AUTHOR=Lenz Shannon M. , Ray Nashone A. , Lema Tsehaynesh , Collins J. Hunter , Thapa Ruby , Girma Selfu , Balagon Marivic , Bobosha Kidist , Hagge Deanna A. , Williams Diana L. , Scollard David M. , Lahiri Ramanuj , Adams Linda B. 

TITLE=Utility of a Mycobacterium leprae molecular viability assay for clinical leprosy: An analysis of cases from the Philippines, Ethiopia, and Nepal

JOURNAL=Frontiers in Tropical Diseases

VOLUME=3

YEAR=2022

URL=https://www.frontiersin.org/journals/tropical-diseases/articles/10.3389/fitd.2022.967351

DOI=10.3389/fitd.2022.967351

ISSN=2673-7515

ABSTRACT=<p><italic>Mycobacterium leprae</italic> is a slow-growing species of mycobacteria that cannot be cultured in axenic media. This presents a number of challenges for monitoring treatment efficacy and advancing new drugs and regimens for treating leprosy. We previously developed a molecular viability assay (MVA) which measures expression of <italic>hsp18</italic> and <italic>esxA</italic> transcripts to determine viability of <italic>M. leprae</italic> directly from infected tissue. The objective of the current study was to determine the utility of the MVA for practical use on clinical specimens. Leprosy cases from the Philippines (N = 199), Ethiopia (N = 40), and Nepal (N = 200) were diagnosed by clinical examination, slit-skin smears (SSS) from index sites, and/or histopathology. Biopsy specimens for MVA were collected from an active lesion and stored in 70% ethanol. DNA and RNA were extracted from the tissue, and <italic>M. leprae</italic> were enumerated on the DNA fraction <italic>via</italic> RLEP qPCR. Based on this count, DNased RNA was normalized to the equivalent of 3x10<sup>3</sup><italic>M. leprae</italic> per reverse transcription reaction, and <italic>hsp18</italic> and <italic>esxA</italic> transcripts were amplified by PCR on the resulting cDNA. There was a strong correlation between RLEP enumeration on the specific biopsy specimen for MVA and the average SSS bacterial index (BI) in all three cohorts (<italic>p</italic> &lt; 0.001). The MVA could be performed on most biopsies with an average SSS BI ≥ 2 and showed a decrease in <italic>M. leprae</italic> viability with increasing duration of leprosy multidrug therapy (<italic>R</italic><sup>2</sup> = 0.81, <italic>p</italic> &lt; 0.001). The MVA also detected viable <italic>M. leprae</italic> in relapse patients where it showed significant correlation with the mouse footpad assay (<italic>p</italic> = 0.018). The MVA is a <italic>M. leprae</italic>-specific, sensitive, and relatively quick test. Clinically, the MVA would likely be most useful to monitor treatment, confirm suspected relapse cases, and determine efficacy of new leprosy drugs in clinical trials.</p>