Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 29
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
J Infect Dis ; 2020 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-32147687

RESUMO

BACKGROUND: Evaluations of HIV curative interventions require reliable and efficient quantification of replication-competent latent reservoirs (LR). The "classic" quantitative viral outgrowth assay (QVOA) has been regarded as "gold standard," although prohibitively resource- and labor-intensive. We compared six "next-gen" VOA employing PCR or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA. METHODS: Next-gen VOA were compared to classic QVOA using single leukapheresis-derived samples from five ART-suppressed HIV+ participants and one HIV- control; each lab tested blinded batches of three frozen and one fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and lab levels. Models also estimated the effect of testing frozen versus fresh samples. RESULTS: Next-gen VOA had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher IUPM than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation (95%CI 2.1,3.5) for next-gen assays. Between-lab variation increased extra-Poisson variation to 3.4-fold (95% CI 2.6,5.4). Frozen storage did not substantially alter IUPM (-18%(-52%,+39%)). CONCLUSIONS: The data offer cautious support for use of next-gen VOA as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of LR in eradication strategies would benefit from high throughput and scalable assays.

2.
Transfusion ; 60(2): 317-325, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31885102

RESUMO

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.

3.
J Infect Dis ; 2019 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-31677350

RESUMO

BACKGROUND: Identification of non-viral markers of HIV infection that increase prior to viral rebound during analytical treatment interruption (ATI) may impact HIV persistence research. We previously showed that HIV RNA is enriched in CD30+CD4+ T cells in many individuals. Here, we studied CD30+CD4+ T cell dynamics prior to ATI, during ATI (prior to detectable plasma RNA), and after HIV rebound. METHODS: PBMC from 23 participants collected longitudinally from five Adult AIDS Clinical Trials Group studies incorporating ATI were included in this study. Flow cytometric characterization of expression of CD30 and markers of T cell activation and exhaustion were performed along with HIV-1 RNA and DNA quantification and measurement of soluble plasma CD30 and CD30 ligand. FINDINGS: The percentage of CD4+ T cells expressing CD30 significantly increased from pre-ATI to post-interruption time points prior to detectible viremia [1.65 mean relative increase, P=0.005]. 77% of participants experienced an increase in CD30+ cells before viral rebound. In contrast, there were no significant differences between pre-ATI and post-interruption pre-rebound time-points in percentages of lymphocytes expressing CD69, CD38/HLA-DR, or PD-1 until after HIV recrudescence. CONCLUSIONS: CD30 may be a surrogate marker of early replication or viral transcriptional activity prior to detection by routine peripheral blood sampling.

5.
BMC Infect Dis ; 19(1): 815, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533639

RESUMO

BACKGROUND: Elite controllers (EC), a small subset of the HIV-positive population (< 1%), suppress HIV viremia below the limit of quantification of clinical viral load assays in the absence of antiretroviral therapy (ART). However, there is a paucity of longitudinal data detailing the viral and immune dynamics or HIV reservoir seeding during acute infection in individuals that go on to become Elite Controllers. CASE PRESENTATION: In this report, we describe a case of a 42 year old woman diagnosed during acute infection who rapidly and permanently suppressed her viremia in the absence of antiretroviral therapy (ART). Rapid antibody/antigen testing was either negative or equivocal during acute infection, despite subsequent viral load testing at that time point with 71,550 plasma HIV RNA copies/mL, making initial diagnosis challenging. The patient subsequently developed detectable anti-HIV antibodies and an increase in HIV-specific CD8+ T cell responses to overlapping subtype C HIV gag peptide; very low-level plasma viremia (0.84 RNA copies/mL) was detected by an ultrasensitive assay 2 years following infection. Subsequently, she was started on ART for multifocal furunculosis despite continued suppression of virus and stable CD4+ T cell counts. Following ART initiation, HIV specific antibody levels and CD8+ T cell responses increased, but no HIV DNA or RNA was able to be isolated from large numbers of peripheral blood CD4+ T cells. CONCLUSION: This case provides important information regarding the establishment of elite HIV control during acute infection and also demonstrates an increase in HIV-specific immune responses following ART despite undetectable peripheral blood cellular measures of HIV persistence. This case also highlights the challenges in diagnosing acute HIV infection without the use of viral load testing in this rare elite controller phenotype.


