A novel mitigation strategy for the prevention of transfusion-transmitted malaria.

BACKGROUND: Malaria is caused by protozoa of the genus Plasmodium and transmitted by Anopheles mosquitos. In the US, blood donors are assessed for malaria risk, including donor travel or previous residence in endemic areas and history of malaria by questionnaire and deferred for three months or three years, respectively. METHODS: The Procleix Plasmodium Assay is a qualitative nucleic acid test based on transcription-mediated amplification (TMA) for the detection of 18S ribosomal RNA of P. falciparum, P. ovale, P. vivax, P. malariae, and P. knowlesi for use on the Procleix Panther system. Analytical sensitivity was evaluated with in vitro transcripts and infected red blood cells. For clinical specificity, 12,800 individual donations and 283 pools of 16 samples from routine US donors were screened. Malaria risk was evaluated by testing 862 donors deferred for 3 years. Reactive results were confirmed with in-house real-time TMA assay and serology. RESULTS: Assay sensitivity was 8.47-11.89 RNA copies/mL and 2.10-6.82 infected red cells/mL. Specificity was 99.99% in 12,800 individual donations and 100% in 283 pools of 16. Of 862 tested deferred donor samples, one donor (0.12%) confirmed positive individually and in pools; he remained confirmed positive for 13 months. The infected donor was a prior resident of a malaria-endemic area in West Africa. CONCLUSIONS: The Procleix Plasmodium Assay showed high sensitivity and specificity and detected Plasmodium RNA in an asymptomatic presenting donor. This assay may prove helpful as a screening test versus the use of risk questions to reduce the number of donors deferred for malaria risk.

Clinical performance of a new multiplex assay for the detection of HIV-1, HIV-2, HCV, HBV, and HEV in blood donations in Catalonia (Spain).

BACKGROUND: Commercial multiplex nucleic acid tests (NATs) for HIV-1/HIV-2/HCV/HBV are widely used in developed countries to screen blood donations. HEV NAT screening has been implemented in some blood banks but is tested with a different assay. STUDY DESIGN AND METHODS: This study describes the clinical sensitivity and specificity of the Procleix® UltrioPlex E (UPxE) assay on the automated Procleix Panther® system for the simultaneous detection of HIV-1/HIV-2/HCV/HBV/HEV. To evaluate routine performance, 10,138 donations were tested in parallel with UPxE (in ID-NAT) and current assays (Procleix Ultrio Elite [UE] assay in ID-NAT and Procleix HEV assay in pool of 16). To assess clinical sensitivity, archived donations positive for HCV, HIV-1, HBV, HEV, or occult HBV infection (OBI) were tested (n = 104-186). RESULTS: Five donations were initially reactive (IR) with UPxE; none of them were reactive with current assays. Two of the three samples IR for HIV-1/HIV-2/HCV/HBV were confirmed positive for HBV (HBV NAT and/or anti-HBV core positive) and classified as OBI. The two samples IR for HEV were confirmed positive (Procleix HEV assay in ID-NAT and in-house RT-PCR HEV assay). One sample IR for HIV-1/HIV-2/HCV/HBV with UPxE and another with UE were not confirmed. UPxE showed a specificity of 99.99% for HIV-1/HIV-2/HCV/HBV and 100% for HEV. Comparable sensitivities were observed for HIV-1, HCV, HBV, OBI, and HEV samples tested in the UPxE, UE, and Procleix HEV assays. DISCUSSION: UPxE may provide an efficient solution for the simultaneous detection of HIV-1, HIV-2, HCV, HBV, and HEV in blood donations in a single test.

Low rate of detection of SARS-CoV-2 RNA in deceased tissue donors.

Given the possibility for disease transmission, this study was performed to determine whether there is detectable SARS-CoV-2 viral RNA in the blood of deceased tissue donors. A retrospective analysis of blood samples from eligible deceased tissue donors from Oct 2019 through June 2020 was performed. Plasma aliquots were initially tested with a SARS-CoV-2 NAT Assay; positive samples were further tested using an alternate NAT and an antibody assay. The proportion of donors with confirmed RNAemia and 95% confidence intervals were computed. Of donor samples collected in 2019, 894 yielded valid results, with 6 initially positive, none of which confirmed positive by alternate NAT. Of donor samples collected in 2020, 2562 yielded valid initial NAT results, with 21 (0.8%) initially positive. Among those, 3 were confirmed by alternate NAT, 17 were not confirmed, and 1 had an invalid alternate NAT result. The rate of SARS-CoV-2 RNAemia in deceased tissue donors is approximately 1 per 1000, and it is unknown whether this RNAemia reflects the presence of infectious virus. Given these results, the risk of transmission through tissue is thought likely to be low.

