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

Ribeiro da Silva, Severino Jefferson, Ferraz de Magalhães, Jurandy Júnior, Matthews, Quinn, Lot Divarzak, Ana Luisa, Germano Mendes, Renata Pessôa, Rodrigues Santos, Bárbara Nazly, Guerra de Albuquerque Cabral, Diego, Bezerra da Silva, Jacilane, Kohl, Alain, Pardee, Keith and Pena, Lindomar (2024) 'Development and field validation of an RT-LAMP assay for the rapid detection of chikungunya virus in patient and mosquito samples'. Clinical Microbiology and Infection. (In Press)

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Abstract

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.

Item Type:	Article
Subjects:	QX Parasitology > Insects. Other Parasites > QX 510 Mosquitoes WC Communicable Diseases > WC 20 Research (General) WC Communicable Diseases > Virus Diseases > Infectious Mononucleosis. Arbovirus Infections > WC 524 Arbovirus infections
Faculty: Department:	Biological Sciences > Department of Tropical Disease Biology Biological Sciences > Vector Biology Department
Digital Object Identifer (DOI):	https://doi.org/10.1016/j.cmi.2024.03.004
Depositing User:	Amy Carroll
Date Deposited:	13 Mar 2024 14:03
Last Modified:	13 Mar 2024 14:03
URI:	https://archive.lstmed.ac.uk/id/eprint/24181

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