Minipool Research Articles

NAT positivity in seronegative voluntary blood donors from western India

Background and objectivesPrevalence and composition of Hepatitis B, Hepatitis C and HIV-1, NAT positive but seronegative voluntary blood donors from western part of India is yet to be documented. Material and methodsOver last 2 1/2 years all the seronegative voluntary blood donors were tested using 10 minipools on a semiautomated NAT testing platform. The positively tested donors were followed up for at least five months for development of seropositivity. Results79532 seronegative donations were tested by 10 minipool (MP) NAT leading to 51 positive sample (44 Hep B, 5 HIV 1 and Hep C positive). All the HIV and Hep C NAT positive donors eventually developed seropositivity and out of 44 Hep B NAT positive donors, 31 developed seropositivity within six months of follow up, following counseling of the donors. This data translate into NAT yield of 1:1559 donors for all virus taken together. NAT yield for Hep B 1:1807 donors were much higher than HIV 1 in 1:15906 and HCV yield of 1:39761. Semiautomated minipool NAT testing system was found to be cost effective way for improving blood safety. Interpretation and conclusionSeronegative NAT yield in voluntary blood donors are quiet high in western part of India and in line with rest of the country is mainly due to Hepatitis B infection. Implementation of strict donor screening, Hep B vaccination of the population and sample mutation of NAT testing should be under taken on war footing.

IMPLEMENTATION OF UNDERWATER GLIDER AND IDENTIFICATION OF ITS PARAMETERS

This paper is part of a series that describe the development and setup of intelligent underwater vehicle. Here we focus on the implementation tests for the identification of the actuating device parameters. An energy efficient type of glider is chosen with its block diagram presented. The assembled model is placed in a mini pool for movement tests with the objective of setting up the motors and pump. When put in neutral, its immersion and surfacing position show that there is interdependent between pump operation time, ballast bag maze, displacement of weight and glider attitude angle. These dependencies are described by linear equations and coefficients which are then identified.

Significance of nucleic acid testing in window period donations: Revisiting transfusion safety in high prevalence-low resource settings

Background: Safe blood transfusion to the people is essential requirement of health care delivery system. Despite the mandatory screening of blood with newest, very sensitive serological test, considerable risk remains for transfusion transmission of virus due to window period infections. Study was aimed to analyze the efficacy of Mini pool Nucleic Acid Amplification Testing as additional donor screening program and its role in improving blood safety in the high prevalence population. Material and Methods: Study was performed at a tertiary-care, accreditated hospital from June 2013 to December 2015 All negative cases for anti-HIV, anti-HCV and HBsAg by ELISA were subjected to MP-NAT to detect HIV-1, HIV-2 and HCV-RNA with HBV DNA.Results: In 31 months, 20470 donations were received of which whole blood donations were 16997 (83.03%) and 3473 (16.97%) apheresis. Out of 16997 donations, 446 (2.61%) were seroreactive. Out of 16551 sero-negative donors subjected to MP-NAT testing, 17 (0.10%) were NAT reactive (NAT yield -1 in 974). Out of 17 cases, 12 were HBV (1 in 1379), four HCV (1 in 4138) and one HIV NAT reactive (1 in 16551). Conclusion: NAT has improvised the blood safety by detecting the virus in the pre-seroconversion, window period thereby providing much higher sensitivity as compared to newest generation serological tests. In countries with high incidence of infection with significant number of window period donations, NAT can serve as a valuable tool along with other serological testing in high prevalence, resource constrained countries to achieve the goal of zero risk of blood.

