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HBV infectivity data were reviewed and the 50% infectious dose (ID

BACKGROUND The residual transfusion-transmitted hepatitis B virus (TT-HBV) risk with different testing strategies depends on the sensitivity of screening assays, the prevalence of hepatitis B surface antigen (HBsAg) compared to HBV-DNA in window period (WP) and occult HBV infections (OBIs), and infectivity of blood in these infection stages. We compared modeled WP and OBI transmission risk in a multiregional individual donation nucleic acid amplification technology (ID-NAT) screening study with observed TT-HBV infection rates in several lookback studies. STUDY DESIGN AND METHODS The WP and OBI risk was estimated from ID-NAT screening data in six geographic regions. The 50% infectious dose (ID50 ), a key factor in the applied risk models, was assumed to be 100-fold higher in OBI than in WP blood. The relative proportion of WP and OBI TT-risk was estimated for different screening scenarios and expressed as a percentage of the ID-NAT yield rate to allow for comparison with observed TT-rates in lookback studies. RESULTS Despite sevenfold to eightfold higher HBV ID-NAT yield rates in OBI than WP in South-East Asia and Europe, our models predicted that 40 (26-55)% of total residual TT-HBV risk was due to OBI, comparable to 37% observed in a Japanese hemovigilance study. Modeled TT-OBI risk was approximately 10-fold higher than observed rates of 2%-8% in five lookback studies but comparable to one other study (36%). CONCLUSION Although the observed TT-OBI rate was generally lower than the modeled risk, the relative risk of WP versus OBI transmission was not incompatible with the observational infectivity data. This supports the validity of our assumptions in the infectivity-based models for estimating worst-case residual risk with different testing scenarios.

Human immunodeficiency virus (HIV) and hepatitis B virus (HBV) are endemic in South Africa while hepatitis C virus (HCV) infection is rare. Two nucleic acid amplification technology platforms, the Procleix Ultrio Elite assay on the Panther instrument (Elite) and the cobas MPX assay on the cobas 6800 or 8800 system (MPX), are used worldwide. In 2015 these were evaluated in South African context. STUDY DESIGN AND METHODS The sensitivity of HIV, HBV, and HCV was evaluated using reference panels and 2-fold dilutions of 51 positive plasma samples tested in 12 to 24 replicates. The 95% and 50% lower limits of detection (LOD) were estimated by probit analysis and window period (WP) risk days by the Weusten model. Specificity was established by testing 3646 blood donations individually and instrument performance by evaluating all runs. RESULTS Specificity was 99.94% for MPX and 99.97% for Elite. The following 95% LODs (95% confidence intervals [CIs]) were estimated for MPX and Elite, respectively: HBV, 17.8 (10.9-33.9) and 47.9 (29.1-92.4) cp/mL; HCV, 21.9 (15.3-34.6) and 13.8 (8.9-24.0) cp/mL; and HIV, 8.3 (5.5-14.7) and 10.4 (6.9-18.2) cp/mL. On SA HBV and HIV dilution panels, relative sensitivity (range) of MPX was 3.20 (1.26-6.50) and 1.42 (0.26-2.72) fold higher than Elite. Downtime on cobas 6800 was 26 hours vs 6.6 hours on Panther (P

Background and objectives Comparison of two models for estimating residual transfusion transmission risk by NAT screened window period (WP) donations in South African repeat donors gave identical results for HIV but not for HBV. In order to understand discrepant HBV modelling outcomes, the values of input parameters in three HBV WP risk models were reviewed and subsequently applied to the same South African screening data generated by HBsAg PRISM and two NAT assays (Ultrio and Ultrio Plus). Two of the models were also compared using individual donation (ID)-NAT screening data from different geographical regions. Methods Values of input parameters were derived from two published data sources and used in three risk models [(1) the incidence rate-WP risk day equivalent model, (2) the NAT yield WP ratio model and (3) the anti-HBc-negative HBsAg yield period ratio model] and subsequently applied to the same ID-NAT screening data. Results The HBV WP transmission risk in South African repeat donations during a one-year Ultrio Plus NAT screening period was estimated as 22, 43 and 17 per million, respectively, for the three models, as compared to 56, 117 and 48 per million for HBsAg PRISM screening. The approximate two-fold higher estimate calculated with the NAT yield WP ratio model was corroborated in repeat donations from three of four regions in a multi-regional study. When another set of model input values (with shorter viraemia periods and a higher proportion of acute occult infections) was applied to the South African screening data, the relative difference in risk estimates between the three models became smaller. Conclusions Window period risk modelling for HBV is more complex than for HIV. Multiple factors affect the modelling outcomes. These include the values used for the length of transient HBsAg and HBV-DNA-positive phases, the proportion of acute occult and vaccine breakthrough infections and the assumption of random appearance of donors throughout the entire acute resolving infection phase. A substantial proportion of HBV WP NAT yields have very low viral load and lack donor follow-up data calling into question their definitive classification into the early acute (infectious) replication stage. Since these possible WP NAT yields most highly impact the NAT yield WP ratio model, we recommend relying on the more conservative estimates of the incidence rate-WP risk day equivalent model.

We prepared severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) working standards and reference panels from a pool of swab fluid samples before and after inactivation by beta-propiolactone and quantified viral load in nucleic acid amplification technology (NAT) detectable RNA copies/mL using limiting dilution analysis. The following 50% lower limits of detection (LOD) were estimated by probit analysis as compared to detection limits of rapid antigen tests on 1.5 fold dilutions of the native material: Roche cobas PCR 1.8 (1.0-3.3), Hologic Aptima TMA 6.6 (4.4-9.9), DRW SAMBA 15 (7-30), Molgen LAMP 23 (13-42), Fluorecare antigen 50,000, Abbott Panbio antigen 75,000 and Roche antigen 100,000 copies/mL. One 50% Tissue Culture Infectious Dose (TCID50)/mL of culture fluid was estimated to be equivalent to approximately 1000 RNA copies/mL (2700-4300 International Units) in our working standard. When assuming this level as start of contagiousness in a log-linear ramp up viremia model with 10-fold rise of viral load per day for the B.1 (Wuhan) type we estimated relative time points of first detectability of early infection by the different SARS-CoV-2 assays from the LODs mentioned above. The four NAT assays would be able to detect early viremia 40-66 hours earlier than the 1000 copies/mL infectivity threshold, whereas the three antigen tests would become positive 41-48 hours later. Our modeling of analytical sensitivity data was found to be compatible with clinical sensitivity data of rapid antigen tests and confirms that NAT assays are more reliable than antigen assays for identifying early infected asymptomatic individuals who are potentially infectious.

The residual transfusion-transmitted hepatitis B virus (TT-HBV) risk with different testing strategies depends on the sensitivity of screening assays, the prevalence of hepatitis B surface antigen (HBsAg) compared to HBV-DNA in window period (WP) and occult HBV infections (OBIs), and infectivity of blood in these infection stages. We compared modeled WP and OBI transmission risk in a multiregional individual donation nucleic acid amplification technology (ID-NAT) screening study with observed TT-HBV infection rates in several lookback studies.The WP and OBI risk was estimated from ID-NAT screening data in six geographic regions. The 50% infectious dose (IDDespite sevenfold to eightfold higher HBV ID-NAT yield rates in OBI than WP in South-East Asia and Europe, our models predicted that 40 (26-55)% of total residual TT-HBV risk was due to OBI, comparable to 37% observed in a Japanese hemovigilance study. Modeled TT-OBI risk was approximately 10-fold higher than observed rates of 2%-8% in five lookback studies but comparable to one other study (36%).Although the observed TT-OBI rate was generally lower than the modeled risk, the relative risk of WP versus OBI transmission was not incompatible with the observational infectivity data. This supports the validity of our assumptions in the infectivity-based models for estimating worst-case residual risk with different testing scenarios.

