Usutu virus infection in Dutch blood donors.

BACKGROUND: The screening of Dutch blood donations for West Nile virus (WNV) may be imminent, as WNV emerges in nearby countries and more donors travel to WNV-affected regions. Since 2016 the related, mosquito-borne Usutu virus (USUV) causes seasonal mortality in Dutch birds. To what extent will human USUV infections affect Dutch WNV donor screening? STUDY DESIGN AND METHODS: From April through September 2018, plasma samples from blood donations in blackbird-rich regions were stored. When increased bird mortality was reported in August, samples from July, August, and September were tested for USUV-RNA in pools of eight, using a home-brew combined WNV/USUV-PCR assay. Reactive pools were deconstructed. Original plasma units and samples of previous and follow-up donations of reactive donors were tested for USUV- and WNV-RNA, and for antibody responses. RESULTS: The number of USUV RNA-positive, WNV RNA-negative donations was 0 of 2688 donations in July, 6 of 4416 in August (1:736), and 1 of 4936 in September. The seven infected donors tested negative for USUV-RNA in preceding and follow-up donations. For 6 donors, seroconversion for USUV-antibodies was demonstrated. All index donations tested positive in a commonly used PCR-assay for WNV donor screening. Three exposed recipients did not show signs of infection. Screening a random subset of 1092 donations from September for USUV-IgG antibodies showed that 22 donors tested reactive; for three donors retrospective testing identified an USUV PCR-positive pre-seroconversion donation. CONCLUSION: Seasonal USUV infection in Dutch blood donors is common. Cross-reactivity in molecular assays for WNV-screening occurs, but can be resolved using USUV- and WNV-specific PCR-primers and sequencing of viral RNA.

Zika virus RNA polymerase chain reaction on the utility channel of a commercial nucleic acid testing system.

BACKGROUND: Several countries have implemented safety strategies to reduce the risk of Zika virus (ZIKV) transmission through blood transfusion. These strategies have included nucleic acid amplification testing (NAT) of blood donations. In this study, a new real-time polymerase chain reaction (PCR) assay including internal control for the detection of ZIKV on the cobas omni Utility Channel (UC) on the cobas 6800 system is presented. STUDY DESIGN AND METHODS: PCR conditions and primer/probe concentrations were optimized on the LightCycler 480 instrument. Optimized conditions were transferred to the cobas omni UC on the cobas 6800 system. Subsequently, the limit of detection (LOD) in plasma and urine, genotype inclusivity, specificity, cross-reactivity, and clinical sensitivity were determined. RESULTS: The 95% LOD of the ZIKV PCR assay on the cobas 6800 system was 23.0 IU/mL (95% confidence interval [CI], 16.5-37.5) in plasma and 24.5 IU/mL (95% CI, 13.4-92.9) in urine. The assay detected African and Asian lineages of ZIKV. The specificity was 100%. The clinical concordance between the newly developed ZIKV PCR assay and the investigational Roche cobas Zika NAT test was 83% (24/29). CONCLUSIONS: We developed a sensitive ZIKV PCR assay on the cobas omni UC on the cobas 6800 system. The assay can be used for large-scale screening of blood donations for ZIKV or for testing of blood donors returning from areas with ZIKV to avoid temporal deferral. This study also demonstrates that the cobas omni UC on the cobas 6800 system can be used for in-house-developed PCR assays.

Validation of new real-time polymerase chain reaction assays for detection of hepatitis A virus RNA and parvovirus B19 DNA.

BACKGROUND: To meet European guidelines for plasma for fractionation, plasma fractionators have implemented parvovirus B19 (B19V) and hepatitis A virus (HAV) nucleic acid test (NAT) screening on test pools. In this study we evaluate recently developed in-house NAT assays for B19V DNA and HAV RNA. The B19V NAT was designed to target two different regions of the B19V genome. STUDY DESIGN AND METHODS: The B19V DNA and HAV RNA tests were validated according to commonly used guidelines. The performance of the B19V and HAV assays was evaluated during routine screening of more than 2 × 10(6) donations. RESULTS: The 95% lower limit of detection (LLD) of the HAV NAT was 1.34 IU/mL. The 95% LLD for B19V was 39.1 IU/mL for the NS1 region and 76.9 IU/mL for the VP2 region. The B19V test showed good accuracy, precision, robustness, and no cross-contamination was observed. Both assays detected B19V Genotypes 1 to 3 and HAV Genotypes I to III. During routine screening 103 donations showed B19V DNA loads of more than 1.25 × 10(6) IU/mL and one donation was reactive in the HAV NAT. CONCLUSION: The dual-target B19V polymerase chain reaction (PCR) showed good accuracy (<0.1 log IU/mL) at the crucial concentration of 10 IU/µL for the NS1 and the VP2 region of the B19V genome and detected all known genotypes with similar sensitivity for each genotype. In addition, the dual target format reduces the chance that molecular variants of B19V are wrongly quantified. The HAV RNA assay showed high sensitivity for Genotypes I to III. Both new PCR assays have been successfully introduced for plasma screening in test pools of 480 or 96 donations.