Nucleic Acid Technology (NAT) testing for blood screening: impact of individual donation and Mini Pool – NAT testing on analytical sensitivity, screening sensitivity and clinical sensitivity

Globally, in a number of countries the donated blood is screened for serology markers for the Human immunodeficiency diseases‐1/2, the Hepatitis C virus (HCV), and the Hepatitis B virus (HBV). Several medium human development index (HDI) countries with reasonably high percentage of transfusion transmitted infections (TTI) are evaluating nucleic acid technology (NAT) assays to detect these viruses. Serology assays are performed on individual samples, while NAT is performed on either the individual donation (ID) or on a wide array of minipool (MP)‐NAT testing formats. The limit of detection of ID‐NAT assays equals the analytical sensitivity and that of pool testing reflects the pool size dependent decreased sensitivity also known as screening sensitivity. To many end users, it is not obvious that pool testing will have less sensitivity. In this review, mathematically predicted pool size dependent increase in risk days which is applicable to assays of all technologies is substantiated with published experimental results with NAT standards, clinical NAT only detected yields and detection misses by MP‐NAT. In the second half, the blood banking system in India, the donor base, and the variables in serology testing are discussed to explain the wide range of reported NAT yields at 1/300 to 1/17 753. Currently, NAT is not mandated in India, and the cost–benefit value of NAT is being seriously debated. As <5 IU/ml of HIV‐1 and HBV have resulted in TTIs, and nearly 83% of the HBV NAT yields in India have <20 IU/ml viral load, the most sensitive assay in the most sensitive format needs to be practiced to ensure maximum blood safety.

Virus de l’hépatite E, implications en transfusion sanguine

Hepatitis E virus (HEV) is a non-enveloped RNA virus transmitted by the fecal-oral route. Autochthonous hepatitis E occurring in developed countries is caused by genotypes 3 and 4 and is a zoonotic infection. Humans are infected mostly after ingestion of undercooked meat from infected animals. Most HEV 3 and 4 infections are clinically inapparent. However, genotype 3 (HEV 3) can lead to chronic hepatitis in immuno-compromised patients such as organ-transplant recipients and patients with haematological malignancies. In Europe, HEV 3 is implicated in transfusion-transmitted HEV infection. In France, as observed in several European countries, prevalence of HEV RNA and specific IgG antibodies are high indicating that viral circulation is important. The systematic HEV NAT screening of blood donations used for preparation of solvent detergent plasma indicate that 1 to 2218 donation is infected by HEV RNA. The need or implementation's impacts of safety measures to prevent HEV transmission by blood transfusion are under reflexion by French's health authorities. The HEV NAT screening is the only available tool of prevention. Alternative strategies are under investigation including individual or mini pool NAT testing all or part of blood donations.

What is the meaning of a nonresolved viral nucleic acid test-reactive minipool?

This study aimed at analyzing the prevalence of hepatitis B virus (HBV) DNA among hepatitis B surface antigen (HBsAg)-negative donations by cobas TaqScreen MPX test (Roche Molecular Systems) and discussing the meaning of a reactive minipool (MP) that does not resolve to an individual donation (ID)-reactive result. Nucleic acid amplification testing (NAT) was performed in 12 Chinese blood centers on 826,044 serologic negative donations in MPs of six. MP-reactive pools that were resolved to ID-reactive donations were confirmed by Roche TaqMan viral load assays. Antibody to hepatitis B surface antigen and antibody to hepatitis B core antigen (anti-HBc) results were also analyzed. Cycle threshold (Ct) values of reactive MPs were analyzed in relation to the probability of pool resolution. A total of 1267 of 137,674 pools were reactive, of which 839 donations were reactive by ID-NAT. The MP6 HBV NAT-yield rate lay between 1 in 1600 and 1 in 1000. At MP Ct values equal or below 37, the probability of pool resolution was approximately 80%. The prevalence of anti-HBc in ID-reactive donations was 81%. The proportion of reactive pools that could not be resolved was 36%. The prevalence of anti-HBc in donations implicated in nonresolved MPs was significantly higher than those in nonreactive MPs (48% vs. 37%, p = 0.016). The anti-HBc data suggest that approximately 10% of nonresolved MPs contain HBV DNA from a low-viral-load occult carrier. We consider ID-NAT resolution testing in duplicate to minimize HBV transmission risk associated with transfusing nonreactive donations implicated in reactive MPs.

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

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.