Surveillance for Zika, Chikungunya, and Dengue Virus Incidence and RNAemia in Blood Donors at 4 Brazilian Blood Centers During 2016-2019.

BACKGROUND: Except for public health case reports, the incidence of Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) infection are not available to assess the potential blood transfusion safety threat in Brazil. METHODS: Pools of 6 donation samples (MP6) left over from human immunodeficiency virus, hepatitis B virus, and hepatitis C virus nucleic acid testing were combined to create MP18 pools (3 MP6 pools). Samples were tested using the Grifols triplex ZIKV, CHIKV, and DENV real-time transcription mediated amplification assay to estimate prevalence of RNAemia and incidence, and to compare these results to case reports in São Paulo, Belo Horizonte, Recife, and Rio de Janeiro, from April 2016 through June 2019. RESULTS: ZIKV, CHIKV, and DENV RNAemia were found from donors who donated without overt symptoms of infection that would have led to deferral. The highest RNAemic donation prevalence was 1.2% (95% CI, .8%-1.9%) for DENV in Belo Horizonte in May 2019. Arbovirus infections varied by location and time of year, and were not always aligned with annual arbovirus outbreak seasons in different regions of the country. CONCLUSIONS: Testing donations for arboviruses in Brazil can contribute to public health. Transfusion recipients were likely exposed to ZIKV, CHIKV, and DENV viremic blood components during the study period.

Clinical evaluation of the Procleix SARS-CoV-2 assay, a sensitive, high-throughput test that runs on an automated system.

Testing is crucial in controlling COVID-19. The Procleix® SARS-CoV-2 assay, a transcription-mediated amplification nucleic acid test that runs on an automated system, was evaluated using inactivated virus and clinical samples. The sensitivity of the assay was assessed using heat-inactivated SARS-CoV-2 and compared to 3 other tests. Clinical validation utilized 2 sets of samples: (1) Nasal, nasopharyngeal and oropharyngeal samples (n = 963) from asymptomatic individuals, and (2) nasopharyngeal samples from symptomatic patients: 100 positive and 100 negative by RT-PCR. The Procleix assay had greater sensitivity (3-fold to 100-fold) than the comparators and had high specificity (100%) in asymptomatic subjects. In symptomatic patients, the Procleix assay detected 100% of PCR-positives and found 24 positives in the initial PCR-negatives. Eighteen of these were confirmed positive and 6 were inconclusive. These studies showed that the Procleix SARS-CoV-2 assay was a sensitive and specific tool for detecting COVID-19.

Background rate of low-level HCV RNA in anti-HCV confirmed-positive and minipool nucleic acid test-nonreactive blood donations.

BACKGROUND: In 2019, the Food and Drug Administration revised the requirement for further testing of anti-HCV-reactive donations testing nucleic acid (NAT)-nonreactive via routine mini-pool (MP)-NAT. Individual donation (ID)-HCV NAT was required as a supplemental test prior to a second FDA-licensed anti-HCV assay; if ID-HCV-NAT is reactive, no further testing is required. This study investigated the rate of low-level RNA in anti-HCV-reactive donation samples prior to and following the implementation of supplemental ID-HCV NAT. STUDY DESIGN/METHODS: A retrospective analysis was conducted on frozen plasma unit samples from 1161 anti-HCV confirmed-positive/HCV-NAT-nonreactive donations collected from December 2014 to January 2020. Samples were tested by multiple replicates on the Grifols Procleix Ultrio Elite (UE) assay and corresponding discriminatory HCV (dHCV) assay on the Procleix Panther System. Prospectively, the prevalence of low-level RNA in 2912 anti-HCV-reactive donations detected through routine screening from April 2020 through March 2021 was determined. RESULTS: In retrospective analyses, 10 (0.86%) of 1161 plasma samples were UE reactive, of which four (0.34%) were dHCV reactive (in all replicates of UE and dHCV). Of 2912 anti-HCV-reactive donation samples testing NAT-nonreactive via routine MP-NAT, three (0.1%; 95% CI: 0.04-0.30) were dHCV reactive; 37% of the remaining samples were reactive by a second anti-HCV assay and thus serologically confirmed. CONCLUSIONS: Retrospective and prospective analysis in comparison to earlier studies revealed that low-level HCV-RNA reactivity is decreasing over time. The very low HCV-RNA rates may be due to the widespread use of highly effective, direct-acting anti-viral treatments for those diagnosed with HCV infection.

