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BACKGROUND: Zika virus (ZIKV), a mosquito-borne flavivirus, causes asymptomatic infections in blood donors and can be transmitted by transfusion. During the 2016 US outbreak, universal individual-donation nucleic acid testing (ID-NAT) was used to screen the blood supply for ZIKV. Testing pooled samples from multiple donations with minipool (MP)-NAT is less sensitive than ID-NAT, which raised questions about its utility in ZIKV outbreaks. STUDY DESIGN AND METHODS: A mathematical model and computer simulation determined the risk of missing ID-NAT-reactive and immunoglobulin (Ig) M-negative donations in a ZIKV outbreak if MP-NAT is used initially instead of ID-NAT. The model calculated the time required for ZIKV RNA to replicate to a concentration detectable by testing donations individually or in pools of 6 (MP6) or 16 (MP16). A computer simulation then randomly selected infection times to determine the probability of detection by the candidate tests. RESULTS: The probability of detecting the first ID-NAT-reactive unit in an outbreak is 92% (2.5th-97.5th percentile, 79%-99%) by MP6 and 85% (2.5th-97.5th percentile, 67%-99%) by MP16. When one donation is detected by MP-NAT, the model predicts that the chance of having missed one or more ID-NAT-reactive donations is 8% to 15%. The probability of missing a unit by MP-NAT is constant over the course of the outbreak (8% by MP6, 15% by MP16). CONCLUSION: The model predicts that the probability that a candidate MP-NAT will detect the first ID-NAT-reactive unit in a ZIKV outbreak is 85% to 92% and remains constant over time.
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Donantes de Sangre , ARN Viral/sangre , Infección por el Virus Zika/sangre , Virus Zika/genética , Transfusión Sanguínea , Simulación por Computador , Humanos , Inmunoglobulina M/sangre , Modelos Teóricos , Puerto Rico/epidemiología , Estados Unidos/epidemiología , Infección por el Virus Zika/epidemiología , Infección por el Virus Zika/prevención & controlRESUMEN
BACKGROUND: The emergence of Zika virus (ZIKV) in 2015 to 2016 created a global public health crisis and an urgent need for accurate detection assays. Nucleic acid testing (NAT) is the most specific and sensitive technology for early detection of ZIKV. Various NAT protocols have been created, but until recently, assessment of assay performance and comparative studies were hampered by the lack of available standards and reference reagents. STUDY DESIGN AND METHODS: The Center for Biologics Evaluation and Research/Food and Drug Administration responded to this crisis with the generation of two ZIKV-RNA reference reagents (ZIKV-RRs) for use in the development, validation, and assessment of performance of ZIKV-NAT assays. These reagents were produced from heat-inactivated (HI) ZIKV culture supernatant stock from two strains (PRVABC59 and FSS13025) diluted in dialyzed, defibrinated human plasma and lyophilized for evaluation in collaborative studies. The liquid, HI stock had been shared with the Paul-Ehrlich-Institute (Germany) and were included in the collaborative validation studies for the World Health Organization International Standard for ZIKV (WHO ZIKV IS). RESULTS: NAT-detectable units (NDUs)/mL were determined in a collaborative study that led to the assignment of 5.77 log NDUs/mL for PRVABC59 and 5.54 log NDUs/mL for FSS13025 as the final concentrations of the FDA ZIKV-RRs. CONCLUSION: We have established well-characterized reference reagents for ZIKV to facilitate evaluation of existing NAT assays and development of novel ZIKV assays which are correlated to that of the First WHO ZIKV IS. Vials of the ZIKV-RRs are available to qualified organizations upon request.