Comment on: One window-period donation in two years of individual donor-nucleic acid test screening for hepatitis B, hepatitis C and human immunodeficiency virus

Since 1999 blood transfusion services around the world have been implementing nucleic acid amplification tests (NAAT) to screen for hepatitis C (HCV) RNA, human immunodeficiency virus (HIV) RNA and hepatitis B (HBV) DNA to improve the safety of the blood supply. Initially HCV RNA and HIV RNA NAAT screening was implemented and more recently HBV DNA NAAT screening commenced. In the beginning mini pools (MP) of 98 were used due to the manual complex systems available which were not suitable for high throughput because of the stringent laboratory requirements(1). Over the years the pool sizes have decreased to either 16 as used in the USA(2) or even smaller such as 6 as used by many European and Asian countries(3-5). South Africa, in 2005, was the first country in the world to implement individual donation (ID) screening for all three viruses of all donations(6). NAAT screening in parallel with serological screening has allowed the classification of HIV positive donations as window period, concordant and elite controller infections. HCV positive donations can be classified as window period, concordant and resolved infections and HBV positive donations can be classified as Window period, HBsAg only, acute concordant, occult and anti-HBc only infections. Since the implementation of NAAT for HBV DNA screening, vaccine breakthrough infections have also been recognized(7). The article in this issue of the Revista Brasileira de Hematologia e Hemoterapia (RBHH) entitled One window-period donation in two years of individual donor-nucleic acid test screening for hepatitis B, hepatitis C and human immunodeficiency virus by Levi et al. describes the first two years of screening for HIV RNA, HCV RNA and HBV DNA using ID NAAT in a small Brazilian blood center(8). From a total of 24,441 donations, no additional yield was obtained for HIV and HCV and they showed a 35% clearance rate for HCV and no elite controllers for HIV. The primary objective of the paper is to describe an unusual HBV yield case. This case tested HBsAg and anti-HBc negative and had an anti-HBs titer of 18 IU/mL. On follow up, the anti-HBs rose to 109 IU/mL, however in four samples over seven months no HBsAg or anti-HBc reactivity occurred. Sequencing confirmed the donation as a genotype A2 vaccine breakthrough infection. This is unusual as most documented breakthrough infections have been of the non-A2 genotype due to the vaccine comprising an A2 virus(7). The authors also showed 19 cases of HBsAg positive with no other HBV marker reactivity. It would be interesting to understand whether the authors viewed these donations as true or false infections in light of the investigations being done in the USA in the context of potentially removing HBsAg screening if HBV NAAT and anti-HBc screening is performed. Although this paper describes a small number of donations screened by ID-NAAT, if extrapolated to the larger Brazilian donor base and the prevalence and incidence is similar to that presented by Levi et al., then ID-NAAT could have a large impact on the safety of the blood supply particularly for HBV and HIV due to the high prevalence and incidence of the diseases, respectively.

West Nile virus infection in blood donors in the New York City area during the 2010 seasonal epidemic

A uniform threshold strategy for converting from minipool (MP)-nucleic acid testing (NAT) to individual donation (ID)-NAT screening for acute West Nile virus (WNV) infection among blood donors is lacking. We report on WNV screening at the New York Blood Center during the 2010 seasonal WNV epidemic, the most severe epidemic in that state since the original outbreak in 1999. Between July 1 and October 31, 2010, blood donations were screened by MP-NAT or ID-NAT and the presence of anti-WNV immunoglobulin (Ig)M and IgG was evaluated among NAT-positive donations. Twenty presumed viremic donations were identified for a frequency of 0.0129% (1 in 7752 donations). Nine donations that could have been missed by MP-NAT were identified. Two of these donations were both IgM and IgG negative, one of which would have been missed if more than one positive donation was required for initiating ID-NAT. Retrospective ID-NAT revealed two positive donations. The majority of the NAT-positive donations in New York (16/19) were from donors who lived in counties that had the highest incidence of human WNV cases in the state. Our data details the identification of WNV NAT-positive blood donations during a severe seasonal epidemic in the New York area. By initiating ID-NAT after one positive donation, using retrospective testing, and triggering ID-NAT regionally, we were able to prevent the release of presumably infectious donations. The detection of NAT-positive donations with retrospective testing, however, may indicate the need for changes in our trigger criteria.

