Publications by authors named "Michael P Busch"

376 Publications

SARS-CoV-2 antibody magnitude and detectability are driven by disease severity, timing, and assay.

medRxiv 2021 Mar 5. Epub 2021 Mar 5.

Serosurveillance studies are critical for estimating SARS-CoV-2 transmission and immunity, but interpretation of results is currently limited by poorly defined variability in the performance of antibody assays to detect seroreactivity over time in individuals with different clinical presentations. We measured longitudinal antibody responses to SARS-CoV-2 in plasma samples from a diverse cohort of 128 individuals over 160 days using 14 binding and neutralization assays. For all assays, we found a consistent and strong effect of disease severity on antibody magnitude, with fever, cough, hospitalization, and oxygen requirement explaining much of this variation. We found that binding assays measuring responses to spike protein had consistently higher correlation with neutralization than those measuring responses to nucleocapsid, regardless of assay format and sample timing. However, assays varied substantially with respect to sensitivity during early convalescence and in time to seroreversion. Variations in sensitivity and durability were particularly dramatic for individuals with mild infection, who had consistently lower antibody titers and represent the majority of the infected population, with sensitivities often differing substantially from reported test characteristics (e.g., amongst commercial assays, sensitivity at 6 months ranged from 33% for ARCHITECT IgG to 98% for VITROS Total Ig). Thus, the ability to detect previous infection by SARS-CoV-2 is highly dependent on the severity of the initial infection, timing relative to infection, and the assay used. These findings have important implications for the design and interpretation of SARS-CoV-2 serosurveillance studies.
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http://dx.doi.org/10.1101/2021.03.03.21251639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941652PMC
March 2021

Pharmacogenomic Profile and Adverse Drug Reactions in a Prospective Therapeutic Cohort of Chagas Disease Patients Treated with Benznidazole.

Int J Mol Sci 2021 Feb 16;22(4). Epub 2021 Feb 16.

Department of Infectious Disease and Institute of Tropical Medicine (IMT-SP), University of São Paulo, Av. Dr. Enéas Carvalho de Aguiar, 470, São Paulo 05403-000, Brazil.

Chagas disease remains a major social and public health problem in Latin America. Benznidazole (BZN) is the main drug with activity against . Due to the high number of adverse drug reactions (ADRs), BZN is underprescribed. The goal of this study was to evaluate the genetic and transcriptional basis of BZN adverse reactions.

Methods: A prospective cohort with 102 Chagas disease patients who underwent BZN treatment was established to identify ADRs and understand their genetic basis. The patients were classified into two groups: those with at least one ADR ( = 73), and those without ADRs ( = 29). Genomic analyses were performed comparing single nucleotide polymorphisms between groups. Transcriptome data were obtained comparing groups before and after treatment, and signaling pathways related to the main ADRs were evaluated.

Results: A total of 73 subjects (71.5%) experienced ADRs. Dermatological symptoms were most frequent (45.1%). One region of chromosome 16, at the gene LOC102724084 (rs1518601, rs11861761, and rs34091595), was associated with ADRs ( = 5.652 × 10). Transcriptomic data revealed three significantly enriched signaling pathways related to BZN ADRs.

Conclusions: These data suggest that part of adverse BZN reactions might be genetically determined and may facilitate patient risk stratification prior to starting BZN treatment.
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http://dx.doi.org/10.3390/ijms22041960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920452PMC
February 2021

Selecting COVID-19 convalescent plasma for neutralizing antibody potency using a high-capacity SARS-CoV-2 antibody assay.

Transfusion 2021 04 18;61(4):1160-1170. Epub 2021 Feb 18.

Vitalant Research Institute, Denver, Colorado, USA.

Background: Efficacy of COVID-19 convalescent plasma (CCP) is hypothesized to be associated with the concentration of neutralizing antibodies (nAb) to SARS-CoV-2. High capacity serologic assays detecting binding antibodies (bAb) have been developed; nAb assays are not adaptable to high-throughput testing. We sought to determine the effectiveness of using surrogate bAb signal-to-cutoff ratios (S/Co) in predicting nAb titers using a pseudovirus reporter viral particle neutralization (RVPN) assay.

Methods: CCP donor serum collected by three US blood collectors was tested with a bAb assay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and a nAb RVPN assay. Prediction effectiveness of various CoV2T S/Co criteria was evaluated for RVPN nAb NT titers using receiver operating characteristics.

Results: Seven hundred and fifty-three CCPs were tested with median CoV2T S/Co and NT of 71.2 of 527.5. Proportions of donors with NT over target nAb titers were 86% ≥1:80, 76% ≥1:160, and 62% ≥1:320. Increasing CoV2T S/Co criterion reduced the sensitivity to predict NT titers, while specificity to identify those below increased. As target NT50 titers increase, the CoV2T assay becomes less accurate as a predictor with a decline in positive predictive value and rise in negative predictive value.

