Development and evaluation of a novel high-throughput image-based fluorescent neutralization test for detection of Zika virus infection.

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus flavivirus that comprises other important public health viruses, such as dengue (DENV) and yellow fever (YFV). In general, ZIKV infection is a self-limiting disease, however cases of Guillain-Barré syndrome and congenital brain abnormalities in newborn infants have been reported. Diagnosing ZIKV infection remains a challenge, as viral RNA detection is only applicable until a few days after the onset of symptoms. After that, serological tests must be applied, and, as expected, high cross-reactivity between ZIKV and other flavivirus serology is observed. Plaque reduction neutralization test (PRNT) is indicated to confirm positive samples for being more specific, however it is laborious intensive and time consuming, representing a major bottleneck for patient diagnosis. To overcome this limitation, we developed a high-throughput image-based fluorescent neutralization test for ZIKV infection by serological detection. Using 226 human specimens, we showed that the new test presented higher throughput than traditional PRNT, maintaining the correlation between results. Furthermore, when tested with dengue virus samples, it showed 50.53% less cross reactivity than MAC-ELISA. This fluorescent neutralization test could be used for clinical diagnosis confirmation of ZIKV infection, as well as for vaccine clinical trials and seroprevalence studies.

Human Antibody Responses to Emerging Mayaro Virus and Cocirculating Alphavirus Infections Examined by Using Structural Proteins from Nine New and Old World Lineages.

Mayaro virus (MAYV), Venezuelan equine encephalitis virus (VEEV), and chikungunya virus (CHIKV) are vector-borne alphaviruses that cocirculate in South America. Human infections by these viruses are frequently underdiagnosed or misdiagnosed, especially in areas with high dengue virus endemicity. Disease may progress to debilitating arthralgia (MAYV, CHIKV), encephalitis (VEEV), and death. Few standardized serological assays exist for specific human alphavirus infection detection, and antigen cross-reactivity can be problematic. Therefore, serological platforms that aid in the specific detection of multiple alphavirus infections will greatly expand disease surveillance for these emerging infections. In this study, serum samples from South American patients with PCR- and/or isolation-confirmed infections caused by MAYV, VEEV, and CHIKV were examined by using a protein microarray assembled with recombinant capsid, envelope protein 1 (E1), and E2 from nine New and Old World alphaviruses. Notably, specific antibody recognition of E1 was observed only with MAYV infections, whereas E2 was specifically targeted by antibodies from all of the alphavirus infections investigated, with evidence of cross-reactivity to E2 of o'nyong-nyong virus only in CHIKV-infected patient serum samples. Our findings suggest that alphavirus structural protein microarrays can distinguish infections caused by MAYV, VEEV, and CHIKV and that this multiplexed serological platform could be useful for high-throughput disease surveillance. IMPORTANCE Mayaro, chikungunya, and Venezuelan equine encephalitis viruses are closely related alphaviruses that are spread by mosquitos, causing diseases that produce similar influenza-like symptoms or more severe illnesses. Moreover, alphavirus infection symptoms can be similar to those of dengue or Zika disease, leading to underreporting of cases and potential misdiagnoses. New methods that can be used to detect antibody responses to multiple alphaviruses within the same assay would greatly aid disease surveillance efforts. However, possible antibody cross-reactivity between viruses can reduce the quality of laboratory results. Our results demonstrate that antibody responses to multiple alphaviruses can be specifically quantified within the same assay by using selected recombinant protein antigens and further show that Mayaro virus infections result in unique responses to viral envelope proteins.

Asian genotype of Chikungunya virus circulating in Venezuela during 2014.

Chikungunya virus emerged on Saint-Martin Island in the Caribbean in late 2013. Since then in July of 2104 Venezuela reported autochthonous cases. This study reports the first phylogenetic characterization of CHIKV autochthonous cases in Venezuela, 2014. The phylogenetic analysis showed that the CHIKV circulating in Venezuela (Aragua state) belong to the Asian genotype (Caribbean clade) and it is related to viruses that circulated in the same year in the Caribbean.