Minipool testing for SARS-CoV-2 RNA in United States blood donors.

BACKGROUND: SARS-CoV-2 RNA prevalence in blood donors from large geographic areas of high community transmission is limited. We tested residual donor plasma minipools (MPs) to determine SARS-CoV-2 RNAemia prevalence in six United States areas. STUDY DESIGN/METHODS: Blood donations collected from 7 March 2020 to 25 September 2020 were tested for SARS-CoV-2 RNA (vRNA) in MP of 6 or 16 donations using the Grifols Procleix SARS-CoV-2 research-use only (RUO) transcription-mediated amplification (TMA) assay. Reactive results were confirmed using an alternate target region TMA assay. Reactive MPs were tested by TMA after serial dilution to estimate viral load. Testing for anti-SARS-CoV-2 antibodies and infectivity was performed. RESULTS: A total of 17,995 MPs corresponding to approximately 258,000 donations were tested for vRNA. Three confirmed reactive MP16 were identified. The estimated prevalence of vRNA reactive donations was 1.16/100,000 (95% CI 0.40, 3.42). The vRNA-reactive samples were non-reactive for antibody, and the estimated viral loads of the (presumed single) positive donations within each MP ranged from <1000 to <4000 copies/ml. When tested, no infectivity was observed in inoculated permissive cell cultures. DISCUSSION: Blood donation MP-nucleic acid testing (NAT) indicated that SARS-CoV-2 RNAemia is infrequent and, when detected, the vRNA was at low concentrations. Only one RNA-reactive MP could be tested for infectivity for operational reasons and was not infectious in cell culture. These findings support current recommendations from international and national regulatory agencies to not screen donors by NAT.

Zika virus RNA and IgM persistence in blood compartments and body fluids: a prospective observational study.

BACKGROUND: Characterisation of the dynamics of Zika virus persistence following acute infection is needed to inform blood donor and diagnostic testing policies and understand the natural history of Zika virus infection. We aimed to characterise the natural history, persistence, and clinical outcomes of Zika virus infection through a prospective study in initially asymptomatic Zika virus RNA-positive blood donors. METHODS: Zika virus-infected blood donors identified through Zika virus nucleic acid amplification test (NAAT) screening at three blood collection organisations in the USA were enrolled into a 1-year follow-up study, with blood and body fluid samples and detailed symptom data collected at up to seven visits. All samples were tested for Zika virus RNA by real-time PCR (rtPCR); follow-up plasma, whole blood, and urine were also tested by replicate NAAT. Plasma was tested for flavivirus-specific IgM and IgG by ELISA. Zika virus RNA persistence for each assay or sample type and plasma antibody persistence from estimated date of plasma NAAT-detectable infection were calculated from follow-up data using survival statistical methods. FINDINGS: Between July 6, 2016 and March 7, 2017, we enrolled 53 participants. From the estimated date of plasma NAAT-detectable infection, Zika virus RNA was detectable in plasma for 9·9 days (95% CI 8·1-12·0), in red blood cells for 95·4 days (62·8-129·1), and in whole blood for 73·5 days (39·8-107·5). Replicate NAATs (one or more of eight replicates positive) extended detection of Zika virus RNA in plasma to 34·8 days (19·9-56·2) and in whole blood (at least one of two tests positive) to 104·8 days (76·7-129·9). Urine was rtPCR reactive up to 14·5 days (10·5-20·3) and saliva up to 26·4 days (19·7-38·7). Zika virus IgM persisted for 237·7 days (128·7-459·5) from estimated time since plasma NAAT-detectable infection. Zika virus RNA fell below detectable limits more rapidly in the saliva of participants with pre-existing dengue virus IgG than in those without. Of 25 donors identified pre-seroconversion with symptom data at the first or second study visit, 16 (64%) developed multiple Zika virus-related symptoms after asymptomatic index donations, compared with nine (36%) of 25 donors detected after seroconversion. INTERPRETATION: Determination of viral marker persistence is enhanced by follow-up of blood donors who are pre-symptomatic or asymptomatic, Zika virus RNA-positive, and antibody negative. Zika virus RNA persists in red blood cells for several months following clearance from plasma and body fluids, and replicate, highly sensitive NAATs extend RNA detection in all compartments. Whole blood testing can extend detection of acute infection for diagnostics and monitoring of pregnant women, sexual partners, and travellers. FUNDING: National Heart, Lung, and Blood Institute, Biomedical Advanced Research and Development Authority.

