Investigational Testing for Zika Virus among U.S. Blood Donors.

BACKGROUND: Because of the potential severe clinical consequences of Zika virus (ZIKV) infection, the large numbers of asymptomatic travelers returning from ZIKV-active areas, the detection of ZIKV nucleic acid in blood, and reports of transmission of ZIKV through transfusion, in 2016 the Food and Drug Administration released recommendations for individual-unit nucleic acid testing to minimize the risk of transmission of ZIKV through blood transfusions. METHODS: The American Red Cross implemented investigational screening of donated blood for ZIKV RNA by means of transcription-mediated amplification (TMA). Confirmatory testing of reactive donations involved repeat TMA, TMA testing in exploratory minipools, real-time reverse-transcriptase polymerase chain reaction, IgM serologic testing, and red-cell TMA. Viral loads in plasma and red cells were estimated by means of end-point TMA. The costs of interdicting a donation that was confirmed to be positive were calculated for the 15-month period between June 2016 and September 2017. RESULTS: Of the 4,325,889 donations that were screened, 393,713 (9%) were initially tested in 24,611 minipools, and no reactive donations were found. Of the 3,932,176 donations that were subsequently tested individually, 160 were initially reactive and 9 were confirmed positive (a 1:480,654 confirmed-positive rate overall; positive predictive value, 5.6%; specificity, 99.997%). Six (67%) of the confirmed-positive donations were reactive on repeat TMA, of which 4 were IgM-negative; of these 4, all 3 that could be tested were reactive on minipool TMA. Two confirmed-positive donors had infections that had been transmitted locally (in Florida), 6 had traveled to ZIKV-active areas, and 1 had received an experimental ZIKV vaccine. ZIKV RNA levels in red cells ranged from 40 to 800,000 copies per milliliter and were detected up to 154 days after donation, as compared with 80 days of detection in plasma at levels of 12 to 20,000 copies per milliliter. On the basis of industry-reported costs of testing and the yield of the tests in our study, the cost of identifying 8 mosquito-borne ZIKV infections through individual-unit nucleic acid testing was $5.3 million per ZIKV RNA-positive donation. CONCLUSIONS: Screening of U.S. blood donations for ZIKV by individual-donation TMA was costly and had a low yield. Among the 9 confirmed ZIKV-positive donations, only 4 were IgM-negative; of these donations, all 3 that were tested were reactive on minipool TMA. (Funded by the American Red Cross and Grifols Diagnostic Solutions.).

Screening for Babesia microti in the U.S. Blood Supply.

BACKGROUND: Babesia microti, a tickborne intraerythrocytic parasite that can be transmitted by means of blood transfusion, is responsible for the majority of cases of transfusion-transmitted babesiosis in the United States. However, no licensed test exists for screening for B. microti in donated blood. We assessed data from a large-scale, investigational product-release screening and donor follow-up program. METHODS: From June 2012 through September 2014, we performed arrayed fluorescence immunoassays (AFIAs) for B. microti antibodies and real-time polymerase-chain-reaction (PCR) assays for B. microti DNA on blood-donation samples obtained in Connecticut, Massachusetts, Minnesota, and Wisconsin. We determined parasite loads with the use of quantitative PCR testing and assessed infectivity by means of the inoculation of hamsters and the subsequent examination for parasitemia. Donors with test-reactive samples were followed. Using data on cases of transfusion-transmitted babesiosis, we compared the proportions of screened versus unscreened donations that were infectious. RESULTS: Of 89,153 blood-donation samples tested, 335 (0.38%) were confirmed to be positive, of which 67 (20%) were PCR-positive; 9 samples were antibody-negative (i.e., 1 antibody-negative sample per 9906 screened samples), representing 13% of all PCR-positive samples. PCR-positive samples were identified all through the year; antibody-negative infections occurred from June through September. Approximately one third of the red-cell samples from PCR-positive or high-titer AFIA-positive donations infected hamsters. Follow-up showed DNA clearance in 86% of the donors but antibody seroreversion in 8% after 1 year. In Connecticut and Massachusetts, no reported cases of transfusion-transmitted babesiosis were associated with screened donations (i.e., 0 cases per 75,331 screened donations), as compared with 14 cases per 253,031 unscreened donations (i.e., 1 case per 18,074 unscreened donations) (odds ratio, 8.6; 95% confidence interval, 0.51 to 144; P=0.05). Overall, 29 cases of transfusion-transmitted babesiosis were linked to blood from infected donors, including blood obtained from 10 donors whose samples tested positive on the PCR assay 2 to 7 months after the implicated donation. CONCLUSIONS: Blood-donation screening for antibodies to and DNA from B. microti was associated with a decrease in the risk of transfusion-transmitted babesiosis. (Funded by the American Red Cross and Imugen; ClinicalTrials.gov number, NCT01528449 .).

Expanded feasibility of ferritin testing: stability of ferritin stored as whole blood and validation of plastic tubes.