Spatial Analysis of Dengue Seroprevalence and Modeling of Transmission Risk Factors in a Dengue Hyperendemic City of Venezuela.

BACKGROUND: Dengue virus (DENV) transmission is spatially heterogeneous. Hence, to stratify dengue prevalence in space may be an efficacious strategy to target surveillance and control efforts in a cost-effective manner particularly in Venezuela where dengue is hyperendemic and public health resources are scarce. Here, we determine hot spots of dengue seroprevalence and the risk factors associated with these clusters using local spatial statistics and a regression modeling approach. METHODOLOGY/PRINCIPAL FINDINGS: From August 2010 to January 2011, a community-based cross-sectional study of 2012 individuals in 840 households was performed in high incidence neighborhoods of a dengue hyperendemic city in Venezuela. Local spatial statistics conducted at household- and block-level identified clusters of recent dengue seroprevalence (39 hot spot households and 9 hot spot blocks) in all neighborhoods. However, no clusters were found for past dengue seroprevalence. Clustering of infection was detected at a very small scale (20-110m) suggesting a high disease focal aggregation. Factors associated with living in a hot spot household were occupation (being a domestic worker/housewife (P = 0.002), lower socio-economic status (living in a shack (P<0.001), sharing a household with <7 people (P = 0.004), promoting potential vector breeding sites (storing water in containers (P = 0.024), having litter outdoors (P = 0.002) and mosquito preventive measures (such as using repellent, P = 0.011). Similarly, low socio-economic status (living in crowded conditions, P<0.001), having an occupation of domestic worker/housewife (P = 0.012) and not using certain preventive measures against mosquitoes (P<0.05) were directly associated with living in a hot spot block. CONCLUSIONS/SIGNIFICANCE: Our findings contribute to a better comprehension of the spatial dynamics of dengue by assessing the relationship between disease clusters and their risk factors. These results can inform health authorities in the design of surveillance and control activities. Focalizing dengue control measures during epidemic and inter-epidemic periods to disease high risk zones at household and neighborhood-level may significantly reduce virus transmission in comparison to random interventions.

[Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics]. / Clonación de secuencias de alfavirus y flavivirus para su uso como controles positivos en el diagnóstico molecular.

The purpose of the study was to obtain a positive control to validate molecular techniques (reverse transcription- polymerase chain reaction [RT-PCR]) used in the diagnosis and research of viral infections. From strains of Chikungunya virus (CHIKV), Zika virus, and Dengue virus (DENV-1, DENV-2, DENV- 3, and DENV-4) viral RNAs were extracted to obtain complementary DNA using RT-PCR from the nsP4 (CHIKV), NS5 (Zika virus), C/prM-M, and 5'UTR-C (DENV-1, DENV-2, DENV-3, DENV-4) sequences, which were cloned into pGEM®-T Easy. Cloning was confirmed through colony PCR, from which plasmid DNA was extracted for fragment cloning verification. Cloning of cDNA corresponding to nsP4, NS5, C/prM-M, and 5'UTR-C of the different viral agents was achieved. In conclusion, recombinant plasmids were obtained with each of the sequences specified for further assessment as positive controls in molecular techniques in an effort to avoid the use of cell cultures, which can be costly, time-consuming, and potentially dangerous.

Clonación de secuencias de alfavirus y flavivirus para su uso como controles positivos en el diagnóstico molecular / Cloning alphavirus and flavivirus sequences for use as positive controls in molecular diagnostics

RESUMEN El objetivo de la investigación fue obtener controles positivos para la validación de técnicas moleculares (RT-PCR) utilizadas en diagnóstico e investigación de infecciones virales. A partir de cepas de CHIKV, Zika, DENV-1, DENV-2, DENV-3 y DENV-4, se extrajeron ARN virales para obtener por RT-PCR los ADN complementarios (ADNc) de las secuencias nsP4 (CHIKV), NS5 (virus Zika), C/prM-M y 5´UTR-C (DENV-1, DENV-2, DENV-3, DENV-4) que fueron clonados en pGEM®-T Easy. La clonación se confirmó mediante PCR de colonias, de las cuales se extrajo el ADN plasmídico para la verificación de la clonación de los fragmentos. Se logró la clonación de ADNc correspondientes a nsP4, NS5, C/prM-M y 5´UTR-C de los distintos agentes virales. En conclusión se obtuvieron los plásmidos recombinantes con cada una de las secuencias especificadas para su posterior valoración como controles positivos en técnicas moleculares, evitando el uso de cultivos celulares que pueden resultar costosos, laboriosos y potencialmente peligrosos.