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Brotes de Enfermedades , Técnicas de Amplificación de Ácido Nucleico/normas , ARN Viral/normas , Infección por el Virus Zika/epidemiología , Virus Zika/genética , Animales , Chlorocebus aethiops , Criopreservación , Liofilización , Humanos , Indicadores y Reactivos , Técnicas de Amplificación de Ácido Nucleico/métodos , Salud Pública , Estabilidad del ARN , ARN Viral/sangre , ARN Viral/aislamiento & purificación , Estándares de Referencia , Sensibilidad y Especificidad , Células Vero , Viremia/sangre , Viremia/diagnóstico , Inactivación de Virus , Organización Mundial de la Salud , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/sangre , Infección por el Virus Zika/diagnósticoRESUMEN
BACKGROUND: Dengue is the most important reemerging mosquito-borne viral disease worldwide. Caused by dengue virus (DENV), a member of the genus Flavivirus in the Flaviviridae family, dengue can be asymptomatic (approx. 80% of cases) or symptomatic, ranging from a flu-like illness known as dengue fever, to a life-threatening form called severe dengue. DENV is primarily transmitted from human to human through the bite of mosquitoes of the genus Aedes; however, it is also transmissible by transfusion of blood and blood components and by solid organ transplant. Nucleic acid test (NAT) assays are considered the most appropriate approach for blood donor screening for recent DENV infections, but there is no Food and Drug Administration-approved assay for the screening of blood for DENV. STUDY DESIGN AND METHODS: An international collaborative study was conducted to assess the suitability of reference reagent (RR) candidates for DENV Types 1 to 4 RNA for use in NAT-based assays. RESULTS: Two sets of RR candidates were prepared for each DENV type, one liquid frozen (Set 1) and one lyophilized (Set 2). A total of 28 laboratories from 20 countries agreed to participate in the study, of which 21 submitted the results for qualitative and/or quantitative assessments. CONCLUSION: The World Health Organization has established the lyophilized materials as international RRs for DENV RNA with a unitage of 13,500, 69,200, 23,400, and 33,900 units/mL for DENV-1 to -4, respectively.
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Seguridad de la Sangre/normas , Virus del Dengue/genética , ARN Viral/sangre , Análisis de Secuencia de ARN/normas , Donantes de Sangre , Liofilización , Congelación , Humanos , Indicadores y Reactivos/normas , Cooperación Internacional , ARN Viral/clasificación , ARN Viral/aislamiento & purificación , Análisis de Secuencia de ARN/métodos , Reacción a la Transfusión , Organización Mundial de la SaludRESUMEN
BACKGROUND: Blood donations are routinely screened for HIV to prevent an infectious unit from being released to the blood supply. Despite improvements to blood screening assays, donations from infected donors remain undetectable during the window period (WP), when the virus has not yet replicated above the lower limit of detection (LOD) of a screening assay. To aid in the quantitative risk assessments of WP donations, a dose-response model describing the probability of transfusion-transmission of HIV over a range of viral RNA copies was developed. METHODS: An exponential model was chosen based on data fit and parsimony. A data set from a HIV challenge study using a nonhuman primate model and another data set from reported human blood transfusions associated with HIV infected donors were separately fit to the model to generate parameter estimates. A Bayesian framework using No-U-Turn Sampling (NUTS) and Monte Carlo simulations was performed to generate posterior distributions quantifying uncertainty in parameter estimation and model predictions. RESULTS: The parameters of the exponential model for both nonhuman primate and human data were estimated with a mean (95% credible intervals) of 2.70 × 10 -2 (7.74 × 10 -3 , 6.06 × 10 -2 ) and 7.56 × 10 -4 (3.68 × 10 -4 , 1.31 × 10 -3 ), respectively. The predicted ID 50 for the animal and human models was 26 (12, 90) and 918 (529, 1886) RNA copies transfused, respectively. CONCLUSION: This dose-response model can be used in a quantitative framework to estimate the probability of transfusion-transmission of HIV through WP donations. These models can be especially informative when assessing risk from blood components with low viral load.
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Infecciones por VIH , Humanos , Animales , Infecciones por VIH/diagnóstico , Teorema de Bayes , Transfusión Sanguínea , Medición de Riesgo , Primates , Donantes de SangreRESUMEN
BACKGROUND: Natural Killer (NK) cells are a crucial component of the host innate immune system with anti-viral and anti-cancer properties. However, the role of NK cells in West Nile virus (WNV) infection is controversial, with reported effects ranging from active suppression of virus to no effect at all. It was previously shown that K562-mb15-41BBL (K562D2) cells, which express IL-15 and 4-1BBL on the K562 cell surface, were able to expand and activate human primary NK cells of normal peripheral blood mononuclear cells (PBMC). The expanded NK cells were tested for their ability to inhibit WNV infection in vitro. RESULTS: Co-culture of PBMC with irradiated K562D2 cells expanded the NK cell number by 2-3 logs in 2-3 weeks, with more than 90% purity; upregulated NK cell surface activation receptors; downregulated inhibitory receptors; and boosted interferon gamma (IFN-gamma) production by approximately 33 fold. The expanded NK (D2NK) cell has strong natural killing activity against both K562 and Vero cells, and killed the WNV infected Vero cells through antibody-dependent cellular cytotoxicity (ADCC). The D2NK cell culture supernatants inhibited both WNV replication and WNV induced cytopathic effect (CPE) in Vero cells when added before or after infection. Anti-IFN-gamma neutralizing antibody blocked the NK supernatant-mediated anti-WNV effect, demonstrating a noncytolytic activity mediated through IFN-gamma. CONCLUSIONS: Co-culture of PBMC with K562D2 stimulatory cells is an efficient technique to prepare large quantities of pure and active NK cells, and these expanded NK cells inhibited WNV infection of Vero cells through both cytolytic and noncytolytic activities, which may imply a potential role of NK cells in combating WNV infection.
