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

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 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 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. Author Summary Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae , an obligate intracellular bacterium. Disease diagnosis is currently performed on skin examinations for clinical signs, bacilli staining in skin smears and invasive skin biopsies. However, the spectrum of clinical manifestations and the low bacterial load can hinder accurate diagnosis, which is critical for providing proper intervention and adequate care as well as for establishing transmission control. Quantitative PCR (qPCR) 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 qPCR that detects two bacterial genes for the diagnosis and a human gene as an internal reaction control. The new qPCR, developed using GMP-grade reagents, is highly sensitive, specific, reproducible, and stable. 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. ### Competing Interest Statement Instituto de Biologia Molecular do Parana (IBMP) produces the PCR mastermix and some of the oligonucleotides used in the qPCR experiments. IBMP had no participation in the present study's design, data collection, analysis, interpretation, or writing of the report and decision to submit for publication. ### Funding Statement This work was funded by a grants from: Banco Nacional de Desenvolvimento Economico e Social (BNDES), contract no. 15.2.0473.1 (Operation #4.816.864) to MAK; and Novartis Foundation and Leprosy Research Initiative (LRI; 703.15.45), Foundation for Research Support of the State of Rio de Janeiro (FAPERJ;E-26/203.053/2016), Brazilian National Council for Scientific and Technological Development (CNPq 421852/2017-2018), Brazilian Coordination for Improvement of Higher Education Personnel (CAPES), and by the National Fund for Health/Brazilian Ministry of Health (MS/SCTIE/DECIT; 404277/2012-8 and TED 145/2018) to MOM. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Ethics Committee of the Oswaldo Cruz Foundation approved this study (CAAE: 38053314.2.0000.5248, number: 976.330; 10/03/2015). All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data is contained in the manuscript.