Assuntos
Infecções por HIV/tratamento farmacológico , HIV-1/isolamento & purificação , Adulto , Antirretrovirais/uso terapêutico , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/patologia , HIV-1/genética , Humanos , RNA Viral/sangue , Carga Viral
6.
Fetal Diagn Ther ; 46(3): 175-186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30661073

RESUMO

INTRODUCTION: Significant limitations with existing treatments for major haemoglobinopathies motivate the development of effective intrauterine therapy. We assessed the feasibility of fetoscopic and ultrasound-guided intrauterine haemopoietic cell transplantation (IUHCT) in macaque fetuses in early gestation when haemopoietic and immunological ontogeny is anticipated to enable long-term donor cell engraftment. MATERIAL AND METHODS: Fluorescent-labelled bone marrow-derived mononuclear cells from 10 pregnant Macaca fascicularis were injected into their fetuses at E71-114 (18.9-170.0E+6 cells/fetus) by fetoscopic intravenous (n = 7) or ultrasound (US)-guided intracardiac injections, with sacrifice at 24 h to examine donor-cell distribution. RESULTS: Operating times ranged from 35 to 118 min. Chorionic membrane tenting and intrachorionic haemorrhage were observed only with fetoscopy (n = 2). Labelled cells were stereoscopically visualised in lung, spleen, liver, and placenta. Donor-cell chimerism was highest in liver, spleen, and heart by flow cytometry, placenta by unique polymorphism qPCR, and was undetected in blood. Chimerism was 2-3 log-fold lower in individual organs by qPCR than by flow cytometry. DISCUSSION: Both fetoscopic and US-guided IUHCT were technically feasible, but fetoscopy caused more intraoperative complications in our pilot series. The discrepancy in chimerism detection predicts the challenges in long-term surveillance of donor-cell chimerism. Further studies of long-term outcomes in the non-human primate are valuable for the development of clinical protocols for IUHCT.


Assuntos
Terapias Fetais/métodos , Fetoscopia/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Modelos Animais , Ultrassonografia de Intervenção/métodos , Animais , Células da Medula Óssea , Macaca
7.
Transfusion ; 59(1): 57-66, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30566231

RESUMO

BACKGROUND: The major aims of the RBC-Omics study were to evaluate the genomic and metabolomic determinants of spontaneous and stress-induced hemolysis during RBC storage. This study was unique in scale and design to allow evaluation of RBC donations from a sufficient number of donors across the spectrum of race, ethnicity, sex, and donation intensity. Study procedures were carefully piloted, optimized, and controlled to enable high-quality data collection. METHODS: The enrollment goal of 14,000 RBC donors across four centers, with characterization of RBC hemolysis across two testing laboratories, required rigorous piloting and optimization and establishment of a quality assurance (QA) and quality control (QC) program. Optimization of WBC elution from leukoreduction (LR) filters, development and validation of small-volume transfer bags, impact of manufacturing and sample-handling procedures on hemolysis parameters, and testing consistency across laboratories and technicians and over time were part of this quality assurance/quality control program. RESULTS: LR filter elution procedures were optimized for obtaining DNA for analysis. Significant differences between standard and pediatric storage bags led to use of an alternative LR-RBC transfer bag. The impact of sample preparation and freezing methods on metabolomics analyses was evaluated. Proficiency testing monitored and documented testing consistency across laboratories and technicians. CONCLUSION: Piloting and optimization, and establishment of a robust quality assurance/quality control program documented process consistency throughout the study and was essential in executing this large-scale multicenter study. This program supports the validity of the RBC-Omics study results and a sample repository that can be used in future studies.