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Interferón gamma/inmunología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/fisiología , Anticuerpos Bloqueadores/farmacología , Técnicas de Cultivo de Célula , Proliferación Celular , Técnicas de Cocultivo , Efecto Citopatogénico Viral/efectos de los fármacos , Efecto Citopatogénico Viral/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Interferón gamma/farmacología , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Receptores de Células Asesinas Naturales/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/inmunología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/patogenicidadRESUMEN
Zika virus (ZIKV) caused a public health threat in the United States in 2016, leading to rapid development and implementation of blood screening assays for ZIKV RNA. Several ZIKV sequences from clinical cases have been reported, but none from asymptomatic/pre-symptomatic infections. We isolated and sequenced ZIKV from asymptomatic/pre-symptomatic blood donor (ABD-ZIKV) samples and compared with reported clinical sequences. Twelve ABD-ZIKV isolates were produced from 67 cultivated samples, and isolates were genetically similar among themselves. Most isolates shared mutations with the clinical isolate PRVABC59 2015, whereas two ABD-ZIKV isolates shared specific mutations with U.S. clinical isolates from 2016. The ABD-ZIKV strains clustered into two distinct subclades: one comprised mostly ABD-ZIKV from Puerto Rico, and another one comprised ABD-ZIKV from Florida and QTX-02 isolate (Puerto Rico). In this study, we showed the circulation of two slightly distinct virus strains among Puerto Rico blood donors, one of which was also reported in Florida.
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Donantes de Sangre , Filogenia , Infección por el Virus Zika/sangre , Virus Zika/genética , Virus Zika/aislamiento & purificación , Adulto , Enfermedades Transmisibles Emergentes/sangre , Enfermedades Transmisibles Emergentes/epidemiología , Florida/epidemiología , Genómica , Humanos , Puerto Rico/epidemiología , Infección por el Virus Zika/diagnóstico , Infección por el Virus Zika/epidemiologíaRESUMEN
In 2015, Zika virus (ZIKV) appeared as an emerging pathogen, generating a global and urgent need for accurate diagnostic devices. During this public health crisis, several nucleic acid testing (NAT)-based Zika assays were submitted to the US Food and Drug Administration (FDA) for Emergency Use Authorization. The FDA's Center for Devices and Radiological Health, in collaboration with the FDA's Center for Biologics Evaluation and Research, responded to this Zika emergency by developing and producing a reference panel (RP) for Zika RNA (Zika FDA-RP) suitable for performance assessment of ZIKV NAT-based in vitro diagnostic devices. Reference panels are a fundamental tool for performance assessment of molecular tests. The panel is composed of five vials: two different heat-inactivated ZIKV strains (PRVABC59 and FSS13025) in concentrated stocks and three blinded concentrations prepared from those strains. The Zika FDA-RP was shared with developers who had devices in the final stages of validation. In vitro diagnostic developers tested the Zika FDA-RP using the FDA-provided protocol. Depending on sample type, 85% (12/14) of the NAT assays had analytical sensitivities between 500 and 5000 RNA NAT-detectable units/mL (NDUs/mL). One device showed better performance (100 NDUs/mL), and another one showed lower performance (10,000 to 30,000 NDUs/mL). Vials of the Zika FDA-RP are available on request to developers who have interacted with the FDA through the review process.