Transcription-mediated amplification blood donation screening for Babesia.

BACKGROUND: Transfusion-transmitted Babesia microti is well recognized in the Northeast and upper Midwestern United States. Blood donation screening in Babesia-endemic states has occurred under investigational protocols prior to US Food and Drug Administration-licensed test availability. Here, we provide a prospective screening summary of nucleic acid testing (NAT) as part of a multicenter Babesia pivotal trial followed by extended investigational use. METHODS: From June 2017 to February 2018, 176,928 donation samples were tested with Procleix Babesia Assay (Grifols Diagnostic Solutions), a blood screening NAT for Babesia species ribosomal RNA detection using whole blood samples. During the pivotal trial, donations were collected in 11 endemic states plus Washington, DC, and Florida (nonendemic). Whole blood lysate samples were either tested in pools of 16 or individually. Reactive samples were confirmed by Babesia microti antibody and polymerase chain reaction (PCR) testing. If unconfirmed, further testing used a second PCR assay capable of detecting multiple Babesia species. Follow-up samples were also tested. Extended investigational testing followed pivotal trial completion. RESULTS: The pivotal trial identified 61 confirmed positives (176,608 donations): 35 (57%) PCR positive, 59 (97%) antibody positive, and two (3%) NAT positive/antibody negative, for a total yield of one positive per 2895 donations, including one Florida resident; others were from seven endemic states. During extended investigational testing of 496,270 donations in endemic states through January 2019, 211 (1:2351) repeat reactive donations were identified. CONCLUSIONS: Babesia was detected in donors from multiple US states, including one previously not associated with positive blood donors. This study supports the use of the Procleix Babesia Assay using individual testing or pools of up to 16.

Prevalence of Babesia in Canadian blood donors: June-October 2018.

BACKGROUND: The erythrocytic protozoan parasite Babesia microti, the cause of human babesiosis, is transmitted not only by tick bites but also via blood transfusion. B. microti is endemic in the northeastern/upper midwestern United States, where partial screening of blood donations has been implemented. In Canada, a 2013 study of approximately 14,000 donors found no B. microti antibody-positive samples, suggesting low risk at that time. METHODS: Between June and October 2018, 50,752 Canadian donations collected from sites near the US border were tested for Babesia nucleic acid by transcription-mediated amplification (TMA). Reactive donations were tested for B. microti by IgG immunofluorescence assay and polymerase chain reaction. A subset of 14,758 TMA nonreactive samples was also screened for B. microti antibody. Donors who tested reactive/positive were deferred, asked about risk factors, and were requested to provide a follow-up sample for supplemental testing. RESULTS: One sample from Winnipeg, Manitoba, was TMA and antibody reactive. Of the 14,758 TMA-nonreactive donations tested for antibody, four reactive donations were identified from southwestern Ontario near Lake Erie. None of the interviewed donors remembered any symptoms, likely tick exposure, or relevant travel within Canada or the United States. CONCLUSIONS: This is the largest B. microti prevalence study performed in Canada. The results indicate very low prevalence, with only one TMA-confirmed-positive donation of 50,752 tested. This donor was from the only region in Canada where autochthonous infection has been reported. Seropositive donations in southwestern Ontario suggest low prevalence; travel should not be ruled out given the proximity to the US border.

The Babesia observational antibody (BAOBAB) study: A cross-sectional evaluation of Babesia in two communities in Kilosa district, Tanzania.