Assuntos
Preservação de Sangue/métodos , Hemólise/fisiologia , Trifosfato de Adenosina/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Humanos , Controle de Qualidade
9.
Transfusion ; 58(12): 2903-2910, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30264498

RESUMO

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.


Assuntos
Babesia microti/metabolismo , Babesiose/sangue , DNA de Protozoário/sangue , Eritrócitos/parasitologia , Parasitemia/sangue , RNA de Protozoário/sangue , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos NOD , Reação em Cadeia da Polimerase em Tempo Real
10.
J Clin Microbiol ; 56(8)2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29793968

RESUMO

Detection of acute HIV infection is critical for HIV public health and diagnostics. Clinical fourth-generation antigen (Ag)/antibody (Ab) combination (combo) and p24 Ag immunoassays have enhanced detection of acute infection compared to Ab-alone assays but require ongoing evaluation with currently circulating diverse subtypes. Genetically and geographically diverse HIV clinical isolates were used to assess clinical HIV diagnostic, blood screening, and next-generation assays. Three-hundred-member panels of 20 serially diluted well-characterized antibody-negative HIV isolates for which the researchers were blind to the results (blind panels) were distributed to manufacturers and end-user labs to assess the relative analytic sensitivity of currently approved and preapproved clinical HIV fourth-generation Ag/Ab combo or p24 Ag-alone immunoassays for the detection of diverse subtypes. The limits of detection (LODs) of virus were estimated for different subtypes relative to confirmed viral loads. Analysis of immunoassay sensitivity was benchmarked against confirmed viral load measurements on the blind panel. On the basis of the proportion of positive results on 300 observations, all Ag/Ab combo and standard sensitivity p24 Ag assays performed similarly and within half-log LODs, illustrating the similar breadth of reactivity and diagnostic utility. Ultrasensitive p24 Ag assays achieved dramatically increased sensitivities, while the rapid combo assays performed poorly. The similar performance of the different commercially available fourth-generation assays on diverse subtypes supports their use in broad geographic settings with locally circulating HIV clades and recombinant strains. Next-generation preclinical ultrasensitive p24 Ag assays achieved dramatically improved sensitivity, while rapid fourth-generation assays performed poorly for p24 Ag detection.


Assuntos
Sorodiagnóstico da AIDS/métodos , Sorodiagnóstico da AIDS/normas , Proteína do Núcleo p24 do HIV/sangue , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/diagnóstico , HIV/isolamento & purificação , Imunoensaio/normas , Carga Viral/normas , Benchmarking , HIV/imunologia , Anticorpos Anti-HIV/sangue , Antígenos HIV/sangue , Antígenos HIV/imunologia , Infecções por HIV/sangue , Humanos , Limite de Detecção , Sensibilidade e Especificidade
11.
Virus res ; 244: 71-74, Jan. 2018. tab
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IIERPROD, Sec. Est. Saúde SP | ID: biblio-1022533

RESUMO

INTRODUCTION: Several genetic polymorphisms may be related to susceptibility or resistance to viral disease outcomes. Immunological or genetic factors may act as major triggers of the immune pathogenesis of HAM/TSP. This study investigated the association of immune related genetic polymorphisms with viral and immunological markers. METHODS: 247 HTLV-1-infected volunteers, drawn from a larger group of HTLV-infected subjects followed at the Institute of Infectious Diseases "Emilio Ribas" (IIER) for up to 19 years, participated in this study, which ran from June 2011 to July 2016. The subjects were classified according to their neurological status into two groups: Group 1 (160 asymptomatic individuals) and Group 2 (87 HAM/TSP patients). Samples were tested for spontaneous lymphocyte proliferation (LPA) and HTLV-1 proviral load (PVL) and for IFN-λ4, HLA-C and KIR genotypes using qPCR. RESULTS: We found associations between LPA (p=0.0001) with HAM/TSP and confirmed the IFN-λ4 polymorphism rs8099917, allele GG, as a protective factor using a recessive model (OR=3.22, CI=1.10-9.47). Polymorphisms in HLA-C and KIR alleles were not associated with risk of developing HAM/TSP. CONCLUSION: We demonstrated that age, LPA and an IFN-λ4 polymorphism were associated with progression to HAM/TSP. Understanding HAM/TSP pathogenesis can provide important markers of prognostic value for clinical management, and contribute to the discovery of new therapeutic interventions in the future


