Multicenter evaluation of a commercial multiplex polymerase chain reaction test for screening plasma donations for parvovirus B19 DNA and hepatitis A virus RNA.

BACKGROUND: Three European laboratories evaluated the TaqScreen DPX test (DPX test), a multiplex nucleic acid test assay for the simultaneous detection and quantitation of parvovirus B19 (B19V) DNA and the detection of hepatitis A virus (HAV) RNA. STUDY DESIGN AND METHODS: The 95% limit of detection of the test for B19V and HAV was determined using the respective WHO International Standards. The reproducibility of the test was evaluated by testing replicate samples of B19V at log 4.0 and 40 IU/mL and HAV at 5 IU/mL. The accuracy of the DPX test for B19V was evaluated by replicate testing of B19V samples containing log 3.0, log 4.0, and log 5.0 IU/mL. Panels of B19V Genotypes 1, 2, and 3 and HAV genotypes were evaluated. Cross-contamination was evaluated. For comparison of the DPX test and the established tests, the sites tested plasma samples in pools of either 96 or 480 donations. RESULTS: The mean 95% lower limits of detection of the three laboratories for B19V and HAV were 20.30 and 1.85 IU/mL. The test showed good reproducibility with the major part of the variance of the test being attributed to intermediate assay variation. The test showed great accuracy for B19V, especially at log 4.0 IU/mL. Spiking of test pools of 480 donations and manufacturing pools with log 4.0 IU/mL B19 DNA and 4 IU/mL HAV RNA showed that the DPX assay was robust. The test was able to detect the three genotypes of B19V and HAV genotypes. No cross-contamination was seen. Test results of routine samples correlated well with those of the established tests. CONCLUSION: The DPX test is a robust and sensitive test for the detection of B19V and HAV in plasma samples. The quantitative B19V results obtained with the test are accurate, and the test is able to detect all the known genotypes of B19V and HAV and fulfills all the European Pharmacopoeia and Food and Drug Administration requirements for a B19V and HAV test for screening of plasma donations and samples from plasma pools for manufacture.

Coxiella burnetii infection among blood donors during the 2009 Q-fever outbreak in The Netherlands.

BACKGROUND: In 2007, 2008, and 2009 outbreaks of Q-fever occurred in The Netherlands with increasing magnitude. The 2009 outbreak with 2354 reported cases is the largest human Q-fever outbreak ever recorded. To assess the extent of infection and the safety of donated blood, we tested local blood donations for presence of Coxiella burnetii antibodies and DNA. STUDY DESIGN AND METHODS: Starting May 2009, more than 40,000 serum samples were collected from all consenting blood donors in the areas with high Q-fever incidence. The 1004 samples from the areas with the highest number of reported cases were tested for C. burnetii DNA by polymerase chain reaction; seroprevalence and incidence were determined using enzyme-linked immunosorbent assay and immunofluorescence assays (IFAs) in the subset of 543 donors of whom a follow-up sample was available. RESULTS: A total of 6 of 1004 donor samples tested reactive for C. burnetii DNA. Confirmatory testing (IFA) on the index and follow-up samples demonstrated seroconversion in two donors, high-level preexisting antibodies in one donor, and no seroconversion in three donors. Immunoglobulin (Ig)G testing of the 543 serum pairs showed that 66 were reactive in the latest sample, of which 10 represented seroconversions. CONCLUSION: In the area with highest incidence during a large Q-fever outbreak, 3 of 1004 blood donations contained C. burnetii DNA (0.3%; 95% confidence interval, 0.1%-1.0%). A total of 66 of 543 (12.2%) donors tested positive for anti-Coxiella IgG. Ten seroconversions were detected, resulting in an incidence of 5.7% per year, which is more than 10-fold higher than the local number of reported clinical cases (0.47% per year).

A probabilistic model for analyzing viral risks of plasma-derived medicinal products.