Conclusion: Selection of a clinically effective nAb titer will impact availability of CCP. Product release with CoV2T assay S/Co criterion must balance the risk of releasing products below target nAb titers with the cost of false negatives. A two-step testing scheme may be optimal, with nAb testing on CoV2T samples with S/Cos below criterion.
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http://dx.doi.org/10.1111/trf.16321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013397PMC
April 2021

Microdrop HIV sequencing for incidence and drug resistance surveillance.

J Infect Dis 2021 Jan 31. Epub 2021 Jan 31.

Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, USA.

Background: Precise and cost-efficient HIV incidence and drug resistance surveillances are in high demand for the advancement of 90-90-90: Treatment for All.

Methods: We developed microdrop HIV sequencing for HIDA (HIV incidence and drug resistance assay), a single blood draw surveillance tool for incidence and drug resistance mutation detection. We amplified full-length HIV envelope and pol gene sequences within micro-droplets and this compartmental amplification with long-read high-throughput sequencing enabled us to recover multiple unique sequences.

Results: We achieved a greater precision in determining the stage of infection than current incidence assays, with a 1.2% false recency rate (proportion of misclassified chronic infections) and 262 days of mean duration of recent infection (average timespan of recent infection classification) from 83 recently infected and 81 chronically infected individuals. Microdrop HIV sequencing demonstrated an increased capacity to detect minority variants and linked drug resistance mutations. By screening all 93 WHO surveillance drug resistance mutations (SDRMs), we detected six pretreatment drug resistance mutations with 2.6% - 13.2% prevalence and cross-linked mutations.

Conclusions: HIDA via microdrop HIV sequencing may promote global HIV real-time surveillance by serving as a precise and high-throughput cross-sectional survey tool that can be generalized for other pathogen surveillances.
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http://dx.doi.org/10.1093/infdis/jiab060DOI Listing
January 2021

Resurgence of COVID-19 in Manaus, Brazil, despite high seroprevalence.

Lancet 2021 02 27;397(10273):452-455. Epub 2021 Jan 27.

Departamento de Molestias Infecciosas e Parasitarias and Instituto de Medicina Tropical da Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP 05403-000, Brazil; MRC Centre for Global Infectious Disease Analysis, J-IDEA, Imperial College London, London, UK; Department of Zoology, University of Oxford, Oxford, UK.

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http://dx.doi.org/10.1016/S0140-6736(21)00183-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906746PMC
February 2021

Serosurveillance for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Incidence Using Global Blood Donor Populations.

Clin Infect Dis 2021 01;72(2):254-256

Vitalant Research Institute, Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA.

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http://dx.doi.org/10.1093/cid/ciaa1116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454349PMC
January 2021

SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques.

Nat Commun 2021 01 22;12(1):541. Epub 2021 Jan 22.

Center for Immunology and Infectious Diseases, UC Davis, Davis, CA, USA.

CD4 T follicular helper (T) cells are important for the generation of durable and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates T cells and stimulates the germinal center (GC) response is an important question as we investigate vaccine induced immunity against COVID-19. Here, we report that SARS-CoV-2 infection in rhesus macaques, either infused with convalescent plasma, normal plasma, or receiving no infusion, resulted in transient accumulation of pro-inflammatory monocytes and proliferating T cells with a T1 profile in peripheral blood. CD4 helper cell responses skewed predominantly toward a T1 response in blood, lung, and lymph nodes. SARS-CoV-2 Infection induced GC T cells specific for the SARS-CoV-2 spike and nucleocapsid proteins, and a corresponding early appearance of antiviral serum IgG antibodies. Collectively, the data show induction of GC responses in a rhesus model of mild COVID-19.
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http://dx.doi.org/10.1038/s41467-020-20642-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822826PMC
January 2021

Analysis of SARS-CoV-2 antibodies in COVID-19 convalescent blood using a coronavirus antigen microarray.

Nat Commun 2021 01 4;12(1). Epub 2021 Jan 4.

Division of Infectious Diseases, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.
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http://dx.doi.org/10.1038/s41467-020-20095-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782488PMC
January 2021

Blood donor exposome and impact of common drugs on red blood cell metabolism.

JCI Insight 2021 Feb 8;6(3). Epub 2021 Feb 8.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, Colorado, USA.

Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures ("exposome") may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell-Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine - though rarely detected in the blood donor population - has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could - through mechanisms involving sphingosine 1-phosphate-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin - improve erythrocyte metabolism and storage quality.
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http://dx.doi.org/10.1172/jci.insight.146175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934844PMC
February 2021

Three-quarters attack rate of SARS-CoV-2 in the Brazilian Amazon during a largely unmitigated epidemic.

Science 2021 01 8;371(6526):288-292. Epub 2020 Dec 8.

Departamento de Molestias Infecciosas e Parasitarias and Instituto de Medicina Tropical da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread rapidly in Manaus, the capital of Amazonas state in northern Brazil. The attack rate there is an estimate of the final size of the largely unmitigated epidemic that occurred in Manaus. We use a convenience sample of blood donors to show that by June 2020, 1 month after the epidemic peak in Manaus, 44% of the population had detectable immunoglobulin G (IgG) antibodies. Correcting for cases without a detectable antibody response and for antibody waning, we estimate a 66% attack rate in June, rising to 76% in October. This is higher than in São Paulo, in southeastern Brazil, where the estimated attack rate in October was 29%. These results confirm that when poorly controlled, COVID-19 can infect a large proportion of the population, causing high mortality.
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http://dx.doi.org/10.1126/science.abe9728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857406PMC
January 2021

Large genome-wide association study identifies three novel risk variants for restless legs syndrome.