Assuntos
Humanos , Vírus Linfotrópico T Tipo 1 Humano , Antígenos HLA
12.
Virus Res ; 244: 71-74, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29129607

RESUMO

INTRODUCTION: Several genetic polymorphisms may be related to susceptibility or resistance to viral disease outcomes. Immunological or genetic factors may act as major triggers of the immune pathogenesis of HAM/TSP. This study investigated the association of immune related genetic polymorphisms with viral and immunological markers. METHODS: 247 HTLV-1-infected volunteers, drawn from a larger group of HTLV-infected subjects followed at the Institute of Infectious Diseases "Emilio Ribas" (IIER) for up to 19 years, participated in this study, which ran from June 2011 to July 2016. The subjects were classified according to their neurological status into two groups: Group 1 (160 asymptomatic individuals) and Group 2 (87 HAM/TSP patients). Samples were tested for spontaneous lymphocyte proliferation (LPA) and HTLV-1 proviral load (PVL) and for IFN-λ4, HLA-C and KIR genotypes using qPCR. RESULTS: We found associations between LPA (p=0.0001) with HAM/TSP and confirmed the IFN-λ4 polymorphism rs8099917, allele GG, as a protective factor using a recessive model (OR=3.22, CI=1.10-9.47). Polymorphisms in HLA-C and KIR alleles were not associated with risk of developing HAM/TSP. CONCLUSION: We demonstrated that age, LPA and an IFN-λ4 polymorphism were associated with progression to HAM/TSP. Understanding HAM/TSP pathogenesis can provide important markers of prognostic value for clinical management, and contribute to the discovery of new therapeutic interventions in the future.


Assuntos
Antígenos HLA-C/genética , Infecções por HTLV-I/genética , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Interleucinas/genética , Paraparesia Espástica Tropical/genética , Receptores KIR/genética , Adulto , Idoso , Doenças Assintomáticas , Proliferação de Células , Progressão da Doença , Feminino , Expressão Gênica , Antígenos HLA-C/imunologia , Infecções por HTLV-I/diagnóstico , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/patologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Interleucinas/imunologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Paraparesia Espástica Tropical/diagnóstico , Paraparesia Espástica Tropical/imunologia , Paraparesia Espástica Tropical/patologia , Polimorfismo Genético , Prognóstico , Receptores KIR/imunologia , Índice de Gravidade de Doença , Linfócitos T/imunologia , Linfócitos T/virologia , Fatores de Tempo , Carga Viral
13.
Cell Med ; 9(3): 117-125, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28713641

RESUMO

The effects of sex on the degree of liver damage and human cell engraftment were investigated in immunodeficient urokinase-type plasminogen activator-transgenic (uPA-NOG) mice. Liver damage, measured by serum alanine transaminase (ALT) levels, was compared in male and female uPA-NOG mice of different ages. Male mice had significantly higher ALT levels than females with a median of 334 versus 158 U/L in transgenic homozygous mice, respectively. Mice were transplanted with human adult hepatocytes or fetal liver cells and analyzed for any correlation of engraftment of hepatocytes, liver sinusoidal endothelial cells (LSECs), and hematopoietic cells with the degree of liver damage. Hepatocyte engraftment was measured by human albumin levels in the mouse serum. Higher ALT levels correlated with higher hepatocyte engraftment, resulting in albumin levels in male mice that were 9.6 times higher than in females. LSEC and hematopoietic cell engraftment were measured by flow cytometric analysis of the mouse liver and bone marrow. LSEC and hematopoietic engraftment did not differ between male and female transplant recipients. Thus, the sex of uPA-NOG mice affects the degree of liver damage, which is reflected in the levels of human hepatocyte engraftment. However, the high levels of LSEC engraftment observed in uPA-NOG mice are not further improved among male mice, suggesting that a lower threshold of liver damage is sufficient to enhance endothelial cell engraftment. Previously described sex differences in human hematopoietic stem cell engraftment in immunodeficient mice were not observed in this model.

