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

Ribeiro da Silva, S. J. et al. (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, (doi: 10.1016/j.cmi.2024.03.004) (PMID:38460820) (Early Online Publication)

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: Articles
• Status: Early Online Publication
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Kohl, Professor Alain
• Authors: Ribeiro da Silva, S. J., Ferraz de Magalhães, J. J., Matthews, Q., Lot Divarzak, A. L., Germano Mendes, R. P., Rodrigues Santos, B. N., Guerra de Albuquerque Cabral 3,, D., Bezerra da Silva, J., Kohl, A., Pardee, K., and Pena, L.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Journal Name: Clinical Microbiology and Infection
• Publisher: Elsevier
• ISSN: 1198-743X
• ISSN (Online): 1469-0691
• Published Online: 08 March 2024
• Copyright Holders: Copyright © 2024 The Authors
• First Published: First published in Clinical Microbiology and Infection 2024
• Publisher Policy: Reproduced under a Creative Commons license

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
172630007		Arthropod-borne infections and emerging virus infections in high risk areas (Programme 4)	Alain Kohl	Medical Research Council (MRC)	MC_UU_12014/8	III - Centre for Virus Research