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ARN Viral/genética , Infección por el Virus Zika/diagnóstico , Virus Zika/genética , Humanos , Técnicas de Diagnóstico Molecular/normas , Salud Pública , Juego de Reactivos para Diagnóstico , Estándares de Referencia , Estados Unidos , United States Food and Drug Administration , Infección por el Virus Zika/virologíaRESUMEN
BACKGROUND: Female hormones are known to play an important role in predisposition for many infectious diseases. Recent work suggests there are gender effects in HIV/AIDS progression. Here we ask whether the sex steroid hormone beta-estradiol affects the replication of HIV-1 or the efficacy of a common anti-retroviral drug, Stavudine (D4T). RESULTS: Human PBL were infected with HIV-1 in the presence or absence of combinations of sex steroid hormones and the anti-retroviral drug, D4T. After seven days in culture, viral supernatants were assayed for HIV-1 p24 protein. beta-estradiol resulted in a modest inhibition of HIV-1 replication of approximately 26%. However, 2 nM beta-estradiol increased the amount of HIV-1 replication in the presence of 50 nM D4T from a baseline of 33% (+/- SE = 5.4) to 74% (+/- SE = 5.4) of control virus levels in the absence of drug. Both results were statistically highly significant (p < 0.001). beta-estradiol did not increase the replication of a D4T-resistant strain of HIV in the presence of D4T. The effects were unlikely to be due to general cell inhibition or toxicity because these concentrations of drug and hormone cause no cytotoxicity in PBL as measured by trypan blue exclusion. CONCLUSION: beta-estradiol inhibited both HIV-1 replication in primary human PBL and the antiretroviral efficacy of D4T in PBL cultures. To optimize antiretroviral drug therapy, it may be necessary to monitor patient hormonal status.
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Fármacos Anti-VIH/farmacología , Estradiol/farmacología , Hormonas Esteroides Gonadales/farmacología , VIH-1/efectos de los fármacos , Estavudina/farmacología , Células Cultivadas , Medios de Cultivo/química , Interacciones Farmacológicas , Femenino , Proteína p24 del Núcleo del VIH/biosíntesis , VIH-1/crecimiento & desarrollo , Humanos , Linfocitos/virología , Replicación Viral/efectos de los fármacosRESUMEN
We report here the complete genome sequences of two Zika virus strains (FSS13025 and PRVABC59) used for formulation of CBER/FDA RNA reference reagents and lot release panels for use with nucleic acid technology (NAT) testing.
RESUMEN
In this investigation, machine-enhanced techniques were applied to bring about scientific insights to identify a minimum set of phenotypic/functional memory-related biomarkers for post-vaccination follow-up upon yellow fever (YF) vaccination. For this purpose, memory status of circulating T-cells (Naïve/early-effector/Central-Memory/Effector-Memory) and B-cells (Naïve/non-Classical-Memory/Classical-Memory) along with the cytokine profile (IFN/TNF/IL-5/IL-10) were monitored before-NV(day0) and at distinct time-points after 17DD-YF primary vaccination-PV(day30-45); PV(year1-9) and PV(year10-11). A set of biomarkers (eEfCD4; EMCD4; CMCD19; EMCD8; IFNCD4; IL-5CD8; TNFCD4; IFNCD8; TNFCD8; IL-5CD19; IL-5CD4) were observed in PV(day30-45), but not in NV(day0), with most of them still observed in PV(year1-9). Deficiencies of phenotypic/functional biomarkers were observed in NV(day0), while total lack of memory-related attributes was observed in PV(year10-11), regardless of the age at primary vaccination. Venn-diagram analysis pre-selected 10 attributes (eEfCD4, EMCD4, CMCD19, EMCD8, IFNCD4, IL-5CD8, TNFCD4, IFNCD8, TNFCD8 and IL-5CD4), of which the overall mean presented moderate accuracy to discriminate PV(day30-45)&PV(year1-9) from NV(day0)&PV(year10-11). Multi-parameter approaches and decision-tree algorithms defined the EMCD8 and IL-5CD4 attributes as the top-two predictors with moderated performance. Together with the PRNT titers, the top-two biomarkers led to a resultant memory status observed in 80% and 51% of volunteers in PV(day30-45) and PV(year1-9), contrasting with 0% and 29% found in NV(day0) and PV(year10-11), respectively. The deficiency of memory-related attributes observed at PV(year10-11) underscores the conspicuous time-dependent decrease of resultant memory following17DD-YF primary vaccination that could be useful to monitor potential correlates of protection in areas under risk of YF transmission.