The Common Specifications/EU 2017/746 regulation for market approval of class D in vitro diagnostic devices (IVDs) intended for detection of blood borne viruses requires testing of the International Standard and 10–30 seroconversion panels to demonstrate ‘state of the art’ assay performance. We examined whether these requirements for performance evaluation are reasonable for HBV-DNA and HBsAg assays. For this purpose, we quantified HBsAg and HBV-DNA (genotype A) in the ramp-up phase of five seroconversion panels and demonstrated a remarkably parallel increase in the Log concentration of both analytes over time. Testing of seroconversion panels by three nucleic acid amplification technology (NAT) methods in multiple replicates and probit analysis with sufficient critical samples from all five panels taken together showed detection limits in copies/mL that were comparable to those on a HBV-DNA genotype A standard dilution panel. This indicates that the viral doubling time in the ramp-up phase is equal above and below the quantification limit of the viral load assay. The geometric mean HBsAg (PRISM) cutoff crossing point was 20 days later than the 50% NAT (Ultrio Plus) conversion point equivalent to 1500 (range: 1100–2200) and 4.8 (CI: 3.7–6.4) HBV-DNA copies/mL, respectively. Analytical sensitivity data of different NAT assay versions obtained over a decade demonstrated that the detection limit on the International Standard is not representative of all genotyped reference samples. From our detailed mathematical analysis, we conclude that HBV-DNA and HBsAg standard dilution series are functionally equivalent to seroconversion panels. A general requirement of a 95% detection limit ≤100 HBV-DNA copies/mL for different viral genotypes would be a better-defined regulation for EU market approval of NAT blood screening assays than the testing of multiple seroconversion panels to claim ‘state of the art’ performance.

Background The prevalence of hepatitis B surface antigen is estimated to be 6.7% in the South African population and in April 1995 the nation introduced universal hepatitis B virus (HBV) vaccination for newborns and infants. We studied the temporal association of this program with HBV prevalence in young blood donors and the contemporary HBV incidence and residual risk of transfusion-transmitted HBV infection (TT-HBV). Methods We used blood donation data from January 2011 to December 2019. Estimation of HBV prevalence donations made by first-time blood donors were analyzed by birth cohort and covariates. To estimate the incidence and residual risk of TT-HBV, mathematical models used data from both first time and repeat donors. Results HBV prevalence in first-time donors decreased from 0.84% (95% confidence interval [CI] 0.78-0.90) in 2011 to 0.66% (95% CI 0.61-0.70) in 2019. The post-1995 birth cohort had a significantly lower HBV prevalence of 0.14% (95% CI 0.13-0.15) than the pre-1985 birth cohort of 1.29% (95% CI 1.25-1.33) and the odds of HBV infection were reduced in a multivariable model (odds ratio [OR] = 0.28, 95% CI 0.24-0.34). The residual risk of TT-HBV occurring from window-period, occult, and possible vaccine breakthrough infections were estimated at 36.9, 5.8, and 2.2 per million red blood cell transfusions, respectively. Conclusion Donors born after the start of routine HBV immunization had significantly lower prevalence of HBV infection, supporting the effectiveness of the vaccination program. The contemporary residual risk of TT-HBV has decreased and should decline further as more vaccinated young people join the donor pool.

The World Health Organization (WHO) recommends the clinical use of a human immunodeficiency virus 1 (HIV-1) viral load (VL) threshold level of 1000 copies (cp)/mL in patients on antiretroviral therapy (ART) to distinguish between viral control (VL 1000 cp/mL) and viral failure or poor adherence (VL 1000 cp/mL). The accuracy of five quantitative HIV-1 RNA assays at this level was compared by replicate testing (n = 24) of 1000 cp/mL samples prepared from the Viral Quality Control (VQC) HIV-1 subtype B standard, which is in use for validation of nucleic acid testing methods since 1995. Until 2004 the VL assays reported geometric mean (95% confidence interval [CI]) values ranging between 449 (188-1067) and 3162 (3057-2367) cp/mL when using the Siemens bDNA 3.0 assay as reference method for an assigned value of 1000 (962-1038) cp/mL. In 2018, the following values (95% CI) were found by 24 replicate tests in each of the VL assays on the 1000 cp/mL samples: Abbott RealTime 1084 (784-1572), BioMerieux EasyQ 1110 (533-2230), Roche CAP/CTM 1277 (892-1828), Hologic Aptima 1616 (1324-1973), and Cepheid GeneXpert 2502 (1713-3655) cp/mL. Calibration studies involving three consecutive WHO replacement standards showed a significant drift in the amount of RNA copies per International Unit overtime. Heat inactivation of HIV-1 standards was found to cause a destandardizing effect. Our study underlines the limitations in HIV-1 RNA assay calibration based on frequently replaced WHO international standards. It is therefore proposed that clinicians interpret the recommended 1000 cp/mL alert level in therapy monitoring with an inaccuracy range of 500 to 2000 cp/mL.

BACKGROUND If anti-hepatitis B core antibody testing is not mandated blood donors with occult hepatitis B infection (OBI) may transmit hepatitis B virus (HBV) to a recipient in spite of the use of nucleic acid amplification technology (NAT) or pathogen inactivation (PI). STUDY DESIGN AND METHODS We developed a model to estimate OBI transmission risk based on three components: the probability distribution of the viral load (VL) in a randomly selected OBI donor, the probability that a given VL remains undetected, and the probability that this VL causes infection in the recipient. A subset of 217 South African OBI samples identified by individual donation (ID)-NAT screening were quantified by replicate testing using an ID-NAT assay (Ultrio Plus) against HBV DNA standard dilution series. The observed log VLs could be described by a Gumbel distribution. A correction was included to compensate for OBI samples missed by initial ID-NAT screening. RESULTS The model estimates that 3.3% of all OBI donations are undetected by ID-NAT (Ultrio Plus) and cause infection by a blood component containing 20 mL plasma, going up to 8.7% when using minipools of 6 (MP6)-NAT. For 200-mL plasma transfusion these risks were estimated at 14 and 28%, respectively, while PI with modest (2 log) reduction capacity would reach 4.8% without NAT and 1.3 or 0.4% when combined with MP6- or ID-NAT. CONCLUSION The model can be used to compare different screening and/or PI strategies in reducing viral transmission risk and could serve as a tool in evaluating efficacy of alternative blood safety scenarios.

Background Transcription-mediated amplification assays for HBV DNA detection have transitioned from the Ultrio to the Ultrio Plus assay, which features increased analytic sensitivity due to inclusion of a target enhancer reagent. The impact on HBV detection for different categories of HBV infection has not been fully evaluated. Study design and methods Hepatitis B virus (HBV) DNA and hepatitis B surface antigen (HBsAg) detection rates as well as viral load (VL) distributions in HBV nucleic acid test (NAT)-yield samples were compared during 1 year of screening of South African blood donors with the Ultrio assay and the subsequent year by the Ultrio Plus version. HBV-DNA concentration at the HBsAg seroconversion point was established by regression analysis using a set of antibody to hepatitis B core antigen-negative acute viremic samples. Results Ultrio Plus detected twofold more window-period (WP) NAT yield donations and 1.7-fold more occult HBV infections than Ultrio. The VL distribution data indicated that Ultrio not only missed samples of less than 100 copies/mL, but also a substantial number higher than this level. The VL at the HBsAg seroconversion point was estimated at 916 copies/mL, whereas the VL at the NAT-conversion points was calculated at 63 and 4.1 copies/mL for Ultrio and Ultrio Plus. This reduced the infectious WP (compared to HBsAg testing) by 10.3 and 20.4 days, respectively. Conclusion The higher-than-expected increase in HBV-NAT yields after introduction of the Ultrio Plus assay is likely attributable to variable sensitivity of the former Ultrio assay for different HBV samples. Therefore, previously published HBV WP reduction and residual risk estimates based on analytical sensitivity of the Ultrio assay need to be revised.

BACKGROUND In 1998 we estimated that 34/million infectious window period donations were entering the blood supply at the South African National Blood Service. Selective use of donations based on donor race-ethnicity reduced this risk to 26/million donations but was deemed unethical. Consequently, in 2005 South African National Blood Service eliminated race-ethnicity-based collection policies and implemented individual-donation nucleic acid testing (ID-NAT). We describe the change in donor base demographics, human immunodeficiency virus (HIV) detection rates, and transfusion-transmissible HIV risk. STUDY DESIGN AND METHODS In ten years 7.7 million donations were tested for anti-HIV and HIV RNA. Number of donations, HIV prevalence, ID-NAT yield rate, serology yield rate and residual transfusion-transmissible HIV risk were analyzed by donor type, race-ethnicity, age, and sex. Multiple regression analysis was performed to investigate the determinants of HIV-positive and nucleic acid testing yield donations. RESULTS The combined strategy of increasing donations from black donors and implementing ID-NAT increased the proportion of donations from black donors from 6% in 2005 to 30% in 2015 (p < 0.00001), and reduced the transfusion-transmissible risk from 24 to 13 per million transfusions. ID-NAT interdicted 481 (1:16,100) seronegative window period donations, while one transfusion-transmissible case (0.13 per million) was documented. Race-ethnicity and donor type were highly significant predictors of HIV positivity, with adjusted odds ratio for first-time donors of 12.5 (95% confidence interval, 11.9-13.1) and for black race-ethnicity of 31.1 (95% confidence interval, 28.9-33.4). The proportion of serology yields among HIV-infected donors increased from 0.27% to 2.4%. CONCLUSION ID-NAT enabled the South African National Blood Service to increase the number of donations from black donors fivefold while enhancing the safety of the blood supply.

