Desarrollo de un método de transcripción inversa seguida de reacción en cadena de la polimerasa para la detección del virus de la fiebre amarilla

Introduction. Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis.Objective. To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples.Materials and methods. RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction.Results. The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach.Conclusion. This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures. doi: http://dx.doi.org/10.7705/biomedica.v33i0.1452Introducción. La fiebre amarilla se considera una enfermedad reemergente y endémica en regiones tropicales de África y Suramérica. Actualmente, no existen estuches estandarizados o comerciales disponibles para la detección del virus de la fiebre amarilla y, por lo tanto, el diagnóstico debe hacerse mediante técnicas de rutina que consumen mucho tiempo y algunas veces no garantizan la detección del virus o de sus proteínas. Además, la cocirculación con otros flavivirus, incluyendo el del dengue, hacen el diagnóstico más complicado.Objetivo. Desarrollar un ensayo específico de amplificación basado en transcripción inversa seguida de reacción en cadena de la polimerasa, con el fin de mejorar la detección y el diagnóstico de la fiebre amarilla, tanto a partir de suero como de tejido fresco.Materiales y métodos. Se diseñaron iniciadores específicos para amplificar un fragmento conservado del virus de la fiebre amarilla. Un segundo par de iniciadores se usó en una reacción de amplificación anidada para incrementar la sensibilidad. Se probaron 33 muestras clínicas con la técnica estandarizada.Resultados. El amplímero esperado se obtuvo en 25 de las 33 muestras analizadas usando este método y 2 más resultaron positivas después de la reacción anidada.Conclusión. Esta técnica mejorada garantiza la detección de todos los genotipos virales de fiebre amarilla y puede incrementar la sensibilidad del ensayo introduciendo una segunda etapa de amplificación, lo cual permite el diagnóstico diferencial con infección por dengue y otros flavivirus, lo cual es de gran importancia para la vigilancia y la toma de medidas epidemiológicas oportunas.doi: http://dx.doi.org/10.7705/biomedica.v33i0.1452

Análisis filogenético del virus del chikungunya en Colombia: evidencia de selección purificadora en el gen E1

Introduction: Chikungunya virus (CHIKV) is a single-stranded positive sense RNA virus that belongs to the Alphavirus genus of the family Togaviridae. Its genome is 11.8 kb in length, and three genotypes have been identified worldwide: Asian, East/Central/South African (ECSA) and West African. Chikungunya fever is an acute febrile disease transmitted by Aedes spp. that usually presents with polyarthralgia and cutaneous eruption. Following introduction of the virus to the Americas in 2013, the first cases in Colombia occurred in September of 2014, and they reached a cumulative total of 399,932 cases by June of 2015. Objective: To identify the genotype or genotypes responsible for the current epidemic in Colombia and to describe the genetic variability of the virus in the country. Materials and methods: Serum samples from patients presenting with symptoms compatible with Chikungunya fever during 2014-2015 were selected for the study. RT-PCR products of the E1 gene from these samples were used for sequencing and subsequent phylogenetic and adaptive evolution analyses. Results: The study identified only the presence of the Asian genotype in Colombia. Comparing the Colombian sequences with other sequences from the Americas revealed an average of 0.001 base substitutions per site, with 99.7% and 99.9% nucleotide identity and 99.9% amino acid identity. The adaptive evolution analysis indicated that the E1 gene is under strong purifying selection. Conclusions: The first epidemic of Chikunguya fever in Colombia was caused by the circulation of the virus Asian genotype. Further genotypic surveillance of the virus in Colombia is required to detect possible changes in its epidemiology, fitness and pathogenicity.Introducción. El virus del chikungunya, perteneciente al género Alphavirus de la familia Togaviridae, es un virus ARN de 11,8 kb, de cadena sencilla y polaridad positiva, transmitido por Aedes spp. Se han identificado tres genotipos a nivel mundial: el de Asia, el del este-centro-sur de África (East/Central/South African, ECSA) y el de África occidental (West African, WA). La fiebre del chikungunya es una enfermedad febril aguda, acompañada principalmente de inflamación en las articulaciones y erupción cutánea. Después de su aparición en las Américas en el 2013, los primeros casos en Colombia ocurrieron en septiembre de 2014 y hasta junio del 2015 se habían notificado 399.932 casos.Objetivo. Identificar el genotipo o los genotipos responsables de la primera epidemia por el virus del chikungunya en Colombia y la variabilidad genética asociada a su dispersión en el territorio nacional.Materiales y métodos. Se seleccionaron muestras de suero de pacientes con síntomas indicativos de fiebre del chikungunya durante 2014 y 2015. Se hizo una transcripción inversa seguida de reacción en cadena de la polimerasa del gen E1, así como su secuenciación, análisis filogenético y análisis de evolución adaptativa.Resultados. Se demostró la presencia exclusiva del genotipo de Asia en Colombia. Se registró un promedio de 0,001 sustituciones de bases por sitio, una identidad de 99,7 a 99,9 % en los nucleótidos y de 99,9 % en los aminoácidos entre las secuencias colombianas y las secuencias de las Américas. Los análisis de evolución adaptativa indicaron una fuerte selección purificadora en el gen E1.Conclusiones. Se determinó la circulación del genotipo de Asia del virus del chikungunya como la causa de la primera epidemia en Colombia. Es necesario continuar con la vigilancia de genotipos, con el fin de detectar posibles cambios en la epidemiología, la eficacia (fitness) viral y la patogenia del virus