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Anticuerpos Antivirales/sangre , Biomarcadores/sangre , Vacunación , Vacuna contra la Fiebre Amarilla/inmunología , Fiebre Amarilla/prevención & control , Virus de la Fiebre Amarilla/inmunología , Adolescente , Adulto , Anciano , Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/sangre , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Fiebre Amarilla/inmunología , Fiebre Amarilla/virología , Adulto JovenRESUMEN
Background. West Nile virus (WNV) is endemic in the United States. It is transmissible by blood transfusion, and the nation's blood supply is currently screened for WNV. Documented transmission of WNV infection through red blood cell (RBC) units in which the plasma co-component had a low viral load could be explained, in at least 1 instance, by cell-association of WNV; in this case, the RBC unit was released as negative by minipool nucleic acid testing (NAT) performed on plasma but was intermittently NAT-positive when subsequently tested as an individual sample. We hypothesized that a proportion of WNV bound to blood cells and was not measured by NAT performed on plasma samples. We have investigated whether WNV binds to RBCs, leading to reduction of WNV RNA detection by NAT performed on plasma samples.Methods. Equal volumes of leukoreduced RBCs and their corresponding plasma components from 20 blood donors with NAT results that were positive for WNV were tested in 5 replicates by reverse-transcriptase polymerase chain reaction TaqMan for WNV. In addition, aliquots from 8 of the RBC units were tested by infectivity assays using Vero cells.Results. The reverse-transcriptase polymerase chain reaction TaqMan assay showed that the viral load in the RBC components exceeded that in the corresponding plasma units by 1 order of magnitude. In addition, viruses associated with the RBCs were infectious in Vero cell cultures.Conclusions. These observations reinforce the notion that extraction of viral RNA from whole blood could improve assay sensitivity for blood donor screening and further reduce the residual risk of WNV transmission through transfusion.
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Eritrocitos/fisiología , Técnicas de Amplificación de Ácido Nucleico , Virus del Nilo Occidental/aislamiento & purificación , Virus del Nilo Occidental/fisiología , Donantes de Sangre , Enfermedades Endémicas , Femenino , Humanos , Masculino , Tamizaje Masivo/métodos , ARN Viral , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Medición de Riesgo , Muestreo , Sensibilidad y Especificidad , Reacción a la Transfusión , Estados Unidos/epidemiología , Carga Viral , Fiebre del Nilo Occidental/epidemiología , Fiebre del Nilo Occidental/prevención & controlRESUMEN
West Nile virus (WNV) has become endemic in the Western Hemisphere since its first introduction in the United States in 1999. An important factor associated with annual reoccurrence of WNV outbreaks in the U.S. is viral adaptation to domestic mosquitoes and birds through accumulation of spontaneous mutations in the WNV genome. Newly emerged mutations in the viral genome can potentially negatively affect the performance of existing diagnostic and screening assays and future vaccines. Therefore, the genetic monitoring of the WNV viral population during annual outbreaks is extremely important for public health and can only be achieved by application of efficient sample preparation methods followed by high throughput genetic analysis. In this study, we developed and evaluated a method for specific isolation of WNV genomic RNA from plasma samples without cultivation of the virus in cells. In combination with the microarray-based genetic analysis of the isolated WNV genomic RNA, this approach is suitable for fast, high throughput genotyping of circulating WNV genetic variants. The methods were evaluated using WNV isolates from the 1999-2012U.S. epidemics.
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Variación Genética , Genoma Viral , Análisis por Micromatrices/métodos , ARN Viral/sangre , ARN Viral/aislamiento & purificación , Virus del Nilo Occidental/genética , Animales , Aves/virología , Culicidae/virología , Epidemias , Genotipo , Mutación , ARN Viral/genética , Estados Unidos/epidemiología , Fiebre del Nilo Occidental/epidemiología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/aislamiento & purificaciónRESUMEN
Molecular diagnostics are increasingly used in the blood bank industry. A device that can combine simultaneous detection of multiple targets with the flexibility of inclusion of emerging pathogens is desirable for testing blood products. A highly multiplexed blood-borne pathogen panel (BBPP) using dual-label probe chemistry (TaqMan assays) was developed for simultaneous detection and discrimination of 17 viral pathogens in human plasma samples and 13 bacterial and protozoan pathogens in human blood samples on the OpenArray platform. The custom BBPP OpenArray plate was tested for specificity and analytical sensitivity with purified nucleic acids from each pathogen and with pathogen-spiked human blood and plasma samples. The results of analytical validation of known samples yielded decision trees for identification of coded samples: pathogens spiked in human plasma or whole blood. Results from coded samples demonstrated no false positives among the plasma or whole blood specimens. Samples not detected were at the lower limit of the detectible range or qualified for retesting as indeterminate. Further demonstration of the performance of the BBPP OpenArray was achieved with clinical samples from a blood donor testing organization. Ninety-five percent of virus-positive samples were correctly identified. These results show that a high-throughput OpenArray PCR platform can be expanded and adapted for higher discrimination and newly emerging agents, enabling consideration for development as a next-generation device for testing blood products.
