Evaluation of automated nucleic acid extraction devices for application in HCV NAT

BACKGROUND: To further improve the safety of the blood supply, various national blood transfusion organizations presently use or are in the process of implementing routine HCV NAT in minipools. According to the Committee for Proprietary Medicinal Products (CPMP) of the European Union, the HCV NAT detection limit of the assay should be 100 IU per mL (270 geq/mL) for testing initial plasma pools. Paul Ehrlich Institute (PEI) regulations stipulate that 5000 IU per mL (13,500 geq/mL) must be detected to calculate the amount contributed by individual donations composing the minipool. The sensitivity for HCV RNA extraction achieved by three commercially available laboratory kits was compared. STUDY DESIGN AND METHODS: Nucleic acids from 1-in-3 serial dilutions of an HCV RNA run control (Pelispy, CLB) were extracted with three kits (Cobas Amplicor, Roche Diagnostic Systems; BioRobot 9604, Qiagen; and NucliSens Extractor, Organon Teknika). HCV PCR of all extracts was performed using a second-generation Cobas Amplicor HCV test and the Cobas Amplicor analyzer. RESULTS: The manual Cobas Amplicor, the BioRobot 9604, and the NucliSens Extractor setups allow a 95-percent HCV RNA detection limit of 129, 82, and 12 geq per mL, respectively. The maximal pool size for the manual Cobas Amplicor, the BioRobot 9604, and the NucliSens Extractor kits that would still meet the PEI criteria for HCV NAT in minipools was calculated at 104, 164, and 1125 donations, respectively. CONCLUSION: All three HCV NAT kits evaluated meet the criteria set by CPMP and PEI. The highest sensitivity for HCV NAT screening can be achieved with the high-volume NucliSens Extractor method in combination with the Cobas Amplicor HCV v2.0 test on the Cobas Amplicor analyzer.     

HCV screening in blood donations using RT-PCR in mini-pool: the experience in Spain after routine use for 2 years

BACKGROUND: The aim of this study is to evaluate the feasibility of implementing a commercial HCV RNA RT-PCR screening method and provide data on the prevalence and incidence rates of hepatitis C in Spain. STUDY DESIGN AND METHODS: Five transfusion centers participated in the study, covering 34.1 percent of the country's total number of donations. All the centers evaluated the sensitivity and characteristics of a commercial RT-PCR reagent kit designed for pool testing (Cobas AmpliScreen HCV v2.0), for which serial dilutions of HCV WHO International Standard 96/790 and preseroconversion samples were used. The data obtained from this technique, employed routinely from May 1999 to June 2001 in 22- to 48-unit mini-pools, are presented in this study. RESULTS: An overall 95-percent detection limit was obtained either at 69 IU per mL when 0.2 mL volume of plasma was extracted (used to analyze individual units), or at 20 IU per mL, when viral particles were pelleted from 1 mL plasma (as used for screening in mini-pool) Three HCV-RNA-positive anti-HCV-negative donations were identified out of 1,015,482 screened donations. One of these had an initially undisclosed risk of HCV sexual transmission and carried a low viral load of 104.2 IU per mL HCV RNA. The analysis of first-time (FT) donations during the period of study (21.3% of the total) indicated an average prevalence rate of 2.05 per 103 FT donors (of which 1.55/103 FT donors were RNA positive); the residual risk calculated on repeat (RPT) donors was 3.91 per 106 donations (serology) or 0.59 per 106 donations (serology + NAT), and the predicted NAT yield estimate was 4.2 per 106 FT + RPT donations. CONCLUSIONS: The commercial RT-PCR reagent kit complies with the current European and FDA recommendations on sensitivity and can be easily implemented on a routine basis. The results obtained by the five transfusion centers on the predicted NAT yield (1/302,000 RPT donations or 1/237,000 FT + RPT donations) are very close to the published estimates corresponding to a larger area of our country (1/237,000 RPT donations) and are somewhat higher than, though in line with, the observed NAT yield (1/338,000 FT + RPT donations).     

