Article
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis
Registro en:
MANTA, Fernanda Saloum de Neves et al. Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. PLOS Neglected Tropical Diseases, p. 1–19, 2022.
1935-2735
10.1371/journal. pntd.0009850
Autor
Manta, Fernanda Saloum de Neves
Jacomasso, Thiago
Rampazzo, Rita de Cássia Pontello
Moreira, Suelen Justo Maria
Zahra, Najua M.
Cole, Stewart T.
Avanzi, Charlotte
Leal-Calvo, Thyago
Vasconcellos, Sidra Ezidio Gonçalves
Suffys, Phillip
Ribeiro-Alves, Marcelo
Krieger, Marco Aurelio
Costa, Alexandre Dias Tavares
Moraes, Milton Ozório
Resumen
Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of
the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and interoperator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.
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