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Patógenos Transmitidos por la Sangre/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Animales , Bacterias/genética , Bacterias/aislamiento & purificación , Infecciones Bacterianas/sangre , Infecciones Bacterianas/microbiología , Humanos , Ácidos Nucleicos/genética , Ácidos Nucleicos/aislamiento & purificación , Infecciones por Protozoos/sangre , Infecciones por Protozoos/parasitología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virosis/sangre , Virosis/virología , Virus/genética , Virus/aislamiento & purificaciónRESUMEN
In this study, strategies for serum biomarker assessment were developed for therapeutic monitoring of HCV patients. For this purpose, serum chemokine/cytokine levels were measured by cytometric-bead-array in HCV patients, categorized according to immunotherapy response as: non-responder/NR, relapser/REL and sustained-virologic-responder/SVR. The results demonstrated an overall increase of serum chemokine/cytokine levels in HCV patients. In general, therapeutic failure was associated with presence of a predominant baseline proinflammatory pattern with enhanced CCL5/RANTES, IFN-α, IFN-γ along with decreased IL-10 levels in NR and increased IL-6 and TNF in REL. SVR displayed lower baseline proinflammatory status with decreased CXCL8/IL-8, IL-12 and IL-17 levels. The inability to uphold IFN-α levels during immunotherapy was characteristic of NR. Serum chemokine/cytokine signatures further support the deleterious effect of proinflammatory baseline status and the critical role of increased/persistent IFN-α levels to guarantee the sustained virologic response. The prominent baseline proinflammatory milieu observed in NR and REL yielded a restricted biomarker network with small number of neighborhood connections, whereas SVR displayed a network with integrated cytokine connectivity. Noteworthy was that SVR presented a shift towards a proinflammatory pattern upon immunotherapy, assuming a pattern similar to that observed in NR and REL at baseline. Moreover, the immunotherapy guided REL towards a profile similar to SVR at baseline. Analysis of baseline-fold changes during treatment pointed out IFN-α and TNF as high-performance biomarkers to monitor immunotherapy outcome. This knowledge may contribute for novel insights into the treatment and control of the continuous public health threat posed by HCV infection worldwide.
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Antivirales/uso terapéutico , Quimiocinas/sangre , Citocinas/sangre , Monitoreo de Drogas/métodos , Hepatitis C Crónica/terapia , Adulto , Anciano , Biomarcadores Farmacológicos/sangre , Femenino , Hepatitis C Crónica/sangre , Humanos , Inmunoterapia , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Interleucina-12/sangre , Interleucina-17/sangre , Interleucina-8/sangre , Masculino , Persona de Mediana Edad , Polietilenglicoles/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Carga Viral/efectos de los fármacos , Adulto JovenRESUMEN
Technological innovations in vaccinology have recently contributed to bring about novel insights for the vaccine-induced immune response. While the current protocols that use peripheral blood samples may provide abundant data, a range of distinct components of whole blood samples are required and the different anticoagulant systems employed may impair some properties of the biological sample and interfere with functional assays. Although the interference of heparin in functional assays for viral neutralizing antibodies such as the functional plaque-reduction neutralization test (PRNT), considered the gold-standard method to assess and monitor the protective immunity induced by the Yellow fever virus (YFV) vaccine, has been well characterized, the development of pre-analytical treatments is still required for the establishment of optimized protocols. The present study intended to optimize and evaluate the performance of pre-analytical treatment of heparin-collected blood samples with ecteola-cellulose (ECT) to provide accurate measurement of anti-YFV neutralizing antibodies, by PRNT. The study was designed in three steps, including: I. Problem statement; II. Pre-analytical steps; III. Analytical steps. Data confirmed the interference of heparin on PRNT reactivity in a dose-responsive fashion. Distinct sets of conditions for ECT pre-treatment were tested to optimize the heparin removal. The optimized protocol was pre-validated to determine the effectiveness of heparin plasma:ECT treatment to restore the PRNT titers as compared to serum samples. The validation and comparative performance was carried out by using a large range of serum vs heparin plasma:ECT 1:2 paired samples obtained from unvaccinated and 17DD-YFV primary vaccinated subjects. Altogether, the findings support the use of heparin plasma:ECT samples for accurate measurement of anti-YFV neutralizing antibodies.