Commun Biol 2020 Nov 25;3(1):703. Epub 2020 Nov 25.

deCODE Genetics, 101, Reykjavik, Iceland.

Restless legs syndrome (RLS) is a common neurological sensorimotor disorder often described as an unpleasant sensation associated with an urge to move the legs. Here we report findings from a meta-analysis of genome-wide association studies of RLS including 480,982 Caucasians (cases = 10,257) and a follow up sample of 24,977 (cases = 6,651). We confirm 19 of the 20 previously reported RLS sequence variants at 19 loci and report three novel RLS associations; rs112716420-G (OR = 1.25, P = 1.5 × 10), rs10068599-T (OR = 1.09, P = 6.9 × 10) and rs10769894-A (OR = 0.90, P = 9.4 × 10). At four of the 22 RLS loci, cis-eQTL analysis indicates a causal impact on gene expression. Through polygenic risk score for RLS we extended prior epidemiological findings implicating obesity, smoking and high alcohol intake as risk factors for RLS. To improve our understanding, with the purpose of seeking better treatments, more genetics studies yielding deeper insights into the disease biology are needed.
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http://dx.doi.org/10.1038/s42003-020-01430-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689502PMC
November 2020

A multi-ancestry sex stratified genome-wide association study of spontaneous clearance of hepatitis C virus.

J Infect Dis 2020 Oct 29. Epub 2020 Oct 29.

Johns Hopkins University, School of Medicine, Baltimore, MD. USA.

Background: Spontaneous clearance of acute hepatitis C virus (HCV) infection is more common in women than in men, independent of known risk factors.

Methods: To identify sex-specific genetic loci, we studied 4423 HCV-infected individuals (2903 males, 1520 females) of European, African and Hispanic ancestry. We performed autosomal, and X-chromosome sex-stratified and combined association analyses in each ancestry group.

Results: A male-specific region near ADP-ribosylation factor-like-5B gene was identified. Individuals with the C allele of rs76398191 were about 30% more likely to have a chronic HCV infection compared to individuals with the T allele (ORMales=0.69,P-valueMales=1.98x10-07) and this was not seen in females. The ARL5B gene encodes an interferon stimulated gene that inhibits immune response to dsRNA viruses. We also identified suggestive associations near Septin6 and Ribosomal Protein L39 genes on the X chromosome. In box sexes, allele G of rs12852885 was associated with a 40% increase in HCV clearance compared to the A allele (OR= 1.4, P-valueAll individuals=2.46 x 10-06). Septin6 facilitates HCV replication via interaction with the HCV NS5b protein and RPL39 acts as an HCV core interactor.

Conclusion: These novel gene associations support differential mechanisms of HCV clearance between sexes and provide biologic targets for treatment or vaccine development.
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http://dx.doi.org/10.1093/infdis/jiaa677DOI Listing
October 2020

Association of proton pump inhibitor and histamine H2-receptor antagonists with restless legs syndrome.

Sleep 2021 Apr;44(4)

RTI International, Atlanta, GA.

Restless legs syndrome (RLS) is a common sensorimotor disorder, which can disrupt sleep and is thought to be caused in part by low cellular iron stores. Proton pump inhibitors (PPI) and histamine H2-receptor antagonists (H2A) are among the most commonly used drugs worldwide and show evidence of causing iron deficiency. We conducted a case/non-case observational study of blood donors in the United States (N = 13,403; REDS-III) and Denmark (N = 50,323; Danish Blood Donor Study, DBDS), both of which had complete blood count measures and a completed RLS assessment via the Cambridge-Hopkins RLS questionnaire. After adjusting for age, sex, race, BMI, blood donation frequency, smoking, hormone use, and iron supplement use, PPI/H2A use was associated with RLS (odds ratio [OR] = 1.41; 95% confidence interval [CI], 1.13-1.76; p = 0.002) in REDS-III for both PPI (OR = 1.43; CI, 1.03-1.95; p = 0.03) and H2A (OR = 1.56; CI, 1.10-2.16; p = 0.01). DBDS exhibited a similar association with PPIs/H2As (OR = 1.29; CI, 1.20-1.40; p < 0.001), and for PPIs alone (OR = 1.27; CI, 1.17-1.38; p < 0.001), but not H2As alone (OR = 1.18; CI, 0.92-1.53; p = 0.2). We found no evidence of blood iron stores mediating this association. The association of PPI, and possibly H2A, consumption with RLS independent of blood iron status and other factors which contribute to RLS risk suggest the need to re-evaluate use of PPI/H2A in populations at particular risk for RLS.
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http://dx.doi.org/10.1093/sleep/zsaa220DOI Listing
April 2021

Multi-ancestry fine mapping of interferon lambda and the outcome of acute hepatitis C virus infection.

