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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

2024; Elsevier BV; Volume: 30; Issue: 6 Linguagem: Inglês

10.1016/j.cmi.2024.03.004

1469-0691

Severino Jefferson Ribeiro da Silva, Jurandy Júnior Ferraz de Magalhães, Quinn Matthews, Ana Luisa Lot Divarzak, Renata Pessôa Germano Mendes, Bárbara Nazly Rodrigues Santos, Diego Guerra de Albuquerque Cabral, Jacilane Bezerra da Silva, Alain Kohl, Keith Pardee, Lindomar Pena,

SARS-CoV-2 detection and testing

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.