Routine HCV PCR screening of blood donations to identify early HCV infection in blood donors lacking antibodies to HCV

BACKGROUND: Detection of early hepatitis C infection of blood donors is still a major problem for blood transfusion. Common anti-HCV screening assays show differences in sensitivity and specificity. The often mild symptoms of acute hepatitis C also cause difficulties in the identification of early HCV infection. The feasibility and efficacy of routine screening of blood donations for HCV RNA were investigated. STUDY DESIGN AND METHODS: Blood donations (n = 251,737) were screened for HCV RNA over 4 years. RNA extraction, amplification, and detection were done by two commercial HCV PCR kits (HCV Cobas Amplicor and HCV Cobas Amplicor 2.0, Roche Diagnostics). Screening was done by pool testing with a maximum pool size of 40 serum samples. RESULTS: Three donations out of 251,737 were HCV RNA positive and anti-HCV negative. ALT levels of these donations were 271, 32, and 10 U per L. The HCV infection of a fourth HCV RNA-positive donor could not be identified by routine, second-generation HCV EIA (Abbott Diagnostika). In this case, two previous donations were also HCV RNA positive, and three second-generation test systems (Abbott) could not detect anti-HCV, whereas third-generation anti-HCV screening assays detected antibody with different sensitivity. The first HCV RNA-positive donation was identified only by the HCV ELISA 3.0 (Ortho Diagnostic Systems). The results of confirmatory assays like RIBA HCV 3.0 (Ortho) and Matrix (Abbott) indicate a restricted immune response to NS3 only. CONCLUSION: HCV RNA detection by PCR can be carried out routinely in blood donor screening without significant delay of release of the components. The residual risk of transmission can be reduced by identification of early infection, which can lead to an improved safety of blood components. RNA screening can also be advantageous in cases of incomplete or lack of antibody response to HCV.     

Effect of viral nucleic acid testing on contamination frequency of manufacturing plasma pools

BACKGROUND: In Europe, all plasma pools used for manufacturing of plasma derivatives must be tested negative for hepatitis C virus (HCV) RNA by nucleic acid amplification techniques (NAT) with a defined minimal sensitivity. For a subset of pools, quantitative B19V DNA NAT is also mandatory. NAT for further viral targets was introduced by most of the manufacturers on a voluntary basis. The contamination frequency of plasma pools with HCV RNA, human immunodeficiency virus (HIV)-1 RNA, and hepatitis B virus (HBV) DNA was investigated with representative pools before and after introduction of NAT. STUDY DESIGN AND METHODS: A total of 873 pools from 1996 and 331 pools from 2006 were analyzed for the detection of HCV RNA, HIV RNA, and HBV DNA with an automated multiplex NAT system. The pools were obtained from different manufacturers and the source material was of European and US origin. RESULTS: HCV RNA, HIV-1 RNA, and HBV DNA were detectable in plasma pools from 1996 with the following frequencies: 17.8 percent (HCV RNA), 0.8 percent (HIV-1 RNA), and 0.5 percent (HBV DNA). Viral genome concentrations were up to 3 x 10(4) IU HCV RNA per mL and 7 x 10(3) IU HIV RNA per mL, whereas HBV DNA was below the quantitation limit of the quantitative NAT assay. Among the pools from 2006, one pool (0.3%) was found HCV RNA-positive at low titer (<10 IU/mL), whereas no HIV RNA or HBV DNA was detectable in any pool. CONCLUSION: The results imply that the introduction of NAT systems for the detection of viral genomes has largely reduced the contamination frequency and viral loads of manufacturing plasma pools and thereby improved the safety margin for human medicinal products manufactured from human plasma. Even with NAT, however, low-titer contamination may occur, which will be coped with by virus inactivation steps included into the manufacturing of plasma derivatives.     

Evaluation of automated RNA-extraction technology and a qualitative HCV assay for sensitivity and detection of HCV RNA in pool-screening systems

