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Evaluation of two molecular methods for the detection of Yellow fever virus genome.

ABSTRACT: Yellow fever virus (YFV), a member of the family Flaviviridae, genus Flavivirus is endemic to tropical areas of Africa and South America and is among the arboviruses that pose a threat to public health. Recent outbreaks in Brazil, Bolivia, and Paraguay and the observation that vectors capable of transmitting YFV are presenting in urban areas underscore the urgency of improving surveillance and diagnostic methods. Two novel methods (RT-hemi-nested-PCR and SYBR(®) Green qRT-PCR) for efficient detection of YFV strains circulating in South America have been developed. The methods were validated using samples obtained from golden hamsters infected experimentally with wild-type YFV strains as well as human serum and tissue samples with acute disease.

SUBMITTER: Nunes MR

PROVIDER: S-EPMC4109279 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Evaluation of two molecular methods for the detection of Yellow fever virus genome.

Nunes Marcio R T MR Palacios Gustavo G Nunes Keley N B KN Casseb Samir M M SM Martins Lívia C LC Quaresma Juarez A S JA Savji Nazir N Lipkin W Ian WI Vasconcelos Pedro F C PF

Journal of virological methods 20110317 1-2

Yellow fever virus (YFV), a member of the family Flaviviridae, genus Flavivirus is endemic to tropical areas of Africa and South America and is among the arboviruses that pose a threat to public health. Recent outbreaks in Brazil, Bolivia, and Paraguay and the observation that vectors capable of transmitting YFV are presenting in urban areas underscore the urgency of improving surveillance and diagnostic methods. Two novel methods (RT-hemi-nested-PCR and SYBR(®) Green qRT-PCR) for efficient dete ...[more]

PMID: 21419803

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Project description: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.

Project description:BackgroundEarly detection of human yellow fever (YF) infection in YF-endemic regions is critical to timely outbreak mitigation. African National Laboratories chiefly rely on serological assays that require confirmation at Regional Reference Laboratories, thus delaying results, which themselves are not always definitive often due to antibody cross-reactivity. A positive molecular test result is confirmatory for YF; therefore, a standardized YF molecular assay would facilitate immediate confirmation at National Laboratories. The WHO-coordinated global Eliminate Yellow Fever Epidemics Laboratory Technical Working Group sought to independently evaluate the quality and performance of commercial YF molecular assays relevant to use in countries with endemic YF, in the absence of stringent premarket assessments. This report details a limited laboratory WHO-coordinated evaluation of the altona Diagnostics RealStar Yellow Fever Virus RT-PCR kit 1.0.Methodology and principal findingsSpecific objectives were to assess the assay's ability to detect YF virus strains in human serum from YF-endemic regions, determine the potential for interference and cross-reactions, verify the performance claims as stated by the manufacturer, and assess usability. RNA extracted from normal human serum spiked with YF virus showed the assay to be precise with minimal lot-to-lot variation. The 95% limit of detection calculated was approximately 1,245 RNA copies/ml [95% confidence interval 497 to 1,640 copies/ml]. Positive results were obtained with spatially and temporally diverse YF strains. The assay was specific for YF virus, was not subject to endogenous or exogenous interferents, and was clinically sensitive and specific. A review of operational characteristics revealed that a positivity cutoff was not defined in the instructions for use, but otherwise the assay was user-friendly.Conclusions and significanceThe RealStar Yellow Fever Virus RT-PCR kit 1.0 has performance characteristics consistent with the manufacturer's claims and is suitable for use in YF-endemic regions. Its use is expected to decrease YF outbreak detection times and be instrumental in saving lives.

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Evaluation of two molecular methods for the detection of Yellow fever virus genome.

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Evaluation of two molecular methods for the detection of Yellow fever virus genome.

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