Genes Immun 2020 11 28;21(5):348-359. Epub 2020 Oct 28.

Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD, USA.

Clearance of acute infection with hepatitis C virus (HCV) is associated with the chr19q13.13 region containing the rs368234815 (TT/ΔG) polymorphism. We fine-mapped this region to detect possible causal variants that may contribute to HCV clearance. First, we performed sequencing of IFNL1-IFNL4 region in 64 individuals sampled according to rs368234815 genotype: TT/clearance (N = 16) and ΔG/persistent (N = 15) (genotype-outcome concordant) or TT/persistent (N = 19) and ΔG/clearance (N = 14) (discordant). 25 SNPs had a difference in counts of alternative allele >5 between clearance and persistence individuals. Then, we evaluated those markers in an association analysis of HCV clearance conditioning on rs368234815 in two groups of European (692 clearance/1 025 persistence) and African ancestry (320 clearance/1 515 persistence) individuals. 10/25 variants were associated (P < 0.05) in the conditioned analysis leaded by rs4803221 (P value = 4.9 × 10) and rs8099917 (P value = 5.5 × 10). In the European ancestry group, individuals with the haplotype rs368234815ΔG/rs4803221C were 1.7× more likely to clear than those with the rs368234815ΔG/rs4803221G haplotype (P value = 3.6 × 10). For another nearby SNP, the haplotype of rs368234815ΔG/rs8099917T was associated with HCV clearance compared to rs368234815ΔG/rs8099917G (OR: 1.6, P value = 1.8 × 10). We identified four possible causal variants: rs368234815, rs12982533, rs10612351 and rs4803221. Our results suggest a main signal of association represented by rs368234815, with contributions from rs4803221, and/or nearby SNPs including rs8099917.
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http://dx.doi.org/10.1038/s41435-020-00115-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657970PMC
November 2020

Estimating the incidence of HIV infection in repeat blood donors with low average donation frequency.

Transfusion 2021 Feb 24;61(2):494-502. Epub 2020 Oct 24.

Department of Pathology, University of British Columbia, Victoria, British Columbia, Canada.

Background: The standard approach to estimating HIV incidence in repeat blood donors includes only donors who made two or more donations in an estimation interval. In China and some other countries, large proportions of repeat donors donate only once in a 1- or 2-year interval. The standard approach may not represent risk among all repeat donors in these areas. Two approaches to including all repeat donors in the incidence estimate were evaluated in a simulation study.

Study Design And Methods: Under one approach, a donor infected at the first donation contributes a partial case to incidence that equals the proportion of time since the preceding donation that is in the estimation interval. Under the other, that donor contributes a full case if at least half the time since the previous donation is in the estimation interval and nothing otherwise. Infections identified at the second or subsequent donations in the interval contribute full cases as usual. The simulations involved proportions with single donations of 11% to 65% combined with a variety of patterns of rising, falling, or constant incidence.

Results: The partial-case approach was unbiased under more test conditions than the whole-case approach and exhibited smaller bias when both were biased. Under both approaches, bias >10% occurred only when rates of single donations >50% were combined with large changes in incidence over time.

Conclusion: The partial-case approach performed better than the whole-case approach. The conditions producing bias >10% are so extreme that they are unlikely to be encountered in the field.
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http://dx.doi.org/10.1111/trf.16144DOI Listing
February 2021

10-year analysis of human immunodeficiency virus incidence in first-time and repeat donors in Brazil.

Vox Sang 2021 Feb 30;116(2):207-216. Epub 2020 Sep 30.

Departamento de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.

Background And Objectives: Incidence in first-time and repeat blood donors is an important measure of transfusion-transmitted HIV infection (TT-HIV) risk. This study assessed HIV incidence over time at four large blood centres in Brazil.

Materials And Methods: Donations were screened and confirmed using serological assays for HIV from 2007 to 2016, and additionally screened by nucleic acid testing from 2011 forward. Limiting antigen (LAg) avidity testing was conducted on HIV seroreactive samples from first-time donors to classify whether an infection was recently acquired. We calculated incidence in first-time donors using the mean duration of recent infection and in repeat donors using classical methods. Time and demographic trends were assessed using Poisson regression.

Results: Over the 10-year period, HIV incidence in first-time donors was highest in Recife (45·1/100 000 person-years (10 py)) followed by São Paulo (32·2/10 py) and then Belo Horizonte (23·3/10 py), and in repeat donors was highest in Recife (33·2/10 py), Belo Horizonte (27·5/10 py) and São Paulo (17·0/10 py). Results from Rio de Janeiro were available from 2013 to 2016 with incidence in first-time donors of 35·9/10 py and repeat donors from 2011 to 2016 of 29·2/10 py. Incidence varied by other donor demographics. When incidence was considered in 2-year intervals, no significant trend was evident. Overall residual risk of TT-HIV was 5·46 and 7·41 per million units of pRBC and FFP transfused, respectively.

Conclusion: HIV incidence in both first-time and repeat donors varied by region in Brazil. Clear secular trends were not evident.
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http://dx.doi.org/10.1111/vox.13002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019535PMC
February 2021

Replicate Aptima Assay for Quantifying Residual Plasma Viremia in Individuals on Antiretroviral Therapy.