ABSTRACT The purpose of the study was to obtain a positive control to validate molecular techniques (reverse transcription- polymerase chain reaction [RT-PCR]) used in the diagnosis and research of viral infections. From strains of Chikungunya virus (CHIKV), Zika virus, and Dengue virus (DENV-1, DENV-2, DENV- 3, and DENV-4) viral RNAs were extracted to obtain complementary DNA using RT-PCR from the nsP4 (CHIKV), NS5 (Zika virus), C/prM-M, and 5′UTR-C (DENV-1, DENV-2, DENV-3, DENV-4) sequences, which were cloned into pGEM®-T Easy. Cloning was confirmed through colony PCR, from which plasmid DNA was extracted for fragment cloning verification. Cloning of cDNA corresponding to nsP4, NS5, C/prM-M, and 5′UTR-C of the different viral agents was achieved. In conclusion, recombinant plasmids were obtained with each of the sequences specified for further assessment as positive controls in molecular techniques in an effort to avoid the use of cell cultures, which can be costly, time-consuming, and potentially dangerous.

Multicountry prospective clinical evaluation of two enzyme-linked immunosorbent assays and two rapid diagnostic tests for diagnosing dengue fever.

We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 febrile individuals from Thailand were also used. Reference testing was performed on all samples using an algorithm involving virus isolation, in-house IgM and IgG capture ELISAs, and plaque reduction neutralization tests (PRNT) to determine the infection status of the individual. The primary endpoints were the clinical sensitivities and specificities of these devices. The SD Bioline Dengue Duo had an overall sensitivity of 87.3% (95% confidence interval [CI], 84.1 to 90.2%) and specificity of 86.8% (95% CI, 83.9 to 89.3%) during the first 14 days post-symptom onset (p.s.o.). The Panbio Dengue Duo Cassette demonstrated a sensitivity of 92.1% (87.8 to 95.2%) and specificity of 62.2% (54.5 to 69.5%) during days 4 to 14 p.s.o. The Panbio IgM capture ELISA had a sensitivity of 87.6% (82.7 to 91.4%) and specificity of 88.1% (82.2 to 92.6%) during days 4 to 14 p.s.o. Finally, the Panbio IgG capture ELISA had a sensitivity of 69.6% (62.1 to 76.4%) and a specificity of 88.4% (82.6 to 92.8%) during days 4 to 14 p.s.o. for identification of secondary dengue infections. This multicountry prospective study resulted in reliable real-world performance data that will facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks.

Dengue seroprevalence and risk factors for past and recent viral transmission in Venezuela: a comprehensive community-based study.

Dengue transmission in Venezuela has become perennial and a major public health problem. The increase in frequency and magnitude of recent epidemics prompted a comprehensive community-based cross-sectional study of 2,014 individuals in high-incidence neighborhoods of Maracay, Venezuela. We found a high seroprevalence (77.4%), with 10% of people experiencing recent infections. Multivariate logistic regression analysis showed that poverty-related socioeconomic factors (place and duration of residence, crowding, household size, and living in a shack) and factors/constraints related to intradomiciliary potential mosquito breeding sites (storing water and used tires) were linked with a greater risk of acquiring a dengue infection. Our results also suggest that transmission occurs mainly at home. The combination of increasingly crowded living conditions, growing population density, precarious homes, and water storage issues caused by enduring problems in public services in Maracay are the most likely factors that determine the permanent dengue transmission and the failure of vector control programs.

Human rhinoviruses and enteroviruses in influenza-like illness in Latin America.