BACKGROUND: Ferritin testing is a recommended strategy to mitigate iron depletion in blood donors. A barrier for some testing platforms is a requirement to complete sample management and testing within a temporal window incompatible with the logistics of many blood collectors. The ability to delay separation of plasma/serum from red cells and subsequent testing would enhance the feasibility of ferritin testing on a broader scale. STUDY DESIGN AND METHODS: Thirty blood donors provided a research donation of 12, 4-mL sample tubes of whole blood. Six pairs of serum and K2 -EDTA-plasma tubes were centrifuged and samples tested in triplicate on day of collection and on each of the next 5 days following storage at 4°C. Comparison of ferritin values for serum versus K2 -EDTA-plasma at baseline was performed to validate plastic EDTA-containing tubes. Variation of ferritin values during storage was assessed for direction and strength of any detectable changes. RESULTS: Ferritin values were comparable between EDTA-plasma and serum, with baseline values from EDTA-plasma samples 7% lower on average than serum (p < 0.0001 by paired t-test). Variability over five storage days was within approved parameters in the manufacturer's instructions. Within-run precision averaged 2% to 3% for each test day and within-subject precision across all samples averaged less than 5% for both serum and EDTA-plasma. Repeated measures showed no difference in changes during storage by tube type or day of testing. CONCLUSION: These results support flexible testing procedures, expanding the opportunity for blood centers to adopt this measure for assessing donor iron status.

Probable and possible transfusion-transmitted dengue associated with NS1 antigen-negative but RNA confirmed-positive red blood cells.

BACKGROUND: In the absence of active blood donation screening, dengue viruses (DENV) have been implicated in only a limited number of transfusion transmissions worldwide. This study attempted to identify if blood from donors testing negative by an NS1-antigen (Ag) enzyme-linked immunosorbent assay (ELISA) but confirmed positive for DENV RNA caused DENV-related disease in recipients during the epidemic years of 2010 to 2012 in Puerto Rico. STUDY DESIGN AND METHODS: Donation aliquots testing negative by an investigational NS1-Ag ELISA were stored frozen and retested retrospectively using a research transcription-mediated amplification assay (TMA) detecting DENV RNA. All RNA-reactive donations were subject to confirmatory RNA and antibody testing. Recipient tracing was conducted for all components manufactured from TMA-reactive components. Medical chart review, recipient interview, and follow-up sampling occurred for 42 recipients transfused with TMA-reactive components. RESULTS: Six of 42 recipients developed new-onset fever in the 2 weeks posttransfusion; three (50%) received RNA confirmed-positive, NS1-Ag-negative red blood cell (RBC) units. One recipient of a high-titer unit (7 × 10(7) DENV-4 RNA copies/mL) developed severe dengue, and a second recipient had only fever recorded but had a negative sepsis work-up. New fever attributable to DENV infection in a third recipient was confounded by fever potentially attributable to posttransfusion sepsis. CONCLUSIONS: In our retrospective study, NS1-Ag detected 20% of all RNA confirmed-positive donations demonstrating limitations of NS1-Ag ELISA for blood donation screening. We identified one recipient with a clinical syndrome compatible with severe dengue who had received an NS1-Ag-negative but RNA confirmed-positive RBC unit. This investigation illustrates the difficulty in confirming transfusion transmission in dengue-endemic areas among severely ill transfusion recipients.

Investigational screening for Babesia microti in a large repository of blood donor samples from nonendemic and endemic areas of the United States.

BACKGROUND: Babesia microti, a transfusion-transmissible intraerythrocytic parasite, is increasing in frequency in the United States with no available FDA-licensed donor screening assay. We utilized investigational arrayed fluorescence immunoassay (AFIA) and polymerase chain reaction (PCR) to detect B. microti antibodies and DNA in blood donors. STUDY DESIGN AND METHODS: AFIA and real-time PCR were performed on frozen paired EDTA plasma (AFIA) and EDTA whole blood (PCR) samples collected from May to September 2010 to 2011 in nonendemic (Arizona [AZ] and Oklahoma [OK]), moderately endemic (Minnesota [MN] and Wisconsin [WI]), and highly endemic (Connecticut [CT] and Massachusetts [MA]) areas of the United States. AFIA utilized B. microti piroplasm as an antigen substrate; PCR primers and probes targeted the B. microti 18S ribosomal RNA gene. Data from AZ and OK were used to calculate specificity. All AFIA- or PCR-positive or -inconclusive donors were deferred, notified, and invited to participate in a follow-up study involving repeat testing and a demographic and risk-factor questionnaire. Recipient tracing was performed for any cellular component transfused at index, at subsequent donation, or within the prior 12 months. RESULTS: Testing of 13,269 paired samples included 4022 from AZ and OK, 4167 from MN and WI, and 5080 from CT and MA. B. microti antibody and/or DNA prevalences were 0.025% (95% confidence interval [CI], 0.00%-0.14%), 0.12% (95% CI, 0.04%-0.28%), and 0.75% (95% CI, 0.53%-1.03%) in the nonendemic, mid-endemic, and high-endemic regions, respectively. Specificities were 99.95% (95% CI, 99.82%-99.99%) at a 1-in-64 AFIA cutoff and 99.98% (95% CI, 99.86%-100.00%) at a 1-in-128 cutoff. CONCLUSIONS: B. microti prevalence followed expected geographical patterns. Screening was feasible with a performance comparable or superior to other infectious disease blood donor screening assays.