BACKGROUND Hepatitis C virus (HCV) antigen and antibody combination assays have been launched as a cost-effective alternative to nucleic acid testing (NAT) for reducing the antibody-negative window period (WP). Later, a HCV antigen chemiluminescence immunoassay (CLIA) became available. STUDY DESIGN AND METHODS A panel composed of 337 HCV NAT–yield samples that were characterized for viral load (VL) and genotype was used to compare the sensitivity of two combination enzyme-linked immunosorbent assays (Monolisa, Bio-Rad; and Murex, formerly Abbott) and a HCV antigen CLIA (Abbott). Analytic sensitivity was compared with HCV RNA detection using Ultrio (Grifols) by testing serial dilutions of 10 genotype (gt)1 to gt4 samples. RESULTS HCV antigen CLIA detected 92.4% of samples, whereas Monolisa and Murex detected 38.3 and 47.5%, respectively. In the HCV RNA VL range of 105 to 107 IU/mL, Monolisa and Murex detected 38% to 56% of gt1, 85% to 78% of gt2, and 21% to 37% of gt3. The overall geometric mean 50% limit of detection (range) of Ultrio on gt1 to gt4 dilution series was 3.5 (1.2-7.7) copies/mL, compared to 3.3 × 106 (4.4 × 105-2.7 × 107), 3.4 × 106 (2.2 × 105-4.2 × 107), and 2728 (415-7243) copies/mL for Monolisa, Murex, and HCV antigen CLIA, respectively. CONCLUSION Analytical sensitivity of NAT was on average 1 million- and 780-fold higher than combination assays and HCV antigen CLIA, respectively. Relative sensitivities of combination assays differed for genotypes with Murex being more sensitive for gt1 and gt3 and Monolisa more sensitive for gt2. Although being less sensitive than NAT, combination assays could be considered in resource-limited settings since they detect 38% to 47% of seronegative WP donations.

Background Sensitivity data from a head-to-head comparison study in South Africa were used to compare the efficacy of the Ultrio Plus assay in individual-donation (ID) and minipool (MP)4 and MP8 formats with that of TaqScreen MP6 in preventing hepatitis B virus (HBV) transmission risk. Study Design and Methods The replicate nucleic acid test (NAT) results on 106 HBV NAT (Ultrio)-yield samples and 29 HBV DNA (Ultrio)-negative, hepatitis B surface antigen (HBsAg)-positive samples were used to determine the viral load in copies/mL against the Eurohep HBV standard by probit analysis. Random viral load distributions were established in 32 pre-HBsAg window period (WP), 15 post-HBsAg WP, and 56 occult HBV infection (OBI) donations. Regression analysis of log viral load and Poisson distribution statistics of infectious HBV particles in blood components was used to predict infectivity and efficacy of NAT options in removing HBV transmission risk. Results For red blood cell transfusions (20 mL of plasma), the modeling predicted an Ultrio Plus ID-NAT efficacy of 68 and 83% in removing WP and (antibody to hepatitis B surface antigen–negative) OBI transmission risk, respectively, compared to 52 and 49% by TaqScreen MP6. For 200 mL of fresh-frozen plasma the estimated efficacy levels by these ID- and MP6-NAT options reduced to 57 and 44% for WP and to 67 and 34% for OBI donations, respectively. Conclusion The efficacy of the currently available commercial NAT systems in reducing HBV transmission risk is mainly driven by the pool size and the transfusion plasma volume. The modeled OBI transmission risk and NAT efficacy levels were in line with those recently reported in three lookback studies and give more insight in the incremental safety provided by HBsAg and antibody to hepatitis B core antigen testing of ID-NAT screened blood.

Background The second triplex transcription-mediated amplification (TMA) assay version (Ultrio Plus, Novartis Diagnostics) uses an additional reagent enhancing the disruption of hepatitis B virus (HBV) particles and release of DNA for the target capture probe. This study compares the performance of this new assay version with the previous one (Ultrio). Study Design and Methods For analytical sensitivity assessment the World Health Organization HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) international standards and various genotype dilution panels were used. Individual donations (IDs) from 9980 first-time donors were screened simultaneously by serology and both TMA assay versions. Results The 50 and 95% limits of detection (LODs) for HBV using Ultrio Plus were 0.8 (0.6-1.0) and 4.6 (3.2-7.2) IU/mL, respectively, 2.4 (1.4-4.8)-fold more sensitive than Ultrio. The TMA assay versions had comparable LODs for HIV-1 and HCV. The improvement factors on analytical sensitivity panels of HBV Genotypes A to G ranged from 1.3 to 7.3 and 50% LODs (95% confidence interval) reduced from 12.5 (10-15) to 3.8 (3.2-4.4) copies/mL. One Ultrio Plus HBV Genotype D yield sample missed by the Ultrio assay in the donor screening study was detected with ninefold higher sensitivity. The specificities of ID nucleic acid test (ID-NAT) and serologic testing in a similar repeat test algorithm were 100 and 99.41%, respectively. Conclusion More efficient target capture chemistry in the new TMA assay version significantly improved sensitivity and diminished variability in detecting HBV strains of various genotypes. We recommend a triplicate ID-NAT repeat test strategy to eliminate discriminatory tests on false-non–repeat-reactive (anti-HBc–nonreactive) donations.

Background After 3 years of individual-donation nucleic acid test (ID-NAT) screening by the South African National Blood Service (SANBS), a repository of 73 human immunodeficiency virus antibody (anti-HIV)-negative window period (WP)-yield samples and 28 anti-HIV–positive, HIV-RNA–negative elite controllers (ECs) became available for comparison of a p24 antigen (p24 Ag) assay (Innogenetics), two viral load assays (Siemens branch DNA [bDNA] 3.0 and Abbott real-time polymerase chain reaction [RT-PCR]), and three triplex NAT assays (Novartis Diagnostics Ultrio and Ultrio-Plus and Roche TaqScreen) by replicate testing of dilutions. Study Design and Methods Viral loads were assessed by bDNA and RT-PCR assays and if below 100 copies (cps)/mL, by Ultrio limiting dilution probit analysis. The probability of virus transmission by WP and EC donations was estimated for different levels of the 50% minimum infectious dose (ID50) using Poisson distribution statistics. Results The equal distribution of WP donations plotted by log HIV-RNA levels indicated a random appearance of donors in the ramp-up phase. The HIV p24 Ag assay detected 45% of WP samples and the cutoff crossing point was estimated at 8140 (bDNA)/22,710 (RT-PCR) cps/mL. On replicate retesting of 40 HIV p24 Ag–negative ID-NAT WP-yield samples Ultrio minipool (MP)8, Ultrio-Plus MP8, and TaqScreen MP6 detected 79, 81, and 78%, respectively. Modeling with an estimated ID50 of 31.6 virions/RBC indicated that 15% of p24 Ag–negative ID-NAT WP-yield donations would have transmitted HIV if MP6-8 NAT had been used. Only 2% of RBC transfusions from ECs are estimated to be infectious with a worst-case ID50 estimate of 316 virions. Conclusion Our analysis of viremia and infectivity of WP and EC donations enables comparison of the efficacy of NAT options in preventing HIV transmission risk.