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Anticoagulantes/sangre , Recolección de Muestras de Sangre/métodos , Celulosa/análogos & derivados , Monitoreo de Drogas/métodos , Heparina/sangre , Pruebas de Neutralización , Vacunación , Vacuna contra la Fiebre Amarilla/administración & dosificación , Fiebre Amarilla/prevención & control , Virus de la Fiebre Amarilla/inmunología , Adolescente , Adulto , Anciano , Biomarcadores/sangre , Celulosa/química , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Ensayo de Placa Viral , Fiebre Amarilla/sangre , Fiebre Amarilla/diagnóstico , Fiebre Amarilla/virología , Adulto JovenRESUMEN
West Nile virus (WNV) is an arbovirus maintained in nature in a bird-mosquito enzootic cycle which can also infect other vertebrates including humans. WNV is now endemic in the United States (U.S.), causing yearly outbreaks that have resulted in an estimated total of 4-5 million human infections. Over 41,700 cases of West Nile disease, including 18,810 neuroinvasive cases and 1,765 deaths, were reported to the CDC between 1999 and 2014. In 2012, the second largest West Nile outbreak in the U.S. was reported, which caused 5,674 cases and 286 deaths. WNV continues to evolve, and three major WNV lineage I genotypes (NY99, WN02, and SW/WN03) have been described in the U.S. since introduction of the virus in 1999. We report here the WNV sequences obtained from 19 human samples acquired during the 2012 U.S. outbreak and our examination of the evolutionary dynamics in WNV isolates sequenced from 1999-2012. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses. Selection pressure analyses were performed with the HyPhy package using the Datamonkey web-server. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. Thirteen of the 19 completely sequenced isolates from 10 U.S. states were genetically similar, sharing up to 55 nucleotide mutations and 4 amino acid substitutions when compared with the prototype isolate WN-NY99. Overall, these analyses showed that following a brief contraction in 2008-2009, WNV genetic divergence in the U.S. continued to increase in 2012, and that closely related variants were found across a broad geographic range of the U.S., coincident with the second-largest WNV outbreak in U.S.
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Donantes de Sangre , Epidemias , Variación Genética , Fiebre del Nilo Occidental/epidemiología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/genética , Sustitución de Aminoácidos , Secuencia de Bases , Teorema de Bayes , Genotipo , Humanos , Mutación , Filogenia , Análisis de Secuencia de ADN , Estados Unidos/epidemiología , Virus del Nilo Occidental/clasificación , Virus del Nilo Occidental/aislamiento & purificaciónRESUMEN
BACKGROUND: Dengue is a mosquito-borne viral disease caused by the four dengue viruses (DENV-1 to 4) that can also be transmitted by blood transfusion and organ transplantation. The distribution of DENV in the components of blood from infected donors is poorly understood. METHODS: We used an in-house TaqMan qRT-PCR assay to test residual samples of plasma, cellular components of whole blood (CCWB), serum and clot specimens from the same collection from blood donors who were DENV-RNA-reactive in a parallel blood safety study. To assess whether DENV RNA detected by TaqMan was associated with infectious virus, DENV infectivity in available samples was determined by culture in mosquito cells. RESULTS: DENV RNA was detected by TaqMan in all tested blood components, albeit more consistently in the cellular components; 78.8% of CCWB, 73.3% of clots, 86.7% of sera and 41.8% of plasma samples. DENV-1 was detected in 48 plasma and 97 CCWB samples while DENV-4 was detected in 21 plasma and 31 CCWB samples. In mosquito cell cultures, 29/111 (26.1%) plasma and 32/97 (32.7%) CCWB samples were infectious. A subset of samples from 29 donors was separately analyzed to compare DENV viral loads in the available blood components. DENV viral loads did not differ significantly between components and ranged from 3-8 log10 PCR-detectable units/ml. CONCLUSIONS: DENV was present in all tested components from most donors, and viral RNA was not preferentially distributed in any of the tested components. Infectious DENV was also present in similar proportions in cultured plasma, clot and CCWB samples, indicating that these components may serve as a resource when sample sizes are limited. However, these results suggest that the sensitivity of the nucleic acid tests (NAT) for these viruses would not be improved by testing whole blood or components other than plasma.