J Clin Microbiol 2020 11 18;58(12). Epub 2020 Nov 18.

Vitalant Research Institute, San Francisco, California, USA.

Detection of residual plasma viremia in antiretroviral therapy (ART)-suppressed HIV-infected individuals is critical for characterizing the latent reservoir and evaluating the impact of cure interventions. Ultracentrifugation-based single-copy assays are sensitive but labor intensive. Fully automated replicate testing using a standard clinical viral load assay was evaluated as a high-throughput alternative for the quantification of low-level viremia. Four plasma samples from blood donors with acute HIV-1 infection and one viral culture supernatant were serially diluted into 25-ml samples to nominal viral loads ranging from 39 to <0.5 copies (cp)/ml. Each dilution was tested with 45 replicates (reps) using 0.5 ml/rep with the Aptima HIV-1 Quant assay. The nominal and estimated viral loads based on the single-hit Poisson model were compared, and a hybrid Poisson digital model for calibrated viral load estimation was derived. Testing performed using 45 reps on longitudinal plasma samples from 50 ART-suppressed individuals in the Reservoir Assay Validation and Evaluation Network (RAVEN) study cohort (range of 1 to 19 years of continuous ART suppression) showed a median viral load of 0.54 cp/ml (interquartile range [IQR], 0.22 to 1.46 cp/ml) and a 14% (95% confidence interval [CI], 9% to 19%) decline in viral load for each additional year in duration suppressed. Within the RAVEN cohort, the expected false-negative rate for detection at lower rep numbers using 9 and 18 reps was 26% and 14%, respectively. Residual plasma viremia levels positively correlated with cell-associated HIV RNA and DNA. The performance characteristics of the replicate Aptima assay support its use for quantifying residual plasma viremia to study the latent HIV reservoir and cure interventions.
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http://dx.doi.org/10.1128/JCM.01400-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685884PMC
November 2020

Automated Multireplicate Quantification of Persistent HIV-1 Viremia in Individuals on Antiretroviral Therapy.

J Clin Microbiol 2020 11 18;58(12). Epub 2020 Nov 18.

Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

Clearance of low-level viremia that persists in most HIV-1-positive individuals on antiretroviral therapy (ART) is an important milestone for efforts to cure HIV-1 infection. The level of persistent viremia on ART is generally below the lower limit of quantification (LOQ) of current FDA-cleared plasma HIV-1 RNA assays (20 to 40 copies/ml) but can be quantified by reverse transcriptase PCR (RT-PCR) assays with single-copy sensitivity. Such assays require multistep manual methods, and their low throughput limits the capacity to monitor the effects of interventions on persistent viremia. Recently, S. Bakkour, X. Deng, P. Bacchetti, E. Grebe, et al. (J Clin Microbiol 58:e01400-20, 2020, https://doi.org/10.1128/JCM.01400-20), reported the use of multiple replicates and Poisson statistics to infer HIV-1 RNA concentrations below the commercial LOQ of an automated platform (Hologic Panther Aptima). Here, we evaluate the detection and quantitation of low-level viremia using the following two adaptions of the automated platform: a multireplicate strategy (9×) and a concentrated single-replicate strategy in which 5 ml of plasma is concentrated by centrifugation (1×, concentrated). We compare these new methods to a recently reported manual -targeting single-copy assay version 2 (iSCA v2). Using laboratory-generated HIV-1 RNA plasma samples at known concentrations, all three methods had similar sensitivity for HIV-1 RNA detection, although iSCA v2 was most sensitive (95% LOD, 2.3 copies/ml), 9× was marginally less sensitive (95% LOD, 3.0 copies/ml), and 1×, concentrated was least sensitive (95% LOD, 3.9 copies/ml). In contrast, for clinical plasma samples, 9× had greater sensitivity than iSCA v2 (82% of samples were quantifiable compared with 62% of samples by iSCA v2). These results support 9× as an acceptable high-throughput alternative to iSCA v2 for quantifying low-level viremia in individuals on ART.
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http://dx.doi.org/10.1128/JCM.01442-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685899PMC
November 2020

SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood.

Nat Commun 2020 09 17;11(1):4698. Epub 2020 Sep 17.

Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA.

Given the limited availability of serological testing to date, the seroprevalence of SARS-CoV-2-specific antibodies in different populations has remained unclear. Here, we report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seroreactivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors in early April 2020. We additionally describe the longitudinal dynamics of immunoglobulin-G (IgG), immunoglobulin-M (IgM), and in vitro neutralizing antibody titers in COVID-19 patients. The median time to seroconversion ranged from 10.3-11.0 days for these 3 assays. Neutralizing antibodies rose in tandem with immunoglobulin titers following symptom onset, and positive percent agreement between detection of IgG and neutralizing titers was >93%. These findings emphasize the importance of using highly accurate tests for surveillance studies in low-prevalence populations, and provide evidence that seroreactivity using SARS-CoV-2 anti-nucleocapsid protein IgG and anti-spike IgM assays are generally predictive of in vitro neutralizing capacity.
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http://dx.doi.org/10.1038/s41467-020-18468-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499171PMC
September 2020

Recently acquired infection among HIV-seropositive donors in the US from 2010-2018.

