Dengue diagnostics: serious inaccuracies are likely to occur if pre-analytical conditions are not strictly followed.

BACKGROUND: The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES: To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS: Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS: Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS: Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.

Undetected Chikungunya virus co-infections in a Brazilian region presenting hyper-endemic circulation of Dengue and Zika.

BACKGROUND: Chikungunya virus (CHIKV) causes a disease characterized by acute onset of fever accompanied by arthralgia. Clinical similarities and co-circulation of other arboviruses such as Dengue virus (DENV) and Zika virus (ZIKV), have complicated their differentiation, making their diagnoses a challenge for the health authorities. Misdiagnosis is a serious issue to the management of patients and development of public health measures. OBJECTIVES: We carried out further screening of CHIKV, DENV and ZIKV cases in Minas Gerais, Brazil, after diagnostics were already issued by a state laboratory and according to the Brazilian Ministry of Health (BMH) policy. Our aim was to look for possible co-infections or previous arboviruses' exposure. STUDY DESIGN: Sera from 193 patients with symptoms of arboviral infections were tested for DEV, ZKV and/or CHIKV by the State laboratory, according to clinical suspicion and following standard BMH guidelines. After an official diagnosis was issued for each patient, we retested samples applying a broader panel of ELISA-based serological tests. RESULTS: We identified 13 patients with concurrent or consecutive infections (IgM positive for more than one arbovirus), including 11 individuals that were positive for CHIKV and other previously confirmed arbovirus infection. DISCUSSION: Guidelines established in many arbovirus-endemic countries prioritizes the diagnosis of Zika and Dengue and no further analyzes are done when samples are positive for those viruses. As a result, possible cases of co-infections with chikungunya are neglected, which affects the epidemiological assessments of virus circulation, patient management, and the development of public health policies.

Development of an enzyme-linked immunosorbent assay using recombinant protein antigen for the diagnosis of Chikungunya virus.

We describe here the development of an in-house enzyme linked immunosorbent assay (ELISA) for the diagnostic of Chikungunya virus (CHIKV) infections using a recombinant protein from CHIKV. The recombinant protein gene was designed based on 154 sequences and we used computational methods to predict its structure and antigenic potential. To confirm predictions, the gene coding for the recombinant CHIKV protein (rCHIKVp) was synthetized and expressed in prokaryotic system. Subsequently, the protein was purified by affinity chromatography and used as antigen in an indirect ELISA. We present data regarding the optimization of the recombinant antigen production and preparation of the ELISA to detect IgG against CHIKV in human sera.

Genomic, epidemiological and digital surveillance of Chikungunya virus in the Brazilian Amazon.

BACKGROUND: Since its first detection in the Caribbean in late 2013, chikungunya virus (CHIKV) has affected 51 countries in the Americas. The CHIKV epidemic in the Americas was caused by the CHIKV-Asian genotype. In August 2014, local transmission of the CHIKV-Asian genotype was detected in the Brazilian Amazon region. However, a distinct lineage, the CHIKV-East-Central-South-America (ECSA)-genotype, was detected nearly simultaneously in Feira de Santana, Bahia state, northeast Brazil. The genomic diversity and the dynamics of CHIKV in the Brazilian Amazon region remains poorly understood despite its importance to better understand the epidemiological spread and public health impact of CHIKV in the country. METHODOLOGY/PRINCIPAL FINDINGS: We report a large CHIKV outbreak (5,928 notified cases between August 2014 and August 2018) in Boa vista municipality, capital city of Roraima's state, located in the Brazilian Amazon region. We generated 20 novel CHIKV-ECSA genomes from the Brazilian Amazon region using MinION portable genome sequencing. Phylogenetic analyses revealed that despite an early introduction of the Asian genotype in 2015 in Roraima, the large CHIKV outbreak in 2017 in Boa Vista was caused by an ECSA-lineage most likely introduced from northeastern Brazil. Epidemiological analyses suggest a basic reproductive number of R0 of 1.66, which translates in an estimated 39 (95% CI: 36 to 45) % of Roraima's population infected with CHIKV-ECSA. Finally, we find a strong association between Google search activity and the local laboratory-confirmed CHIKV cases in Roraima. CONCLUSIONS/SIGNIFICANCE: This study highlights the potential of combining traditional surveillance with portable genome sequencing technologies and digital epidemiology to inform public health surveillance in the Amazon region. Our data reveal a large CHIKV-ECSA outbreak in Boa Vista, limited potential for future CHIKV outbreaks, and indicate a replacement of the Asian genotype by the ECSA genotype in the Amazon region.