Background Twenty-one blood organizations from five geographical regions provided HIV individual donation (ID)-NAT and serology data on 11,787,610 donations. Infections were classified as anti-HIV-/RNA+ window period (WP), anti-HIV+/RNA+ concordant positive (CP) or anti-HIV+/RNA- elite controller (EC). Residual risk and efficacy of several screening scenarios were estimated for first time, lapsed and repeat donations. Methods WP residual risk estimates assumed a 50% infectious dose of 3.16 virions and a 50% detection limit of 2.7 HIV RNA copies/mL for ID-NAT and 10,000 copies/mL for p24Ag. Infectivity for CP (100%) and EC (2.2%) donations was estimated based on viral load distributions and 100-fold reduced infectivity by antibody neutralization as reported elsewhere. Efficacy was calculated as proportion of transmission risk removed from baseline (i.e. in absence of any screening). Results There was no significant difference in transmission risk between lapsed and repeat donations in any region. Risk was 3.8-fold higher in first time than combined lapsed/repeat donations in South Africa but not in other regions. Screening strategies were most efficacious at interdicting infectious transfusions in first time (98.7-99.8%) followed by lapsed (97.6-99.7%) and repeat (86.8-97.7%) donations in all regions combined. In each donor category the efficacy of ID-NAT alone (97.7-99.8%) was superior to that of minipool (MP)-NAT/anti-HIV (95.0-99.6%) and p24 Ag/anti-HIV (89.8-99.1%). Conclusions Efficacy patterns were similar by donor/donation status in each region despite large differences in HIV prevalence and transmission risk. As similar data become available for HBV and HCV, this modeling may be useful in cost effectiveness analyses of alternative testing scenarios.

Background Several comparison studies showed that the Ultrio assay (Novartis Diagnostics) used in individual-donation nucleic acid amplification testing (ID-NAT) format was as sensitive as the TaqScreen assay (Roche) on minipools of six donations (MP6), but the sensitivity of HBV DNA detection has been improved in the new Ultrio Plus version of the assay. A head-to-head comparison study was designed to compare the clinical sensitivity of the Ultrio and Ultrio Plus assay in ID, MP4, and MP8 formats using TaqScreen MP6 as a reference assay. Study Design and Methods Plasma samples of 107 hepatitis B surface antigen (HBsAg)-negative, HBV ID-NAT (Ultrio) positive-yield samples and 29 HBV DNA–negative, HBsAg-positive samples were used for comparison of NAT options in replicate testing of dilutions. Viral loads and relative sensitivities were determined by probit analysis against the Eurohep standard. Results Ultrio Plus detected a significantly (p

Background Occult hepatitis B virus (HBV) infection (OBI) is identified in 1:1000 to 1:50,000 European blood donations. This study intended to determine the infectivity of blood products from OBI donors. Study Design and Methods Recipients of previous donations from OBI donors were investigated through lookback (systematic retrieval of recipients) or traceback (triggered by clinical cases). Serologic and genomic studies were undertaken on consenting donors and recipients. Multiple variables potentially affecting infectivity were examined. Results A total of 45 of 105 (42.9%) donor-recipients pairs carried antibodies to HBV core (anti-HBc) as evidence of previous HBV infection. Subtracting 15% of anti-HBc population background, the adjusted transmission rate was 28%. Anti-HBc prevalence increased to 28 of 44 (63.8%) in unvaccinated recipients receiving anti-HBs–negative OBI blood products. In contrast, four of 26 (15.4%) recipients of anti-HBs–positive products were anti-HBc positive. Transmission with anti-HBs–negative products depended on volume of plasma transfused (85%-100% with 200 mL of fresh frozen plasma [FFP], 51% with 50 mL in platelet concentrates [PCs], and 24% with 20 mL in red blood cells [RBCs], p

If anti-hepatitis B core antibody testing is not mandated blood donors with occult hepatitis B infection (OBI) may transmit hepatitis B virus (HBV) to a recipient in spite of the use of nucleic acid amplification technology (NAT) or pathogen inactivation (PI).We developed a model to estimate OBI transmission risk based on three components: the probability distribution of the viral load (VL) in a randomly selected OBI donor, the probability that a given VL remains undetected, and the probability that this VL causes infection in the recipient. A subset of 217 South African OBI samples identified by individual donation (ID)-NAT screening were quantified by replicate testing using an ID-NAT assay (Ultrio Plus) against HBV DNA standard dilution series. The observed log VLs could be described by a Gumbel distribution. A correction was included to compensate for OBI samples missed by initial ID-NAT screening.The model estimates that 3.3% of all OBI donations are undetected by ID-NAT (Ultrio Plus) and cause infection by a blood component containing 20 mL plasma, going up to 8.7% when using minipools of 6 (MP6)-NAT. For 200-mL plasma transfusion these risks were estimated at 14 and 28%, respectively, while PI with modest (2 log) reduction capacity would reach 4.8% without NAT and 1.3 or 0.4% when combined with MP6- or ID-NAT.The model can be used to compare different screening and/or PI strategies in reducing viral transmission risk and could serve as a tool in evaluating efficacy of alternative blood safety scenarios.

BACKGROUND: Since October 2005, a total of 2,921,561 blood donations have been screened by the South African National Blood Service for hepatitis B virus (HBV) by individual-donation nucleic acid testing (ID-NAT). Over 4 years, 149 hepatitis B surface antigen–negative acute-phase HBV NAT–positive donations were identified (1:19,608). The lookback program identified one probable HBV transmission. STUDY DESIGN AND METHODS: The complete genomes of HBV isolated from the donor and recipient were sequenced, cloned, and analyzed phylogenetically. The HBV window period (WP) transmission risk was estimated assuming a minimum infectious dose of 3.7 HBV virions and an incidence rate correction factor of 1.34 for transient detectability of HBV DNA. RESULTS: Of 149 acute-phase HBV NAT yields, 114 (1:25,627) were classified as pre–antibody to hepatitis B core antigen (anti-HBc) WP and 35 (1:83,473) as post–anti-HBc WP. The acute-phase transmission risk in the HBV DNA–negative pre- and post–anti-HBc WPs (of 15.3 and 1.3 days, respectively) was estimated at 1:40,000 and 1:480,000, respectively. One HBV transmission (1:2,900,000) was identified in a patient who received a transfusion from an ID-NAT–nonreactive donor in the pre–anti-HBc WP. Sequence analysis confirmed transmission of HBV Subgenotype A1 with 99.7% nucleotide homology between donor and recipient strains. The viral burden in the infectious red blood cell unit was estimated at 32 (22-43) HBV DNA copies/20 mL of plasma. CONCLUSION: We report the first known case of transfusion-transmitted HBV infection by blood screened using ID-NAT giving an observed HBV transmission rate of 0.34 per million. The estimated pre–acute-phase transmission risk in the ID-NAT screened donor population was 73-fold higher than the observed WP transmission rate.

BACKGROUND: In minipool nucleic acid test (MP-NAT) screening protocols, the donations implicated in reactive test pools are released for transfusion when they are nonreactive in a repeat test on the individual samples, but in individual-donation (ID)-NAT screening algorithms the release of nonrepeatable reactive (NRR) donations is under discussion. STUDY DESIGN AND METHODS: A previously developed window phase (WP) transmission risk model for NAT-screened blood transfusions has been refined to take the effect of repeat tests of initially reactive (IR) MP- or ID-NAT results into account. The model has then been applied to simulate the effect of different screening algorithms with ULTRIO and the new-generation ULTRIO Plus assay (Novartis Diagnostics) on transmission risk for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). RESULTS: We calculated WP risk-day equivalents for MP16-, MP8-, and ID-NAT with and without duplicate retesting of IR results of 3.1, 2.7, 1.5, and 1.3 days for HCV; 6.3, 5.5, 3.3, and 2.9 days for HIV; and 24.4, 22.2, 15.6, and 14.1 days for HBV, respectively. These latter infectious HBV WPs reduced to 20.4, 18.2, 11.6, and 10.3 days, respectively, with the more sensitive ULTRIO Plus assay. CONCLUSION: ULTRIO Plus ID-NAT screening reduces the virus transmission risk in the WP by 54% to 58% compared to ULTRIO MP16-NAT, while the incremental risk caused by releasing donations with duplicate ID-NAT NRR results is 5% to 6%. To achieve maximum safety and specificity a similar repeat test algorithm can be applied to ID-NAT as used for serologic assays.