Transfusion 2020 Oct 28;60(10):2340-2347. Epub 2020 Aug 28.

Vitalant Research Institute, San Francisco, California, USA.

Background: Monitoring of transfusion-transmissible infections in the blood supply is essential for blood safety, as the donor population is not static, and changes in policy, donor behavior, or other factors could increase the risk of recipient infection. We assessed patterns of recently acquired HIV infection in US blood donors, including before and after the implementation of the 12-month deferral for men who have sex with men (MSM).

Study Design And Methods: A large convenience sample of donations from donors testing HIV-1 nucleic acid testing (NAT) and serology-reactive were further tested with the Sedia HIV-1 Limiting Antigen enzyme immunoassay. Samples were analyzed across available demographic and donation data to provide an assessment of recently acquired HIV infection in US blood donors from 2010 to 2018.

Results: Overall, 317 of 1154 (27.5%; 95% confidence interval, 24.9%-30.1%) donations from HIV NAT and serology-reactive donors had recently acquired HIV infection. There was no evidence of change in the percentages of recent HIV infection by year over the study period, either in all donors or in male donors, including after the MSM policy change. In multivariable logistic regression analysis, donors aged 24 years or younger were over 2.7 times more likely and repeat donors 2.2 times more likely to have recently acquired HIV infection compared to donors aged 55 years or older and first-time donors, respectively.

Conclusion: Patterns of recently acquired HIV infection varied by demographics but not over time. These findings suggest no impact of the MSM policy change on recently acquired HIV infection in US blood donors.
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http://dx.doi.org/10.1111/trf.16032DOI Listing
October 2020

Everolimus, an mTORC1/2 inhibitor, in ART-suppressed individuals who received solid organ transplantation: A prospective study.

Am J Transplant 2020 Aug 11. Epub 2020 Aug 11.

Department of Surgery, University of California San Francisco, San Francisco, California, USA.

Pharmacologic inhibition of the mammalian target of rapamycin (mTOR) in the setting of renal transplantation has previously been associated with lower human immunodeficiency virus 1 (HIV-1) DNA burden, and in vitro studies suggest that mTOR inhibition may lead to HIV transcriptional silencing. Because prospective clinical trials are lacking, we conducted an open-label, single-arm study to determine the impact of the broad mTOR inhibitor, everolimus, on residual HIV burden, transcriptional gene expression profiles, and immune responses in HIV-infected adult solid organ transplant (SOT) recipients on antiretroviral therapy. Whereas everolimus therapy did not have an overall effect on cell-associated HIV-1 DNA and RNA levels in the entire cohort, participants who maintained everolimus time-averaged trough levels >5 ng/mL during the first 2 months of therapy had significantly lower RNA levels up to 6 months after the cessation of study drug. Time-averaged everolimus trough levels significantly correlated with greater inhibition of mTOR gene pathway transcriptional activity. Everolimus treatment also led to decreased PD-1 expression on certain T cell subsets. These data support the rationale for further study of the effects of mTOR inhibition on HIV transcriptional silencing in non-SOT populations, either alone or in combination with other strategies. Trial Registration: ClinicalTrials.gov NCT02429869.
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http://dx.doi.org/10.1111/ajt.16244DOI Listing
August 2020

Proteome-Wide Zika Virus CD4 T Cell Epitope and HLA Restriction Determination.

Immunohorizons 2020 08 4;4(8):444-453. Epub 2020 Aug 4.

Department of Medicine, University of Washington, Seattle, WA 98195;

Zika virus (ZIKV) is a mosquito-borne pathogen that caused an epidemic in 2015-2016. ZIKV-specific T cell responses are functional in animal infection models, and helper CD4 T cells promote avid Abs in the vaccine context. The small volumes of blood available from field research limit the determination of T cell epitopes for complex microbes such as ZIKV. The goal of this project was efficient determination of human ZIKV CD4 T cell epitopes at the whole proteome scale, including validation of reactivity to whole pathogen, using small blood samples from convalescent time points when T cell response magnitude may have waned. Polyclonal enrichment of candidate ZIKV-specific CD4 T cells used cell-associated virus, documenting that T cells in downstream peptide analyses also recognize whole virus after Ag processing. Sequential query of bulk ZIKV-reactive CD4 T cells with pooled/single ZIKV peptides and molecularly defined APC allowed precision epitope and HLA restriction assignments across the ZIKV proteome and enabled discovery of numerous novel ZIKV CD4 T cell epitopes. The research workflow is useful for the study of emerging infectious diseases with a very limited human blood sample availability.
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http://dx.doi.org/10.4049/immunohorizons.2000068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839664PMC
August 2020

Sepsis from an apheresis platelet contaminated with Acinetobacter calcoaceticus/baumannii complex bacteria and Staphylococcus saprophyticus after pathogen reduction.

Transfusion 2020 Sep 1;60(9):1960-1969. Epub 2020 Aug 1.

