The geographical conditions of Colombia favor the re-emergence and propagation of infectious tropical diseases. Among them, the Dengue virus is highly endemic throughout the country, thereby locating this arbovirus as one of the major pathologies of the public health system. Therefore, there is a global challenge to generate novel strategies to predict and control dengue virus transmission. In particular, during the Colombian 2016 Dengue outbreak, more than 17 thousand Dengue cases were reported by the health authorities of Medellin, Antioquia. In this paper, we present a machine learning approach for the early detection of dengue outbreaks in the city of Medellin. We use an artificial neural network as the core of the machine learning algorithm, with environmental, meteorological and epidemiological data from the National Institute of Health -SIVIGILA- and the Aburra Valley Early Warning System SIATA. Our objective is to identify possible Dengue outbreaks, i.e. to create an early warning system, to provide a preventive timeline to the health authorities to design contingency plans and to mitigate the impact of dengue on the population of Medellin. Our results indicate that a artificial neural network forecasting for time series shows a trend for the correct prediction of dengue cases up to the first four weeks with a deterioration in precision as the forecast is pushed for additional ten to twenty weeks.

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.

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.

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.

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.

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.

Flavivirus and alphavirus affect the health of humans. In Venezuela and particularly in Aragua, the communities have been affected by some members of these genus, such as dengue (DENV), and Chikungunya (CHIKV). DENV circulating in Aragua since 1989 generating outbreaks of clinical importance, while CHIKV made its appearance in 2014. In Aragua state, the diagnosis had been directed toward the detection of DENV, however the situation with CHIKV generates the need to expand the diagnosis spectrum to other viral agents. In the LARDIDEV we adapted two methodology of RT-PCR's previously de scribed to detect members of these genus using flavivirus (DENV y Zika) and alphavirus (CHIKV) control strains. Both techniques were modified in the concentration of some reactants (MgCl2, dNTP's, and primers) in the second reaction of PCR. The other conditions are kept equal to those originally described. Both techniques allowed amplified up to 1 fg of RNA of viral strains (Zika and CHIKV virus), but it could to amplify smaller amounts. In all cases, sharp bands according to the expected sizes using previously identified strains of DENV, CHIKV and Zika virus were obtained. The implementation of these methodologies will strengthen the timely diagnosis of members of this genus within the system of epidemiological surveillance of viral diseases.

ABSTRACTWe 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.

Varios mecanismos inmuno-patogenicos se han propuesto para explicar el incremento masivo de la permeabilidad vascularobservada en las formas severas de la infeccion por el Virus del Dengue (DENV). El objetivo del estudio fue determinar loscambios cineticos de mediadores inflamatorios (IL-8, TNF-α), marcadores soluble de activacion linfocitica temprana (sIL-2R,sTNF-Rp75) y fracciones solubles de moleculas de adhesion celular (sICAM-1, sVCAM-1) como marcadores tempranos deseveridad en pacientes con dengue. Veinte pacientes clasificados como Dengue (Dengue±Signos de Alarma,D±WS) y treintapacientes con Dengue Severo (DS) fueron incluidos en el estudio. Suero de individuos aparentemente saludables fueronincluidos como controles. En comparacion con los individuos controles, los casos con Dengue mostraron niveles de IL-8 yTNF con diferencias no significativas en la fase febril o critica de la enfermedad; sin embargo, un incremento significativo desICAM-1 y sVCAM-1 ocurrio en ambas fases, mientras que los niveles de sIL2R y sTNF-p75 se elevaron significativamentesolo en la fase critica de la enfermedad. En comparacion con los casos con dengue y controles, los pacientes con DS mostraron diferencias significativas en los niveles de IL-8 y TNF-α durante la fase critica y un incremento significativo demoleculas de adhesion en ambas fases, pero los niveles mas elevados de sVCAM-1 y sIL-2R fueron observados en la fasefebril. En conclusion, sIL-2R y sVCAM-1, como marcadores tempranos de activacion linfocitica y endotelial, servirian comoindicadores de severidad en la fase aguda de la infeccion por el virus del dengue. Dinamic changes of pro-inflammatory cytokines, adhesion molecules and lymphocytesactivation markers as early indicators of diseases severity in patients with Dengue Abstract Several immunopathogenic mechanisms have been proposed to explain the massive increase of vascular permeabilityobserved in the severe forms of infection by Dengue Virus (DENV). Our aim was to determine the kinetic changes ofinflammatory mediators (IL-8, TNF- α), soluble early lymphocyte activation markers (sIL-2R, sTNF-Rp75) and solublefractions of cell adhesion molecules (sICAM-1 and sVCAM-1) as indicators for early recognition of disease severity inpatients with laboratory-confirmed dengue. Twenty patients classified as Dengue±Warning Signs (D±WS) and thirty patientswith Severe Dengue (SD) were included in the study. Serums of apparently healthy individuals were included as controls.Compared with normal subjects, D±WS cases did not show significant differences in the levels of IL-8 or TNF-α during theacute nor in the critical stages of the disease; however, in D±WS cases levels of sICAM-1 and sVCAM-1 were higher thancontrols during both phases; in contrast, significant increase of sTNF-p75 and sIL2R levels were observed during the criticalphase of the disease. Compared with both dengue patients and controls, patients with SD showed significant rise in thelevels of IL-8 and TNF-α during the critical phase of the disease and a significant increase in adhesion molecules weredetected in both phases, but the highest levels of sVCAM-1 and sIL-2R were observed only during the acute stage of thedisease. In conclusion, sIL-2R and sVCAM-1, as early markers of lymphocyte and endothelial activation, would serves asindicators of severity during the acute phase of dengue infection.

