Author | Bertão-Santos, Amanda | |
Author | Dias, Larisse da Silva | |
Author | Ribeiro-Alves, Marcelo | |
Author | Pinheiro, Roberta Olmo | |
Author | Moraes, Milton Ozório | |
Author | Manta, Fernanda Saloum de Neves | |
Author | Costa, Alexandre Dias Tavares | |
Access date | 2024-10-16T18:01:41Z | |
Available date | 2024-10-16T18:01:41Z | |
Document date | 2024 | |
Citation | BERTÃO-SANTOS, Amanda et al. Validation of the performance of a point of care molecular test for leprosy: from a simplified DNA extraction protocol to a portable qPCR. PLoS Neglected Tropical Diseases, v. 18, n. 10, p. 1-19, 7 Oct. 2024. | |
ISSN | 1935-2727 | |
URI | https://www.arca.fiocruz.br/handle/icict/66564 | |
Description | Produção científica do Laboratório de Hanseníase. | pt_BR |
Description | Author summary: The diagnosis of tropical or neglected infectious diseases, such as leprosy, is highly dependent on techniques that are not very sensitive or very laborious, such as optical microscopy or culture. More sensitive and specific molecular tests are only available in urban centers, where there is a specialized workforce and adequate infrastructure. However, a large portion of the population does not have access to such medical services, either due to financial or distance issues. Thus, performing diagnostic tests at the point of care (POC) provides agility in diagnosis, avoiding the need for patients to travel to medical centers or transport biological samples to clinical analysis centers, ensuring that this population has access to diagnostic tests, even in remote areas or with poor infrastructure. In this work, we optimize and validate, on a laboratory scale, simplified versions of all the procedures necessary to perform molecular tests, from collection and DNA extraction to the detection of genomic targets using portable qPCR equipment. The results presented here are the basis of a novel tool with potential to increase a leprosy screening test in remote areas, facilitating the active search of positive cases and monitoring by health authorities responsible for underserved populations. | en_US |
Sponsorship | This study was supported by grants from Carlos Chagas Filho Research Support Foundation of the State of Rio de Janeiro (Faperj, E-26/203.913/2022, www.faperj.br, to Fernanda Saloum de Neves Manta) Carlos Chagas Institute Research Stimulus Program (ICC 008 FIO 21 – SUB 22, www.icc.fiocruz.br, to Alexandre Dias Tavares Costa) and by the National Fund for Health/Brazilian Ministry of Health (TED 69/2021, www.gov.br/saude/pt-br, to Milton Ozório Moraes). Larisse da Silva Dias is a CNPq fellowship holder, Fernanda Saloum de Neves Manta is a Faperj fellowship holder, and Alexandre Dias Tavares Costa is a CNPq productivity fellow (level 2). | |
Language | eng | en_US |
Publisher | Public Library of Science | |
Rights | open access | |
Subject in Portuguese | RNA ribossômico | pt_BR |
Subject in Portuguese | Dispositivos e equipamentos médicos | pt_BR |
Subject in Portuguese | Mycobacterium leprae | pt_BR |
Subject in Portuguese | Extração de DNA | pt_BR |
Subject in Portuguese | Hanseníase | pt_BR |
Subject in Portuguese | Biópsia | pt_BR |
Subject in Portuguese | Genômica | pt_BR |
Subject in Portuguese | Ureia | pt_BR |
Title | Validation of the performance of a point of care molecular test for leprosy: from a simplified DNA extraction protocol to a portable qPCR | en_US |
Type | Article | |
DOI | 10.1371/journal.pntd.0012032 | |
Abstract | The study aimed to optimize qPCR reactions using oligonucleotides from the first Brazilian molecular diagnostic kit for leprosy on a portable platform (Q3-Plus). In addition, we sought to develop a simplified protocol for DNA extraction that met point-of-care criteria. During optimization on the Q3-Plus, optical parameters, thresholds, and cutoffs for the 16S rRNA and RLEP targets of M. leprae were established using synthetic DNA, purified DNA from M. leprae, and pre-characterized clinical samples. For the simplified extraction protocol, different lysis solutions were evaluated using chaotropic agents, and purification was carried out by transferring the lysed material to FTA cards. The complete protocol (simplified extraction + qPCR on the portable platform) was then evaluated with pre-characterized clinical skin biopsy samples and compared with standard equipment (QuantStudio-5). LOD95% for the optimized reactions was 113.31 genome-equivalents/μL for 16S rRNA and 17.70 genome-equivalents/μL for RLEP. Among the lysis solutions, the best-performing was composed of urea (2 M), which provided good dissolution of the skin fragment and a lower Ct value, indicating higher concentrations of DNA. The complete technological solution showed a sensitivity of 52% in reactions. Our results highlight the need for additional optimization to deal with paucibacillary samples, but also demonstrate the feasibility of the portable platform for the qPCR detection of M. leprae DNA in low infrastructure settings. | en_US |
Affilliation | Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Ciências e Tecnologias Aplicadas em Saúde. Curitiba, PR, Brasil. | |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil. | |
Affilliation | Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em DST e AIDS. Rio de Janeiro, RJ, Brasil. | |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil. | |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil. | |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil. | |
Affilliation | Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Ciências e Tecnologias Aplicadas em Saúde. Curitiba, PR, Brasil. | |
Subject | Ribosomal RNA | en_US |
Subject | Medical devices and equipment | en_US |
Subject | Mycobacterium leprae | en_US |
Subject | DNA extraction | en_US |
Subject | Leprosy | en_US |
Subject | Biopsy | en_US |
Subject | Genomics | en_US |
Subject | Urea | en_US |
e-ISSN | 1935-2735 | |
xmlui.metadata.dc.subject.ods | 03 Saúde e Bem-Estar | |