Development and field validation of an RT-LAMP assay for the rapid detection of chikungunya virus in patient and mosquito samples

Objectives

We aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform for the rapid detection of CHIKV in both patient and mosquito samples from Brazil.

Methods

We optimized an RT-LAMP assay, then evaluated the sensitivity and specificity 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.

Results

Our 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 to the gold 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% to 100.00%), clinical specificity of 96.72% (95% CI, 88.65% to 99.60%), and overall accuracy of 98.00% (95% CI, 92.96% to 99.76%).

Conclusions

Taken 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.