english) Several immunopathogenic mechanisms have been proposed to explain the massive increase of vascular permeability observed in the severe forms of infection by Dengue Virus (DENV). Our aim was to determine the kinetic changes of inflammatory mediators (IL-8, TNF- α), soluble early lymphocyte activation markers (sIL-2R, sTNF-Rp75) and soluble fractions of cell adhesion molecules (sICAM-1 and sVCAM-1) as indicators for early recognition of disease severity in patients with laboratory-confirmed dengue. Twenty patients classified as Dengue±Warning Signs (D±WS) and thirty patients with Severe Dengue (SD) were included in the study. Serums of apparently healthy individuals were included as controls. Compared with normal subjects, D±WS cases did not show significant differences in the levels of IL-8 or TNF-α during the acute nor in the critical stages of the disease; however, in D±WS cases levels of sICAM-1 and sVCAM-1 were higher than controls during both phases; in contrast, significant increase of sTNF-p75 and sIL2R levels were observed during the critical phase of the disease. Compared with both dengue patients and controls, patients with SD showed significant rise in the levels of IL-8 and TNF-α during the critical phase of the disease and a significant increase in adhesion molecules were detected in both phases, but the highest levels of sVCAM-1 and sIL-2R were observed only during the acute stage of the disease. In conclusion, sIL-2R and sVCAM-1, as early markers of lymphocyte and endothelial activation, would serves as indicators of severity during the acute phase of dengue infection.

Varios mecanismos inmuno-patogenicos se han propuesto para explicar el incremento masivo de la permeabilidad vascular observada en las formas severas de la infeccion por el Virus del Dengue (DENV). El objetivo del estudio fue determinar los cambios cineticos de mediadores inflamatorios (IL-8, TNF-α), marcadores soluble de activacion linfocitica temprana (sIL-2R, sTNF-Rp75) y fracciones solubles de moleculas de adhesion celular (sICAM-1, sVCAM-1) como marcadores tempranos de severidad en pacientes con dengue. Veinte pacientes clasificados como Dengue (Dengue±Signos de Alarma,D±WS) y treinta pacientes con Dengue Severo (DS) fueron incluidos en el estudio. Suero de individuos aparentemente saludables fueron incluidos como controles. En comparacion con los individuos controles, los casos con Dengue mostraron niveles de IL-8 y TNF con diferencias no significativas en la fase febril o critica de la enfermedad; sin embargo, un incremento significativo de sICAM-1 y sVCAM-1 ocurrio en ambas fases, mientras que los niveles de sIL2R y sTNF-p75 se elevaron significativamente solo en la fase critica de la enfermedad. En comparacion con los casos con dengue y controles, los pacientes con DS

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.

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.

SUMMARY Dengue is a mosquito-borne disease caused by four closely related dengue virus (genus Flavivirus) serotypes (DENV-1–4). The clinical outcomes vary from mild febrile illness to life-threatening haemorrhagic manifestations. DENVs are endemic in the tropics and subtropics globally and currently no specific treatment or vaccines are available. In Venezuela, the American-Asian genotype of DENV-2 is the most prevalent and has been associated with severe disease outcomes. We aimed to follow-up the molecular epidemiology of DENV-2 in Venezuela to investigate if the evolution of the virus has remained the same throughout time or if the same dynamics documented in Brazil (hyperendemic co-circulation) also occurred. The results show that whereas the epidemiology of DENV in several endemic areas is characterized by serotype replacements through time, in Venezuela the American-Asian genotype DENV-2 has evolved into several genetic lineages and has remained in hyperendemic co-circulation with the other serotypes.

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.