Vitalant Research Institute, San Francisco, California, USA.

Background: Strategies to reduce platelet (PLT) bacterial contamination include donor screening, skin disinfection, sample diversion, bacterial culture, pathogen reduction (PR), and day-of-transfusion tests. We report bacterial sepsis following a pathogen-reduced PLT transfusion.

Case Report: An adult male with relapsed acute lymphoblastic leukemia was successfully treated for central catheter-associated Staphylococcus aureus bacteremia. A peripherally inserted central catheter (PICC) was placed. Chills, rigors, and flushing developed immediately after PICC-infused pathogen-reduced PLTs, progressing to septic shock requiring intensive care management.

Methods: PICC and peripheral blood (PB), transfused bag saline flushes (TBFs), environmental samples, and the pathogen-reduced untransfused co-component (CC) were cultured. Plasma metagenomic and bacterial isolate whole-genome sequencing; PLT mitochondrial DNA (mtDNA) testing of untransfused CC and TBF; CC testing for amotosalen (S-59)/S-59 photoproducts; isolate PR studies (INTERCEPT); and TBF polymerase chain reaction for recipient Y-chromosome DNA were performed.

Results: PB and PICC cultures grew Acinetobacter calcoaceticus/baumannii complex (ACBC). TBF was gram-positive; mass spectrometry identified ACBC and Staphylococcus saprophyticus (SS). CC Gram stain and cultures were negative. Environmental cultures, some done after decontamination, were ACBC/SS negative. Posttransfusion patient plasma and TBF ACBC sequences were genetically identical. No Y-chromosome signal was detected in TBF. S-59 photoproducts and evidence of mtDNA amplification inhibition were found in the CC. Spiking PR studies showed >5.9-log inactivation for both isolates. Donor skin cultures for Acinetobacter were negative.

Conclusion: CC sterility, PR studies, residual S-59 photoproducts, and mtDNA amplification inhibition suggest successful PR. Unidentified environmental sources and inherent or acquired bag defects may have contributed to postmanufacturing pathogen-reduced PLT contamination.
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http://dx.doi.org/10.1111/trf.15951DOI Listing
September 2020

HIV incidence in US first-time blood donors and transfusion risk with a 12-month deferral for men who have sex with men.

Blood 2020 09;136(11):1359-1367

Vitalant Research Institute, San Francisco, CA.

In 2015, the US Food and Drug Administration published revised guidance that recommended a change in blood donor deferral of men who have sex with men (MSM) from an indefinite to a 12-month deferral since the donor last had sex with a man. We assessed whether HIV incidence in first-time blood donors or associated transfusion risk increased. Donations in 4 major blood collection organizations were monitored for 15 months before and 2 years after implementation of the 12-month MSM deferral policy. HIV-positive donations were classified as recently acquired or long-term using a recent infection testing algorithm and incidence in both periods estimated. Residual transfusion transmission risk was estimated by multiplying incidence by the length of the infectious window period. The latter was estimated using a model based on infectious dose and the sensitivity of nucleic acid testing. Factors associated with incident infection in each period were assessed using Poisson regression. Overall HIV incidence in first-time donors before implementation of the 12-month MSM deferral was estimated at 2.62 cases per 100 000 person-years (105 PY) (95% credible interval [CI], 1.53-3.93 cases/105 PY), and after implementation at 2.85 cases/105 PY (95% CI, 1.96-3.93 cases/105 PY), with no statistically significant change. In male first-time donors, the incidence difference was 0.93 cases/105 PY (95% CI, -1.74-3.58 cases/105 PY). The residual risk of HIV transfusion transmission through components sourced from first-time donors was estimated at 0.32 transmissions per million (106) packed red blood cell transfusions (95% CI, 0.29-0.65 transmissions/106 transfusions) before and 0.35 transmissions/106 transfusions (95% CI, 0.31-0.65 transmissions/106 transfusions) after implementation. The difference was not statistically significant. Factors associated with incident infection were the same in each period. We observed no increase in HIV incidence or HIV transfusion transmission risk after implementation of a 12-month MSM deferral policy.
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http://dx.doi.org/10.1182/blood.2020007003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483431PMC
September 2020

SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques.

bioRxiv 2020 Jul 8. Epub 2020 Jul 8.

CD4 T follicular helper (T ) cells are important for the generation of long-lasting and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates T cells and stimulates the germinal center response is an important question as we investigate vaccine options for the current pandemic. Here we report that, following infection with SARS-CoV-2, adult rhesus macaques exhibited transient accumulation of activated, proliferating T cells in their peripheral blood on a transitory basis. The CD4 helper cell responses were skewed predominantly toward a T 1 response in blood, lung, and lymph nodes, reflective of the interferon-rich cytokine environment following infection. We also observed the generation of germinal center T cells specific for the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, and a corresponding early appearance of antiviral serum IgG antibodies but delayed or absent IgA antibodies. Our data suggest that a vaccine promoting Th1-type Tfh responses that target the S protein may lead to protective immunity.
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http://dx.doi.org/10.1101/2020.07.07.191007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359530PMC
July 2020

Evolving viral and serological stages of Zika virus RNA-positive blood donors and estimation of incidence of infection during the 2016 Puerto Rican Zika epidemic: an observational cohort study.

