Development, verification, and validation of an RT-qPCR-based protocol for Yellow Fever diagnosis.

INTRODUCTION:Yellow fever (YF) is a public health threat with frequent outbreaks in tropical and subtropical areas, despite the existence of a safe and effective vaccine. The diagnosis of acute infection of the etiologic agent relies mainly on real-time reverse transcription-polymerase chain reaction (RT-qPCR)-based assays. OBJECTIVES:The aim of this study was to evaluate and compare this novel protocol for yellow fever virus (YFV) diagnosis against assays developed in-house by reference laboratories for arboviruses. METHODS:We developed a novel molecular protocol for the detection of YFV that includes an Internal Control to validate the reaction and an External Control to monitor the RNA extraction efficiency. RESULTS AND DISCUSSION:Our assay detects one viral genome per reaction and displays no cross-reactions with dengue (1-4), Zika, or Chikungunya viruses. This novel assay yielded 95% of agreement with the reference method recommended by the Pan American Health Organization when analyzing 204 clinical samples and cultured viruses, these samples were analyzed in 3 different diagnosis centers for arboviruses in Brazil. The data suggest the use of the proposed multiplex assay protocol to do routine tests in a clinical laboratory. This product adds higher specificity and sensitivity in addition to reduced cost per test due to hands-on time and reagent spending.