BACKGROUND: The objective of this study was the evaluation of NAT technology for the detection of HCV RNA in plasma pools according to the recommendations of the Paul Ehrlich Institute (5000 IU/mL/donation) and the Committee for Proprietary Medical Products (100 IU/mL/manufacturing pool). STUDY DESIGN AND METHODS: Serial dilutions of both the EUROHEP standard (3,800 genome equivalents [geq]/mL; HCV genotype 1) and the World Health Organization (WHO) international standard (100,000 IU/mL; HCV genotype 1) were made in S/D plasma (ESPEP plasma, OctaPharma), which was nonreactive in serologic tests. Serial dilutions of plasma (2 mL) were used for extraction of HCV RNA with an automated version of a nucleic acid isolation method (NucliSens Extractor, Organon Teknika). HCV RNA was co-extracted from 2 mL of plasma, together with 84 copies of an in vitro-synthesized single-strand RNA serving as internal extraction control (IC) to monitor the efficiency of extraction and PCR. Amplification and detection of both HCV RNA and IC RNA were performed with an automated PCR system and a qualitative HCV assay (COBAS Amplicor 2.0 HCV, Roche Diagnostics). RESULTS: A cutoff value of 16 geq per mL (10/10 runs [100% hit rate]) was found by using the EUROHEP standard, whereas the WHO international standard had a cutoff value of approximately 12 IU per mL (10/10 runs [100% hit rate]). The IC had a cutoff value of approximately 17.5 copies per mL (6/6 runs [100% hit rate]). Forty-two copies per mL of IC RNA were found in 282 of 284 runs (99% hit rate). The negative controls (ESDEP plasma) were negative in all experiments. Experiments with pool sizes of 12, 24, 48, and 96 using serial dilutions of the WHO international standard revealed a cutoff value of 8 IU per mL (100% hit rate). The EUROHEP standard and the WHO international standard were detected with a 50 percent detection endpoint of 5.2 geq per mL and 1.5 IU per mL, respectively. CONCLUSION: This test system (NucliSens Extractor, and the COBAS Amplicor 2.0 HCV assay) revealed a high sensitivity for HCV RNA; considering the proposed requirements for sensitivity of NAT assays for the detection of HCV RNA in donor plasma, pool sizes of about 400 donors are possible. These endpoint results indicated that 1 IU is equal to about 3.4 geq.     

Sensitivity of HCV core antigen and HCV RNA detection in the early infection phase

BACKGROUND: Various countries have introduced HCV NAT to exclude infectious donations collected during the preseroconversion window phase (PWP). For the same purpose, an ELISA has also been developed to detect HCV core antigen (cAg). STUDY DESIGN AND METHODS: Using sequential samples from regular plasma donors with very recent HCV infections, a total of 494 samples from 52 anti-HCV-negative donors were collected. These panels were used for direct comparison of the performance of PCR and ELISA in detecting viral markers (RNA and cAg) during the PWP of HCV infection. The panels were genotyped, and each sample was analyzed by qualitative and quantitative HCV PCR and by cAg ELISA. The HCV RNA doubling time was calculated from quantitation of viral RNA in consecutive samples during the earliest outbreak of viremia. RESULTS: Concurrent detection of HCV RNA and cAg in 218 and nondetection in 185 samples yielded 81.6-percent concordance in the results of 494 samples. Unidirectional discrepancy of results (i.e., PCR positive and cAg negative) was seen in 91 of 494 (18.4%) samples, which was consistent with 65 specimens with RNA concentrations ranging between 300 and 100,000 IU per mL and 26 specimens with less than 300 IU per mL (limit of quantitative PCR). Individual genotyped panels had different kinetics and courses of viremia. The mean doubling time in the early PWP at the onset of viremia was derived to be 10.8 (range, 5.8-21.0) hours. CONCLUSION: A majority of HCV RNA-positive samples were also cAg-positive during the PWP. The current cAg detection corresponds to 100,000 IU per mL of HCV RNA. Since low-titer samples would be identified only by single-donation NAT, which is often affordable only in developed countries, the cAg ELISA could offer a practical alternative for some countries. The doubling time for HCV RNA at the onset of viremia corresponds to a calculated mean delay of cAg detection during the virus burst phase of 2 or 5 days, when compared with minipool (5000 IU/mL) or single-donation NAT (50 IU/mL), respectively.     

Magnetic bead technology in viral RNA and DNA extraction from plasma minipools

BACKGROUND: Nucleic acid testing (NAT) of pooled plasma samples from individual blood donations for viral nucleic acids has become widely established. Full automation of such sample processing can overcome many of the problems associated with methods used so far. STUDY DESIGN AND METHODS: In this study an automated extraction method for viral nucleic acids (parvovirus [PAV] B19 DNA, hepatitis B virus [HBV] DNA, and hepatitis A virus [HAV] RNA), starting directly from the minipool sample (n = 96, 9.6 mL), was evaluated. A magnetic separation module I (chemagic, Polymer Laboratories) in combination with the chemagic viral DNA and RNA kit special based on the use of magnetic beads was used for this purpose. More than 144 pools spiked with defined concentrations of reference material and an additional 102 pools negative for the analyte were extracted and amplified. The isolated viral nucleic acids were detected by polymerase chain reaction (PCR). RESULTS: The assays were highly specific and obtained a 95 percent detection limit of 875 IU per mL of pooled single donation for PAV B19, 260 IU per mL for HAV, and 1274 IU per mL for HBV, respectively. The crossing points showed variation coefficients from 1.49 to 2.76 percent. The turnaround time for the whole process was 3 hours. Testing of subpools to determine an infected single donation would be possible with the same general extraction method. A total of 102 unspiked minipools (96 x 100 microL per donation) were analyzed and none tested positive. CONCLUSION: The automated magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donations for viremic donors to further increase the safety of blood products. Minipools as well as subpools can be directly processed.     

