Development of in-house serological methods for diagnosis and surveillance of chikungunya / Desarrollo de métodos serológicos propios para el diagnóstico y la vigilancia del chikungunya

ABSTRACT Objective To develop and evaluate serological methods for chikungunya diagnosis and research in Nicaragua. Methods Two IgM ELISA capture systems (MAC-ELISA) for diagnosis of acute chikungunya virus (CHIKV) infections, and two Inhibition ELISA Methods (IEM) to measure total antibodies against CHIKV were developed using monoclonal antibodies (mAbs) and hyperimmune serum at the National Virology Laboratory of Nicaragua in 2014-2015. The sensitivity, specificity, predictive values, and agreement of the MAC-ELISAs were obtained by comparing the results of 198 samples (116 positive; 82 negative) with the Centers for Disease Control and Prevention's IgM ELISA (Atlanta, Georgia, United States; CDC-MAC-ELISA). For clinical evaluation of the four serological techniques, 260 paired acute and convalescent phase serum samples of suspected chikungunya cases were used. Results All four assays were standardized by determining the optimal concentrations of the different reagents. Processing times were substantially reduced compared to the CDC-MAC-ELISA. For the MAC-ELISA systems, a sensitivity of 96.6% and 97.4%, and a specificity of 98.8% and 91.5% were obtained using mAb and hyperimmune serum, respectively, compared with the CDC method. Clinical evaluation of the four serological techniques versus the CDC real-time RT-PCR assay resulted in a sensitivity of 95.7% and a specificity of 88.8%-95.9%. Conclusion Two MAC-ELISA and two IEM systems were standardized, demonstrating very good quality for chikungunya diagnosis and research demands. This will achieve more efficient epidemiological surveillance in Nicaragua, the first country in Central America to produce its own reagents for serological diagnosis of CHIKV. The methods evaluated here can be applied in other countries and will contribute to sustainable diagnostic systems to combat the disease.

RESUMEN Objetivo Elaborar y evaluar métodos serológicos para el diagnóstico y la investigación del chikungunya. Métodos Se elaboraron dos sistemas de ELISA de captura de IgM (MAC-ELISA por sus siglas en inglés) para el diagnóstico de la infección aguda por el virus de (CHIKV) y dos métodos de ELISA de inhibición (MEI) para determinar el valor cuantitativo de los anticuerpos totales contra el CHIKV, en el Laboratorio Nacional de Virología de Nicaragua en 2014-2015, para lo cual se utilizaron anticuerpos monoclonales (AcMo) y sueros hiperinmunes. Se determinó la sensibilidad, la especificidad y los valores predictivos, así como la concordancia de los MAC-ELISA, comparando los resultados de 198 muestras (116 positivas y 82 negativas) con el ELISA de los Centros para el Control y la Prevención de Enfermedades de los Estados Unidos (Atlanta; MAC-ELISA-CDC). Para la evaluación clínica de las cuatro técnicas serológicas, se emplearon 260 muestras de suero obtenidas en la fase aguda y en la fase de convalecencia de presuntos casos de chikungunya. Resultados Se estandarizaron los cuatro métodos analíticos determinando las concentraciones óptimas de los diferentes reactivos. La duración del procesamiento se redujo sustancialmente en comparación con el MAC-ELISA-CDC. Con los sistemas de MAC-ELISA, se obtuvo una sensibilidad del 96,6% y del 97,4% y una especificidad del 98,8% y del 91,5% al utilizar AcMo y suero hiperinmune, respectivamente, en comparación con el método de los CDC. La evaluación clínica de las cuatro técnicas serológicas, en comparación con la PCR en tiempo real de los CDC, arrojó una sensibilidad del 95,7% y una especificidad del 88,8%-95,9%. Conclusiones Se estandarizaron dos sistemas de ELISA-MAC y dos de MEI y se comprobó que poseen la calidad adecuada para el diagnóstico y las investigaciones del chikungunya, con lo cual mejorará la eficiencia de la vigilancia epidemiológica en Nicaragua, el primer país centroamericano que produce sus propios reactivos para el diagnóstico serológico del CHIKV. Los métodos estudiados en este trabajo pueden aplicarse en otros países y contribuyen al desarrollo de sistemas de diagnóstico sostenibles para combatir la enfermedad.