Lancet Infect Dis 2020 12 13;20(12):1437-1445. Epub 2020 Jul 13.

Vitalant Research Institute, San Francisco, CA, USA.

Background: Puerto Rico began screening blood donations for Zika virus RNA with nucleic acid amplification tests (NAATs) on April 3, 2016, because of an emerging Zika virus outbreak. We followed up positive donors to assess the dynamics of viral and serological markers during the early stages of Zika virus infection and update the estimate of infection incidence in the Puerto Rican population during the outbreak.

Methods: Blood donations from volunteer donors in Puerto Rico were screened for the presence of Zika virus RNA using the cobas Zika NAAT. Positive donations were further tested to confirm infection, estimate viral load, and identify Zika virus-specific IgM antibodies. Individuals with positive blood donations were invited to attend follow-up visits. Donations with confirmed infection (defined as detection of Zika virus RNA or IgM on additional testing of index or follow-up samples) were assessed for stage of infection according to Zika virus RNA detectability in simulated minipools, viral load, and Zika virus IgM status. A three-step process was used to estimate the mean duration of NAAT reactivity of Zika virus in human plasma from individuals identified pre-seroconversion with at least one follow up visit and to update the 2016 incidence estimate of Zika virus infection.

Findings: Between April 3 and Dec 31, 2016, 53 112 blood donations were screened for Zika virus, of which 351 tested positive, 339 had confirmed infections, and 319 could be staged. Compared with IgM-positive index donations (n=110), IgM-negative index donations (n=209) had higher mean viral loads (1·1 × 10vs 8·3 × 10 international units per mL) and were more likely to be detected in simulated minipools (93% [n=194] vs 26% [n=29]). The proportions of donations with confirmed infections that had viral RNA detected only in individual-donation NAATs (ie, not in simulated minipools) and were IgM positive increased as the epidemic evolved. The estimated mean duration of NAAT detectability in the 140 donors included in the follow-up study was 11·70 days (95% CI 10·06-14·36). Applying this detection period to the observed proportion of donations that were confirmed NAAT positive yielded a Zika virus seasonal incidence estimate of 21·1% (95% CI 18·1-24·1); 768 101 infections in a population of 3 638 773 in 2016.

Interpretation: Characterisation of early Zika virus infection has implications for blood safety because infectivity of blood donations and utility of screening methods likely correlate with viral load and serological stage of infection. Our findings also have implications for diagnostic testing, public health surveillance, and epidemiology, and we estimate that around 21% of the Puerto Rican population was infected during the 2016 outbreak.

Funding: Biomedical Advanced Research and Development Authority, National Heart, Lung, and Blood Institute.
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http://dx.doi.org/10.1016/S1473-3099(19)30706-6DOI Listing
December 2020

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

Lancet Infect Dis 2020 12 13;20(12):1446-1456. Epub 2020 Jul 13.

Vitalant Research Institute, San Francisco, CA, USA; Department of Laboratory Medicine, University of California, San Francisco, CA, USA.

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.
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http://dx.doi.org/10.1016/S1473-3099(19)30708-XDOI Listing
December 2020

HIV antiretroviral therapy and prevention use in US blood donors: a new blood safety concern.

Blood 2020 09;136(11):1351-1358

Vitalant Research Institute, San Francisco, CA.

Antiretroviral therapy (ART) to treat and pre-exposure prophylaxis (PrEP) to prevent HIV infection are effective tools to help end the HIV epidemic. However, their use could affect HIV transfusion-transmission risk. Three different ART/PrEP prevalence analyses in blood donors were conducted. First, blood samples from HIV-positive and a comparison group of infection-nonreactive donors were tested under blind using liquid chromatography-tandem mass spectrometry for ART. Second, blood donor samples from infection-nonreactive, 18- to 45-year-old, male, first-time blood donors in 6 US locations were tested for emtricitabine and tenofovir. Third, in men who have sex with men (MSM) participating in the 2017 Centers for Disease Control and Prevention National HIV Behavioral Surveillance (NHBS) from 5 US cities, self-reported PrEP use proximate to donation was assessed. In blind testing, no ART was detected in 300 infection-nonreactive donor samples, but in 299 HIV confirmed-infected donor samples, 46 (15.4%; 95% confidence interval [CI], 11.5% to 20.0%) had evidence of ART. Of the 1494 samples tested from first-time male donors, 9 (0.6%; 95% CI, 0.03% to 1.1%) had tenofovir and emtricitabine. In the NHBS MSM survey, 27 of 591 respondents (4.8%; 95% CI, 3.2% to 6.9%) reported donating blood in 2016 or 2017 and PrEP use within the same time frame as blood donation. Persons who are HIV positive and taking ART and persons taking PrEP to prevent HIV infection are donating blood. Both situations could lead to increased risk of HIV transfusion transmission if blood screening assays are unable to detect HIV in donations from infected donors.
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http://dx.doi.org/10.1182/blood.2020006890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483439PMC
September 2020