Blood donation screening for hepatitis B virus markers in the era of nucleic acid testing: are all tests of value?

The American Red Cross implemented hepatitis B virus (HBV) minipool (MP)-nucleic acid testing (NAT) in June 2009, in addition to existing tests for hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B core antigen (anti-HBc). The value of all three tests was evaluated. HBsAg, anti-HBc, and HBV DNA (Ultrio MP-NAT, Gen-Probe/Novartis) donation results were analyzed during a 12-month period (July 1, 2009-June 30, 2010). Additional testing by individual-donation (ID) polymerase chain reaction (PCR) to confirm donor infection was performed when any HBV screening test was reactive or positive, except in the case of HBsAg neutralization-positive, anti-HBc-reactive samples. Numbers of blood donations identified as reactive or positive versus nonreactive or negative were compared. Of about 6.5 million donations, 699 were defined as from HBV-infected donors, of which 64% (444) were reactive for all three markers. More than 99% (697) had reactivity to one or both serologic tests with 68% (477) showing reactivity by MP-NAT. Only two donations were DNA-positive, seronegative NAT-yield donations (1 per 3.23 million), fewer than expected (p = 0.0075). Among MP-NAT-reactive donors, only small numbers represented early infection (2 or 0.4% with negative serology and 10 or 2.1% who were HBsAg confirmed positive, anti-HBc nonreactive). Of the 142 occult HBV-infected donors, 85% were MP-NAT nonreactive requiring ID-PCR for detection (121 or 54.5% of all MP-NAT nonreactives vs. 21 or 4.4% of all MP-NAT reactives). The HBV DNA-positive yield rate from MP-NAT was lower than expected, likely representing the rarity of such findings even in very large studies. With the implementation of HBV MP-NAT, the value of maintaining anti-HBc for the detection of low-level HBV DNA-positive donors was confirmed; however, HBsAg screening showed no blood safety value.

Recent insights in testing for transfusion transmissible viral infections

Since the advent of HIV in the early 80s there has been arapid evolution in the detection of transfusion transmissibleviral infections (TTVIs), first based on serologic screeningmethods and more recently on molecular diagnostics ornucleic acid amplification technology (NAT). For manyyears, transfusion centres have relied on serologic testingfor HBsAg, anti-HIV and anti-HCV to minimize the risk ofTTVIs, but since the late 1990s NAT for detection of HIV-RNA, HCV-RNA and HBV-DNA has been introduced in agrowing number of countries to provide a second layer ofsafety. Initially, NAT was performed on larger minipools(MPs) of typically 16–96 donations, but since 2005 auto-mated triplex NAT systems have become available for test-ing donations in smaller test pools ( 10% of the popula-tion has been exposed to the virus. Nevertheless, as anti-HBc-positive blood is known to be infectious some of thetime (<10%) if anti-HBs is not detectable [10,11] anti-HBcscreening has been introduced in a number of countrieswith a low HBV prevalence, well before NAT was intro-duced. More recently, a few European countries have intro-duced anti-HBc testing after MP-NAT had already beenintroduced [12], when it became clear that HBV transmis-sion cases by blood from donors with occult HBV infection(OBI) could still occur [13]. HBV-DNA levels in donors withOBI are generally low and can fluctuate around the detec-tion limit of the NAT systems in use. Recently, more sensi-tive ID-NAT technology for detection of HBV has beenintroduced, but it is unclear which proportion of HBVtransmission cases caused by occult HBV carriers wouldhave been detectable with these assays. HBV transmissionreports have shown that HBV-DNA positive blood in OBIcan be infectious if anti-HBs titres are below 10 mIU⁄ml[13], but the few systematic look back studies that havebeen performed so far indicate that the risk of HBV trans-mission from occult HBV carriers is relatively low [11,14].If anti-HBs antibodies are detectable the risk of anti-HBcpositive blood to be infectious is negligible [10,11], unlessviraemia breaks at relatively high level through a low anti-HBs titer as was observed in one case reports [15]. Thislatter case report showed that re-appearance of persistentlypresent HBV-DNA in OBI when it increased to approxi-mately 900 copies⁄ml at the time anti-HBs titres haddeclined to 12 mIU⁄ml was infectious in two recipients.