Efficient SARS-CoV-2 Surveillance during the Pandemic-Endemic Transition Using PCR-Based Genotyping Assays.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOC) pose an increased risk to public health due to higher transmissibility and/or immune escape. In this study, we assessed the performance of a custom TaqMan SARS-CoV-2 mutation panel consisting of 10 selected real-time PCR (RT-PCR) genotyping assays compared to whole-genome sequencing (WGS) for identification of 5 VOC circulating in The Netherlands. SARS-CoV-2 positive samples (N = 664), collected during routine PCR screening (15 ≤ CT ≤ 32) between May-July 2021 and December 2021-January 2022, were selected and analyzed using the RT-PCR genotyping assays. VOC lineage was determined based on the detected mutation profile. In parallel, all samples underwent WGS with the Ion AmpliSeq SARS-CoV-2 research panel. Among 664 SARS-CoV-2 positive samples, the RT-PCR genotyping assays classified 31.2% as Alpha (N = 207); 48.9% as Delta (N = 325); 19.4% as Omicron (N = 129), 0.3% as Beta (N = 2), and 1 sample as a non-VOC. Matching results were obtained using WGS in 100% of the samples. RT-PCR genotyping assays enable accurate detection of SARS-CoV-2 VOC. Furthermore, they are easily implementable, and the costs and turnaround time are significantly reduced compared to WGS. For this reason, a higher proportion of SARS-CoV-2 positive cases in the VOC surveillance testing can be included, while reserving valuable WGS resources for identification of new variants. Therefore, RT-PCR genotyping assays would be a powerful method to include in SARS-CoV-2 surveillance testing. IMPORTANCE The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome changes constantly. It is estimated that there are thousands of variants of SARS-CoV-2 by now. Some of those variants, variants of concern (VOC), pose an increased risk to public health due to higher transmissibility and/or immune escape. Pathogen surveillance helps researchers, epidemiologists, and public health officials to monitor the evolution of infectious diseases agents, alert on the spread of pathogens, and develop counter measures like vaccines. The technique used for the pathogen surveillance is called sequence analysis which makes it possible to examine the building blocks of SARS-CoV-2. In this study, a new PCR method based on the detection of specific changes of those building blocks is presented. This method enables a fast, accurate and cheap determination of different SARS-CoV-2 VOC. Therefore, it would be a powerful method to include in SARS-CoV-2 surveillance testing.

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.

Novel strategy for the selection of human recombinant Fab fragments to membrane proteins from a phage-display library.

Traditionally, the selection of phage-display libraries is performed on purified antigens (Ags), immobilized to a solid substrate. However, this approach may not be applicable for some Ags, such as membrane proteins, which for structural integrity strongly rely on their native environment. Here we describe an approach for the selection of phage-libraries against membrane proteins. The envelope glycoproteins (Env) of the Human Immunodeficiency Virus type-1 (HIV-1) were used as a model for a type-1 integral membrane protein. HIV-1IHI Env, expressed on the surface of Rabbit Kidney cells (RK13) with a recombinant vaccinia virus (rVV), was solubilized using the non-ionic detergent n-Octyl beta-D-glucopyranoside (OG). Membrane associated Env was reconstituted into vesicles by the simultaneous removal of detergent and free monomeric Env subunits by gel-filtration. The resulting antigen preparation, termed OG-P1IHI, was captured on microtiter plates coated with Galanthus nivalis agglutinin (GNA) and used for rounds of selection (panning) of a well-characterized phage-display library derived from an HIV-1 seropositive donor. Simultaneously, an identical experiment was performed with OG-P1IHI vesicles disrupted by Nonidet P-40 (NP-P1IHI). Both membrane-associated and soluble Ags were selected for vaccinia-specific clones (OG-P1IHI: 59/75 and NP-P1IHI: 1/75) and HIV-1-specific clones (OG-P1IHI: 11/75 and NP-P1IHI: 65/75) using our approach. Hence, the novel panning strategy described here may be applicable for selection of phage-libraries against membrane proteins.

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.

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.

Quantitative real-time detection of parvovirus B19 DNA in plasma.

BACKGROUND: As of 2004, the European Pharmacopoeia demands that plasma pools for production of anti-D immunoglobulin should not contain more than 104 IU per mL of parvovirus B19 (B19V) DNA. Hence, before pooling, highly viremic donations have to be identified, and after pooling the level of B19V DNA must be determined. The performance of a new real-time B19V DNA PCR test (Roche, Mannheim, Germany) was studied, using a DNA extractor (NucliSens, bioMerieux, Boxtel, the Netherlands) for isolation of nucleic acid, and using a DNA quantification test (LightCycler apparatus, Roche, Mannheim, Germany) for amplification and detection. STUDY DESIGN AND METHODS: Dilutions of the international B19V DNA standard and reference preparations were tested to determine the precision, linear range, and accuracy of the assay and to calculate the factor for conversion of B19V DNA copies to IUs. The internal control signals, invalid test results, and the effect of cryo-poor plasma were studied as a measure for robustness. Routine performance was assessed by testing 164 manufacturing pools (not screened for B19V) and 1048 test pools of 480 donations each. RESULTS: The copies-to-IU conversion factor was calculated to be 3.34 (95% CI, 3.07-3.63). The assay appears linear between 10(3) and 10(7) IU per mL. Between 10(3) and 10(5) IU per mL, the test can discriminate samples differing a factor two in B19V DNA content. Overall, 0.78 percent of the test results were invalid. Of 127 B19V DNA negative control plasma samples, 7 were contaminated with low levels of B19V DNA. Of 164 nonscreened manufacturing plasma pools, 92 contained B19V DNA (56%); 13 contained more than 10(4) IU per mL. Of 503,040 donations, 29 contained more than 5 x 10(6) IU per mL B19V DNA (1:17,346). CONCLUSION: The B19V DNA quantification test (LightCycler, Roche ) is suitable for quantitative, routine, in-process measurement of B19V DNA levels in plasma pools, using the DNA extractor (NucliSens, bioMerieux) for nucleic acid isolation.