Analytical and clinical performance of molecular assay used by the Brazilian public laboratory network to detect and discriminate Zika, Dengue and Chikungunya viruses in blood

ABSTRACT In response to the Zika epidemics in Brazil, the ZDC molecular assay (Bio-Manguinhos) was developed and registered at the Brazilian Regulatory Agency of Health Surveillance - ANVISA. The circulation of Zika (ZIKV) Dengue (DENV) and Chikungunya (CHIKV) viruses and their clinical similarities are challenges to correctly diagnose these viruses. The simultaneous detection of ZIKV, DENV and CHIKV is an important tool for diagnosis and surveillance. Here, we present the analytical and clinical performance evaluation of ZDC molecular assay (Bio-Manguinhos) at the public health laboratories three years after its registration at ANVISA. The clinical performance demonstrates the ZDC molecular assay (Bio-Manguinhos) has 100% sensitivity and 100% specificity to detect and discriminate ZIKV, CHIKV, and DENV from clinical plasma samples. The ZDC molecular assay (Bio-Manguinhos) results were highly reproducible and no cross-reactivity was seen during testing with a panel of other infectious agents. In conclusion, the ZDC molecular assay (Bio-Manguinhos) is an accurate and reliable tool to monitor Zika, dengue and chikungunya infections in countries like Brazil with simultaneous circulation of the three viruses.

Genomic surveillance of the Chikungunya Virus (CHIKV) in Northeast Brazil after the first outbreak in 2014

Abstract INTRODUCTION: We performed an epidemiological surveillance of the Chikungunya (CHIKV) lineages in Bahia after the 2014 East/Central/South African (ECSA) genotype outbreak. METHODS: Reverse-transcription polymerase chain reaction (RT-PCR), viral isolation, and phylogenetic analyses were conducted on serum samples from 605 patients with CHIKV-like symptoms during 2014-2018. RESULTS: Of the 605 samples, 167 were CHIKV-positive. Viral isolation was achieved for 20 samples; their phylogenetic analysis (E2 protein) revealed the presence of ECSA lineage and reinforced the phylogenetic relationship between ECSA and Indian Ocean lineages. CONCLUSIONS: The genomic surveillance of CHIKV showed that only ECSA lineage circulated in Bahia since the 2014 outbreak.

Chikungunya in a kidney transplant recipient: a case report / Chikungunya em um receptor de transplante renal: relato de caso

ABSTRACT In 2004, a global spread of Chikungunya fever affected most tropical and subtropical regions of the world. In 2016, an outbreak occurred in Northeast Brazil with hundreds of cases documented. Solid organ transplant recipients have a modified immune response to infection and the clinical course is usually different from immunocompetent patients. The diagnosis can be challenging in this population. Most reports describe patients residing in endemic areas, although we must emphasize the importance of differential diagnosis in kidney transplanted travelers who visit endemic regions, such as Northeast Brazil. Here, we reported a case of a kidney transplant recipient that acquired Chikungunya fever after a trip to an endemic region at Northeast Brazil during the outbreak in 2016, with a good clinical evolution. We also present warning recommendations for travelers to endemic areas as additional measures to prevent disease outbreaks.

RESUMO Em 2004, um surto global de Chikungunya afetou a maioria das regiões tropicais e subtropicais do mundo. Em 2016, um surto ocorreu no Nordeste do Brasil com centenas de casos documentados. Receptores de transplantes de órgãos sólidos têm uma resposta imune modificada à infecção, e o curso clínico é geralmente diferente daquele em pacientes imunocompetentes. O diagnóstico pode ser desafiador nessa população. A maioria dos relatos descreve pacientes residentes em áreas endêmicas, embora devamos enfatizar a importância do diagnóstico diferencial em viajantes transplantados renais que visitam regiões endêmicas, como o Nordeste do Brasil. Aqui, nós relatamos o caso de um receptor de transplante renal que adquiriu febre Chikungunya após uma viagem a uma região endêmica no Nordeste do Brasil durante o surto de 2016, com uma boa evolução clínica. Também apresentamos recomendações de alerta para viajantes em áreas endêmicas, como medidas adicionais para prevenir surtos de doenças.

A localized outbreak of Chikungunya virus in Salvador, Bahia, Brazil

A localized Chikungunya virus (CHIKV; East/Central/South African genotype) outbreak (50 cases, 70% laboratory-confirmed; attack rate: 5.3 confirmed cases/100 people) occurred in a Salvador, Brazil neighborhood, between Apr-Jun/2017. Highly clustered cases in space and time, mostly along a single street, highlight an increased risk of CHIKV transmission among pockets of susceptible populations. This finding underscores the need for ongoing local level surveillance for arboviral outbreaks.

