. Despite the extraordinary high level of safety reached, vigilance is rec- ommended to identify undesirable behavior of blood donors that could potentially impact blood safety. ACKNOWLEDGMENTS The authors thank the members of the German Red Cross (GRC) NAT Study Group for contributing to the study throughout the years. Members of the GRC NAT Study Group are U. Bauerfeind, W. Sireis, E.-M. Zayc-Schmidt, T.H. Müller, J. Schnabl, D. Hedges, K. Körner, W. Weichert, G. Elbert, A. Himmel, M. Loehr, G. Neumann, A. Vornwald, T. Dengler, A. Karl, and K. Frank. This study was supported by the DRK Forschungsgemeinschaft. REFERENCES 1. Busch MP. Transfusion-transmitted viral infections: build- ing bridges to transfusion medicine to reduce risks and understand epidemiology and pathogenesis. Transfusion 2006;46:1624-40. 2. Kleinman S, Busch MP, Korelitz JJ, Schreiber GB. The incidence/window period model and its use to assess the risk of transfusion-transmitted human immunodeficiency virus and hepatitis C virus infection. Transfus Med Rev 1997;11:155-72. 3. Cardoso MS, Koerner K, Kubanek B. 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