West Nile virus in 2006 and 2007: the Canadian Blood Services' experience

The Canadian blood supply has been screened for West Nile virus (WNV) since 2003. A strategy for targeted individual-donation nucleic acid testing (ID-NAT) was implemented in 2004 to identify potentially infectious donations that may be missed by minipool (MP) testing. In 2007, Canada experienced a larger epidemic than in previous years providing an opportunity to evaluate the ID-NAT triggering algorithm in higher-risk areas. A specially created database and internal-external communication identified regions for targeted ID-NAT using MP and community triggers. WNV-positive donations identified by ID-NAT were reexamined in MP to assess the efficacy of targeted ID-NAT in identifying potentially infectious donations that may have been missed by MP testing. WNV-positive donation data from 2006 and 2007 were analyzed to examine temporal and geographic trends. A telephone survey about symptoms was carried out after the 2007 season. In total 78 WNV-positive donations were identified (66 true-positives and four false-positives being in 2007). Most positive donations were in the late summer, concentrated in the same western provinces as community cases. Fifty-two donations were identified by ID-NAT and 46% were consistently positive in MP. Of the other 54%, 74% were immunoglobulin (Ig)M- and/or IgG-positive. Fifty-six percent of donors experienced mostly mild symptoms before or after donation (but all said they were well at the time of donation). WNV-positive donations correspond geographically with the epidemic. MP testing identifies most potentially infectious donations with a smaller potential benefit from targeted ID-NAT. Mild symptoms are common but may not deter donation.

Experience of mandatory nucleic acid test (NAT) screening across all blood organizations in Germany: NAT yield versus breakthrough transmissions

Mandatory nucleic acid test (NAT) blood screening was introduced in Germany in 1999 for hepatitis C virus (HCV) RNA and in 2004 for human immunodeficiency virus Type 1 (HIV-1) RNA. Minimal sensitivity limits of 5000 IU HCV RNA/mL and 10,000 IU HIV-1 RNA/mL were defined for the individual donation facilitating testing of minipools (MPs). The NAT yield obtained from all blood organizations is summarized. Transfusion-associated virus transmissions despite NAT screening ("breakthrough transmissions") are analyzed. In Germany, a variety of NAT assays is applied for NAT screening pool sizes of up to 96 donations. Subsets of NAT yield cases were characterized with regard to viral loads by quantitative NAT and with regard to viral genotypes. Confirmed breakthrough transmissions were analyzed using different molecular and serologic assays. Ninety-two HCV NAT yield cases among 40.8 million and 11 HIV-1 NAT yield cases among 17.1 million donations were identified. During this period, one transmission case was confirmed for HCV and one for HIV-1. The two incidents escaped NAT detection because of low-level viremia and/or suboptimal amplification efficiency. Evidence was obtained for a case of HIV-1 nontransmission by a low-level HIV-1 contaminated red blood cell unit. NAT screening of MPs identified the vast majority of window-phase donations. A significant number of transmission cases was interdicted; breakthrough transmissions may still occur as rare events, even with individual-donation NAT in place. Sensitivity limits might be adapted to the current "state of the art" taking account of viral dynamics during early infection, incidence rates, and costs.