BACKGROUND: Babesia, a tick-borne genus of intraerythrocytic parasites, is understudied in humans outside of established high-endemic areas. There is a paucity of data on Babesia in Africa, despite evidence that it is regionally present. A pilot study suggested that Babesia was present in a rural district of Tanzania. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional study was conducted July-August 2017: residents in a case hamlet that had clustering of subjects with high signal-to-cut off (S/CO) ratios for antibodies against B. microti in the pilot study, and a control hamlet that had lacked significant signal, were evaluated for B. microti. Subjects aged ≥15yrs (n = 299) underwent clinical evaluation and household inspections; 10ml whole blood was drawn for Babesia transcription mediated amplification (TMA), B. microti indirect fluorescent antibody testing (IFA) and rapid diagnostic testing (RDT) for Plasmodium spp. Subjects aged <15yrs (n = 266) underwent a RDT for Plasmodium and assessment by ELISA for B. microti antibodies. A total of 570 subjects participated (mean age 22 [<1 to 90yrs]) of whom 50.7% were female and 145 (25.5%) subjects were Plasmodium RDT positive (+). In those <15yrs, the median ELISA S/CO was 1.11 (IQR 0.80-1.48); the median S/CO in the case (n = 120) and control (n = 146) hamlets was 1.19 (IQR 0.81-1.48) and 1.06 (IQR 0.80-1.50) respectively (p = 0.4). Children ≥5yrs old were more likely to have a higher S/CO ratio than those <5yrs old (p<0.001). One hundred (38%) subjects <15yrs were Plasmodium RDT+. The median S/CO ratio (children <15yrs) did not differ by RDT status (p = 0.15). In subjects ≥15yrs, no molecular test was positive for Babesia, but four subjects (1.4%) were IFA reactive (two each at titers of 128 and 256). CONCLUSIONS/SIGNIFICANCE: The findings offer further support for Babesia in rural Tanzania. However, low prevalence of seroreactivity questions its clinical significance.

Probable transmission of hepatitis E virus (HEV) via transfusion in the United States.

BACKGROUND: Hepatitis E virus (HEV) can inapparently infect blood donors. To assess transfusion transmission of HEV in the United States, which has not been documented, a donor-recipient repository was evaluated. STUDY DESIGN AND METHODS: To identify donations that contained HEV RNA and were linked to patient-recipients with antibody evidence of HEV exposure, we assayed samples from the Retrovirus Epidemiology Donor Study (REDS) Allogeneic Donor and Recipient repository that represents 13,201 linked donations and 3384 transfused patients. Posttransfusion samples, determined to contain IgG anti-HEV by enzyme-linked immunosorbent assay, were reassayed along with corresponding pretransfusion samples for seroconversion (incident exposure) or at least fourfold IgG anti-HEV increase (reexposure). HEV-exposed patients were linked to donations in which HEV RNA was then detected by reverse-transcription quantitative polymerase chain reaction, confirmed by transcription-mediated amplification, and phylogenetically analyzed as subgenomic cDNA sequences. RESULTS: Among all patients, 19 of 1036 (1.8%) who had IgG anti-HEV before transfusion were reexposed; 40 of 2348 (1.7%) without pretransfusion IgG anti-HEV seroconverted. These 59 patients were linked to 257 donations, 1 of which was positive by reverse-transcription quantitative polymerase chain reaction and transcription-mediated amplification. Plasma from this donation contained 5.5 log IU/mL of HEV RNA that grouped with HEV genotype 3, clade 3abchij. The patient-recipient of RBCs from this donation had a greater than eightfold IgG increase; however, clinical data are unavailable. CONCLUSIONS: This is the first report of probable HEV transmission via transfusion in the United States, although it has been frequently observed in Europe and Japan. Additional data on the magnitude of the risk in the United States are needed.

Minimal infectious dose and dynamics of Babesia microti parasitemia in a murine model.

BACKGROUND: Babesia microti is a parasite that infects red blood cells (RBCs) in mammals. It is transmitted to humans by tick bites, transfusion, organ transplantation, and congenital acquisition. Although the Babesia natural history and seroprevalence in donors have been well described, gaps in knowledge relevant to transfusion remain. STUDY DESIGN AND METHODS: Mice were infected with dilutions of parasitized blood to address the minimal infectious dose and the kinetics of parasitemia by quantitative polymerase chain reaction (qPCR) and of antibodies by enzyme immunoassay. RESULTS: In immunocompetent DBA/2 mice infected with 100 parasitized RBCs (pRBCs) and in immunodeficient NSG mice infected with 63 pRBCs, parasitemia was detectable in five of five mice each. Peak parasitemia up to 2 × 107 pRBCs/mL at 2 to 3 weeks or 5 × 108 pRBCs/mL at 6 weeks was observed for DBA/2 and NSG mice, respectively. Protracted fluctuating parasitemia was observed for 8 months in DBA/2 mice, whereas NSG mice exhibited a high-plateau parasitemia. Antibody titers continued to increase until 6 to 18 weeks in DBA/2 mice and remained high through 6 months. This study also investigated the analytical performance of Babesia assays that detect parasite DNA or RNA using a blinded panel. A Babesia assay targeting parasite RNA was approximately 10-fold more sensitive compared to qPCR targeting DNA. CONCLUSION: The mice in this study were highly susceptible to Babesia infection using as few as 1 to 2 log pRBCs and maintained chronic parasitemia. If the infectious dose in human transfusion recipients is comparably low, a highly sensitive assay targeting parasite RNA may safeguard the blood supply, particularly before antibody detection.