Co-infección por los virus del dengue y chikungunya: revisión narrativa / Coinfection of dengue and chikungunya viruses: narrative review

Resumen Introducción: Los virus del dengue y chikungunya son transmitidos por la hembra de los mosquitos Aedes aegypti y Aedes albopictus, ampliamente distribuidos en zonas tropicales y subtropicales, lo que facilita la co-infección. Objetivo: Determinar la magnitud, la distribución geográfica y el cuadro clínico de la co-infección por dengue y chikungunya. Material y Métodos: Revisión narrativa. Búsqueda en las bases de datos PubMed y Lilacs, utilizando los términos MeSH "Chikungunya", "CHIKV", "DENV", "Dengue" y "coinfection. Se incluyeron los artículos de los últimos 20 años. Resultados: Se incluyeron 45 artículos. El mayor reporte de co-infección fue en Asia seguido de África. En las Américas la información es limitada por la reciente circulación del chikungunya. La magnitud de la co-infección varió entre 0 y 31,9%. No se encontraron diferencias en la distribución de la co-infección por sexo y edad. El cuadro clínico de la mono-infección y la co-infección fue similar. Algunos reportes de caso exponen cuadros graves con afección del sistema nervioso central, manifestaciones hemorrágicas y enfermedad de Still. Conclusión: Las manifestaciones clínicas de la co-infección por dengue y chikungunya son similares a la mono-infección, situación que dificulta el diagnóstico y la medición de su magnitud.

Background: Dengue and chikungunya viruses are transmitted by the female Aedes aegypti and Aedes albopictus, which are widely distributed in tropical and subtropical areas, facilitating coinfection. Aim: To determine the magnitude, geographical distribution and clinical picture of dengue and chikungunya coinfection. Material and Methods: Narrative review. A search in the PubMed and Lilacs databases was made, using the MeSH terms "Chikungunya", "CHIKV", "DENV", "Dengue" and "coinfection. The articles of the last 20 years were included. Results: A total of 45 articles were included. The largest coinfection report was in Asia followed by Africa. In the Americas, the information is limited because of the recent circulation of chikungunya. The magnitude of coinfection varies between 0% and 31.9%. No differences were found in the distribution of coinfection by sex and age. The clinical picture of monoinfection and coinfection was similar. Some case reports show severe cases with central nervous system involvement, hemorrhagic manifestations and Still's disease. Conclusion: The clinical manifestations of coinfection by dengue and chikungunya viruses are similar to those due to monoinfection, which difficult the diagnosis and measurement of its magnitude.

Potential role of dengue virus, chikungunya virus and Zika virus in neurological diseases

This study showed that laboratory markers of recent infection by dengue, Zika or chikungunya arboviruses were detected in the biological samples of approximately one-third of patients with encephalitis, myelitis, encephalomyelitis or Guillain-Barré syndrome, in a surveillance programme in Piauí state, Brazil, between 2015-2016. Fever and myalgia had been associated with these cases. Since in non-tropical countries most infections or parainfectious diseases associated with the nervous system are attributed to herpesviruses, enteroviruses, and Campylobacter jejuni, the present findings indicate that in tropical countries, arboviruses may now play a more important role and reinforce the need for their surveillance and systematic investigation in the tropics.

Sensitivity and detection of chikungunya viral genetic material using several PCR-based approaches

Abstract INTRODUCTION: Chikungunya fever is a condition resulting from infection by chikungunya virus (CHIKV), an Aedes sp.-transmitted virus. This disease has been diagnosed in thousands of cases in the Americas, particularly in Brazil, in recent years, and there is an ongoing epidemic of chikungunya fever in Brazil that began in 2014. Clinical diagnosis is difficult; only a few cases have been confirmed by laboratory tests due to the low number of specific, efficient tests available for virus or antibody detection. Here, we aimed to evaluate different polymerase chain reaction (PCR) approaches for detection of CHIKV genetic material. METHODS: Specific primers and probes within the viral capsid gene region were designed for this work. To evaluate the analytic sensitivity of detection, human sera were spiked with serial dilutions of the viral stock. Several PCR protocols were performed to investigate the sensitivity of CHIKV RNA detection in serum dilutions ranging from 106 to 1 PFU equivalents. RESULTS: The technique showing the greatest sensitivity was a real-time PCR assay using specific probes that could detect the genetic material of the virus at all dilutions, followed by conventional PCR. Digital PCR showed low sensitivity and was much more expensive than other technologies. Digital PCR should be used for specific purposes other than clinical diagnosis. CONCLUSIONS: Although quantitative PCR using probes was more expensive than the use of intercalating dyes or conventional PCR, it had the highest sensitivity out of all tested PCR approaches.

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.

Phylogenetic analyses of chikungunya virus among travelers in Rio de Janeiro, Brazil, 2014-2015

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that emerged in Brazil by late 2014. In the country, two CHIKV foci characterized by the East/Central/South Africa and Asian genotypes, were established in North and Northeast regions. We characterized, by phylogenetic analyses of full and partial genomes, CHIKV from Rio de Janeiro state (2014-2015). These CHIKV strains belong to the Asian genotype, which is the determinant of the current Northern Brazilian focus, even though the genome sequence presents particular single nucleotide variations. This study provides the first genetic characterisation of CHIKV in Rio de Janeiro and highlights the potential impact of human mobility in the spread of an arthropod-borne virus.

Emerging alphaviruses in the Americas: Chikungunya and Mayaro

Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are emergent arthropod-borne viruses that produce outbreaks of acute febrile illness with arthropathy. Despite their different continental origins, CHIKV and MAYV are closely related and are components of the Semliki Forest Complex of the Alphavirus (Togaviridae). MAYV and, more recently, CHIKV, which are both transmitted by Aedes mosquitoes, have resulted in severe public health problems in the Americas, including Brazil. In this review, we present aspects of the pathogenesis, clinical presentation and treatment of febrile illnesses produced by CHIKV and MAYV. We also discuss the epidemiological aspects and effects related to the prophylaxis of infections by both viruses.