14.
Emerg Infect Dis ; 23(8): 1360-1363, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28514227

RESUMO

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.


Assuntos
Anticorpos Antivirais/sangue , Surtos de Doenças , Infecção por Zika virus/epidemiologia , Zika virus , Adolescente , Adulto , Feminino , Honduras/epidemiologia , Humanos , Masculino , Fatores de Tempo , População Urbana , Adulto Jovem , Zika virus/imunologia , Infecção por Zika virus/sangue
15.
Transfusion ; 57(5): 1199-1207, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28236306

RESUMO

BACKGROUND: Biochemical analyses of mechanisms triggered in platelets (PLTs) upon pathogen inactivation (PI) are crucial to further understand the impact of PI on PLT functionality and, subsequently, quality. STUDY DESIGN AND METHODS: PLT concentrates (PCs) were split into four small illumination bags: 1) untreated control, 2) treated with riboflavin and ultraviolet light (RF/UV), and spiked with 3) solvent control dimethyl sulfoxide and 4) p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 before RF/UV treatment. Flow cytometry was used to monitor PLT mitochondrial potential (ΔΨm ); generation of intracellular reactive oxygen species (ROS); and release of microvesicles (MVs), mitochondria (MT), and MVs containing MT (MVs/MT). Quantitative polymerase chain reaction (qPCR) was used to quantify extracellular mitochondrial DNA (mtDNA). Translocation of selected mitochondrial proteins was analyzed in subcellular fractions by immunoblot. RESULTS: RF/UV treatment triggered an increased mitochondrial translocation of both Bax and Bid (p < 0.05, Day 7) and cytochrome c release (p < 0.01, Day 7), loss of ΔΨm (p < 0.05, Day 5 and Day 7), and ROS generation (p < 0.01, Day 5 and Day 7) in PCs compared to the untreated control during storage. These PI-triggered changes were inhibited by SB203580 (p < 0.05). The release of MVs, MT, and MVs/MT was increased upon the RF/UV treatment during storage (p < 0.05) and, with the exception of MT, the release was decreased by the inhibitor (p < 0.05). qPCR analysis showed that RF/UV does not trigger mtDNA release during storage. CONCLUSION: These findings further our understanding of mechanisms in PLTs initiated by the RF/UV treatment, demonstrating that this treatment induces p38 MAPK-dependent mitochondrial signaling and MV release in apheresis PCs.


Assuntos
Plaquetas/enzimologia , Mitocôndrias/fisiologia , Plaquetoferese/métodos , Riboflavina/farmacologia , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Plaquetas/efeitos dos fármacos , Plaquetas/efeitos da radiação , Plaquetas/ultraestrutura , Micropartículas Derivadas de Células/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/efeitos da radiação , Espécies Reativas de Oxigênio
16.
Transfusion ; 57(3pt2): 734-747, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28194799

RESUMO

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.


Assuntos
Doadores de Sangue , Seleção do Doador/métodos , RNA Viral/sangue , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecção por Zika virus , Zika virus , Adulto , Feminino , Humanos , Masculino , Infecção por Zika virus/sangue , Infecção por Zika virus/diagnóstico
17.
Comput Biol Chem ; 68: 1-5, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28213308

RESUMO

Digital polymerase chain reaction (dPCR) is a refinement of the conventional PCR approach to nucleic acid detection and absolute quantification. Digital PCR works by partitioning a sample of DNA or cDNA into many individual, parallel PCR reactions. Current quantification methods rely on the assumption that the PCR reactions are always able to detect single target molecules. When the assumption does not hold, the copy numbers will be severely underestimated. We developed a novel dPCR quantification method which determines whether the single copy assumption is violated or not by simultaneously estimating the assay sensitivity and the copy numbers using serial dilution data sets. The implemented method is available as an R package "digitalPCR".