West Nile virus testing experience in 2007: evaluation of different criteria for triggering individual‐donation nucleic acid testing

In 2007, clients served by Blood Systems Laboratories used variable approaches for triggering West Nile virus (WNV) RNA individual-donation (ID) nucleic acid testing (NAT). These included two minipool (MP) NAT-reactive donations and a greater than 1:1000 rate in a 7-day interval (primary trigger), criteria based on one MP-NAT-reactive donation when there was WNV activity in overlapping and/or adjacent geographic areas (neighbor trigger), or zero MP-NAT-reactive donation (self-trigger). The Procleix WNV assay was used in either a 16-sample MP or an ID format. NAT-repeat reactivity or anti-immunoglobulin M (IgM) positivity defined true positives (TPs). TPs that were negative on 1:16 dilution testing were considered ID-NAT yield cases. WNV NAT performed on 1,217,929 donations identified 162 TPs; 87 were detected by MP (rate of 0.008%) and 75 by ID (rate of 0.10%; p < 0.0001). There were 34 ID-NAT yield cases, including 4 IgM/immunoglobulin G (IgG)-negative and 9 IgM-positive/IgG-negative donations. Rates of yield cases by primary, neighbor, and self-triggering were 0.077, 0.052, and 0.004% (p = 0.0003). None of 11 ID-NAT yield cases detected by the neighbor trigger would have been detected if the primary trigger had been used. Primary triggering criteria identified 21 viremic donations that would have been missed by MP testing; however, 11 other low-level viremic donations required more stringent criteria (e.g., neighbor trigger) for detection. It is reasonable to adopt more stringent ID-NAT triggers, including elimination of the rate criterion and triggering on one NAT-reactive donation for regions adjacent to centers which have already triggered.

Two HBV DNA+/HBsAg− blood donors identified by HBV NAT in Shenzhen, China

Background In order to further improve blood safety, mini-pool (MP) nucleic acid testing (NAT) was implemented to screen samples negative for hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (anti-HCV), anti-human immunodeficiency virus (anti-HIV), syphilis (anti-Treponemal antibody) and with normal ALT. Study design and methods From August 2006 to February 2008, 41,301 donations were screened using commercial HIV/HCV RNA and HBV DNA Real-Time PCR NAT assays in pools of 8. Reactive pools were re-tested as individual samples using the appropriate screening test and confirmed using an alternate commercial NAT assay. Donors reactive on both NAT assays were considered ‘confirmed’ positive for the virus concerned and recalled for additional follow-up testing and counseling. Results Of the 41,301 samples screened, no HIV or HCV RNA-positive/seronegative donations were detected but two HBV DNA positive/HBsAg negative blood donors (Donors 1 and 2) were identified. Their respective hepatitis immunological markers were: Donor 1 – anti-HBc positive/anti-HBe positive/HBeAg negative/ALT normal and HBV DNA viral load of 112 IU/ml; Donor 2 – anti-HBc positive/anti-HBe negative/HBeAg negative/ALT normal and HBV DNA viral load 2750 IU/ml. Conclusions MP NAT identified two HBsAg negative donors with presumed occult infection but no HIV or HCV seronegative/NAT positive (yield) donors. The HBV yield rate of 1 in 20,650 (95%CI – 1 in 5663 to 1 in 75,303) is comparatively high, exceeds the predicted rate based on previous modeling for the population and demonstrates the incremental blood safety value of NAT in countries where HBV is highly epidemic. The low viral load of the two yield samples underscores the importance of optimizing the sensitivity of the HBV NAT assay selected for screening.