BACKGROUND: The risk of transfusion-transmitted hepatitis B virus (HBV) in Switzerland by testing blood donors for hepatitis B surface antigen (HBsAg) alone has been historically estimated at 1:160,000 transfusions. The Swiss health authorities decided not to introduce mandatory antibody to hepatitis B core antigen (anti-HBc) testing but to evaluate the investigation of HBV nucleic acid testing (NAT). STUDY DESIGN AND METHODS: Between June 2007 and February 2009, a total of 306,000 donations were screened routinely for HBsAg and HBV DNA by triplex individual-donation (ID)-NAT (Ultrio assay on Tigris system, Gen-Probe/Novartis Diagnostics). ID-NAT repeatedly reactive donors were further characterized for HBV serologic markers and viral load by quantitative polymerase chain reaction. The relative sensitivity of screening for HBsAg, anti-HBc, and HBV DNA was assessed. The residual HBV transmission risk of NAT with or without anti-HBc and HBsAg was retrospectively estimated in a mathematical model. RESULTS: From the 306,000 blood donations, 31 were repeatedly Ultrio test reactive and confirmed HBV infected, of which 24 (77%) and 27 (87%) were HBsAg and anti-HBc positive, respectively. Seven HBV-NAT yields were identified (1:44,000), two pre-HBsAg window period (WP) donations (1:153,000) and five occult HBV infections (1:61,000). Introduction of ID-NAT reduced the risk of HBV WP transmission in repeat donors from 1:95,000 to 1:296,000. CONCLUSIONS: Triplex NAT screening reduced the HBV WP transmission risk approximately threefold. NAT alone was more efficacious than the combined use of HBsAg and anti-HBc. The data from this study led to the decision to introduce sensitive HBV-NAT screening in Switzerland. Our findings may be useful in designing more efficient and cost-effective HBV screening strategies in low-prevalence countries.

Background: Standard preparations for HBsAg are required for quality control of test kits and clinical studies on HBsAg quantitation. WHO provides purified heat inactivated HBsAg diluted in negative defribinated plasma as 2nd International Standard (IS) for quality control of tests. Objective: Study of possible alterations of antigenicity, protein composition, size and density of the heat inactivated source material (SM) for the 2nd IS. Study design: Native HBsAg and SM were examined by quantitative immune electrophoresis (QIE), SDS-PAGE, ultracentrifugation and gel chromatography. HBV DNA was sequenced and the HBsAg geno/subtype derived. Results: The SM contained 97,600 International Units HBsAg/ml in QIE which agreed very well with the previous evaluations by WHO using 10 different assays. In SDS-PAGE, SM showed on a strong background the small HBs proteins but no preS proteins. SM had a more heterogeneous density than native HBsAg and contained particle aggregates. The HBsAg geno/subtype of SM was A2/adw2. Conclusions: The IS has very good HBs antigenicity, but it lacks the preS domains, has modified HBs proteins and is partially aggregated. While it has been proven very useful for quality control of tests, certain inconsistencies due to the altered structure of its HBsAg cannot be excluded.

Blood screening by NAT for major transfusion transmitted viral infections (TTIs) was originally intended to complement serology for detection of infected donations. Reports from developed countries showed limited marginal value to NAT blood screening in improving blood safety. Reports on NAT results from Europe indicated yield of 1:0.6 million donations for HBV

BACKGROUND: In 2005, the South African National Blood Service introduced individual-donation (ID) nucleic acid test (NAT) screening for human immunodeficiency virus (HIV) RNA, hepatitis C virus (HCV) RNA, and hepatitis B virus (HBV) DNA. At the same time the use of ethnic origin to prioritize the transfusion of blood according to a hierarchy of residual risk was discontinued. STUDY DESIGN AND METHODS: ID-NAT (Ultrio on Procleix Tigris, Chiron) and serology (PRISM, Abbott) repeat test and confirmation testing algorithms were designed to enable differentiation between false-positive and true-NAT and -serology yields. After 1 year, the NAT and serology yield rates in first-time, lapsed, and repeat donors were analyzed and used to estimate the residual risk of HIV, HBV, and HCV infections by blood transfusion. RESULTS: The HIV, HBV, and HCV ID-NAT window phase yield rates in 732,250 blood donations were 1:45,765, 1:11,810, and 1:732,200, respectively. Seven of 16 HIV window phase donations with viral loads above 16,000 copies/mL were HIV p24 antigen enzyme-linked immunosorbent assay positive. PRISM detected anti-HIV and hepatitis B surface antigen (HBsAg) in 89.4 and 73.9% of early infections in repeat donors. The Procleix assay detected viremia in 99.7 and 95.5% of anti-HIV– and HBsAg-positive first-time donors. In these donors, the occult HBV DNA carrier rate was 1:5200. The residual transmission risk of ID-NAT HIV, HBV, and HCV window phase donations was estimated at 1:479,000, 1:61,500, and 1:21,000,000 respectively. CONCLUSION: One-year ID-NAT screening of 732,250 donations interdicted 16 HIV, 20 HBV, and 1 HCV window phase donations and 42 anti-hepatitis B core antigen–reactive infections during an early recovery or a later stage of occult HBV infection.

Background/Aims Occult hepatitis B infection (OBI) in blood donations is not considered infectious when anti-HBs is present. Methods Four months after transfusion of eight blood components during coronary arterial bypass surgery, a 59-year-old patient developed acute hepatitis B. A second 71-year-old patient transfused with a red cell concentrate (RCC) from one of these donations had early HBV infection 7months post-transfusion. Samples were tested for HBV serological markers and HBV DNA was quantified and sequenced. Results One implicated donation contained anti-HBc, anti-HBs (12 IU/L) and 180IU/ml of HBV DNA. Previous and subsequent samples contained 3–10 times lower viral load and slightly variable anti-HBs. Two previous donations did not cause HBV infection. Recipients of the FFP and RCC from the index donation were both HBV infected and carried genotype D strains with sequences identical to the donor strain. Conclusions Despite anti-HBs, an OBI carrier transmitted HBV to two immunocompetent transfusion recipients.

Hepatitis C virus (HCV) antigen and antibody combination assays have been launched as a cost-effective alternative to nucleic acid testing (NAT) for reducing the antibody-negative window period (WP). Later, a HCV antigen chemiluminescence immunoassay (CLIA) became available.A panel composed of 337 HCV NAT-yield samples that were characterized for viral load (VL) and genotype was used to compare the sensitivity of two combination enzyme-linked immunosorbent assays (Monolisa, Bio-Rad; and Murex, formerly Abbott) and a HCV antigen CLIA (Abbott). Analytic sensitivity was compared with HCV RNA detection using Ultrio (Grifols) by testing serial dilutions of 10 genotype (gt)1 to gt4 samples.HCV antigen CLIA detected 92.4% of samples, whereas Monolisa and Murex detected 38.3 and 47.5%, respectively. In the HCV RNA VL range of 10(5) to 10(7) IU/mL, Monolisa and Murex detected 38% to 56% of gt1, 85% to 78% of gt2, and 21% to 37% of gt3. The overall geometric mean 50% limit of detection (range) of Ultrio on gt1 to gt4 dilution series was 3.5 (1.2-7.7) copies/mL, compared to 3.3 × 10(6) (4.4 × 10(5) -2.7 × 10(7) ), 3.4 × 10(6) (2.2 × 10(5) -4.2 × 10(7) ), and 2728 (415-7243) copies/mL for Monolisa, Murex, and HCV antigen CLIA, respectively.Analytical sensitivity of NAT was on average 1 million- and 780-fold higher than combination assays and HCV antigen CLIA, respectively. Relative sensitivities of combination assays differed for genotypes with Murex being more sensitive for gt1 and gt3 and Monolisa more sensitive for gt2. Although being less sensitive than NAT, combination assays could be considered in resource-limited settings since they detect 38% to 47% of seronegative WP donations.

Twenty-nine laboratories in 12 countries participated in a study to assess the performance of various human papillomavirus (HPV) detection assays through the use of a recombinant HPV DNA standard reagent panel. The panel was designed by a group of HPV experts, and samples were prepared and distributed by the World Health Organization International Laboratory for Standards and Biologicals in The Netherlands. Each panel consisted of 24 coded samples including a dilution series for HPV types 16 and 18, alone or in combination with five other high-risk (HR) HPV types including HPV types 31, 33, 35, 45, and 52, the low-risk HPV type 6, and a negative control. Qualitative assays were generally consistent across laboratories, and most invalid results reflected a lack of HPV test sensitivity. The combined data sets had a proficiency for HPV 16 of 62.5% (15/24) and for HPV 18 of 73.9% (17/23). HPV 31 was the least accurately detected by participating laboratories. Approximately half of participating laboratories failed to detect high concentrations of HPV 31 and, to a lesser extent, to detect HPV types 35, 52, and 6. The panel sample materials offer a source of renewable and reproducible material that could be used in the future development of international standard reagents for calibration of HPV DNA assays and kits.