Assuntos
DNA/genética , Reação em Cadeia da Polimerase , Algoritmos
18.
Transfusion ; 57(3pt2): 762-769, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28164310

RESUMO

BACKGROUND: Zika virus (ZIKV) has spread in the Americas, including parts of the southern United States, and infection can be associated with serious complications, including congenital brain abnormalities. Probable transfusion transmission of ZIKV has been documented in Brazil. STUDY DESIGN AND METHODS: Preemptive testing of blood donations for ZIKV RNA was implemented in southern US states at risk of local transmission using a test approved under a Food and Drug Administration (FDA) investigational new drug application, cobas Zika. Screening was expanded after issuance of an updated FDA guidance. Donations reactive on initial screening were further tested by nucleic acid and antibody tests to determine the donor status. RESULTS: Of 358,786 donations from US states screened by individual donation testing, 23 were initially reactive on cobas Zika. Fourteen of these represented probable ZIKV infection based on reactivity on additional nucleic acid testing or anti-Zika immunoglobulin M. Ten of the 14 donors reported travel to an identified ZIKV-active area within 90 days before donation (median time from end of travel to donation, 25 days; range, 6-71 days). Three donors with travel history also had a potential sexual exposure. Only seven of the 14 donations with probable ZIKV infection were detectable upon 1:6 dilution to simulate minipool testing. The estimated specificity of the cobas Zika test was 99.997%. CONCLUSION: Screening of donations for ZIKV RNA can interdict ZIKV-infected donors. Donor risk factors include travel more than 4 weeks before donation and sexual exposure. Minipool screening would have detected only 50% of the RNA-positive donations.


Assuntos
Anticorpos Antivirais/sangue , Doadores de Sangue , Seleção do Doador , RNA Viral/sangue , Infecção por Zika virus/sangue , Zika virus , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estados Unidos
19.
Transfusion ; 57(3pt2): 770-778, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28229475

RESUMO

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.


Assuntos
Doadores de Sangue , RNA Viral/sangue , Infecção por Zika virus , Zika virus , Adulto , Região do Caribe/epidemiologia , América Central/epidemiologia , Feminino , Humanos , Masculino , Infecção por Zika virus/sangue , Infecção por Zika virus/etnologia , Infecção por Zika virus/transmissão
20.
PLoS One ; 12(1): e0171148, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28141843

RESUMO

Animal models of Zika virus (ZIKV) are needed to better understand tropism and pathogenesis and to test candidate vaccines and therapies to curtail the pandemic. Humans and rhesus macaques possess similar fetal development and placental biology that is not shared between humans and rodents. We inoculated 2 non-pregnant rhesus macaques with a 2015 Brazilian ZIKV strain. Consistent with most human infections, the animals experienced no clinical disease but developed short-lived plasma viremias that cleared as neutralizing antibody developed. In 1 animal, viral RNA (vRNA) could be detected longer in whole blood than in plasma. Despite no major histopathologic changes, many adult tissues contained vRNA 14 days post-infection with highest levels in hemolymphatic tissues. These observations warrant further studies to investigate ZIKV persistence and its potential clinical implications for transmission via blood products or tissue and organ transplants.


Assuntos
Infecção por Zika virus/sangue , Infecção por Zika virus/virologia , Zika virus/fisiologia , Doença Aguda , Envelhecimento/patologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Feminino , Macaca mulatta , Especificidade de Órgãos , RNA Viral/sangue , RNA Viral/urina , Saliva/virologia , Distribuição Tecidual , Viremia/sangue , Zika virus/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA