Abstract
BackgroundYellow fever (YF) is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV), is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control.MethodologyThe aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA) assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay) to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal.Conclusion/SignificanceThe assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction) and rapid processing time (<20 min). Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for YFV detection in low-resource settings.
Highlights
Yellow fever (YF) has been one of the most feared diseases during the past centuries, its historical impact ranking next to plague and smallpox
Primer and probe design for recombinase polymerase amplification (RPA) By analyzing the alignment of all available full genome sequences of yellow fever virus (YFV), the conserved 59-non-coding region (NCR) of the YFV genome was chosen for primer and probe design (Table 3)
Real-time RT-polymerase chain reaction (PCR) detected as low as 8 GC/rxn while real-time and lateral-flow stripes (LFS) RT-RPA assays could detect as low as 44 GC/rxn in YFV RNA extracts and 21 GC/rxn for the testing of YFV-spiked human plasma samples (Figure 2)
Summary
Yellow fever (YF) has been one of the most feared diseases during the past centuries, its historical impact ranking next to plague and smallpox. Despite the use of an effective vaccine since the 1930s, the World Health Organization (WHO) estimates that the disease affects more than 200,000 persons causing 30,000 deaths per year [1]. Severe outbreaks have occurred in regions of Africa that have long been free of the virus, such as Darfur in Sudan or South Omo in Ethiopia which experienced the worst YF outbreak in Africa in 20 years in 2012 [4]. The causative agent, the yellow fever virus (YFV), is found in tropical and subtropical areas of South America and Africa. A vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