BACKGROUND: The performance of the recently launched Procleix Ultrio (Chiron/Gen-Probe) human immunodeficiency virus-1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) blood screening assay was evaluated in a European multicenter study. STUDY DESIGN AND METHODS: Serial dilutions of reference materials were tested to determine the detection limits. Robustness and specificity were assessed by testing alternating high-load HCV RNA–positive and –negative samples, and 2912 test pools of eight donations. The added value of minipool and single-donation HBV nucleic acid testing protocols was compared to the currently used Prism (Abbott GmbH & Co. KG) hepatitis B surface antigen (HBsAg) and Auszyme (Abbott GmbH & Co. KG) dynamic HBsAg tests in 15 HBV seroconversion panels. RESULTS: The 95 percent detection limits (and 95% confidence interval [CI]) on the WHO International Standards was 26 (16-58) IU per mL for HIV-1 RNA, 4.6 (3.7-6.5) IU per mL for HCV RNA, and 11 (7.3-22) IU per mL for HBV DNA. No cross-contamination was observed. Testing 2912 pools of eight donations revealed 16 initial reactive samples; 11 were confirmed. The specificity after initial testing and percentage of invalid results were 99.83 and 0.48 percent, respectively. The HBV window-period (WP) reductions relative to HBsAg seroconversion in Prism and Auszyme dynamic HBsAg were, respectively, 6 days (95% CI, 3-8) and 9 days (95% CI, 7-12) in 1:8 minipool (MP) testing. CONCLUSION: The performance characteristics of Procleix Ultrio assay and the Procleix HIV-1 and HCV assay are comparable. The sensitivity for HIV-1 and HCV met the directives of the Paul-Ehrlich Institute and the FDA. The assay can reduce the WP for HBV by 6 days to 2 weeks when used in small MP (

The relative contribution of serologic screening and nucleic acid testing (NAT) to prevent hepatitis C virus (HCV) transmission has not been rigorously addressed.Twenty-one blood organizations in seven geographical regions performing individual-donation (ID)-NAT in parallel with anti-HCV screening provided data from 10,897,105 donations to establish HCV infection rates in first-time, lapsed, and repeat donations. Screening efficacy was modeled for: anti-HCV alone, HCV antigen/antibody (combo), minipool (MP)-NAT in pools of 8 and 16 with anti-HCV, ID-NAT and anti-HCV, and ID-NAT alone. Probabilities of infectivity for red blood cell transfusions were estimated as 100% from window period (WP) and concordant HCV RNA/antibody-positive (concordantly positive [CP]) donations and 0.028% from anti-HCV-positive and RNA-negative probable resolved (PR) donations.There were 5146 confirmed infections (30 WP, 3827 CP, and 1289 PR). Infection rates and transmission risks varied substantially across regions and by donation status. Residual risk with ID-NAT and serology screening was estimated at one in 250,000 in Egypt and at one in 10,000,000 in other regions combined; risk would increase to one in 7300 and one in 312,000, respectively, if NAT had not been performed. ID-NAT with or without anti-HCV testing showed higher efficacy than either MP-NAT or combo assays, particularly in lapsed or repeat donors in whom 99.2, 98.5, and 93.2% of infectious donations were estimated to be interdicted by these respective testing strategies.The incremental efficacy of anti-HCV testing when ID- NAT screening is performed was minimal, particularly for screening lapsed and repeat donations.

BACKGROUND The Ultrio Elite assay (Hologic/Grifols) runs on the Panther blood screening system and is comparable to the Ultrio Plus assay apart from the addition of oligonucleotides for human immunodeficiency virus Type 2 (HIV-2) detection. In this multicenter evaluation study the analytical sensitivity and genotype detection efficiency of the two assay versions were compared. STUDY DESIGN AND METHODS The analytical sensitivity and genotype detection efficiency were analyzed by replicate (18-303) testing of 27 hepatitis B virus (HBV), hepatitis C virus (HCV), HIV-1, and HIV-2 standard dilution panels calibrated in international units (IUs) and copies/mL. A wider range of subgenotypes was tested at 25 copies/mL. Specificity was evaluated in 30,756 donor samples. RESULTS The 95% lower limits of detection (LODs) in Ultrio Elite assay on WHO standards were 4.6, 7.3, 23.5, and 23.3 IU/mL for HBV, HCV, HIV-1, and HIV-2, respectively, and ranged from 13 to 44, 7 to 23, 6 to 15, and 9 copies/mL on genotype panels of the respective viruses. Comparable LODs had been previously found on the same panels with the Ultrio Plus assay. The specificity was 99.95% on initial test and 100% in the repeat test algorithm. CONCLUSION The change in the oligonucleotide design of the Ultrio Elite assay to enable HIV-2 detection has not affected the analytical sensitivity for the other viruses regardless of the genotype. Genotype reference panels are instrumental to compare the sensitivity of nucleic acid test assay versions and could serve as an alternative to seroconversion panels.

The prevalence of hepatitis G virus (HGV)-RNA and HGV-E2 antibodies was studied in a cohort of Dutch hemophilia patients in relation to clotting products used, age, and coinfection with hepatitis C. Between 1991 and 1995, blood samples were taken from 294 patients with hemophilia A, B, or von Willebrand disease. From each patient one fresh frozen sample was tested for HGV cDNA polymerase chain reaction (PCR) and HCV cDNA PCR. Alanine aminotransferase (ALT) tests were performed on plasma samples of all patients. The presence of HGV-E2 antibodies was tested on plasma samples from a subset of 169 patients representing all age groups. Based on the origin and viral safety of the products used, three subgroups of patients were distinguished. Group A: patients who used viral noninactivated factors derived from small and large donor pools; group B: patients who used factors prepared with inadequate viral inactivation techniques derived from small and large donor pools; and group C: patients treated only with optimally viral inactivated large pool clotting factor or recombinant clotting factor concentrate. The prevalence of HGV-RNA was 18%. In group A patients the prevalence was 71%, in group B 50%, and in group C 6%. When related to age, the highest prevalence of HGV-RNA (35%) was seen in patients born between 1980 and 1989. The prevalence of HGV-E2 antibodies increased with age. Of HGV-RNA–negative patients born before 1950, 96% tested positive. HGV viremia did not affect ALT levels, neither in HCV-RNA positive nor in HCV-RNA negative patients. HGV infection is frequently seen in patients with hemophilia. In older age groups a lower rate of HGV-RNA positivity is seen coinciding with a higher rate of antienvelope antibodies. © 1998 by The American Society of Hematology.

Sensitivity data from a head-to-head comparison study in South Africa were used to compare the efficacy of the Ultrio Plus assay in individual-donation (ID) and minipool (MP)4 and MP8 formats with that of TaqScreen MP6 in preventing hepatitis B virus (HBV) transmission risk.The replicate nucleic acid test (NAT) results on 106 HBV NAT (Ultrio)-yield samples and 29 HBV DNA (Ultrio)-negative, hepatitis B surface antigen (HBsAg)-positive samples were used to determine the viral load in copies/mL against the Eurohep HBV standard by probit analysis. Random viral load distributions were established in 32 pre-HBsAg window period (WP), 15 post-HBsAg WP, and 56 occult HBV infection (OBI) donations. Regression analysis of log viral load and Poisson distribution statistics of infectious HBV particles in blood components was used to predict infectivity and efficacy of NAT options in removing HBV transmission risk.For red blood cell transfusions (20 mL of plasma), the modeling predicted an Ultrio Plus ID-NAT efficacy of 68 and 83% in removing WP and (antibody to hepatitis B surface antigen-negative) OBI transmission risk, respectively, compared to 52 and 49% by TaqScreen MP6. For 200 mL of fresh-frozen plasma the estimated efficacy levels by these ID- and MP6-NAT options reduced to 57 and 44% for WP and to 67 and 34% for OBI donations, respectively.The efficacy of the currently available commercial NAT systems in reducing HBV transmission risk is mainly driven by the pool size and the transfusion plasma volume. The modeled OBI transmission risk and NAT efficacy levels were in line with those recently reported in three lookback studies and give more insight in the incremental safety provided by HBsAg and antibody to hepatitis B core antigen testing of ID-NAT screened blood.

