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

• Published in: International journal of infectious diseases 2022-06, Vol.119, p.34-37
• Main Authors: Rampazzo, Rita de Cássia Pontello, Zambenedetti, Miriam Ribas, Alexandrino, Fabiana, Jacomasso, Thiago, Tschá, Marcel Kruchelski, de Fillipis, Ana Maria Bispo, Morello, Luis Gustavo, Marchini, Fabricio Klerynton
• Format: Article
• Language: English
• Subjects: Arboviruses; Chikungunya Fever - diagnosis; Dengue; Dengue Virus - genetics; Humans; Molecular diagnosis; RT-qPCR; viral load; Yellow Fever; Yellow Fever - prevention & control; Yellow fever virus - genetics; Zika Virus - genetics; Zika Virus Infection - diagnosis
• ISSN: 1201-9712; 1878-3511
• DOI: 10.1016/j.ijid.2021.12.361

•Yellow Fever continues to cause disease in tropical and subtropical areas.•Yellow Fever may mislead with other diseases presenting the same clinical symptoms.•Our reaction was sensitive presenting LoD95% of approximately 1 virus per reaction.•The reaction did not present cross-reactions with other viruses. 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. 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. 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. 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.