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.

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.

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.

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.

Entomological study of chikungunya infections in the State of Kelantan, Malaysia.

Background & objectives: Chikungunya infection has become a public health threat in Malaysia since the 2008 nationwide outbreaks. Aedes albopictus Skuse has been identified as the chikungunya vector in Johor State during the outbreaks. In 2009, several outbreaks had been reported in the State of Kelantan. Entomological studies were conducted in Kelantan in four districts, namely Jeli, Tumpat, Pasir Mas and Tanah Merah to identify the vector responsible for the virus transmission. Methods: CHIKV cases records were obtained from State Health Department, Kelantan and localities involved were identified. Larva survey was conducted to collect the immature mosquito stages. Modified aspirators were used to collect the adult mosquitoes. All samples on dry ice were transferred to laboratory and the presence of the virus was detected using reverse transcriptase PCR. Results: A total of 1,245 mosquito larvae were collected during larval survey and 2,019 adult mosquitoes were collected using aspirator. From these collections, 640 mosquito pools were tested for the presence of CHIKV by RT-PCR but none found positive. Ae. albopictus was the most abundant mosquito collected, followed by Culex sp., Armigeres sp. and Anopheles sp. A total of 2, 814 artificial containers were inspected during the study. Interpretation & conclusions: Since none of the mosquito samples was found to be positive for chikungunya virus, the vector(s) of chikungunya virus in these localities could not be identified.

Entomo-epidemiological investigations on chikungunya outbreak in the Lakshadweep islands, Indian Ocean.

Background & objectives: An outbreak of chikungunya fever characterized by prolonged incapacitation in the Lakshadweep islands in Indian Ocean occurred in November 2006. We undertook the entomological and epidemiological investigations on the Chikungunya outbreak in the Lakshadweep islands, Indian Ocean. Methods: Epidemic information in the affected places was obtained from the local hospital records. Entomological observations like larval survey and the adult resting/landing collections by the hand collection methods were individually conducted in the two affected islands Andrott and Kalpeni in November and December 2006. Results: The main breeding sources of the mosquitoes were the rodent-devoured coconuts, coconut shells, areca nut soaking mud and plastic pots, discarded containers, grinding stones, metal containers and plastic containers. Aedes albopictus was pre-dominantly present in the Lakshadweep islands. Interpretation & conclusion: It is concluded that the routine campaigns need to be organized regularly within the community highlighting the potential breeding grounds of mosquitoes and the possible control methods. Source reduction strategies like destruction of coconut shells and rodent-devoured coconuts by burning or by burying them inside the ground. Ae. albopictus played major role as the vector mosquitoes responsible for the chikungunya transmission.

Clinical profile of chikungunya fever in patients in a tertiary care centre in Maharashtra, India.

Background & objectives: In India a chikungunya fever outbreak started in December 2005 when the country experienced more than 13 lakhs of chikungunya infected cases. We undertook this study to study detailed clinical profile of chikungunya fever in both indoor and outdoor patients in a tertiary care hospital in Nagpur, Maharashtra in 2006. Methods: Suspected cases of chikungunya fever (n=405) during the period of July to September 2006, having clinical triad of fever, arthralgia and/or rashes were included in the study. Clinical profile was studied in all the cases. Of the 405 samples collected, 166 were tested for serum CHIK IgM antibodies. Results: Of the 166 samples tested for CHIKV IgM antibodies, 87 (52.4%) were positive (confirmed cases). Male: female ratio was 2.3:1. Fever and arthralgia were present in all cases. Rash was present in 27(31%) confirmed and 38(12%) suspected cases. Lymphadenopathy was present in 12 (13.8 %) confirmed and 4 of suspected cases. Chronic polyarthritis was seen in 22 (25.3%) confirmed and 75 (23.6%) suspected cases. Neurological manifestations were observed in 08 (9%) confirmed and 10(3.14%) suspected cases. Mortality was 7(2.2%) in 318 suspected cases and 3 (3.4%) in 87 confirmed cases. Interpretation &conclusions: Our findings showed that about half of the serum samples for CHIKV IgM antibody tested positive from cases suspected to have chikungunya fever. Fever, joint pain and headache were major symptoms. Certain rare manifestations like lymphodenopathy, oral ulcers and encephalitis were also seen. Mortality in confirmed cases was about 3.4 per cent.

A case of bilateral presumed chikungunya neuroretinitis.

Chikungunya fever is a relatively rare from of vector-borne viral fever caused by chikungunya virus and spread by bites of the Aedes aegypti and Aedes albopictus mosquito. Epidemics of chikungunya fever have been reported in the past from different parts of the world. Although the virus had been passive for quite some time, recent reports of outbreaks of chikungunya fever in several parts of Southern India have confirmed the re-emergence of this virus. Symptoms of this infection include abrupt onset of fever, chills, and headache, rash, severe joint pain, conjunctival injection and photophobia. Ocular manifestations have been recently reported with this infection. We report a case of a 48-year-old female patient, who presented with defective vision two weeks after a serology proven chikungunya infection. There was bilateral neuroretinitis with peripapillary cotton wool spots. These findings should be kept in mind as an ocular manifestation of chikungunya virus infection.

Chikungunya: an overview.

Chikungunya (CHIK),a mosquito borne debilitating disease,is caused by CHIK virus,an alphavirus belonging to the family Togaviridae.The sudden onset of very high fever along with rash, and severe arthralgia especially in the small joints of hands and toes are the characteristics of the disease. It was first reported from Tanzania in 1952-53 and spread subsequently to sub-Saharan Africa, South East Asia and Pacific causing large epidemics. The virus exists in three genotypes, the Asian, West African and East Central South African that are responsible for outbreaks in the respective areas.The first outbreak in Asia was in Bangkok in 1958 followed by other Asian countries. India experienced massive outbreaks of CHIK in the 1960s and early 70s mainly in cities. After a gap of 32 years an explosive outbreak of CHIK devastated the country affecting more than 1.4 million people in 13 states.The epidemic also witnessed many unusual clinico-pathological complications including CHIK associated deaths and mother to child transmission. High morbidity with severe arthralgia persisted for several months made the people mentally and physically weak. This review describes CHIK in general and highlights the various clinico-pathological aspects observed during the recent outbreak.

Expression and Evaluation of Chikungunya Virus E1 and E2 Envelope Proteins for Serodiagnosis of Chikungunya Virus Infection

PURPOSE: Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIKV fever, which is a mosquitoe-transmitted viral disease in Africa, India, South-East Asia, and recently Southern Europe. Currently, serological diagnostic tests such as hemagglutination inhibition test (HI test), in-house IgM capture enzyme-linked immunosorbent assays (ELISA), and indirect immunofluorescence test were used for diagnosis of chikungunya fever, which are based on whole virus antigens. MATERIALS AND METHODS: CHIKV E1, and E2 envelope proteins for the CHIKV-specific serodiagnostic reagents for chikungunya fever were expressed in baculovirus expression system. The seroreactivity of recombinant CHIKV E1 and E2 envelope proteins were evaluated using sera panels of patients from Laboratoire Marcel Merieux by indirect IgM capture ELISA. RESULTS: The recombinant CHIKV E1 and E2 envelope protein showed sensitivity of 77.5% and 90%, respectively. The specificities of both CHIKV E1 and E2 envelope proteins were 100%. CONCLUSION: The recombinant CHIKV E1 and E2 envelope proteins could be a useful diagnostic reagent for CHIKV infection.