RESUMO
BACKGROUND: Infections by dengue virus (DENV) and Zika virus (ZIKV) have some similar symptoms and a cross-reactive immune response, although with different risk populations and outcomes. Here, we evaluated the virological characteristics and the nonstructural protein 1 (NS1)-specific antibody responses to DENV and ZIKV in children suspected of dengue in different epidemiological moments in Colombia. METHODS: Viral RNA, circulating NS1 and IgM/IgG specific for DENV and ZIKV were performed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) in 301 children suspected of dengue enrolled in a hospital setting during the ZIKV epidemic and a primary healthcare setting during a DENV epidemic. For the detection of DENV and ZIKV-specific IgM, an NS1-based ELISA was validated using characterized pediatric samples. Clinical and laboratory parameters were also evaluated. RESULTS: DENV RNA or NS1 antigen was detected in the plasma of 62% of children, and in none, the ZIKV RNA was found. NS1-based ELISA for DENV and ZIKV IgM showed a sensitivity/specificity of 90/84% and 73/98%, respectively. Of 114 children without detectable viremia or antigenemia, 30.7%, 17.5%, 22% and 30% were IgM-DENV+, IgM-ZIKV+, IgM-DENV+ZIKV+ and IgM-DENV-ZIKV-, respectively. The ZIKV/DENV IgM-NS1 ratio allows the identification of the infecting ortho flavivirus in 88% of the children with IgM-DENV+ZIKV+, confirming a high predominance of DENV infections in the 2 pediatric settings. CONCLUSION: Overall, 88% of the children with clinical suspicion of dengue had an identifiable ortho flaviviral infection, with 80% caused by DENV, 7% by ZIKV and 0.7% classified as recent infections or coinfection, demonstrating active viral cocirculation in the pediatric population of southern Colombia. The IgM-NS1 detection improved the identification of ortho flaviviral infections in children without viremia or antigenemia, suggesting it is a helpful complementary tool for medical personnel in tropical regions with high viral cocirculation and different clinical scenes.
RESUMO
The isolation of nucleic acids is a critical and limiting step for molecular assays, which prompted the arrival in Colombia of automated purification instruments during the SARS-CoV-2 pandemic. The local application of this technology in the study of tropical diseases, such as dengue and zika, is beginning to be tested. We evaluated the efficiency of the automated extraction of viral RNA for studies of pediatric dengue and zika. Clinical samples of children with dengue that were well characterized through RNA isolation by silica columns and serotype-specific nested RT-PCR (DENV-1 n = 7, DENV-2 n = 5, and negatives n = 8) in addition to 40 pediatric plasma samples spiked with ZIKV (strain PRVA BC59) and 209 from negative pre-epidemic children were analyzed. RNA from patients was extracted by two automated standard and high-throughput protocols on the KingFisher™ Flex instrument. The isolated RNA was evaluated for concentration and purity by spectrophotometry, for structural and functional integrity by electrophoresis and expression of the RNase P gene, and usefulness in serotype-specific DENV detection by conventional and real-time RT-PCR. For the evaluation of ZIKV RNA, the commercial TaqMan Triplex® assay was used, along with a well-tested in-house RT-qPCR assay. The concentration of RNA (5.2 vs. 7.5 ng/µL, P=0.03) and the number of integral bands (9 vs. 11) were higher with the high-throughput protocol. However, the number of specimens serotyped for DENV by RT-qPCR was comparable for both protocols. The cycle thresholds of the TaqMan Triplex® commercial kit and the in-house assay for the detection of plasma ZIKV RNA isolated with the standard protocol showed a strong association (r = 0.93, P < 0.0001) and a Cohen Kappa index of 0.98 when all 249 samples were analyzed. These preliminary results suggest that automated instruments could be used in studies of cocirculating flaviviruses that have represented a public health problem in recent decades in Colombia. They boast advantages such as efficiency, precision, time savings, and lower risk of cross-contamination.