BACKGROUND: Human rhinoviruses (HRVs) belong to the Picornaviridae family with high similarity to human enteroviruses (HEVs). Limited data is available from Latin America regarding the clinical presentation and strains of these viruses in respiratory disease. METHODS: We collected nasopharyngeal swabs at clinics located in eight Latin American countries from 3,375 subjects aged 25 years or younger who presented with influenza-like illness. RESULTS: Our subjects had a median age of 3 years and a 1.2:1.0 male:female ratio. HRV was identified in 16% and HEV was identified in 3%. HRVs accounted for a higher frequency of isolates in those of younger age, in particular children < 1 years old. HRV-C accounted for 38% of all HRVs detected. Phylogenetic analysis revealed a high proportion of recombinant strains between HRV-A/HRV-C and between HEV-A/HEV-B. In addition, both EV-D68 and EV-A71 were identified. CONCLUSIONS: In Latin America as in other regions, HRVs and HEVs account for a substantial proportion of respiratory viruses identified in young people with ILI, a finding that provides additional support for the development of pharmaceuticals and vaccines targeting these pathogens.

Sequential waves of gene expression in patients with clinically defined dengue illnesses reveal subtle disease phases and predict disease severity.

BACKGROUND: Dengue virus (DENV) infection can range in severity from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Changes in host gene expression, temporally through the progression of DENV infection, especially during the early days, remains poorly characterized. Early diagnostic markers for DHF are also lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated host gene expression in a cohort of DENV-infected subjects clinically diagnosed as DF (n = 51) and DHF (n = 13) from Maracay, Venezuela. Blood specimens were collected daily from these subjects from enrollment to early defervescence and at one convalescent time-point. Using convalescent expression levels as baseline, two distinct groups of genes were identified: the "early" group, which included genes associated with innate immunity, type I interferon, cytokine-mediated signaling, chemotaxis, and complement activity peaked at day 0-1 and declined on day 3-4; the second "late" group, comprised of genes associated with cell cycle, emerged from day 4 and peaked at day 5-6. The up-regulation of innate immune response genes coincided with the down-regulation of genes associated with viral replication during day 0-3. Furthermore, DHF patients had lower expression of genes associated with antigen processing and presentation, MHC class II receptor, NK and T cell activities, compared to that of DF patients. These results suggested that the innate and adaptive immunity during the early days of the disease are vital in suppressing DENV replication and in affecting outcome of disease severity. Gene signatures of DHF were identified as early as day 1. CONCLUSIONS/SIGNIFICANCE: Our study reveals a broad and dynamic picture of host responses in DENV infected subjects. Host response to DENV infection can now be understood as two distinct phases with unique transcriptional markers. The DHF signatures identified during day 1-3 may have applications in developing early molecular diagnostics for DHF.

Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus.

Early diagnosis of dengue virus (DENV) infection represents a key factor in preventing clinical complications attributed to the disease. The aim of this study was to evaluate the amplification efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, using the non-structural conserved genomic region protein-5 (NS5) versus two genomic regions usually employed for virus detection, the capsid/pre-membrane region (C-prM) and the 3'-noncoding region (3'NC). One-hundred sixty seven acute phase serum samples from febrile patients were used for validation purposes. Results showed that the three genomic regions had similar amplification profiles and correlation coefficients (0.987-0.999). When isolated viruses were used, the NS5 region had the highest qPCR efficiencies for the four serotypes (98-100%). Amplification from acute serum samples showed that 41.1% (67/167) were positive for the universal assay by at least two of the selected genomic regions. The agreement rates between NS5/C-prM and NS5/3'NC regions were 56.7% and 97%, respectively. Amplification concordance values between C-prM/NS5 and NS5/3'NC regions showed a weak (kappa = 0.109; CI 95%) and a moderate (kappa = 0.489; CI 95%) efficiencies in amplification, respectively. Serotyping assay using a singleplex NS5-TaqMan format was much more sensitive than the C-prM/SYBR Green I protocol (76%). External evaluation showed a high sensitivity (100%), specificity (78%) and high agreement between the assays. According to the results, the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples.

Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus / Análisis de las eficiencias de amplificación por PCR en tiempo real de tres regiones genómicas del virus dengue

Early diagnosis of dengue virus (DENV) infection represents a key factor in preventing clinical complications attributed to the disease. The aim of this study was to evaluate the amplification efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, using the non-structural conserved genomic region protein-5 (NS5) versus two genomic regions usually employed for virus detection, the capsid/pre-membrane region (C-prM) and the 3’-noncoding region (3’NC). One-hundred sixty seven acute phase serum samples from febrile patients were used for validation purposes. Results showed that the three genomic regions had similar amplification profiles and correlation coefficients (0.987-0.999). When isolated viruses were used, the NS5 region had the highest qPCR efficiencies for the four serotypes (98-100%). Amplification from acute serum samples showed that 41.1% (67/167) were positive for the universal assay by at least two of the selected genomic regions. The agreement rates between NS5/C-prM and NS5/3’NC regions were 56.7% and 97%, respectively. Amplification concordance values between C-prM/NS5 and NS5/3’NC regions showed a weak (k= 0.109; CI 95%) and a moderate (k= 0.489; CI 95%) efficiencies in amplification, respectively. Serotyping assay using a singleplex NS5-TaqMan® format was much more sensitive than the C-prM/SYBR Green® I protocol (76%). External evaluation showed a high sensitivity (100%), specificity (78%) and high agreement between the assays. According to the results, the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples.

El diagnóstico precoz de la infección por el virus dengue (DENV) constituye un elemento clave para la prevención de las complicaciones clínicas propias de la enfermedad. El objetivo del estudio fue evaluar la detección de DENV mediante un ensayo cuantitativo de PCR-tiempo real (qPCR), desarrollado localmente, utilizando la región no-estructural-5 (NS5), versus dos regiones tradicionalmente empleadas para la detección del virus, la región cápside/pre-membrana (C-prM), y la región noncodificante-3’ (3’NC). Se recolectaron 167 muestras de suero de pacientes en fase aguda de la enfermedad. Las tres regiones génicas tuvieron perfiles de amplificación/coeficientes de correlación similares (0,987-0,999). Sin embargo, la región NS5 tuvo la eficiencia de amplificación más elevada para los cuatro serotipos (98-100%). Durante el proceso de validación, 41,1% (67/167) de las muestras de suero resultaron positivas para DENV al menos por dos de las regiones genómicas empleadas. Los valores de concordancia entre las regiones NS5/C-prM y NS5/3’NC fueron de 56,7% y 97%, respectivamente. La concordancia fue débil entre las regiones NS5/C-prM (k= 0,109; CI 95%), sin embargo, fue moderada entre las regiones NS5/3’NC (k= 0,489; CI 95%). El ensayo de tipificación uniplex en formato NS5/TaqMan® mostró alta sensibilidad (100%) que el protocolo C-prM/SYBRGreen®-I (76%). La validación externa del ensayo mostró una alta sensibilidad (100%), especificidad (78%) y acuerdo alto entre los ensayos utilizados. De acuerdo a los resultados obtenidos, la región NS5 ofrece la mayor opción para la detección y serotipificación del DENV en muestras clínicas.

Sentinel surveillance of influenza-like illness in two hospitals in Maracay, Venezuela: 2006-2010.