Hepatitis C infection: recent insights relevant to transfusion safety

Hepatitis C virus (HCV) infects over 100 million persons worldwide, and is hence one of the most prevalent and clinically significant blood‐borne pathogens. The introduction of HCV antibody screening in the early 1990s led to detection of large numbers of infected donors and dramatic increases in blood safety. The seroprevalence of HCV in blood donors varies, ranging from rates as low as 0·01% in South Africa to ~0·5% in most developed countries, to &gt; 5% in focal endemic areas such as Egypt. Studies of HCV in infected donors and recipients were instrumental in the discovery of HCV, and have yielded ongoing insights into HCV epidemiology, natural history and pathogenesis. Recent examples include detailed characterization of the dynamics of acute HCV infection based on studies of plasma donor panels, and important contributions to understanding viral and host factors influencing HCV transmission and disease outcome based on linked donor‐recipient cohort studies. The addition of nucleic acid testing (NAT) for HCV RNA to routine HCV antibody screening of donors in 1999 further reduced the risk of transfusion‐transmitted HCV. Mini‐pool (MP)‐NAT screening has led to detection of well over 500 donors in the viraemic, preseroconversion phase of primary HCV infection, as well as to discrimination of seropositive donors into those with resolved and chronic infections. Studies of donors with acute, chronic/persistent and resolved HCV infections have further contributed to our understanding of viral and host genetic and immunological determinants of spontaneous clearance of HCV and short‐ and long‐term disease outcomes. This review will highlight these past scientific contributions, and present the results of a recent survey of the yield of HCV MP‐NAT screening and estimated residual risks of MP‐NAT screened transfusions in different regions of the world. Finally, the review will discuss available evidence for the infectivity of very low‐level viraemia that may be missed by MP‐NAT, information that is pivotal to modelling the residual risk of MP‐NAT screened blood transfusions, and which will guide further strategies to reduce risk by further enhancing screening or implementation of pathogen‐inactivation technologies.

Sensitivity of two hepatitis B virus, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) nucleic acid test systems relative to hepatitis B surface antigen, anti‐HCV, anti‐HIV, and p24/anti‐HIV combination assays in seroconversion panels

Accurate determination of the infectious window period (IWP) that remains with individual-donation (ID) or minipool (MP) NAT compared to those with serology assays is essential for residual risk estimations. The relative sensitivity of the Procleix Tigris system (Gen-Probe/Chiron) used in ID-NAT format and cobas s 201 (Roche Molecular Systems) applied in 1:6 diluted samples to mimic six-minipool (MP6) nucleic acid test (NAT) was assessed by quadruplicate testing of five seroconversion panels per marker. A mathematical analysis based on the log-linear increase of viremia in the ramp-up phase, as established with bDNA 3.0 assays enabled estimation of the IWP for human immunodeficiency virus (HIV) and hepatitis B virus (HBV) assays. The mean IWPs were Tigris HIV RNA 5.5 days, s 201 (1:6) HIV RNA 7.4 days, GenScreen Plus p24/anti-HIV 17.8 days, PRISM anti-HIV 19.0 days, Tigris HBV DNA 20.6 days, s 201 (1:6) HBV DNA 22.6 days, Bio-Rad hepatitis B surface antigen (HBsAg) 37.8 days, and PRISM HBsAg 35.5 days. At estimated 50 percent NAT seroconversion rates, s 201 (1:6) and Tigris showed mean window-period reduction times (WPRTs) of 30.5 to 35.5 days to hepatitis C virus antibody (anti-HCV) assays, 10.4 to 13.5 days to anti-HIV, or combination p24/anti-HIV assays and 12.8 to 17.2 days to HBsAg assays. Tigris ID-NAT detected HIV RNA 2 days earlier than s 201 MP6-NAT, but the difference in sensitivity between the two NAT systems was not significant in HBV seroconversion panels. Insufficient seroconversion samples were available for reliable modeling of WPRT in early HCV infection, but 1.4 to 2.0 days could be predicted by translating analytical sensitivity data. Both multiplex NAT systems demonstrate significant WPRTs compared to (combined) antigen and antibody assays.

Comparison of the analytical and operational performance of two viral nucleic acid test blood screening systems: Procleix Tigris and cobas s 201