Intraamniotic Zika virus inoculation of pregnant rhesus macaques produces fetal neurologic disease.

Zika virus (ZIKV) infection of pregnant women can cause fetal microcephaly and other neurologic defects. We describe the development of a non-human primate model to better understand fetal pathogenesis. To reliably induce fetal infection at defined times, four pregnant rhesus macaques are inoculated intravenously and intraamniotically with ZIKV at gestational day (GD) 41, 50, 64, or 90, corresponding to first and second trimester of gestation. The GD41-inoculated animal, experiencing fetal death 7 days later, has high virus levels in fetal and placental tissues, implicating ZIKV as cause of death. The other three fetuses are carried to near term and euthanized; while none display gross microcephaly, all show ZIKV RNA in many tissues, especially in the brain, which exhibits calcifications and reduced neural precursor cells. Given that this model consistently recapitulates neurologic defects of human congenital Zika syndrome, it is highly relevant to unravel determinants of fetal neuropathogenesis and to explore interventions.

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.).

Real-Time Evolution of Zika Virus Disease Outbreak, Roatán, Honduras.

A Zika virus disease outbreak occurred in Roatán, Honduras, during September 2015-July 2016. Blood samples and clinical information were obtained from 183 patients given a clinical diagnosis of suspected dengue virus infection. A total of 79 patients were positive for Zika virus, 13 for chikungunya virus, and 6 for dengue virus.

First cases of Zika virus-infected US blood donors outside states with areas of active transmission.

BACKGROUND: Zika virus (ZIKV) is transmitted by Aedes mosquitos and can result in severe congenital and adult neurologic abnormalities. ZIKV has rapidly spread northward through Central America and the Caribbean and autochthonous cases have been identified in the continental United States. High rates of ZIKA RNA positivity were detected in blood donors during previous epidemics. ZIKV transmission by transfused blood from healthy donor components has been a growing concern. STUDY DESIGN AND METHODS: Individual-donation aliquots of plasma from volunteer blood donors were tested individually with an investigational Procleix ZIKV assay. Initially reactive samples were tested for ZIKV RNA in plasma and red blood cells (RBCs) and for ZIKV-specific antibodies in serum. A confirmed positive classification required confirmation of RNA and/or detection of ZIKV antibodies in index and/or follow-up samples. RESULTS: Between September 19 and November 30, 2016, a total of 466,834 donations were screened for ZIKV RNA. Five donors (one in approx. 93,000) were reactive for ZIKV RNA by both the Procleix ZIKV assay and supplemental testing. The donations were collected outside areas considered as having active transmission, and all five donors had travel exposures. A lookback case demonstrated no infection despite transfusion of a Zika IgG-positive platelet (PLT) component with probable low levels of ZIKV RNA. CONCLUSIONS: This report describes the first ZIKV-positive donors detected outside areas with active transmission. These donors most likely represent travel-acquired "tail-end infections" with prolonged RBC-associated ZIKV RNA. The lack of transmission to the recipient of an apheresis PLT may suggest that these units are not infectious.

Relative analytical sensitivity of donor nucleic acid amplification technology screening and diagnostic real-time polymerase chain reaction assays for detection of Zika virus RNA.

