TY - JOUR
T1 - Development and field validation of an RT-LAMP assay for the rapid detection of chikungunya virus in patient and mosquito samples
AU - Silva, Severino Jefferson Ribeiro da
AU - Magalhães, Jurandy Júnior Ferraz de
AU - Matthews, Quinn
AU - Divarzak, Ana Luisa Lot
AU - Mendes, Renata Pessôa Germano
AU - Santos, Bárbara Nazly Rodrigues
AU - Cabral, Diego Guerra de Albuquerque
AU - Silva, Jacilane Bezerra da
AU - Kohl, Alain
AU - Pardee, Keith
AU - Pena, Lindomar
PY - 2024/3/7
Y1 - 2024/3/7
N2 - ObjectivesWe 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.MethodsWe 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.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 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%).ConclusionsTaken 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.
AB - ObjectivesWe 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.MethodsWe 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.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 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%).ConclusionsTaken 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.
KW - CHIKV
KW - Diagnostic
KW - Mosquitoes
KW - Patients
KW - Point-of-care
KW - RT-LAMP
U2 - 10.1016/j.cmi.2024.03.004
DO - 10.1016/j.cmi.2024.03.004
M3 - Article
SN - 1198-743X
VL - 30
SP - 810
EP - 815
JO - Clinical Microbiology and Infection
JF - Clinical Microbiology and Infection
IS - 6
ER -