Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis

Author summaryLeprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Diagnosis of leprosy often relies on skin examinations for clinical signs, bacilli staining from skin smears and invasive skin biopsies. However, the spectrum of clinical manifestations and, often, low bacilli numbers can hinder accurate diagnosis. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and requiring trained health professionals. Proper intervention for adequate care and transmission control depends on early and reliable pathogen detection. Quantitative PCR methods for detecting bacterial DNA are more sensitive and could aid in differentially diagnosing leprosy from other dermatological conditions. In this work, we present a new multiplex PCR that was assessed for quality control standards, and the data indicate that the assay is stable and reproducible. The results presented here are the basis of a novel and robust tool with potential to increase the accuracy of leprosy diagnosis in routine or reference laboratories. 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 inter-operator C-p 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.