Ultrio Plus Research Articles

Early Dynamics of Hepatitis B Virus (HBV)-DNA and Surface Antigen (HBsAg) in Ramp-Up Phase of Viremia: Implications for Performance Evaluation of Blood Screening Assays.

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.

Alternative hepatitis B virus DNA confirmatory algorithm identified occult hepatitis B virus infection in Chinese blood donors with non-discriminatory nucleic acid testing.

Multiplex viral nucleic acid testing (NAT) and a discriminatory testing algorithm have been used to detect viral infections in blood donors. Non-discriminated reactive (NDR) results may arise from low hepatitis B virus (HBV) DNA levels and are challenging for donor management by blood services. The aim of this study was to evaluate the performance and feasibility of alternative viral particle concentration methods to confirm and to characterise HBV infection status in NDR donors from Dalian, China, in order to improve routine donor management according to the potential residual risk estimate. Individual donations were tested with ULTRIO Plus, and discriminated when reactive. Virions were concentrated from 12 and 6 mL plasma samples by ultracentrifugation (UC) and polyethylene glycol (PEG) precipitation, respectively. HBV DNA was detected with four nested polymerase chain reactions (95% limit of detection: 5-25 IU/mL). Amplified products were sequenced for definitive confirmation. Anti-HBc and anti-HBs were tested. Of 77,556 donors, 79 (0.1%) were NAT NDR. After viral particle concentration by UC and PEG precipitation, HBV DNA was detected in 46 (58.2%) and 34 (43.0%) NDR donors, respectively, including 61.7% of samples that were repeatedly non-reactive with multiple NAT testing. Anti-HBc and anti-HBs (median titre: 37 mIU/mL) were detected in 87.3% and 46.8% of NDR donors, respectively. Sequencing confirmed HBV DNA in 65.8% of NDR donors, of whom 96.2% were occult HBV carriers with rare mutations in S and core proteins. A HBV DNA confirmatory procedure with limited technical constraints was implemented successfully. The majority of NDR donors had occult HBV infections with extremely low viral DNA levels, which may constitute a potential residual threat for blood safety. Only a minority of anti-HBc+ NDR donors had anti-HBs levels high enough to consider their reinstatement as donors. The data support the permanent deferral of NDR donors to ensure maximum blood safety in areas of high HBV endemicity.

A methodology for deriving the sensitivity of pooled testing, based on viral load progression and pooling dilution

BackgroundPooled testing, in which biological specimens from multiple subjects are combined into a testing pool and tested via a single test, is a common testing method for both surveillance and screening activities. The sensitivity of pooled testing for various pool sizes is an essential input for surveillance and screening optimization, including testing pool design. However, clinical data on test sensitivity values for different pool sizes are limited, and do not provide a functional relationship between test sensitivity and pool size. We develop a novel methodology to accurately compute the sensitivity of pooled testing, while accounting for viral load progression and pooling dilution. We demonstrate our methodology on the nucleic acid amplification testing (NAT) technology for the human immunodeficiency virus (HIV).MethodsOur methodology integrates mathematical models of viral load progression and pooling dilution to derive test sensitivity values for various pool sizes. This methodology derives the conditional test sensitivity, conditioned on the number of infected specimens in a pool, and uses the law of total probability, along with higher dimensional integrals, to derive pooled test sensitivity values. We also develop a highly accurate and easy-to-compute approximation function for pooled test sensitivity of the HIV ULTRIO Plus NAT Assay. We calibrate model parameters using published efficacy data for the HIV ULTRIO Plus NAT Assay, and clinical data on viral RNA load progression in HIV-infected patients, and use this methodology to derive and validate the sensitivity of the HIV ULTRIO Plus Assay for various pool sizes.ResultsWe demonstrate the value of this methodology through optimal testing pool design for HIV prevalence estimation in Sub-Saharan Africa. This case study indicates that the optimal testing pool design is highly efficient, and outperforms a benchmark pool design.ConclusionsThe proposed methodology accounts for both viral load progression and pooling dilution, and is computationally tractable. We calibrate this model for the HIV ULTRIO Plus NAT Assay, show that it provides highly accurate sensitivity estimates for various pool sizes, and, thus, yields efficient testing pool design for HIV prevalence estimation. Our model is generic, and can be calibrated for other infections.

