Development and field validation of a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for the rapid detection of chikungunya virus in patient and mosquito samples

Abstract

ObjectivesWe aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform for the rapid detection of chikungunya virus (CHIKV) in both patient and mosquito samples from Brazil. MethodsWe optimized an RT-LAMP assay and then evaluated the specificity and sensitivity using visual detection. In comparison with the RT-qPCR reference method, we validated the utility of this assay as a molecular diagnostic test in a reference laboratory for arbovirus diagnostics using 100 serum samples collected from suspected CHIKV cases. ResultsOur RT-LAMP assay specifically detected CHIKV without cross-reactivity against other arboviruses. The limit of detection of our RT-LAMP was estimated in −1.18 PFU (confidence interval [CI] ranging from –2.08 to 0.45), resulting in a similar analytical sensitivity when directly compared with the reference standard RT-qPCR assay. Then, we demonstrate the ability of our RT-LAMP assay to detect the virus in different human specimens (serum, urine, and saliva), and crude lysate of Aedes aegypti mosquitoes in as little as 20–30 minutes and without a separate RNA isolation step. Lastly, we showed that our RT-LAMP assay could be lyophilized and reactivated by adding water, indicating potential for room-temperature storage. Our RT-LAMP had a clinical sensitivity of 100% (95% CI, 90.97–100.00%), clinical specificity of 96.72% (95% CI, 88.65–99.60%), and overall accuracy of 98.00% (95% CI, 92.96–99.76%). DiscussionTaken together, these findings indicate that the RT-LAMP assay reported here solves important practical drawbacks to the deployment of molecular diagnostics in the field and can be used to improve testing capacity, particularly in low- and middle-income countries.