Genomic epidemiology supports multiple introductions and cryptic transmission of Zika virus in Colombia

BACKGROUND: Colombia was the second most affected country during the American Zika virus (ZIKV) epidemic, with over 109,000 reported cases. Despite the scale of the outbreak, limited genomic sequence data were available from Colombia. We sought to sequence additional samples and use genomic epidemiology to describe ZIKV dynamics in Colombia. METHODS: We sequenced ZIKV genomes directly from clinical diagnostic specimens and infected Aedes aegypti samples selected to cover the temporal and geographic breadth of the Colombian outbreak. We performed phylogeographic analysis of these genomes, along with other publicly-available ZIKV genomes from the Americas, to estimate the frequency and timing of ZIKV introductions to Colombia. RESULTS: We attempted PCR amplification on 184 samples; 19 samples amplified sufficiently to perform sequencing. Of these, 8 samples yielded sequences with at least 50% coverage. Our phylogeographic reconstruction indicates two separate introductions of ZIKV to Colombia, one of which was previously unrecognized. We find that ZIKV was first introduced to Colombia in February 2015 (95%CI: Jan 2015 - Apr 2015), corresponding to 5 to 8 months of cryptic ZIKV transmission prior to confirmation in September 2015. Despite the presence of multiple introductions, we find that the majority of Colombian ZIKV diversity descends from a single introduction. We find evidence for movement of ZIKV from Colombia into bordering countries, including Peru, Ecuador, Panama, and Venezuela. CONCLUSIONS: Similarly to genomic epidemiological studies of ZIKV dynamics in other countries, we find that ZIKV circulated cryptically in Colombia. More accurately dating when ZIKV was circulating refines our definition of the population at risk. Additionally, our finding that the majority of ZIKV transmission within Colombia was attributable to transmission between individuals, rather than repeated travel-related importations, indicates that improved detection and control might have succeeded in limiting the scale of the outbreak within Colombia

Role of coinfection by the different serotypes of the Dengue virus in patients died during the 2010-2011 epidemic in Colombia