Nearly half of Ultrio plus NAT non-discriminated reactive blood donors were identified as occult HBV infection in South China

BackgroundBlood donor plasma samples were detected by the Ultrio Plus NAT system for HBV, HCV and HIV-1 in Shenzhen blood center, China. Reactive samples underwent further discriminatory testing of a single virus by the same methodology. A large number of cases of non-discriminated reactive (NDR) donors were found, leaving potential risk of transmitting HBV if not deferrals. This study identified those non-discriminated samples.MethodsThe NDR plasma samples from blood donation screening were detected and classified by additional molecular and serological tests. Molecular characterizations of DNA+ NDR were determined by sequencing analysis.ResultsA number of 259 (0.21%) NDR plasma samples from screening of 123,280 eligible blood donors were detected, which presented a higher rate (91.1%) of anti-HBc reactivity and nearly half (46.7%) of HBV DNA+ that classified as occult HBV infection (OBI). Most OBI strains were wild-type HBV, but some substitutions V168A, S174 N, V177A, Q129R/L/H, G145A/R in S region of genotype B (OBIB) and T47K/V/A, P49H/L, Q101R/H/K, S174 N, L175S, V177A, T118 M/R/K, G145R/A/K/E, R160K/N in S region of genotype C (OBIC) strains were identified in high frequency.ConclusionNearly half of NDR blood samples were identified as OBI, in which a number of important mutations were detected. NDR donation might have potential risk for HBV transmission, but need to be further investigated.

Nucleic acid amplification test: Bridging the gap in blood safety & re-evaluation of blood screening for cryptic transfusion-transmitted infection among Indian donors.

Background & objectives:Nucleic acid amplification test (NAT) in blood donor screening not only detects window period (WP) donors but also those with chronic occult infections which are negative by routine serological screening. This study was conducted to determine the time trend of NAT positivity and seroprevalence of transfusion-transmitted infections (TTIs) through a period of six years and evaluate the strength of NAT as a supplementary test in identifying the cryptic carriers in blood donor population.Methods:A total of 1,01,411 blood donations were screened between January 2011 and December 2016 by the ELISA and individual donor (ID) NAT Procleix Ultrio Plus Assay. Additional molecular and serological assays were done on the NAT yield samples to differentiate the type of cryptic carriers.Results:NAT yields comprised 0.05 per cent (50/101411) of the total samples tested with a yield rate of 1/2028. Hepatitis B virus (HBV) contributed to 80 per cent of the total NAT yields and the rest 20 per cent due to hepatitis C virus (HCV). Majority of HBV NAT yields (75%) were from chronic occult donors and 25 per cent were WP donors. Both HBV and HCV NAT yields had a wide range of viral count. There was no HIV NAT yield. A significant decline in the prevalence rate of TTIs through the study period of six years was observed.Interpretation & conclusions:The cryptic infections found in blood donors increase the risk of TTIs. Blood screening by both serology and NAT can reduce this threat.

Incidence of hepatitis B virus infection in young Chinese blood donors born after mandatory implementation of neonatal hepatitis B vaccination nationwide.