Fluctuation of HCV viral load before seroconversion in a healthy volunteer blood donor

BACKGROUND: Newly implemented NAT has been shown to be able to effectively identify HCV-positive blood donated during the preseroconversion period. STUDY DESIGN AND METHODS: EDTA-plasma pools of 24 donations were tested using an HIV-1/HCV multiplex NAT under an FDA-approved IND application. Samples in a positive pool were retested individually. Positive samples were further tested by two discriminatory assays to determine specific viral reactivity. Upon obtaining informed consent, seronegative donors with positive NAT results were enrolled into a follow-up study for risk factor analysis and laboratory testing. RESULTS: A donation by a 29 year old female was identified as HCV NAT-positive with negative serology and an elevated ALT. Her two previous donations, 5 and 12 months earlier, were both seronegative and with normal ALT. Her husband tested positive for HCV RNA. The donor remained seronegative for at least 36 days. The index donation had a viral RNA concentration of >500,000 copies per mL while the first seropositive sample was NAT-negative. Laboratory data on serial follow-up samples showed 100- to 1,000-fold fluctuations in viral load during a period of 48 days prior to seroconversion. CONCLUSIONS: This case suggests that, at least in some newly infected individuals, the HCV viral load can fluctuate dramatically prior to seroconversion, and that NAT, even on individual samples, will not totally prevent HCV transmission.     

Comparison of two automated nucleic acid testing systems for simultaneous detection of human immunodeficiency virus and hepatitis C virus RNA and hepatitis B virus DNA

BACKGROUND: Recently developed nucleic acid testing (NAT) assays incorporating simultaneous detection of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) have made HBV NAT screening more feasible for blood services. This study compared the performance of two "multiplex" NAT assays and their automated testing platforms. STUDY DESIGN AND METHODS: The HBV NAT yield rate was estimated by testing 10,397 Hong Kong (HK) donor samples concurrently on the PROCLEIX ULTRIO (Ultrio) assay as individual donor samples with the TIGRIS and on the cobas TaqScreen multiplex (cobas MPX) test in pools of 6 with the cobas s 201. Analytical sensitivity was assessed by probit analysis of diluted international standards and operational performance was compared. RESULTS: Each system detected two different HBV NAT yield samples for a combined rate of 0.04 percent. One additional sample was reactive on the cobas MPX test but remained unresolved. The 95 percent detection limits for HIV-1, HBV, and HCV were 42.2, 12.2, and 2.0 IU per mL, respectively, for Ultrio and 50.5, 8.4, and 6.0 IU per mL for the cobas MPX. The invalid test and failed run rates were 0.05 and 2.92 percent, respectively, for the TIGRIS and 2.39 and 5.53 percent for the cobas s 201. CONCLUSION: Clinical sensitivity for HBV in HK blood donors was equivalent, as was the analytical sensitivity for HIV-1 and HBV; however, the Ultrio assay had a higher analytical sensitivity for HCV. Despite a shorter downtime and mean time of repair for the cobas s 201, the TIGRIS demonstrated better overall operational performance.     

Detection of HCV core antigen in human serum and plasma with an automated chemiluminescent immunoassay

BACKGROUND: Currently, the detection of HCV infection in blood donors relies on the ability of immunoassays to detect circulating HCV antibodies. However, a significant delay exists between the time of infection and the development of antibodies. This delay (window period) can last up to 70 days. The introduction of NAT for the detection of HCV RNA has reduced this window period dramatically. However, NAT is labor intensive, prone to contamination, and expensive as compared with standard serologic tests. STUDY DESIGN AND METHODS: An automated, microparticle-based chemiluminescent assay for the detection of HCV core antigen in human serum and plasma was developed. The specificity and sensitivity of this prototype assay were evaluated by testing a population of normal blood donors and commercially available seroconversion panels. RESULTS: The HCV core antigen assay exhibited a 99.9-percent specificity by detecting a single repeatably reactive sample out of 1004 normal donors tested. Assay sensitivity was determined by comparing the HCV core antigen detection rate with the antibody seroconversion profile and the rate of HCV RNA detection. Among 15 seroconversion panels examined, core antigen was detected in 69 of 70 antibody-negative and/or RNA-positive samples for a sensitivity relative to NAT of 98.6 percent. CONCLUSION: These data indicate that the automated, microparticle-based chemiluminescent HCV core antigen assay can reduce the window period for detection of potentially infected blood donors by 32.7 days, and it represents a viable alternative to HCV RNA testing.     