BACKGROUND: Limited information exists on the epidemiology of acute febrile respiratory illnesses in tropical South American countries such as Venezuela. The objective of the present study was to examine the epidemiology of influenza-like illness (ILI) in two hospitals in Maracay, Venezuela. METHODOLOGY/PRINCIPAL FINDINGS: We performed a prospective surveillance study of persons with ILI who presented for care at two hospitals in Maracay, Venezuela, from October 2006 to December 2010. A respiratory specimen and clinical information were obtained from each participant. Viral isolation and identification with immunofluorescent antibodies and molecular methods were employed to detect respiratory viruses such as adenovirus, influenza A and B, parainfluenza, and respiratory sincytial virus, among others. There were 916 participants in the study (median age: 17 years; range: 1 month--86 years). Viruses were identified in 143 (15.6%) subjects, and one participant was found to have a co-infection with more than one virus. Influenza viruses, including pandemic H1N1 2009, were the most frequently detected pathogens, accounting for 67.4% (97/144) of the viruses detected. Adenovirus (15/144), parainfluenza virus (13/144), and respiratory syncytial virus (11/144) were also important causes of ILI in this study. Pandemic H1N1 2009 virus became the most commonly isolated influenza virus during its initial appearance in 2009. Two waves of the pandemic were observed: the first which peaked in August 2009 and the second--higher than the preceding - that peaked in October 2009. In 2010, influenza A/H3N2 re-emerged as the most predominant respiratory virus detected. CONCLUSIONS/SIGNIFICANCE: Influenza viruses were the most commonly detected viral organisms among patients with acute febrile respiratory illnesses presenting at two hospitals in Maracay, Venezuela. Pandemic H1N1 2009 influenza virus did not completely replace other circulating influenza viruses during its initial appearance in 2009. Seasonal influenza A/H3N2 was the most common influenza virus in the post-pandemic phase.

Epidemiología molecular de los virus Dengue / Molecular epidemiology of Dengue virus

El dengue es la enfermedad viral más importante transmitida por mosquitos a humanos por su alta morbimortalidad y el potencial de diseminación de su vector Aedes aegypti. Además, la falta de una vacuna y medicamentos antivirales específicos, así como el incremento progresivo de las infecciones secundarias y la hiperendemicidad en diferentes países, hacen de esta enfermedad un problema de salud pública. Existen cuatro serotipos del virus del dengue (DENV), dentro de cada serotipo se han descrito varios genotipos, constituidos a su vez por diferentes linajes o clados. La epidemiología molecular combina los análisis filogenéticos de los DENV detectados en un área geográfica, en un tiempo definido, con la información clínica y epidemiológica disponible. El objetivo de estos estudios es tratar de establecer asociaciones entre genotipos o linajes virales con el origen (ancestros), procedencia geográfica, ruta de transmisión viral, severidad de la enfermedad, grupos poblacionales afectados, y la intensidad y extensión de los brotes epidémicos. La epidemiología molecular ha generado información relevante como la etiología del DENV genotipo Asiático en los casos graves de dengue de la epidemia ocurrida en Venezuela en 1989, y la identificación de cambios nucleotídicos puntuales en el genoma viral asociados a propiedades biológicas fundamentales. En la actualidad se hace necesario realizar análisis exhaustivos del genoma viral completo, conjuntamente con el análisis bioinformático, biológico, clínico y epidemiológico de los cuatro serotipos circulantes en los países endémicos, así como instaurar en los laboratorios adscritos a los sistemas de vigilancia epidemiológica del dengue, la vigilancia molecular para la identificación de genotipos (o linajes) circulantes, lo que contribuiría entre otros aspectos al control efectivo de la enfermedad por DENV.

Dengue is the most important viral disease transmitted by mosquitoes to humans in tropical and subtropical regions of the world. This is the result of its high morbidity and mortality, the spread potential of the vector Aedes aegypti, the lack of effective vaccines and specific antiviral drugs, the gradual increase in secondary infections and hyperendemicity differences in distinct countries. There are four serotypes of dengue virus which are phylogenetically grouped in genotypes and subdivided in lineages or clades. Molecular epidemiology combines phylogenetic analysis of DENV detected in particular geographic areas within a defined time with the available clinical and epidemiologic information. The objective of these studies is to look for relationships between genotypes or lineages, viral origin, geographical spreading and routes of viral transmission, disease severity, population groups affected, and the intensity, speed and extent of outbreaks. Also, molecular epidemiology has generated relevant information such as the Asian genotype DENV etiology in cases of the severe dengue epidemic in Venezuela in 1989, and the identification of specific nucleotide changes in the viral genome associated with its fundamental biological properties. However, analysis of the complete viral genome, together with bioinformatic, biological, clinical and epidemiological analysis corresponding to the four serotypes circulating in endemic countries should be performed. Molecular surveillance for the identification of genotypes (or strains) circulating should be implemented in the laboratories responsible for the epidemiological surveillance of dengue, which would improve the effective control of DENV.

