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

Area of Science:

• Virology
• Molecular Diagnostics
• Public Health

Background:

• Yellow fever (YF) remains a significant public health concern, characterized by recurrent outbreaks in tropical and subtropical regions.
• Current diagnosis of acute YF infection primarily relies on real-time reverse transcription-polymerase chain reaction (RT-qPCR) assays.

Purpose of the Study:

• To evaluate and compare a novel molecular protocol for yellow fever virus (YFV) detection.
• To assess the performance of the new assay against established methods used by arbovirus reference laboratories.

Main Methods:

• Development of a novel molecular protocol for YFV detection.
• Inclusion of an Internal Control for reaction validation and an External Control for RNA extraction efficiency monitoring.

Main Results:

• The novel assay demonstrated high specificity, with no cross-reactivity observed with dengue, Zika, or Chikungunya viruses.
• Analysis of 204 clinical samples and cultured viruses across three Brazilian diagnostic centers showed 95% agreement with the Pan American Health Organization's recommended reference method.
• The assay can detect as little as one viral genome per reaction.

Conclusions:

• The developed multiplex assay is suitable for routine clinical laboratory use.
• This novel protocol offers enhanced specificity and sensitivity compared to existing methods.
• The assay reduces costs per test through optimized hands-on time and reagent usage.