Twenty-one blood organizations from five geographical regions provided HIV individual donation (ID)-NAT and serology data on 11,787,610 donations. Infections were classified as anti-HIV-/RNA+ window period (WP), anti-HIV+/RNA+ concordant positive (CP) or anti-HIV+/RNA- elite controller (EC). Residual risk and efficacy of several screening scenarios were estimated for first time, lapsed and repeat donations.WP residual risk estimates assumed a 50% infectious dose of 3.16 virions and a 50% detection limit of 2.7 HIV RNA copies/mL for ID-NAT and 10,000 copies/mL for p24Ag. Infectivity for CP (100%) and EC (2.2%) donations was estimated based on viral load distributions and 100-fold reduced infectivity by antibody neutralization as reported elsewhere. Efficacy was calculated as proportion of transmission risk removed from baseline (i.e. in absence of any screening).There was no significant difference in transmission risk between lapsed and repeat donations in any region. Risk was 3.8-fold higher in first time than combined lapsed/repeat donations in South Africa but not in other regions. Screening strategies were most efficacious at interdicting infectious transfusions in first time (98.7-99.8%) followed by lapsed (97.6-99.7%) and repeat (86.8-97.7%) donations in all regions combined. In each donor category the efficacy of ID-NAT alone (97.7-99.8%) was superior to that of minipool (MP)-NAT/anti-HIV (95.0-99.6%) and p24 Ag/anti-HIV (89.8-99.1%).Efficacy patterns were similar by donor/donation status in each region despite large differences in HIV prevalence and transmission risk. As similar data become available for HBV and HCV, this modeling may be useful in cost effectiveness analyses of alternative testing scenarios.

The second triplex transcription-mediated amplification (TMA) assay version (Ultrio Plus, Novartis Diagnostics) uses an additional reagent enhancing the disruption of hepatitis B virus (HBV) particles and release of DNA for the target capture probe. This study compares the performance of this new assay version with the previous one (Ultrio).For analytical sensitivity assessment the World Health Organization HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) international standards and various genotype dilution panels were used. Individual donations (IDs) from 9980 first-time donors were screened simultaneously by serology and both TMA assay versions.The 50 and 95% limits of detection (LODs) for HBV using Ultrio Plus were 0.8 (0.6-1.0) and 4.6 (3.2-7.2) IU/mL, respectively, 2.4 (1.4-4.8)-fold more sensitive than Ultrio. The TMA assay versions had comparable LODs for HIV-1 and HCV. The improvement factors on analytical sensitivity panels of HBV Genotypes A to G ranged from 1.3 to 7.3 and 50% LODs (95% confidence interval) reduced from 12.5 (10-15) to 3.8 (3.2-4.4) copies/mL. One Ultrio Plus HBV Genotype D yield sample missed by the Ultrio assay in the donor screening study was detected with ninefold higher sensitivity. The specificities of ID nucleic acid test (ID-NAT) and serologic testing in a similar repeat test algorithm were 100 and 99.41%, respectively.More efficient target capture chemistry in the new TMA assay version significantly improved sensitivity and diminished variability in detecting HBV strains of various genotypes. We recommend a triplicate ID-NAT repeat test strategy to eliminate discriminatory tests on false-non-repeat-reactive (anti-HBc-nonreactive) donations.

Several comparison studies showed that the Ultrio assay (Novartis Diagnostics) used in individual-donation nucleic acid amplification testing (ID-NAT) format was as sensitive as the TaqScreen assay (Roche) on minipools of six donations (MP6), but the sensitivity of HBV DNA detection has been improved in the new Ultrio Plus version of the assay. A head-to-head comparison study was designed to compare the clinical sensitivity of the Ultrio and Ultrio Plus assay in ID, MP4, and MP8 formats using TaqScreen MP6 as a reference assay.Plasma samples of 107 hepatitis B surface antigen (HBsAg)-negative, HBV ID-NAT (Ultrio) positive-yield samples and 29 HBV DNA-negative, HBsAg-positive samples were used for comparison of NAT options in replicate testing of dilutions. Viral loads and relative sensitivities were determined by probit analysis against the Eurohep standard.Ultrio Plus detected a significantly (p0.00001) higher proportion of replicate assays on HBV NAT yields (77%) than Ultrio ID (62%) and TaqScreen MP6 (47%), whereas Ultrio Plus MP4 and MP8 detected 53 and 41%, respectively. On HBsAg-yield samples missed by Ultrio screening, the reactivity rate increased significantly (p0.0001) from 23% in Ultrio to 65% in Ultrio Plus and further to 72% (p = 0.10) in the TaqScreen assay. The overall improvement factor of the analytical sensitivity offered by the target enhancer reagent in the Ultrio Plus assay was 2.5 (2.0-3.1)-fold on the Ultrio yield samples, but 43 (11-350)-fold on the HBsAg yields. In ID-NAT format the analytical sensitivity of TaqScreen relative to Ultrio Plus was 2.0 (1.0-4.2), 0.9 (0.7-1.3), and 1.6 (0.9-3.0) on the Eurohep standard, HBV NAT-, and HBsAg-yield samples respectively.The clinical sensitivity of the currently available commercial NAT methods is mainly driven by the pool size.

Since October 2005, a total of 2,921,561 blood donations have been screened by the South African National Blood Service for hepatitis B virus (HBV) by individual-donation nucleic acid testing (ID-NAT). Over 4 years, 149 hepatitis B surface antigen-negative acute-phase HBV NAT-positive donations were identified (1:19,608). The lookback program identified one probable HBV transmission.The complete genomes of HBV isolated from the donor and recipient were sequenced, cloned, and analyzed phylogenetically. The HBV window period (WP) transmission risk was estimated assuming a minimum infectious dose of 3.7 HBV virions and an incidence rate correction factor of 1.34 for transient detectability of HBV DNA.Of 149 acute-phase HBV NAT yields, 114 (1:25,627) were classified as pre-antibody to hepatitis B core antigen (anti-HBc) WP and 35 (1:83,473) as post-anti-HBc WP. The acute-phase transmission risk in the HBV DNA-negative pre- and post-anti-HBc WPs (of 15.3 and 1.3 days, respectively) was estimated at 1:40,000 and 1:480,000, respectively. One HBV transmission (1:2,900,000) was identified in a patient who received a transfusion from an ID-NAT-nonreactive donor in the pre-anti-HBc WP. Sequence analysis confirmed transmission of HBV Subgenotype A1 with 99.7% nucleotide homology between donor and recipient strains. The viral burden in the infectious red blood cell unit was estimated at 32 (22-43) HBV DNA copies/20 mL of plasma.We report the first known case of transfusion-transmitted HBV infection by blood screened using ID-NAT giving an observed HBV transmission rate of 0.34 per million. The estimated pre-acute-phase transmission risk in the ID-NAT screened donor population was 73-fold higher than the observed WP transmission rate.

In minipool nucleic acid test (MP-NAT) screening protocols, the donations implicated in reactive test pools are released for transfusion when they are nonreactive in a repeat test on the individual samples, but in individual-donation (ID)-NAT screening algorithms the release of nonrepeatable reactive (NRR) donations is under discussion.A previously developed window phase (WP) transmission risk model for NAT-screened blood transfusions has been refined to take the effect of repeat tests of initially reactive (IR) MP- or ID-NAT results into account. The model has then been applied to simulate the effect of different screening algorithms with ULTRIO and the new-generation ULTRIO Plus assay (Novartis Diagnostics) on transmission risk for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV).We calculated WP risk-day equivalents for MP16-, MP8-, and ID-NAT with and without duplicate retesting of IR results of 3.1, 2.7, 1.5, and 1.3 days for HCV; 6.3, 5.5, 3.3, and 2.9 days for HIV; and 24.4, 22.2, 15.6, and 14.1 days for HBV, respectively. These latter infectious HBV WPs reduced to 20.4, 18.2, 11.6, and 10.3 days, respectively, with the more sensitive ULTRIO Plus assay.ULTRIO Plus ID-NAT screening reduces the virus transmission risk in the WP by 54% to 58% compared to ULTRIO MP16-NAT, while the incremental risk caused by releasing donations with duplicate ID-NAT NRR results is 5% to 6%. To achieve maximum safety and specificity a similar repeat test algorithm can be applied to ID-NAT as used for serologic assays.