核酸扩增技术在献血者血液HBV DNA、HCV RNA及HIV-1 RNA筛查中的应用研究

目的探讨在我国无偿献血者的血液筛查中引进核酸扩增技术(NAT)的必要性,了解献血者血清学病毒标志物检测阴性、NAT检测阳性的感染状况。方法应用Roche PCR、PCR-微流芯片、实时荧光PCR方法对深圳市131 174人(次)血清学检测病毒标志物阴性的献血者进行HBV DNA、HCV RNA和HIV-1 RNA检测,对NAT阳性献血者追踪检测并做定量分析。结果HBV DNA阳性22例,阳性率为1/5 962,其中15例为抗-HBc阳性,阳性率为1/8 745;HCV RNA阳性1例,阳性率1/131 174;HIV-1 RNA未检出阳性。对14名HBV DNA阳性者的追踪发现,8人发生了血清转换现象。结论采用高灵敏度的NAT筛查献血者血液中的HBV和HCV,有助于提高输血及血液安全。     

Multicenter performance evaluation of a transcription-mediated amplification assay for screening of human immunodeficiency virus-1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA in blood donations

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

Yield of HCV and HIV-1 NAT after screening of 3.6 million blood donations in central Europe

BACKGROUND: HCV and HIV-1 NAT of all blood donations was initiated at our institutions in January 1997 to reduce the residual risk of transfusion-transmitted virus infections. The yield of NAT after testing more than 3.6 million donations in central Europe is reported. STUDY DESIGN AND METHODS: Automated pipetting instruments were used to pool up to 96 donor samples including those that were antibody reactive. To compensate for dilution of the individual donor samples by pooling, viruses were enriched from the pools by centrifugation at 48,000 x g. A commercial PCR (Cobas Amplicor, Roche) and an in-house PCR were applied for HCV and HIV-1 amplification, respectively. RESULTS: Six HCV and 2 HIV-1 PCR confirmed-positive, antibody-negative donations (yield, 1 in 600,000 and 1 in 1.8 million, respectively) were identified. Thirty-nine and 11 multiple-time donors seroconverted for HCV and HIV, respectively, and look-back procedures were initiated. Archived samples from preseroconversion donations were thawed and retested by single-sample PCR and remained negative. The recipients of the blood components were traced and tested. All traced recipients were negative for HCV and HIV antibodies. CONCLUSION: The yield of NAT in central European Red Cross blood donors was less than expected from theoretical calculations for American and German multiple-time donors. Look-back procedures for HCV and HIV indicated that no donation given before seroconversion of the donor was missed by minipool PCR. Sensitivity of minipool PCR testing after virus enrichment seems to be sufficiently high to close the diagnostic window almost completely.     

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

BACKGROUND: 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.
STUDY DESIGN AND METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.     

Current prevalence and incidence of infectious disease markers and estimated window-period risk in the American Red Cross blood donor population

BACKGROUND: There has been continuing progress in measures to reduce the risk of transfusion-transmitted infection, including introduction of serologic tests of increased sensitivity and the recent implementation of investigational NAT in small pools of samples. STUDY DESIGN AND METHODS: Data relating to all blood donations to the American Red Cross have been consolidated into a single database. The prevalence of confirmed-positive test results for HBsAg, HCV, HIV, and HTLV were evaluated for each year for first-time donors from 1995 through 2001. Incidence rates for these infections were evaluated among repeat donors having at least two donations in a 2-year period. The frequencies of HIV-1 RNA- and HCV RNA-positive, seronegative donations were assessed for first-time and repeat donations. The relationship risk = (window period) x (incidence) was used to assess residual risk among repeat donations and to evaluate the incidence of HCV and HIV infection among first-time donors. RESULTS: During the study period, prevalence rates for all markers declined significantly over time: in 2001, the rates per 100,000 were 75.6 for HBsAg, 299 for HCV, 9.7 for HIV, and 9.6 for HTLV; the corresponding incidence rates (/100,000 person-years) were 1.267, 1.889, 1.554, and 0.239, respectively. Estimates of residual risk in donations from repeat donors (after NAT) for HCV and HIV were 1 per 1,935,000 and 1 per 2,135,000, respectively. However, incidence rates for these agents are approximately two times greater among first-time donors. For both HCV and HIV, NAT yield was concordant with that predicted by current window-period models. CONCLUSION: These data cover about half of all the whole blood collected in the United States. They suggest increasing improvement in transfusion safety and clearly define the benefit of pooled NAT.     