BACKGROUND: Zika virus (ZIKV) has spread rapidly in the Pacific and throughout the Americas and is associated with severe congenital and adult neurologic outcomes. Nucleic acid amplification technology (NAT) assays were developed for diagnostic applications and for blood donor screening on high-throughput NAT systems. We distributed blinded panels to compare the analytical performance of blood screening relative to diagnostic NAT assays. STUDY DESIGN AND METHODS: A 25-member, coded panel (11 half-log dilutions of a 2013 French Polynesia ZIKV isolate and 2015 Brazilian donor plasma implicated in transfusion transmission, and 3 negative controls) was sent to 11 laboratories that performed 17 assays with 2 to 12 replicates per panel member. Results were analyzed for the percentage reactivity at each dilution and by probit analysis to estimate the 50% and 95% limits of detection (LOD50 and LOD95 , respectively). RESULTS: Donor-screening NAT assays that process approximately 500 µL of plasma into amplification reactions were comparable in sensitivity (LOD50 and LOD95 , 2.5 and 15-18 copies/mL) and were approximately 10-fold to 100-fold more sensitive than research laboratory-developed and diagnostic reverse transcriptase-polymerase chain reaction tests that process from 10 to 30 µL of plasma per amplification. Increasing sample input volume assayed with the Centers for Disease Control and Prevention reverse transcriptase-polymerase chain reaction assays increased the LODs by 10-fold to 30-fold. CONCLUSIONS: Blood donor-screening ZIKV NAT assays demonstrate similar excellent sensitivities to assays currently used for screening for transfusion-transmitted viruses and are substantially more sensitive than most other laboratory-developed and diagnostic ZIKV reverse transcriptase-polymerase chain reaction assays. Enhancing sensitivities of laboratory-developed and diagnostic assays may be achievable by increasing sample input.

Hepatitis E viral loads in plasma pools for fractionation.

BACKGROUND: It is now recognized that blood donors may be silently infected with hepatitis E virus (HEV) and that plasma pools used in the manufacture of plasma-derived medicinal products may also contain detectable virus RNA. The occurrence of HEV-infected blood and plasma donors can vary considerably depending on local epidemiology. STUDY DESIGN AND METHODS: Manufacturing plasma pools from North America, Europe, the Middle East, and Asia were examined for the presence of HEV using transcription-mediated amplification of HEV RNA; confirmatory testing was performed using real-time reverse transcription polymerase chain reaction and sequencing. RESULTS: A total of 484 pools were tested. Asian pools were most frequently positive for HEV RNA and had higher viral loads, although none exceeding 300 IU/mL, and the sequenced strains (n = 5) clustered with Genotype 4, including one significantly divergent sequence. Only HEV Genotype 3 was identified in North American (n = 5) and European (n = 5) pools. There was no evidence of HEV in any pools tested from the Middle East. CONCLUSIONS: HEV was detected in manufacturing plasma pools from three different continents; viral loads were low-consistent with large pool sizes and moderate levels of HEV viremia at the individual donation level-but are nevertheless informative for risk assessment of plasma-derived medicinal products. Where sequencing was possible, analysis confirmed the presence of viruses consistent with locally circulating genotypes in the respective regions. The absence of HEV in Middle Eastern pools is consistent with the low prevalence of HEV in this region, likely due to low pork consumption.

Duration of Dengue Viremia in Blood Donors and Relationships Between Donor Viremia, Infection Incidence and Clinical Case Reports During a Large Epidemic.

BACKGROUND: Dengue viruses (DENV-1-4) pose a transfusion-transmission risk. This study estimated the dengue RNA detection period in asymptomatic blood donors and relationships between donor viremia and dengue incidence during a large epidemic. METHODS: Donor samples from the 2012 dengue transmission season in Rio de Janeiro, Brazil, were tested for DENV RNA by a transcription-mediated amplification (TMA) assay, with DENV types and viral loads determined by polymerase chain reaction. Samples collected during the first and last weeks of enrollment were tested for DENV immunoglobulin (Ig) G and IgM to estimate incidence during the study period, which was analyzed relative to nucleic acid amplification technology (NAT) yield to estimate the duration of NAT-detectable viremia and compared with reported clinical dengue cases in Rio. RESULTS: Samples from 16 241 donations were tested; 87 (0.54%) were confirmed as DENV-4 RNA positive. Dengue IgM-positive/IgG-positive reactivity increased from 2.8% to 8.8%, indicating a 6.2% incidence (95% confidence interval [CI], 3.2%-9.1%) during the study period. Based on these data, we estimated a 9.1-day period (95% CI, 4.4-13.9 days) of RNA detectable with TMA. With 100 475 reported cases of clinical dengue, 1 RNA-positive donation was identified per 800 DENV cases. CONCLUSIONS: These parameters allow projections of dengue incidence from donor NAT yield data and vice versa, and suggest that viremic donations will be rare relative to clinical disease cases.