ilustraciones, gráficas, mapas, tablasLa infección por virus del dengue es considerada una de las principales arbovirosis de interés en salud pública en todo el mundo. Colombia se considera endémico para la enfermedad, con alrededor de 50 años donde la circulación viral ha sido constante. En el país se informan al menos 15.000 casos nuevos anualmente. La circulación de los 4 serotipos, la variabilidad genética de las cepas, la alta infestación del vector, y las condiciones sociodemográficas han favorecido que en Colombia se presenten grandes brotes de la enfermedad con picos epidémicos aproximadamente cada dos a cuatro años. En el año 2010-2011 se documentó en el país una gran epidemia de la enfermedad donde 187.896 casos fueron notificados al sistema de vigilancia. Durante este periodo se detectó la circulación de los 4 serotipos del virus en la mayoría de las regiones y la severidad de la enfermedad fue estimada en alrededor de 5% con 534 muertes probables. En concordancia con los hallazgos de la literatura, las características de esta epidemia pudieron favorecer la ocurrencia de infecciones por más de un serotipo en el mismo individuo. La evidencia en varios países sugiere la controversia de este fenómeno respecto a la posibilidad de asociarse a formas más severas de la enfermedad. El presente trabajo decidió investigar: i) si estas infecciones por más de un serotipo en efecto se presentaron durante este periodo epidémico; ii) la frecuencia de estas infecciones; y iii) su posible asociación con las manifestaciones clínicas en casos fatales de la enfermedad. Un total de 160 mortalidades por dengue se confirmaron por detección viral, y entre ellas, tan solo 9 correspondieron a infecciones simultáneas por más de un serotipo. Estos 160 casos fueron seleccionados para investigar diferencias asociadas a severidad entre infecciones por un solo serotipo y coinfecciones, al comparar los cambios histopatológicos en 6 tejidos de interés asociados a la fisiopatología del dengue en humanos. Los resultados mostraron la tendencia a que, en las infecciones por un solo serotipo, se presentaran más alteraciones histopatológicas y en mayor frecuencia, comparado con las coinfecciones. Sin embargo, la baja frecuencia de estas últimas (5.6%) no permitió definir si estos hallazgos pudieran ser significativamente distintos. Este trabajo sugiere la necesidad de vigilar este fenómeno con mayor detalle durante lapsos epidemiológicos más prolongados con la inclusión de periodos endémicos y epidémicos en el país. (Texto tomado de la fuente).Dengue is considered one of the most important Arbovirus disease in public health around the world. Colombia is defined like an endemic country with constant viral circulation in the last 50 years, and 15.000 annual new cases at least. Four serotypes circulation, genetic variability, high mosquitoes infestation, and socio-demographic factors have lidered large dengue epidemics occurrence in the last years in Colombia. During 2010-2011, a dengue epidemic with 187.896 cases was reported, with the four serotypes circulation in several colombian regions, high disease severity, and 534 estimated fatal cases. According with other investigation findings, this colombian epidemic characteristics could lead to a simultaneal serotype infection in the same subject. The evidence obtanied in several countries suggests some controversies over this phenomenum about its relationship with dengue severity. his work researched if these multiple serotypes infection ocurred during the same epidemic period, the simultaneal infection frequency, and the possible association with severity manifestations in fatal cases. 160 dengue fatal cases were confirmed by viral detection, and among them only 9 simultaneal infection were detected. Those 160 cases were selected to investigate the severity differences between one serotype infections and coinfections, by comparing histopathologic changes using 6 tissues associated to human dengue physiopathology. Results showed that severity histopathologic findings were more frequent in single serotype infections, compared with coinfections. However, it should be noted that low coinfections frequency have not allowed statistics difference estimations. This work suggests that this surveillance must be executed in longer time lapses including endemic and epidemic periods in Colombia.Incluye anexosMaestríaMagíster en Infecciones y Salud en el TrópicoEs un estudio de casos empleando la metodología de análisis de casos, de fuente secundaria conformada por la información de las bases de datos de los casos fallecidos con resultado positivo por el Laboratorio de Arbovirus y los resultados de análisis histopatológicos realizados por el Grupo de Patología de la Dirección de Redes en Salud Pública del INS, en la cual se consignaron datos sociodemográficos, clínicos y paraclínicos de estos pacientes

Phylogenetic analysis of Chikungunya virus in Colombia: Evidence of purifying selection in the E1 gene

Introduction: Chikungunya virus (CHIKV) is a single-stranded positive sense RNA virus that belongs to the Alphavirus genus of the family Togaviridae. Its genome is 11.8 kb in length, and three genotypes have been identified worldwide: Asian, East/Central/South African (ECSA) and West African. Chikungunya fever is an acute febrile disease transmitted by Aedes spp. that usually presents with polyarthralgia and cutaneous eruption. Following introduction of the virus to the Americas in 2013, the first cases in Colombia occurred in September of 2014, and they reached a cumulative total of 399,932 cases by June of 2015. Objective: To identify the genotype or genotypes responsible for the current epidemic in Colombia and to describe the genetic variability of the virus in the country. Materials and methods: Serum samples from patients presenting with symptoms compatible with Chikungunya fever during 2014-2015 were selected for the study. RT-PCR products of the E1 gene from these samples were used for sequencing and subsequent phylogenetic and adaptive evolution analyses. Results: The study identified only the presence of the Asian genotype in Colombia. Comparing the Colombian sequences with other sequences from the Americas revealed an average of 0.001 base substitutions per site, with 99.7% and 99.9% nucleotide identity and 99.9% amino acid identity. The adaptive evolution analysis indicated that the E1 gene is under strong purifying selection. Conclusions: The first epidemic of Chikunguya fever in Colombia was caused by the circulation of the virus Asian genotype. Further genotypic surveillance of the virus in Colombia is required to detect possible changes in its epidemiology, fitness and pathogenicity

