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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 Silva, Severino Jefferson Ribeiro da Magalhães, Jurandy Júnior Ferraz de Matthews, Quinn Divarzak, Ana Luísa Lot Mendes, Renata Pessôa Germano Santos, Bárbara Nazly Rodrigues Cabral, Diego Guerra de Albuquerque Silva, Jacilane Bezerra da Kohl, Alain Pardee, Keith Pena, Lindomar José ORGANISMOS [B]::Vírus::Vírus de RNA::Vírus de RNA de Cadeia Positiva::Togaviridae::Alphavirus::Vírus Chikungunya TÉCNICAS E EQUIPAMENTOS ANALÍTICOS, DIAGNÓSTICOS E TERAPÊUTICOS [E]::Diagnóstico ORGANISMOS [B]::Eucariotos::Animais::Invertebrados::Artrópodes::Insetos::Pterigotos::Neópteros::Holometábolos::Dípteros::Nematóceros::Culicomorfos::Culicidae DENOMINAÇÕES DE GRUPOS [M]::Pessoas::Pacientes ASSISTÊNCIA À SAÚDE [N]::Administração de Serviços de Saúde::Administração dos Cuidados ao Paciente::Sistemas Automatizados de Assistência Junto ao Leito Severino Jefferson Ribeiro da Silva and Lindomar José Pena are the inventors of the RT-LAMP protocol described throughout this study and both authors have other patents related to this technology (BR 10 2019 027711 4, filed 23 December 2019). This work was supported by funds to Lindomar José Pena and Keith Pardee from the International Development Research Centre (IDRC, Grant No. 109434). Lindomar José Pena is funded by the Fiocruz Inova Programme, Canadian International Development Research Centre (108410-001) and the Foundation for Science and Technology of Pernambuco – FACEPE, Brazil (Grant No. APQ-0154-2.12/16). Severino Jefferson Ribeiro da Silva was supported by a Ph.D. fellowship sponsored by the Foundation for Science and Technology of Pernambuco (FACEPE), reference number IBPG-1321-2.12/18, and currently is supported by a Postdoctoral Fellowship sponsored by the University of Toronto, Canada. Jurandy Júnior Ferraz de Magalhães is funded by the Foundation for Science and Technology of Pernambuco – FACEPE, Brazil (Grant No. APQ-0741-4.01/22). Alain Kohl is funded by the UK Medical Research Council (MC_UU_12014/8, MC_UU_00034/4). Objectives: We aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform for the rapid detection of chikungunya virus (CHIKV) in both patient and mosquito samples from Brazil. Methods: We optimized an RT-LAMP assay and then evaluated the specificity and sensitivity 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 with the reference 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, 90.97–100.00%), clinical specificity of 96.72% (95% CI, 88.65–99.60%), and overall accuracy of 98.00% (95% CI, 92.96–99.76%). Discussion: 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. 2025-10-24T16:29:53Z 2024-06 journal article SILVA, Severino Jefferson Ribeiro da et al. 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. Clinical Microbiology and Infection, v. 30, n. 6, p. 810-815, June 2024. 1198-743X https://arca.fiocruz.br/handle/icict/73057 10.1016/j.cmi.2024.03.004 1469-0691 eng Clinical Microbiology and Infection open access application/pdf Elsevier B.V.