The risk of transfusion-transmitted hepatitis B virus (HBV) in Switzerland by testing blood donors for hepatitis B surface antigen (HBsAg) alone has been historically estimated at 1:160,000 transfusions. The Swiss health authorities decided not to introduce mandatory antibody to hepatitis B core antigen (anti-HBc) testing but to evaluate the investigation of HBV nucleic acid testing (NAT).Between June 2007 and February 2009, a total of 306,000 donations were screened routinely for HBsAg and HBV DNA by triplex individual-donation (ID)-NAT (Ultrio assay on Tigris system, Gen-Probe/Novartis Diagnostics). ID-NAT repeatedly reactive donors were further characterized for HBV serologic markers and viral load by quantitative polymerase chain reaction. The relative sensitivity of screening for HBsAg, anti-HBc, and HBV DNA was assessed. The residual HBV transmission risk of NAT with or without anti-HBc and HBsAg was retrospectively estimated in a mathematical model.From the 306,000 blood donations, 31 were repeatedly Ultrio test reactive and confirmed HBV infected, of which 24 (77%) and 27 (87%) were HBsAg and anti-HBc positive, respectively. Seven HBV-NAT yields were identified (1:44,000), two pre-HBsAg window period (WP) donations (1:153,000) and five occult HBV infections (1:61,000). Introduction of ID-NAT reduced the risk of HBV WP transmission in repeat donors from 1:95,000 to 1:296,000.Triplex NAT screening reduced the HBV WP transmission risk approximately threefold. NAT alone was more efficacious than the combined use of HBsAg and anti-HBc. The data from this study led to the decision to introduce sensitive HBV-NAT screening in Switzerland. Our findings may be useful in designing more efficient and cost-effective HBV screening strategies in low-prevalence countries.

A semiautomated dot blot assay and cDNA polymerase chain reaction (PCR) were used to study longitudinal anti-hepatitis C virus (HCV) recognition patterns in relation to presence of HCV-RNA in transfusion recipients and their infectious donors. In 9 recipients, 4 different patterns of HCV infection were observed: (A) persistent HCV carriage accompanied by chronic hepatitis in 6, (B) acute resolved hepatitis, but persistent HCV replication in one, and (C) continuous HCV replication without hepatitis in one and (D) acute resolved hepatitis with clearance of infection in one. This last self-limited infection was characterized by the disappearance of HCV-RNA as well as anti-HCV reactivity. In contrast, antibody reactivity persisted in 7 of 8 patients with chronic HCV infection who could be followed until 1990. Seven of the 9 recipients developed antibodies to all recombinant peptides in dot blot assay; one became positive for anti-C33 and anti-core and one developed anti-core only. The sequence of appearance of antibodies differed among individual patients. In 7 patients with full anti-HCV recognition patterns, the sequence of events was (mean and limits in days after transfusion): onset of hepatitis at day 50 (22-74), seroconversion of anti-C33 at day 91 (59-129), anti-core at day 133 (54-203), and anti-C100 at day 143 (59-365). The incorporation of C33 and core proteins, in addition to C100, in the second generation anti-HCV ELISA enhanced the detection rate in the HCV-infected transfusion recipients from 7/9 (78%) to 9/9 (100%).(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND: Screening of blood units for hepatitis B virus (HBV) DNA identifies donations collected during the window period (WP) of the acute infection and may improve viral safety of the blood supply. It also leads to the detection of occult hepatitis B infection (OBI). STUDY DESIGN AND METHODS: From January 2005 to December 2006, a total of 383,267 blood units were screened for hepatitis B surface antigen (HBsAg) and HBV DNA in two transfusion centers in Madrid, using either individual-donation nucleic acid testing (ID-NAT) or minipool (MP-NAT) of eight donations (MP8). Samples positive for HBV DNA and negative for HBsAg were confirmed by a second molecular test, the viral DNA was quantified, and a genome fragment including the region encoding the major hydrophilic region (MHR) of HBsAg was sequenced. RESULTS: The overall yield of HBV DNA–positive, HBsAg-negative units was 1 in 21,282 (18 cases), higher when using ID-NAT than MP8-NAT (1:9862 vs. 1:51,011; p

BACKGROUND: Screening of blood donors for viral nucleic acids has recently been introduced in several countries. With the use of transcription-mediated amplification, a blood donor was detected who had 90,000 copies of hepatitis B virus (HBV) DNA/mL but no hepatitis B surface antigen (HBsAg) or antibody to hepatitis B core antigen (anti-HBc). One month later, anti-HBc and hepatitis B surface antibody (anti-HBs) appeared; HBV DNA disappeared after 2 months. This study asked why HBsAg was undetectable in this rare case of transient occult HBV infection. STUDY DESIGN AND METHODS: The HBV DNA in the first sample was cloned and sequenced to identify mutations. The physical nature of the virus was examined by polyethylene glycol precipitation, DNase digestion, density gradient centrifugation, and immunoprecipitation. RESULTS: Several mutations were found all over the genome, but the HBs antigen loop was unchanged. A stop mutation in the precore region led to loss of hepatitis B e antigen (HBeAg) expression. No HBV DNA–containing immune complexes were present. The plasma did not contain nonencapsidated HBV DNA that could explain the absence of HBsAg. The virus was immune precipitated by antibodies against HBsAg or preS1 antigen. The ratio of HBV to HBsAg subviral particles was estimated to be 1 in less than 20 whereas in overt cases the ratio is 1 in more than 1000. CONCLUSION: The acute resolving occult HBV infection was caused by an HBeAg-negative variant, which otherwise was almost normal. The negative HBsAg result was probably due to an unusually low production of surplus HBsAg. The absence of the viral immunomodulator HBeAg and the early appearance of anti-HBs suggested a rapid noncytolytic HBsAg-specific T-cell response leading to low expression of HBsAg.

The performance of the recently launched Procleix Ultrio (Chiron/Gen-Probe) human immunodeficiency virus-1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) blood screening assay was evaluated in a European multicenter study.Serial dilutions of reference materials were tested to determine the detection limits. Robustness and specificity were assessed by testing alternating high-load HCV RNA-positive and -negative samples, and 2912 test pools of eight donations. The added value of minipool and single-donation HBV nucleic acid testing protocols was compared to the currently used Prism (Abbott GmbHCo. KG) hepatitis B surface antigen (HBsAg) and Auszyme (Abbott GmbHCo. KG) dynamic HBsAg tests in 15 HBV seroconversion panels.The 95 percent detection limits (and 95% confidence interval [CI]) on the WHO International Standards was 26 (16-58) IU per mL for HIV-1 RNA, 4.6 (3.7-6.5) IU per mL for HCV RNA, and 11 (7.3-22) IU per mL for HBV DNA. No cross-contamination was observed. Testing 2912 pools of eight donations revealed 16 initial reactive samples; 11 were confirmed. The specificity after initial testing and percentage of invalid results were 99.83 and 0.48 percent, respectively. The HBV window-period (WP) reductions relative to HBsAg seroconversion in Prism and Auszyme dynamic HBsAg were, respectively, 6 days (95% CI, 3-8) and 9 days (95% CI, 7-12) in 1:8 minipool (MP) testing.The performance characteristics of Procleix Ultrio assay and the Procleix HIV-1 and HCV assay are comparable. The sensitivity for HIV-1 and HCV met the directives of the Paul-Ehrlich Institute and the FDA. The assay can reduce the WP for HBV by 6 days to 2 weeks when used in small MP (1:8) or single-donation screening protocols.

Many laboratories use working reagents/run controls to monitor the performance of their nucleic acid amplification techniques (NAT) for the measurement of HIV-1 RNA. A collaborative study was carried out in order to calibrate seven internationally available working reagents, QC105 (National Serology Reference Laboratory [NRL], Australia), B5 and B10 (Center for Biological Evaluation and Research [CBER], USA), Pelispy (Central Laboratory of the Netherlands Blood Transfusion Service [CLB], The Netherlands), PWS-1 and PWS-3 (National Institute for Biological Standards and Control [NIBSC], UK) and IRC (Virology Networks [VN], The Netherlands) against the 1st International Standard for HIV-1 RNA (code 97/656). Twenty-one laboratories from 12 different countries participated in the collaborative study and from the results it was determined that QC105 contained 4.0 log(10) International Units (IU)/ml, B5 2.2 log(10) IU/ml, B10 3.8 log(10) IU/ml, Pelispy 4.4 log(10) IU/ml, PWS-1 3.6 log(10) IU/ml, PWS-3 2.7 log(10) IU/ml and IRC 4.3 log(10) IU/ml. The seven working reagents calibrated in this international study may be used to validate and standardise the large number of qualitative and quantitative, commercial and in-house NAT assays that are currently being applied in the fields of blood safety and patient management. They will also help laboratories to comply with the sensitivity requirements that may be brought in by the regulatory authorities and may contribute to further harmonisation of guidelines on NAT published by organisations such as the European Medicines Evaluation Agency (EMEA), Paul-Ehrlich Institute and CBER, FDA.