Clinical indicators of fatal dengue in two endemic areas of Colombia: a hospital-based case-control study

According to the World Health Organization, 98% of fatal dengue cases can be prevented; however, endemic countries such as Colombia have recorded higher case fatality rates during recent epidemics. We aimed to identify the predictors of mortality that allow risk stratification and timely intervention in patients with dengue. We conducted a hospital-based, case-control (1:2) study in two endemic areas of Colombia (2009-2015). Fatal cases were defined as having either 1) positive serological test (IgM or NS1), 2) positive virological test (RT-PCR or viral isolation), or 3) autopsy findings compatible with death from dengue. Controls (matched by state and year) were hospitalized nonfatal patients and had a positive serological or virological dengue test. Exposure data were extracted from medical records by trained staff. We used conditional logistic regression (adjusting for age, gender, disease's duration, and health-care provider) in the context of multiple imputation to estimate exposure to case-control associations.Weevaluated 110 cases and 217 controls (mean age: 35.0 versus 18.9; disease's duration pre-admission: 4.9 versus 5.0 days). In multivariable analysis, retro-ocular pain (odds ratios [OR] = 0.23), nausea (OR = 0.29), and diarrhea (OR = 0.19) were less prevalent among fatal than nonfatal cases, whereas increased age (OR = 2.46 per 10 years), respiratory distress (OR = 16.3), impaired consciousness (OR = 15.9), jaundice (OR = 32.2), and increased heart rate (OR = 2.01 per 10 beats per minute) increased the likelihood of death (AUC: 0.97, 95% confidence interval: 0.96, 0.99). These results provide evidence that features of severe dengue are associated with higher mortality, which strengthens the recommendations related to triaging patients in dengue-endemic areas

Genomic epidemiology supports multiple introductions and cryptic transmission of Zika virus in Colombia

BACKGROUND: Colombia was the second most affected country during the American Zika virus (ZIKV) epidemic, with over 109,000 reported cases. Despite the scale of the outbreak, limited genomic sequence data were available from Colombia. We sought to sequence additional samples and use genomic epidemiology to describe ZIKV dynamics in Colombia. METHODS: We sequenced ZIKV genomes directly from clinical diagnostic specimens and infected Aedes aegypti samples selected to cover the temporal and geographic breadth of the Colombian outbreak. We performed phylogeographic analysis of these genomes, along with other publicly-available ZIKV genomes from the Americas, to estimate the frequency and timing of ZIKV introductions to Colombia. RESULTS: We attempted PCR amplification on 184 samples; 19 samples amplified sufficiently to perform sequencing. Of these, 8 samples yielded sequences with at least 50% coverage. Our phylogeographic reconstruction indicates two separate introductions of ZIKV to Colombia, one of which was previously unrecognized. We find that ZIKV was first introduced to Colombia in February 2015 (95%CI: Jan 2015 - Apr 2015), corresponding to 5 to 8 months of cryptic ZIKV transmission prior to confirmation in September 2015. Despite the presence of multiple introductions, we find that the majority of Colombian ZIKV diversity descends from a single introduction. We find evidence for movement of ZIKV from Colombia into bordering countries, including Peru, Ecuador, Panama, and Venezuela. CONCLUSIONS: Similarly to genomic epidemiological studies of ZIKV dynamics in other countries, we find that ZIKV circulated cryptically in Colombia. More accurately dating when ZIKV was circulating refines our definition of the population at risk. Additionally, our finding that the majority of ZIKV transmission within Colombia was attributable to transmission between individuals, rather than repeated travel-related importations, indicates that improved detection and control might have succeeded in limiting the scale of the outbreak within Colombia

Severe Neurologic Disorders in 2 Fetuses with Zika Virus Infection, Colombia

We report the results of pathologic examinations of 2 fetuses from women in Colombia with Zika virus infection during pregnancy that revealed severe central nervous system defects and potential associated abnormalities of the eye, spleen, and placenta. Amniotic fluid and tissues from multiple fetal organs tested positive for Zika virus