LAUSR

In Brazil and other tropical countries, febrile illnesses caused by arboviruses are common. Zika, Chikungunya, Mayaro, Oropouche, Yellow Fever, and Dengue viruses are transmitted by mosquitoes and cause similar symptoms. Viral RNA detection tests enable the precise identification of the virus responsible for patients’ symptoms, providing rapid support for medical decision-making. Therefore, this study aims to validate an assay for the simultaneous detection of these viruses while also distinguishing the dengue virus subtypes. For assay validation, 95 EDTA-plasma samples from routine laboratory testing and 18 samples from proficiency testing providers were used, including 16 positive for Zika, 11 for Chikungunya, 10 for Mayaro, 13 for Oropouche, one for Yellow Fever, and 58 for Dengue (25 for type 1, 27 for type 2, 12 for type 3, and 4 for type 4). Additionally, 15 samples tested negative for all six viruses. Some samples were positive for multiple targets. Total nucleic acids were extracted from 300 µL of EDTA-plasma using Maxwell automated extractor. A synthetic RNA not found in nature was used as an exogenous internal control (IC). Two primer sets were used for Zika detection and one set for each of the other viruses, with primers and probes obtained from the literature. Six multiplex reactions were performed for target detection: Zika 1 (FAM) and Zika 2 (HEX); Dengue 1 (FAM) and Dengue 2 (HEX); Dengue 3 (FAM) and Dengue 4 (HEX); Chikungunya (FAM) and Mayaro (HEX); Yellow Fever (FAM); and Oropouche (FAM). In all reactions, IC was detected on the Cy5 channel. Accuracy, reproducibility, precision, and specificity assays were conducted. The limit of detection (LOD) was determined using probit regression analysis with serial dilutions. Test accuracy was assessed by comparing results with previous RT-qPCR findings. The accuracy obtained was 100% for all targets. Reproducibility and precision assays showed consistent intra- and inter-assay results. Specificity assays confirmed that the test is highly specific, with no cross-reactivity observed. The detection limits obtained were: Zika (126 copies/mL), Chikungunya (110 copies/mL), Mayaro (55 copies/mL), Oropouche (72 copies/mL), Yellow Fever (30 copies/mL), Dengue type 1 (54 copies/mL), type 2 (75 copies/mL), type 3 (69 copies/mL), and type 4 (46 copies/mL). The simultaneous assay for the detection of Zika, Chikungunya, Mayaro, Oropouche, Yellow Fever (YFV), and Dengue viruses demonstrated high sensitivity, with detection limits below 130 copies for all targets. The overall agreement was 100%. Since its implementation, this methodology has enabled the diagnosis of 515 patients with febrile syndromes, including the detection of emerging pathogens such as Oropouche and Mayaro. Additionally, dengue subtyping plays a crucial role in epidemiological surveillance.