Discovery proteomics and nonparametric modeling pipeline in the development of a candidate biomarker panel for dengue hemorrhagic fever.

Secondary dengue viral infection can produce capillary leakage associated with increased mortality known as dengue hemorrhagic fever (DHF). Because the mortality of DHF can be reduced by early detection and intensive support, improved methods for its detection are needed. We applied multidimensional protein profiling to predict outcomes in a prospective dengue surveillance study in South America. Plasma samples taken from initial clinical presentation of acute dengue infection were subjected to proteomics analyses using ELISA and a recently developed biofluid analysis platform. Demographics, clinical laboratory measurements, nine cytokines, and 419 plasma proteins collected at the time of initial presentation were compared between the DF and DHF outcomes. Here, the subject's gender, clinical parameters, two cytokines, and 42 proteins discriminated between the outcomes. These factors were reduced by multivariate adaptive regression splines (MARS) that a highly accurate classification model based on eight discriminant features with an area under the receiver operator curve (AUC) of 0.999. Model analysis indicated that the feature-outcome relationship were nonlinear. Although this DHF risk model will need validation in a larger cohort, we conclude that approaches to develop predictive biomarker models for disease outcome will need to incorporate nonparametric modeling approaches.

A three-component biomarker panel for prediction of dengue hemorrhagic fever.

Dengue virus infections are a major cause of morbidity in tropical countries. Early detection of dengue hemorrhagic fever (DHF) may help identify individuals that would benefit from intensive therapy. Predictive modeling was performed using 11 laboratory values of 51 individuals (38 DF and 13 DHF) obtained on initial presentation using logistic regression. We produced a robust model with an area under the curve of 0.9615 that retained IL-10 levels, platelets, and lymphocytes as the major predictive features. A classification and regression tree was developed on these features that were 86% accurate on cross-validation. The IL-10 levels and platelet counts were also identified as the most informative features associated with DHF using a Random Forest classifier. In the presence of polymerase chain reaction-proven acute dengue infections, we suggest a complete blood count and rapid measurement of IL-10 can assist in the triage of potential DHF cases for close follow-up or clinical intervention improving clinical outcome.

Influencia de las variables climáticas en la casuística de dengue y la abundancia de Aedes aegypti (Diptera: Culicidae) en Maracay, Venezuela / Influence of climatic variables on dengue cases and abundance of Aedes aegypti (Diptera: Culicidae) in Maracay, Venezuela

Se realizó un estudio retrospectivo y longitudinal en el área metropolitana de Maracay para el período 1997-2005, a fin de determinar si las variables climáticas y entomológicas influyen sobre la casuística de dengue. Se obtuvieron los datos climatológicos diarios y mensuales de la Estación Climatológica de la Universidad Central de Venezuela y los datos epidemiológicos basados en casos confirmados y muy sugestivos reportados por el Laboratorio Regional de Diagnóstico e Investigación del Dengue y otras Enfermedades Virales (LARDIDEV). Los datos entomológicos se obtuvieron de colectas de adultos de Aedes aegypti (L.) realizadas durante el período Noviembre 2000-Diciembre 2001. La abundancia del vector varió de 3,6 Aedes/casa durante el mes de Abril (período de sequía) a 14,7 Aedes/casa durante el mes de Agosto (período de lluvia). El análisis de correlación de Pearson mostró correlación positiva entre el número de casos reportados con la precipitación (r= 0,7183, P = 0,0038) y la abundancia de Ae. aegypti (r= 0,677, P = 0,0078), pero no con la temperatura ni la humedad relativa para el período Noviembre 2000-Diciembre 2001. El análisis de regresión mostró que para dos meses de rezago existe una regresión lineal altamente significativa (P < 0,0001) con un ajuste de R2= 88,7%, indicando que en promedio, la mayor casuística de dengue ocurre dos meses después de ocurrido el pico de precipitación. Los resultados obtenidos podrían ser utilizados para diseñar e implementar programas para la vigilancia epidemiológica y entomológica del dengue, así como establecer un sistema de alerta temprana para la prevención de brotes y/o epidemias de la enfermedad.