BACKGROUND: The prevention of transmission of viral infections by plasma-derived medicinal products is of concern to manufacturers, legislators, and patient representative groups. Recent European legislation requires a viral risk assessment for all new marketing applications of such products. STUDY DESIGN AND METHODS: A discrete event Monte Carlo model was developed to determine the viral transmission risks of the plasma-derived medicinal products. The model incorporates donor epidemiology, donation intervals, efficiency of screening tests for viral markers, inventory hold period, size and composition of the manufacturing pool, production time, process virus reduction capacity, and product yield. With the model, the human immunodeficiency virus (HIV) and hepatitis C virus (HCV) contamination risks of a typical hypothetical plasma product were calculated, and the sensitivity of the risk to various model variables was analyzed. RESULTS: The residual HIV and HCV risks of the finished products are linear in change with viral incidence rate and inversely linear with product yield and process virus reduction capacity. For the product analyzed in this article, the residual risk is less sensitive to changes in screening test pool size, donation frequency, and inventory hold period. There is only a limited dependency on the donation type (apheresis or whole-blood donations) and a negligible dependency on the manufacturing pool size. CONCLUSIONS: The use of probabilistic model simulation techniques is indispensable when estimating realistic residual viral risks of plasma-derived medicinal products. In contrast to conventional deterministic residual risk estimations, the probabilistic approach allows incorporation of specific manufacturing decisions and therefore provides the only feasible alternative for a correct assessment of residual risks.

Diversity and origin of hepatitis C virus infection among unpaid blood donors in the Netherlands.

BACKGROUND: To improve transfusion policy and to increase understanding of the spread of hepatitis C virus (HCV) in the general population, HCV infections among voluntary Dutch blood donors were examined with molecular epidemiologic techniques. STUDY DESIGN AND METHODS: During 6 years, 1997 through 2002, confirmed anti-HCV-positive donors were interviewed on HCV-associated risk behavior with a standardized questionnaire. Additionally, HCV isolates were genotyped, partially sequenced, and compared to sequences obtained from Dutch injecting drug users (IDUs). RESULTS: HCV prevalence and incidence rates among Dutch donors were extremely low; the residual risk of transmitting HCV was calculated to be 1 in 30 million donations. Former IDUs (21%), transfusion recipients (30%), and immigrants (>12%) were identified as major HCV risk groups. Cryptogenic transmission caused 18 percent of infections among new donors and all infections among repeat donors. Compared to IDUs, genotype distribution among donors was highly diverse; major subtypes were 3a (27%), 1a (24%), 1b (24%), 2a/b (10%), and 4 (9%). Half of the donors were infected with IDU-related subtypes 1a and 3a, whereas subtype 1b mainly spread via blood transfusion and various other nosocomial modes of transmission in the past. HCV infections acquired in endemic countries could be clearly identified based on genotype. CONCLUSION: Different modes of transmission are linked to infections with certain HCV subtypes, suggesting separate HCV epidemics, but spillover between different risk groups underlines the value of molecular epidemiologic techniques to gain insight into the origin and dynamics of HCV infections on a population level.

Validation of the NucliSens Extractor combined with the AmpliScreen HIV version 1.5 and HCV version 2.0 test for application in NAT minipool screening.

BACKGROUND: Routine HCV NAT minipool screening (48 donations) of all blood donations was implemented in July 1999 and was combined with HIV NAT in November 2000. This report describes the validation of the NAT methods and the results of quality control testing. STUDY DESIGN AND METHODS: Nucleic acid was extracted from 2-mL plasma samples by using an automated silica-based extraction method (NucliSens Extractor, Organon Teknika). Eluates were tested with RT-PCR (AmpliScreen HIV-1 version 1.5 and AmpliScreen HCV version 2.0 test, Roche Diagnostic Systems). HIV-1 and HCV RNA reference panels and run controls (PeliCheck and PeliSpy, respectively, Sanquin-CLB) and human plasma minipools were used for NAT validation. RESULTS: The 95-percent detection limit (and 95% CI) for HIV-1 RNA genotype B, HIV-1 RNA genotype E, and HCV RNA genotype 1 was 32 (19-76), 30 (17-72), and 21 (13-44) genome equivalents (geq) per mL, respectively. During initial validation, 2332 samples for HIV-1 RNA and 2644 samples for HCV RNA were analyzed, with 13 (0.56%) and 12 (0.45%) invalid test results, respectively. Thereafter, over 19,600 samples (minipools and run controls) were analyzed during the first 11 months of routine screening. Invalid test results for HIV-1 RNA and HCV RNA were found in 1.1 and 1.07 percent of the samples tested, respectively. HIV-1 RNA minipool testing resulted in 27 (0.16%) initial false-positive results and 3 (0.02%) confirmed positive results. HCV RNA minipool testing resulted in four (0.02%) initial false-positive results and five (0.02%) confirmed positive results. CONCLUSION: Routine HIV and HCV NAT minipool screening using the NucliSens Extractor, AmpliScreen HIV-1 version 1.5, and AmpliScreen HCV version 2.0 meets the sensitivity criteria set by the regulatory bodies and provides sufficient specificity and robustness for timely release of blood donations.