Correlates of hepatitis C virus (HCV) RNA negativity among HCV-seropositive blood donors

BACKGROUND: Approximately 20 percent of persons infected with hepatitis C virus (HCV) clear viremia. Factors associated with resolution of viremia are not well defined. Implementation of routine nucleic acid testing (NAT) of blood donors has yielded a large data set for analysis of demographic correlates of resolved viremia. STUDY DESIGN AND METHODS: HCV antibody and NAT data, liver enzyme (alanine aminotransferase [ALT]) results, and donor demographic characteristics were compiled for 2,579,290 allogeneic donations given at five large blood centers after NAT implementation in 1999 through December 2001. Donation HCV RNA status was compared between first-time donors categorized by ALT levels, sex, age, race and/or ethnicity, country of birth, level of education, blood center location, and blood group, with chi-square tests and multivariable logistic regression methods. RESULTS: Of 35 confirmed-seropositive repeat donors, 19 (54.3%) tested negative for the presence of HCV RNA; there was no association between RNA status and preseroconversion intervals (p = 0.74). Of 2105 RIBA-positive, first-time donors, 402 (19.1%) tested negative for the presence of HCV RNA by NAT (presumptive resolved infections). There were significant differences in the frequency of RNA negativity among first-time donors categorized by ALT levels and by race and/or ethnicity. ALT levels were more likely to be elevated in RNA-positive, first-time donors (p < 0.0001). Viremia was less likely to resolve in Asian (8.2%) and black non-Hispanic (14.4%) donors than in white non-Hispanic (20.7%), Hispanic (22.1%), and other race and/or ethnicity (22.1%) donors (p = 0.02). No significant associations were found for age, sex, country of origin, level of education, blood type, and donor center location. CONCLUSION: These results confirm that the frequency of HCV RNA negativity among seropositive persons differs by race and/or ethnicity. Follow-up studies of donors with resolved viremia are warranted to further elucidate viral, immunologic, and genetic factors underlying spontaneous viral clearance.     

Impact of nucleic acid testing for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus on the safety of blood supply in Italy: a 6-year survey

BACKGROUND: Nucleic acid testing (NAT) for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) has been implemented in several European countries and in the United States, while hepatitis B virus (HBV) NAT is still being questioned by opinions both in favor and against such an option, depending on the HBV endemicity, health care resources, and expected benefits. STUDY DESIGN AND METHODS: This survey was aimed to assess the NAT impact in improving the safety of blood supply in Italy, 6 years after implementation. The study involved 93 Italian transfusion centers and was carried out in 2001 through 2006. A total of 10,776,288 units were tested for the presence of HCV RNA, 7,932,430 for HIV RNA, and 3,405,497 for HBV DNA, respectively. RESULTS: Twenty‐seven donations or 2.5 per million tested were HCV RNA–positive/anti‐HCV–negative; 14 or 1.8 per million units tested were HIV RNA–positive/anti‐HIV–negative; and 197 or 57.8 per million donations tested were HBV DNA–positive/hepatitis B surface antigen–negative. Of the latter, 8 (2.3/10⁶) were collected from donors in the window phase of infection and 189 (55.5/10⁶) from donors with occult HBV. Sixty‐eight percent of the latter donors had hepatitis B surface antibody, 74.5 percent of whom with concentrations considered protective (≥10 mIU/mL). CONCLUSION: NAT implementation has improved blood safety by reducing the risk of entering 2.5 HCV and 1.8 HIV infectious units per million donations into the blood supply. The yield of NAT in detecting infectious blood before transfusion was higher for HBV than for HCV or HIV. However, the benefit of HBV NAT in terms of avoided HBV‐related morbidity and mortality in blood recipients needs to be further evaluated.