Tipificación molecular del virus dengue 3 durante el brote epidémico de dengue clásico en Lima, Perú, 2005 / Molecular typing of dengue 3 virus during the classic dengue outbreak in Lima-Peru, 2005

Objetivos: Identificar mediante trascripción reversa-reacción en cadena de la polimerasa (RT-PCR) y sitios específicos de restricción - reacción en cadena de la polimerasa (RSS-PCR) al agente causal del brote epidémico presentado en el distrito de Comas, Lima en abril del año 2005. Materiales y métodos: veinte muestras de suero colectadas durante el brote de dengue fueron procesados por RT-PCR para determinar el serotipo, esta técnica se realizó en un solo paso. Luego se aplicó la técnica RSS-PCR para la identificación del genotipo circulante y se corroboraron los resultados posteriormente con aislamiento viral y secuenciamiento. Resultados: El análisis del RTPCR del ARN extraído de las muestras presentó un producto amplificado de 290pb que corresponden al dengue serotipo 3 (DEN 3). El análisis de los productos de RSS-PCR del ARN extraído a partir de aislamientos de DEN 3 correspondió al patrón C, incluido en el genotipo III. Los aislamientos de los virus dengue 3 en líneas celulares C6/36, tipificadas por IFI y el secuenciamiento genético confirmaron los resultados obtenidos por las pruebas previamentedescritas. Conclusión: Durante el brote epidémico de dengue clásico en Lima, circuló el genotipo III del virus DEN 3.

Objectives: To identify the causative agent of the outbreak that occurred in Comas, Lima in April 2005 using reverse transcription-polymerase chain reaction (RT-PCR) and specific restriction site-PCR (RSS-PCR). Materials and methods: 20 serum samples collected during the dengue outbreak were assessed using RT-PCR for identifying serotypes, this technique was performed in a single step. Later, the RSS-PCR method was used to identify the circulating serotypes, and the results were corroborated by viral isolation and sequencing. Results: RT-PCR of viral RNA taken from the samples showed a 290-pb amplified product corresponding to dengue virus serotype 3 (DEN 3). RSS-PCR product analysis of RNA from DEN 3 isolates corresponded to pattern C, included in genotype III. Dengue 3 virus isolates in C6/36 cell lines identified using indirect immunofluorescence and gene sequencing confirmed the results obtained using the aforementioned tests. Conclusion: During the classic dengue fever outbreak in Lima, DEN 3 genotype III circulated.

Molecular typing of dengue virus type 2 in Brazil

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype)

Emergence and Global Spread of a Dengue Serotype 3; Subtype III Virus

Over the past two decades; dengue virus serotype 3 (DENV-3) has caused unexpected epidemics of Dengue hemorrhagic fever (DHF) in Sri Lanka; East Africa; and Latin America. We used a phylogenetic approach to evaluate the roles of virus evolution and transport in the emergence of these outbreaks. Isolates from these geographically distant epidemics are closely related and belong to DENV-3; subtype III; which originated in the Indian subcontinent. The emergence of DHF in Sri Lanka in 1989 correlated with the appearance there of a new DENV-3; subtype III variant. This variant likely spread from the Indian subcontinent into Africa in the 1980s and from Africa into Latin America in the mid-1990s. DENV-3; subtype III isolates from mild and severe disease outbreaks formed genetically distinct groups; which suggests a role for viral genetics in DHF

Complete nucleotide sequence analysis of a Brazilian dengue virus type 2 strain

In the last decade, dengue fever (DF) in Brazil has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2) into the country in 1990. In order to analyze the complete genome sequence of a DEN-2 Brazilian strain (BR64022/98), we designed primers to amplify contiguous segments of approximately 500 base pairs across the entire sequence of the viral genome. Twenty fragments amplified by reverse transcriptase-PCR were cloned, and the complete nucleotide and the deduced amino acid sequences were determined. This constitutes the first complete genetic characterization of a DEN-2 strain from Brazil. All amino acid changes differentiating strains related to the Asian/American-Asian genotype were observed in BR64022/98, indicating the Asiatic origin of the strain