Infectivity of Hepatitis B Virus Surface Antigen-Positive Plasma With Undetectable HBV-DNA: Can HBsAg Screening Be Discontinued in Egyptian Blood Donors?

Hepatitis B Virus (HBV) infectivity data were reviewed and the 50% infectious dose (ID50) was reassessed in different HBsAg-positive infection stages enabling modelling of transfusion-transmitted (TT)-HBV infection risk if HBsAg donor screening was replaced by individual donation nucleic acid amplification technology (ID-NAT). Quantitative HBsAg and HBV-DNA assays were performed against international standards to compare the ratio between potential infectious HBV virions and subviral HBsAg particles in Egyptian HBsAg-positive blood donors as well as in Japanese chimpanzee samples of known infectivity. HBV-DNA load below the quantification limit of detection was estimated against a reference standard by replicate NAT testing (n = 25). Infectivity of chimpanzee samples collected during ramp-up and declining viremic phase were tested in a human liver chimeric mice (HLCM) model and compared with published infectivity data from different HBsAg-positive infection stages. Lowest estimates of ID50 in HBsAg-positive plasma were 3-6 HBV virions in chimpanzee studies. Infectivity decreased approximately 10-100-fold in the declining viremic phase using HLCM. In acute phase samples, HBV to HBsAg particle ratios varied between 1:102-104 but in HBsAg-positive blood donors this particle ratio reached 1:106-1012 when viral load was below 100 HBV-DNA copies/mL. Modelled TT-HBV risk of an HBsAg-positive/ID-NAT nonreactive blood transfusion was estimated at 5.5%-27% for components containing 20-200 mL of plasma when assuming an ID50 of 316 (point estimate between 100 and 1000) virions. It cannot be ensured that discontinuation of HBsAg donor screening and reliance on ID-NAT alone is safe.

Infectivity of hepatitis B virus (HBV) surface antigen (HBsAg) positive plasma with undetectable HBV-DNA: Can HBsAg screening be discontinued in Egyptian blood donors?

HBV infectivity data were reviewed and the 50% infectious dose (ID50 ) was reassessed in different HBsAg positive infection stages enabling modelling of transfusion-transmitted (TT)-HBV infection risk if HBsAg donor screening was replaced by individual donation nucleic acid amplification technology (ID-NAT). Quantitative HBsAg and HBV-DNA assays were performed against international standards to compare the ratio between potential infectious HBV virions and subviral HBsAg particles in Egyptian HBsAg positive blood donors as well as in Japanese chimpanzee samples of known infectivity. HBV-DNA load below the quantification limit of detection was estimated against a reference standard by replicate NAT testing (n = 25). Infectivity of chimpanzee samples collected during ramp-up and declining viremic phase were tested in a human liver chimeric mice (HLCM) model and compared with published infectivity data from different HBsAg positive infection stages. Lowest estimates of ID50 in HBsAg positive plasma were 3-6 HBV virions in chimpanzee studies. Infectivity decreased approximately 10-100-fold in the declining viremic phase using HLCM. In acute-phase samples, HBV to HBsAg particle ratios varied between 1:102 -104 but in HBsAg positive blood donors this particle ratio reached 1:106 -1012 when viral load was below 100 HBV-DNA copies/ml. Modelled TT-HBV risk of an HBsAg positive/ID-NAT nonreactive blood transfusion was estimated at 9%-46% for components containing 20-200 ml of plasma assuming an ID50 of 316 (point estimate between 100 and 1000) virions. In the Egyptian setting, discontinuation of HBsAg donor screening and reliance on ID-NAT alone seems to be unsafe.

Residual risk of transfusion-transmitted hepatitis B virus (TT-HBV) infection by NAT-screened blood components: A review of observed versus modeled infectivity from donors with window period and occult HBV infections.

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.

An assessment of hepatitis B virus prevalence in South African young blood donors born after the implementation of the infant hepatitis B virus immunization program: Implications for transfusion safety.

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.

Accuracy of quantitative HIV-1 RNA test methods at 1000 copies/mL and the potential impact of differences in assay calibration on therapy monitoring of patients.

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.

Comparison of two nucleic acid amplification technology systems for detection of human immunodeficiency virus, hepatitis B virus, and hepatitis C virus.

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 < .001). We estimated infectious WPs for HBV, HCV, and HIV-1 at 13.8, 1.8, and 2.6 days for Elite and 10.3, 2.1, and 2.4 days for MPX. CONCLUSION: Although MPX was significantly more sensitive for HBV, Elite was implemented due to instrument reliability during evaluation.

Direct comparison of three residual risk models for hepatitis B virus window period infections using updated input parameters.

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.

Reassessment of hepatitis B virus window periods for two transcription-mediated amplification assays using screening data of South African blood donors.

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.

Assessment of HIV transfusion transmission risk in South Africa: a 10-year analysis following implementation of individual donation nucleic acid amplification technology testing and donor demographics eligibility changes.

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.

A mathematical model for estimating residual transmission risk of occult hepatitis B virus infection with different blood safety scenarios.

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.

Detection of different categories of hepatitis B virus (HBV) infection in a multi-regional study comparing the clinical sensitivity of hepatitis B surface antigen and HBV-DNA testing.

BACKGROUND: Twenty-two users of individual donation nucleic acid amplification technology (ID-NAT) in six geographical regions provided detailed hepatitis B virus (HBV) infection data in first-time, lapsed, and repeat donations and classified confirmed HBV-positive donors into different infection categories. These data were used to compare the clinical sensitivity of hepatitis B surface antigen (HBsAg) and HBV-DNA testing. STUDY DESIGN AND METHODS: In total 10,981,776 donations from South Africa, Egypt, the Mediterranean, North and Central Europe, South East Asia, and Oceania were screened for HBV-DNA using the Ultrio assay (Grifols/Hologic) and for HBsAg using a chemiluminescence immunoassay, and 9455 HBV-infected donations were identified. HBsAg-negative window period (WP), HBsAg-positive and occult HBV infection (OBI) stages were determined using supplemental serology, quantitative polymerase chain reaction, and replicate multiplex and discriminatory HBV NAT test strategies. For two regions, additional data sets using the more sensitive Ultrio Plus assay were assessed. RESULTS: Regional HBV detection rates in first-time donors varied between 0.08% and 1.07%, with WP NAT yield rates varying between 1:7700 and 1:294,000 and OBI NAT yield rates varying from 1:3900 to 1:59,000. HBsAg CLIA detected 97.0% of infections in first-time donors, 62.7% in lapsed donors, and 41.0% in repeat donors; whereas Ultrio detected 93.1%, 95.0%, and 98.3% of infections in these respective groups. HBV-DNA detection rates in HBsAg-positive donors varied from 90.2% to 96.3% between regions but increased significantly (range, 95.2-98.2%) with the Ultrio Plus assay. CONCLUSION: ID-NAT and serology are complementary in detecting HBV infection in first-time donors, but HBV-DNA is superior to HBsAg detection in repeat donors.

Relative efficacy of nucleic acid amplification testing and serologic screening in preventing hepatitis C virus transmission risk in seven international regions.

BACKGROUND: The relative contribution of serologic screening and nucleic acid testing (NAT) to prevent hepatitis C virus (HCV) transmission has not been rigorously addressed. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSIONS: The incremental efficacy of anti-HCV testing when ID- NAT screening is performed was minimal, particularly for screening lapsed and repeat donations.

Viremia levels in hepatitis C infection among Egyptian blood donors and implications for transmission risk with different screening scenarios.

BACKGROUND: Knowledge about the viral load (VL) distributions in different stages of hepatitis C virus (HCV) infection is essential to compare the efficacy of serologic screening and nucleic acid testing (NAT) in preventing transfusion transmission risk. We studied HCV-RNA levels in Egyptian blood donors in the preseroconversion window period (WP) and in later anti-HCV-positive stages of infection. STUDY DESIGN AND METHODS: Subsets of individual-donation (ID)-NAT and anti-HCV-yield samples from a screening study among 119,756 donors were tested for VL by quantitative polymerase chain reaction (qPCR). Low viremia levels below the quantification limit of qPCR were determined by probit analysis using the proportion of reactive results on replicate NATs. Poisson distribution statistics were used to estimate transmission risk in different stages of HCV infection based on 50% minimum infectious doses (MID50 ) of 3.2 (1-10) and 316 (100-1000) virions in the absence and presence of anti-HCV, respectively. RESULTS: Rates of total HCV infections and WP-NAT-yield donations in two Egyptian blood centers varied between 2.6% to 4.5% and 1:3100 to 1:9500, respectively. VLs ranged from 82 to 3 × 10(7) copies/mL in WP and from fewer than 1600 to 1.6 × 10(6) copies/mL in anti-HCV-positive carrier donations. Only two (1.1%) of 175 donors with probable resolved infection had detectable RNA on replicate testing (estimated VLs of 0.5 and 1.8 copies/mL). This translates to an estimated transmission risk of 0.028% if ID-NAT-nonreactive, anti-HCV-positive donations would be used for RBC transfusions. CONCLUSION: Almost 99% of anti-HCV-reactive donations without detectable HCV-RNA on initial ID-NAT screening had eradicated the virus from the circulation, while 1% had extremely low VLs and are likely not infectious. The incremental safety offered by serologic testing of ID-NAT-screened blood seems minimal.

Inclusion of human immunodeficiency virus Type 2 (HIV-2) in a multiplex transcription-mediated amplification assay does not affect detection of HIV-1 and hepatitis B and C virus genotypes: a multicenter performance evaluation study.

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.

Sensitivity of hepatitis C virus core antigen and antibody combination assays in a global panel of window period samples.

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 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. 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.

A mathematical approach to estimate the efficacy of individual-donation and minipool nucleic acid amplification test options in preventing transmission risk by window period and occult hepatitis B virus infections.

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.

Enhanced detection of hepatitis B virus in Hong Kong blood donors after introduction of a more sensitive transcription-mediated amplification assay.

BACKGROUND: A total of 517,072 and 399,326 consecutive donations were screened for hepatitis B virus (HBV) by individual-donation nucleic acid testing (ID-NAT) using Ultrio and Ultrio Plus assays (Novartis Diagnostics), respectively. The impact of more sensitive HBV detection by the latter assay version was established by comparing NAT yield and transmission risk. STUDY DESIGN AND METHODS: Donations were screened simultaneously for HBV serologic markers and ID-NAT, followed by discriminatory assay and confirmatory test algorithms. Window period (WP) reduction and residual HBV transmission risk were computed using mathematical modeling. RESULTS: HBV NAT-yield rates for both WP and occult HBV infection (OBI) increased significantly from 1:34,471 to 1:17,362 (p = 0.036) and from 1:5120 to 1:2450 (p < 0.0001), despite a 1.2- and 1.6-fold decrease in hepatitis B surface antigen (HBsAg) incidence and prevalence rates respectively. After adjusting for this bias, the WP and OBI NAT-yield improvement factors were 2.3 and 3.4, respectively, higher than a less than 1.5-fold increase estimated from analytical sensitivity studies on HBV Genotype A and C standards. The current WP transmission risk with Ultrio Plus screening was estimated at 1:55,000 compared to 1:22,000 with HBsAg testing. CONCLUSION: The observed greater than twofold enhanced WP NAT yield with the Ultrio Plus assay can be explained by greater than 10-fold increased analytical sensitivity in detecting the HBV Genotype B and C strains in Hong Kong. Direct comparison studies of the two assay versions on dilutions of HBV NAT-yield samples are required to confirm this hypothesis.

Prevalence of human immunodeficiency virus RNA and antibody in first-time, lapsed, and repeat blood donations across five international regions and relative efficacy of alternative screening scenarios.

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.

Sensitivity of individual-donation and minipool nucleic acid amplification test options in detecting window period and occult hepatitis B virus infections.

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 < 0.00001) 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 (p < 0.0001) 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. CONCLUSION: The clinical sensitivity of the currently available commercial NAT methods is mainly driven by the pool size.

Comparison of human immunodeficiency virus assays in window phase and elite controller samples: viral load distribution and implications for transmission risk.

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.