In order to determine the influence of climatic and entomological variables on the number of dengue cases, a retrospective and longitudinal study was conducted in the metropolitan area of Maracay during the period 1997-2005. Daily and monthly climatological data was obtained from the Climatological Station at the Universidad Central de Venezuela and epidemiological data based on confirmed and highly suggestive cases reported by the Regional Laboratory for Diagnosis and Research on Dengue and other Viral Infections (LARDIDEV). Aedes aegypti (L.) adults were collected monthly between November 2000 and December 2001. Vector abundance varied from 3,6 Aedes/house during April (dry season) to 14,7 Aedes/house (rainy season). Pearson correlation analysis showed a positive correlation between the number of dengue cases and rainfall (r= 0,7182, P =0,0038) and abundance of Ae. aegypti (r= 0,667, P =0,0078), but no correlation was found with temperature or relative humidity. Regression analysis showed that there was a highly significant relation (R2 = 88,7%, P< 0,0001) between rainfall and dengue cases at a lag of 2 months. The present results might be used to design and implement programs for the epidemiological and entomological dengue surveillance as well as to establish an early warning system to prevent epidemics.

Circulating strains of human respiratory syncytial virus in central and south America.

Human respiratory syncytial virus (HRSV) is a major cause of viral lower respiratory tract infections among infants and young children. HRSV strains vary genetically and antigenically and have been classified into two broad subgroups, A and B (HRSV-A and HRSV-B, respectively). To date, little is known about the circulating strains of HRSV in Latin America. We have evaluated the genetic diversity of 96 HRSV strains by sequencing a variable region of the G protein gene of isolates collected from 2007 to 2009 in Central and South America. Our results show the presence of the two antigenic subgroups of HRSV during this period with the majority belonging to the genotype HRSV-A2.

Evolution of dengue virus type 3 genotype III in Venezuela: diversification, rates and population dynamics.

BACKGROUND: Dengue virus (DENV) is a member of the genus Flavivirus of the family Flaviviridae. DENV are comprised of four distinct serotypes (DENV-1 through DENV-4) and each serotype can be divided in different genotypes. Currently, there is a dramatic emergence of DENV-3 genotype III in Latin America. Nevertheless, we still have an incomplete understanding of the evolutionary forces underlying the evolution of this genotype in this region of the world. In order to gain insight into the degree of genetic variability, rates and patterns of evolution of this genotype in Venezuela and the South American region, phylogenetic analysis, based on a large number (n = 119) of envelope gene sequences from DENV-3 genotype III strains isolated in Venezuela from 2001 to 2008, were performed. RESULTS: Phylogenetic analysis revealed an in situ evolution of DENV-3 genotype III following its introduction in the Latin American region, where three different genetic clusters (A to C) can be observed among the DENV-3 genotype III strains circulating in this region. Bayesian coalescent inference analyses revealed an evolutionary rate of 8.48 x 10⁻4 substitutions/site/year (s/s/y) for strains of cluster A, composed entirely of strains isolated in Venezuela. Amino acid substitution at position 329 of domain III of the E protein (A→V) was found in almost all E proteins from Cluster A strains. CONCLUSIONS: A significant evolutionary change between DENV-3 genotype III strains that circulated in the initial years of the introduction in the continent and strains isolated in the Latin American region in recent years was observed. The presence of DENV-3 genotype III strains belonging to different clusters was observed in Venezuela, revealing several introduction events into this country. The evolutionary rate found for Cluster A strains circulating in Venezuela is similar to the others previously established for this genotype in other regions of the world. This suggests a lack of correlation among DENV genotype III substitution rate and ecological pattern of virus spread.

Genetic analysis of influenza A/H1N1 of swine origin virus (SOIV) circulating in Central and South America.

Since the first detection of swine origin virus (SOIV) on March 28, 2009, the virus has spread worldwide and oseltamivir-resistant strains have already been identified in the past months. Here, we show the phylogenetic analysis of 63 SOIV isolates from eight countries in Central and South America, and their sensitivity to oseltamivir.