The operational and analytical performance of two automated triplex hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) nucleic acid test (NAT) systems were compared in four screening laboratories of the French Blood Service. Two laboratories evaluated the Procleix Tigris system (Chiron/Gen-Probe) in individual donation (ID) format and two sites used the cobas s 201 system (Roche Molecular Systems) on minipools (MPs) of six donations. The analytical sensitivity, the specificity, and operational performance were compared. The ID to MP-NAT relative sensitivity factors in standard dilution panels of different genotypes varied between 8.7 and 21.9 for HCV RNA, 6.7 and 14.8 for HIV RNA, and 0.71 and 11.6 for HBV DNA. Tigris was 800-fold more sensitive than cobas s 201 (1:6) for a HIV group O sample, but did not detect the HIV-2 sample picked up by cobas s 201 with equal sensitivity as the HIV-1 group M samples. The specificity of both NAT systems after initial screening of 10,520 donations with Tigris and 1444 test pools on s 201 was 99.9 percent for both systems, but reached 100 percent after the repeat and pool resolution test algorithms. A higher throughput of the pool test protocol on cobas s 201 became apparent when the daily workload was more than 400 donations. Tigris ID-NAT format was significantly more sensitive than cobas s 201 MP-NAT in detecting HCV RNA and HIV RNA dilution panels, but despite the 1:6 dilution factor in s 201 the difference in sensitivity was not significant for some of the HBV genotype panels. Both NAT systems demonstrated acceptable operational performance, but for routine use further improvement in system reliability is desirable.

Performance characteristics of the Food and Drug Administration–licensed Roche Cobas TaqScreen West Nile virus assay

The cobas TaqScreen West Nile virus (WNV) test (Roche Molecular Systems) was licensed by the Food and Drug Administration (FDA) in August 2007 for detecting WNV RNA in pools of six or in individual donations (IDs). A series of studies established the performance characteristics of the assay and test system before FDA licensure. Analytic sensitivity was determined by probit analysis using multiple source materials. Clinical sensitivity was determined by testing a panel of 315 known WNV RNA-positive specimens. A large clinical specificity study was conducted by five laboratories during months when WNV activity was not expected. The 95 percent limit of detection for ID testing using the Lineage 1 Health Canada WNV reference standard was 40.3 copies per mL (95% individual donation, 35.1-47.8 copies per mL). Clinical sensitivity was 100 percent (95% confidence interval [CI], 98.8%-100%) for ID testing and 97.5 percent (95% CI, 95.1-98.9%) for minipool (MP) testing. Clinical specificity, when resolved to the ID, was 100 percent for both formats and was 99.986 percent at the MP level. The cobas TaqScreen WNV test performed on the cobas s 201 system is a fully automated test system with excellent clinical sensitivity and specificity that offers the benefits of automated sample preparation and a secure environment for donor testing information.

Cost-Effectiveness of HIV Screening of Blood Donations in Accra (Ghana)

Objectives Areas with high HIV-incidence rates compared to the developed world may benefit from additional testing in blood banks and may show more favorable cost-effectiveness ratios. We evaluated the cost-effectiveness of adding p24 antigen, mini pool nucleic acid amplification testing (MP-NAT), or individual donation NAT (ID-NAT) to the HIV-antibody screening at the Korle Bu Teaching Hospital (Accra, Ghana), where currently only HIV-antibody screening is undertaken. Methods The residual risk of HIV transmission was derived from blood donations to the blood bank of the Korle Bu Teaching Hospital in 2004. Remaining life expectancies of patients receiving blood transfusion were estimated using the World Health Organization life expectancies. Cost-effectiveness ratios for adding the tests to HIV-antibody screening only were determined using a decision tree model and a Markov model for HIV. Results The prevalence of HIV was estimated at 1.51% in 18,714 donations during 2004. The incremental cost per disability-adjusted life-year (DALY) averted was US$1237 for p24 antigen, US$3142 for MP-NAT and US$7695 compared to the next least expensive strategy. HIV-antibody screening itself was cost-saving compared to no screening at all, gaining US$73.85 and averting 0.86 DALY per transfused patient. Up to a willingness-to-pay of US$2736 per DALY averted, HIV-antibody screening without additional testing was the most cost-effective strategy. Over a willingness-to-pay of US$11,828 per DALY averted, ID-NAT was significantly more cost-effective than the other strategies. Conclusions Adding p24 antigen, MP-NAT, or ID-NAT to the current antibody screening cannot be regarded as a cost-effective health-care intervention for Ghana.