Asunto(s)
Virus del Dengue/aislamiento & purificación , Dengue/virología , ARN Viral/sangre , Animales , Donantes de Sangre/estadística & datos numéricos , Culicidae/virología , Dengue/sangre , Virus del Dengue/clasificación , Virus del Dengue/genética , Humanos , Puerto Rico , ARN Viral/clasificación , ARN Viral/genéticaRESUMEN
Since its initial isolation in Uganda in 1937 through the present, West Nile virus (WNV) has become an important cause of human and animal disease worldwide. WNV, an enveloped virus of the genus Flavivirus, is naturally maintained in an enzootic cycle between birds and mosquitoes, with occasional epizootic spillover causing disease in humans and horses. The mosquito vectors for WNV are widely distributed worldwide, and the known geographic range of WNV transmission and disease has continued to increase over the past 77 years. While most human infections with WNV are asymptomatic, severe neurological disease may develop resulting in long-term sequelae or death. Surveillance and preventive measures are an ongoing need to reduce the public health impact of WNV in areas with the potential for transmission.
Asunto(s)
Culicidae/fisiología , Insectos Vectores/fisiología , Fiebre del Nilo Occidental/epidemiología , Fiebre del Nilo Occidental/transmisión , Virus del Nilo Occidental/fisiología , Animales , Culicidae/virología , Humanos , Insectos Vectores/virologíaRESUMEN
The use of nucleic acid tests for detection of pathogens has improved the safety of blood products. However, ongoing pathogen emergence demonstrates a need for development of devices testing for multiple pathogens simultaneously. One approach combines two proven technologies: Taqman chemistry for target identification and quantification and the OpenArray nanofluidic real-time PCR platform for spatial multiplexing of assays. A panel of Taqman assays was developed to detect nine blood-borne pathogens (BBPs): four viral, two bacterial, and three protozoan parasites. The custom BBP OpenArray plate with 18 assays was tested for specificity and analytical sensitivity for nucleic acid from each purified pathogen and with pathogen-spiked human blood and plasma samples. For most targets, the limits of detection (10 to 10,000 copies/mL) were comparable with existing real-time platforms. The testing of the BBP OpenArray with pathogen-spiked coded human plasma or blood samples and negative control specimens demonstrated no false-positive results among the samples tested and correctly identified pathogens with the lowest concentration detected ranging from 10 cells/mL (Trypanosoma cruzi) to 10,000 cells/mL (Escherichia coli). These results represent a proof of concept that indicated the BBP OpenArray platform in combination with Taqman chemistry may provide a multiplex real-time PCR pathogen detection method that points the way for a next-generation platform for infectious disease testing in blood.
Asunto(s)
Infecciones Bacterianas/sangre , Patógenos Transmitidos por la Sangre , Reacción en Cadena de la Polimerasa/métodos , Infecciones por Protozoos/sangre , Virosis/sangre , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/genética , ADN Bacteriano/análisis , ADN Protozoario/análisis , ADN Viral/análisis , Humanos , Límite de Detección , Técnicas de Diagnóstico Molecular/métodos , Infecciones por Protozoos/diagnóstico , Infecciones por Protozoos/genética , Polimerasa Taq , Virosis/diagnóstico , Virosis/genéticaRESUMEN
West Nile virus (WNV), an arbovirus maintained in a bird-mosquito enzootic cycle, can infect other vertebrates including humans. WNV was first reported in the US in 1999 where, to date, three genotypes belonging to WNV lineage I have been described (NY99, WN02, SW/WN03). We report here the WNV sequences obtained from two birds, one mosquito, and 29 selected human samples acquired during the US epidemics from 2006-2011 and our examination of the evolutionary dynamics in the open-reading frame of WNV isolates reported from 1999-2011. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses and selection pressure analyses were conducted with the HyPhy package. Phylogenetic analysis identified human WNV isolates within the main WNV genotypes that have circulated in the US. Within genotype SW/WN03, we have identified a cluster with strains derived from blood donors and birds from Idaho and North Dakota collected during 2006-2007, termed here MW/WN06. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. The mean nucleotide substitution rate for WNV isolates obtained from humans was calculated to be 5.06×10(-4) substitutions/site/year (s/s/y). The Bayesian skyline plot shows that after a period of high genetic variability following the introduction of WNV into the US, the WNV population appears to have reached genetic stability. The establishment of WNV in the US represents a unique opportunity to understand how an arbovirus adapts and evolves in a naïve environment. We describe a novel, well-supported cluster of WNV formed by strains collected from humans and birds from Idaho and North Dakota. Adequate genetic surveillance is essential to public health since new mutants could potentially affect viral pathogenesis, decrease performance of diagnostic assays, and negatively impact the efficacy of vaccines and the development of specific therapies.