This study was carried out to determine the incidence of hepatitis B virus (HBV) infection in the young generation born after mandatory implementation of hepatitis B vaccination since 1992. Repeat blood donors born between 1992 and 1997 were enrolled, who gave blood at least twice during the past 3years. Donors were tested for HBV infection markers of HBsAg, anti-HBc, anti-HBs and viral DNA by immunoassays (EIAs) and nucleic acid tests (NAT). A total of 14937 pre-donation screening qualified young repeat donors aged 18-23years were tested with 9 (0.06%) being HBsAg by EIA and 10 (1:1494) HBV DNA positive by Ultrio NAT (10.4 IU/mL), respectively. HBV DNA was further detected in 1:192 (9/1732) anti-HBc+ repeat donors with Ultrio Plus NAT (3.4 IU/mL). Most cases were identified as occult HBV infection (OBI). Of 14937 repeat donors, 20.9% were anti-HBc+ positive, while approximately 50% of 12024 repeat donors were anti-HBs negative or had levels <100 IU/L. HBsAg+ or OBI strains were classified as wild type of genotype B or genotype C. Incident HBV infection in repeat donors was approximately 1:18.5 person-years (1.1%/year) but significantly less frequent in donors with confirmed HBV vaccination (2.4%-3.3%) than those unsure of vaccination status (10.5%; P=.0023). Hepatitis B virus vaccination appears largely protective of HBV infection, but incidence of infections increases in young adults with mostly undetectable or low anti-HBs or occasionally high anti-HBs. A boost of hepatitis B vaccine for adolescents prior to age 18years may reduce HBV infection, and implementation of more sensitive NAT in blood donation screening may improve HBV safety in blood transfusion.

The validation and implementation of donor screening for hepatitis B virus, hepatitis C virus, human immunodeficiency virus 1/2 and syphilis by ultrio elite assay (Panther system) and chemiluminescence assay (Abbott Architect i2000SR system) in Namibia

Infectious disease screening of Namibia blood donations for infectious diseases was contracted to the South African National Blood Service (SANBS) since 2004 and needed to be relocated back to Namibia. A cost analysis conducted showed that by introducing certain strategies, it was cost‐effective to implement ID‐NAT in Namibia. NAMBTS chose Ultrio Elite assay/Panther System (Gen‐Probe and Novartis, USA) for ID‐NAT for HIV 1/2 RNA, HBV DNA and HCV RNA and Chemiluminescent immunoassay done on the Architect i2000SR system (Abbott, Delkenheim, Germany) for HIV 1/2 Ag/Ab, anti‐HCV, HBsAg and Syphilis. For performance evaluation, the Panther System was compared with the Procleix TIGRIS system—Ultrio Plus assay (Gen‐Probe and Novartis, USA) while the Architect system was compared with the Abbott PRISM. The results of this evaluation demonstrated a 100% sensitivity and 100% specificity of the Ultrio Elite assay while the Architect also demonstrated 100% sensitivity and specificity of above 99% for detecting viral markers for HBV, HIV and HCV, and 97% sensitivity and 99·7% specificity for detecting syphilis. We found both the Procleix Panther and Architect systems to be flexible, robust and effective in screening blood for transfusion in our setting. Donor screening for HIV 1/2, HBV, HCV and syphilis for ID‐NAT and serology using the Procleix Panther and Architect systems was successfully relocated from South Africa and implemented in February 2014 in Namibia.

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

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 (2log) 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.

Comparison of two algorithms to confirm and discriminate samples initially reactive for nucleic acid amplification tests.

BACKGROUND:Blood centers in India have published individual donor nucleic acid testing (ID-NAT) data based on an algorithm (Algorithm A) where serologically negative, NAT reactive sample was subsequently tested with discriminatory NAT (d-NAT), and on the basis of d-NAT, initial reactive samples were classified as “NAT yield” or inconclusive. We followed Algorithm B based on replicate testing and Ultrio Plus assay and compared the results with Algorithm A with Ultrio assay.MATERIALS AND METHODS:Results of ID-NAT using two algorithms were analyzed.RESULTS:A total of 88,583 (31,844 with Algorithm A and 56,739 with Algorithm B) samples were tested. Among serology nonreactive donations, NAT inconclusive results came down from 95.2% in Algorithm A to 73.1% in Algorithm B (P = 0.0001). Discriminated yield (DY) rate went up from 4.7% in Algorithm A to 21.9% in Algorithm B (P = 0.001).CONCLUSION:The study data suggest that replicate testing strategy and Ultrio Plus reduce the number of “inconclusive results” seen with earlier commonly used algorithm. We recommend a replicate testing strategy in ID-NAT testing since it will increase the DY and will eliminate the unnecessary discriminatory tests.

Three-Year Experience in NAT Screening of Blood Donors for Transfusion Transmitted Viruses in Croatia

Background: Croatia implemented individual donation (ID)-NAT testing of blood donors in 2013 for three viruses HBV, HCV, and HIV-1 as a mandatory test for all blood donors. This study assessed the impact of NAT screening 3 years after its implementation. Methods: A total of 545,463 donations were collected and screened for HBV, HCV, and HIV-1 using the Procleix Ultrio Plus Assay. All initially reactive (IR) NAT samples were retested in triplicate and, if repeatedly reactive (RR), NAT discriminatory assay (dNAT) was performed. ID-NAT positive donations were confirmed by RT-PCR on the COBAS AmpliPrep/TaqMan platform. Results: Out of 545,463 samples tested, 108 (0.02%) were RR in NAT. There were 82 (75,9%) HBV reactive, 16 (14.8%) HCV reactive, and 10 (9.3%) HIV-1 reactive samples. 51 (47.2%) samples were ID-NAT positive only. Out of these 51 NAT yield cases, 1 window period HIV-1 and 50 occult HBV infections (OBI) were determined. There were only two potential HBV DNA transmissions from OBI donors. Conclusion: The implementation of NAT screening for three viruses has improved blood safety in Croatia. During the 3-year period, 1 window period HIV-1 and a number of occult HBV donations were identified.

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

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. 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. 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. 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.

Evaluation of the Procleix Ultrio Plus ID NAT assay for detection of HIV 1, HBV and HCV in blood donors.

Introduction:The Procleix Ultrio Plusassay is a new-generation qualitative in vitro nucleic acid amplification test used to screen for human immunodeficiency virus type 1 (HIV-1) RNA, hepatitis C virus (HCV) RNA and hepatitis B virus (HBV) DNA in blood donors. This study was performed to compare the Procleix Ultrio assay with the new-generation Procleix Ultrio Plus Nucleic Acid Test (NAT) assays.Materials and Methods:Ten thousand three hundred and two donor samples were run in parallel for ID NAT using the Procleix Ultrio and the Procleix Ultrio Plus assay. Simultaneously, enzyme-linked immunosorbent assay testing was performed on an EVOLIS Walk away System for HIV, HCV, HBsAg and anti-HBc. Reactive samples were confirmed using polymerase chain reaction.Results:In the 10,302 samples tested during the study period, we identified 15 NAT yields, and all these revealed HBV DNA in the discriminatory assays. Eight of these were exclusive yields from the Ultrio Plus assay and the remaining seven cases were determined as HBV NAT yield, both by the Procleix Ultrio as well as the Ultrio Plus assays, i.e. “Combined” yields. No HCV or HIV 1 yields were detected during the study period by either of two assays.Conclusion:With an overall yield rate of 1 in 687 and an exclusive yield rate of 1 in 1287, the Procleix Ultrio Plus assay proved to be highly sensitive in detecting occult HBV infections.

Sensitivity of individual and mini-pool nucleic acid testing assessed by dilution of hepatitis B nucleic acid testing yield samples

Introduction:For nucleic acid testing (NAT) of blood donations, either the blood samples can be pooled together in a batch of six or eight prior to testing (mini-pool-NAT [MP-NAT]), or the tests can be run on every individual sample (individual donor-NAT [ID-NAT]). It has been debated in various studies whether pooling of samples results in decreased sensitivity of detection as the volume of individual samples gets lesser in a pool. The objective of this study was to investigate the effect of dilution on the sensitivity of tests.Materials and Methods:The study was performesd on nine plasma samples which were hepatitis B reactive exclusively by Procleix Ultrio Plus and not by Procleix Ultrio or serology. These nine exclusive UltrioPlus ID-NAT yield samples were diluted in 1:2, 1:4. 1:6 and 1:8 dilutions using previously tested negative plasma and each dilution of every sample along with archived undiluted sample were retested in three replicates with Procleix Ultrio Plus Assay.Results:Among NAT yield samples, 88.88% of the samples were detected when retested in ID-NAT in undiluted form. Samples with higher viral load (sample 5 and 6) were detected by all dilutions. When samples with viral load below 20 IU/mL were tested in dilutions of 1:6 or 1:8, only 9 out of 27 replicates (33.33%) were detected. This means that more than 67% of low viral load samples were missed by MP-NAT of 1:6 or 1:8 dilution out of total NAT yield samples.Conclusion:Individual Donor NAT is ideal methodology for NAT as dilution due to pooling may miss samples with low viral load as evident in this study.

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

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 (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. The clinical sensitivity of the currently available commercial NAT methods is mainly driven by the pool size.

Comparative analysis of triplex nucleic acid test assays in United States blood donors

This study assessed the clinical sensitivity of three fully automated, human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) triplex nucleic acid test (NAT) assays by individual donation (ID-NAT) and at operational minipool (MP-NAT) sizes used worldwide. MPX, Ultrio, and Ultrio Plus were used to test 2222 pedigreed, marker-positive samples with varying viral loads, each from a unique US blood donor. NAT-positive, seronegative yield samples (16 HBV, 156 HCV, and 23 HIV) were tested in replicates of three; undiluted; and in 1:6, 1:8, and 1:16 dilutions (MP6, MP8, and MP16), simulating various MP sizes. Seropositive samples (1276 HBV, 488 HCV, and 263 HIV) were tested by ID-NAT in singlet. MPX-MP6 and Ultrio Plus-MP16 had equivalent HCV sensitivity. Although Ultrio Plus-MP16 for HIV trended toward lesser sensitivity, this was not corroborated in a large substudy of low-viral-load samples in which Ultrio Plus-MP8/MP16 showed 100% reactivity. MPX-ID and Ultrio Plus-ID HBV clinical sensitivity were identical, but MPX-MP6 was significantly more sensitive than Ultrio Plus-MP16; the differential yield projected to one HBV NAT yield per 4.72 million US donations. Ultrio Plus HBV sensitivity did not increase at MP8 versus MP16. Ultrio Plus versus Ultrio sensitivity was significantly increased in HBV-infected donors with early acute, late acute or chronic, and occult infections. No difference in sensitivity was noted for any virus for MPX-MP6 versus Ultrio Plus-ID. Our data support US donation screening with MPX-MP6 or Ultrio Plus-MP16 since the HBV DNA detection of Ultrio Plus was significantly enhanced (vs. Ultrio) without compromising HIV or HCV RNA detection.

Head‐to‐head comparison of two transcription‐mediated amplification assay versions for detection of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus Type 1 in blood donors

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.

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

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. 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. 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. 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.

Ultrio and Ultrio Plus non‐discriminating reactives: false reactives or not?

The low, fluctuating levels of DNA characteristic of occult hepatitis B infection make its detection by nucleic acid testing (NAT) a challenge. Four year's routine use of the Ultrio and Ultrio Plus assays in blood donations in New Zealand was analysed. 0·09% of donations tested with Ultrio and Ultrio Plus assays showed reactivity in the multiplex assay, but non-reactivity in all three discriminatory assays and relevant mandatory serological assays (anti-HIV, anti-HCV, HBsAg). These donations were more likely to be anti-HBc reactive (Ultrio, 13%; Ultrio Plus, 57%; random donors, 6·8%). Thirty-four per cent of these anti-HBc-reactive donations were also reactive in either an alternate NAT assay or on repeat multiplex testing. Thirteen per cent of the donors of the discriminatory-negative, anti-HBc-reactive donations who had given other Ultrio- or Ultrio Plus-tested donations had at least one other multiplex reactive donation. These findings suggest that their HBV DNA levels are around the assay's limit of detection, that false reactivity cannot be presumed when a donor fails to discriminate and that caution should be applied when deciding whether to continue accepting donations from such donors.

Refinement of a viral transmission risk model for blood donations in seroconversion window phase screened by nucleic acid testing in different pool sizes and repeat test algorithms

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.