Development, verification, and validation of an RT-qPCR-based protocol for Yellow Fever diagnosis. (June 2022)

• Record Type: Journal Article
• 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
• Is Part Of: International journal of infectious diseases. Volume 119(2022)
• Page Start: 34
• Page End: 37
• Publication Date: 2022-06
• Subjects: Yellow Fever -- Molecular diagnosis -- RT-qPCR -- viral load; Communicable diseases -- Periodicals; Communicable Diseases -- Periodicals; Communicable diseases; Periodicals; Electronic journals; 616.9
• DOI: 10.1016/j.ijid.2021.12.361
• Languages: English
• ISSNs: 1201-9712

Abstract

Highlights: 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. Abstract: 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.