One-Step reverse transcriptase PCR for detection of arboviruses in serum samples of patients assisted in Basic health Units in the State of Maranhão, Brazil / PCR de transcriptase reversa em uma etapa para detecção de arbovírus em amostras de soro de pacientes atendidos em Unidades Básicas de Saúde no estado do Maranhão, Brasil

polymerase chain reaction to detect acute infections caused by dengue, zika, chikungunya, and mayaro virus in clinical samples. Methods: We evaluated 620 sera samples collected from March 2016 to March 2018 and provided by the Central Health Laboratory of Maranhão (LACEN-MA). Total RNA was isolated from clinical specimens and used as the template for one-step RT-PCR assays with specific-primers designed for this study. Results: Of the 620 sera evaluated, 386 (62.2%) were positive, among them 330 (85.5%) amplified a specific fragment for chikungunya, 55 (14.2%) showed a fragment compatible with dengue serotype 4, and 1 (0.3%) exhibited profile for mayaro virus. Conclusions: The results obtained here were more sensitive than IgM-ELISA because the viral RNA was detected in serum samples from patients, not only from 1 to 6 days but also from 7 to 10 days after the beginning of clinical signs (convalescent period). Besides, the mayaro virus was detected in one serum sample that was IgM-ELISA negative for dengue, zika, and chikungunya. 

Detection and differentiation of dengue virus serotypes by one-step multiplex reverse transcription PCR assays / Detecção e diferenciação de sorotipos do vírus da dengue por ensaios de PCR com transcrição reversa multiplexada em uma etapa

Background: Dengue infections are a severe public health problem in Brazil. The Ministry of Health recommends an immunosorbent assay (ELISA) for the capture of IgM (MAC-ELISA) to diagnose dengue. However, it detects antibodies that cross-react with other flaviviruses and requires confirmation in reference laboratories. Methods: One-step multiplex RT-PCR assay was used to amplify RNA of 197 serum from patients with clinical suspicion of dengue infection. The samples had been screened with the IgM ELISA kit in the Central Public Health Laboratory of the State of Maranhão. Results: Of the 197 samples evaluated by IgM ELISA, 135 were positive; of these, 96 (71.1%) were from patients in the acute phase of the infection. The one-step multiplex RT-PCR detected viral RNA in 88 (91.7%) of this serum. Among the 62-negative serum by ELISA, 29 samples (46.8%) were amplified using the molecular method. Conclusions: One-step multiplex RT-PCR was sensitive in the detection of viral particles from the first day until the sixth day after the onset of the feverish period. Moreover, it was specific and 100% reproducible. Based on these results, we recommend the use of this molecular assay to diagnose and differentiate the DENV serotypes in the acute phase of the disease.Background: Dengue infections are a severe public health problem in Brazil. The Ministry of Health recommends an immunosorbent assay (ELISA) for the capture of IgM (MAC-ELISA) to diagnose dengue. However, it detects antibodies that cross-react with other flaviviruses and requires confirmation in reference laboratories. Methods: One-step multiplex RT-PCR assay was used to amplify RNA of 197 serum from patients with clinical suspicion of dengue infection. The samples had been screened with the IgM ELISA kit in the Central Public Health Laboratory of the State of Maranhão. Results: Of the 197 samples evaluated by IgM ELISA, 135 were positive; of these, 96 (71.1%) were from patients in the acute phase of the infection. The one-step multiplex RT-PCR detected viral RNA in 88 (91.7%) of this serum. Among the 62-negative serum by ELISA, 29 samples (46.8%) were amplified using the molecular method. Conclusions: One-step multiplex RT-PCR was sensitive in the detection of viral particles from the first day until the sixth day after the onset of the feverish period. Moreover, it was specific and 100% reproducible. Based on these results, we recommend the use of this molecular assay to diagnose and differentiate the DENV serotypes in the acute phase of the disease