Assessment of esxA, hsp18, and 16S transcript expression as a measure of Mycobacterium leprae viability: A comparison with the mouse footpad assay

Abstract

Objectives Shepard’s mouse footpad (MFP) assay is the definitive method for ascertaining Mycobacterium leprae viability; however, this technique is laborious and prolonged. Alternate methods that alleviate burden and time while maintaining sensitivity would benefit clinical and experimental studies. We compared a molecular viability assay (MVA), which measures normalized hsp18 and esxA transcript expression, with growth in the MFP as a determinant of M. leprae viability. Methods Receiver operating characteristic curve analyses were performed to determine viability cut-off values for M. leprae expression of hsp18, esxA, and 16S transcripts. To verify these values, immunocompetent BALB/c mice were inoculated in the footpads with a high dose of viable M. leprae. Footpads were harvested on Day 1 and at various timepoints up to 12 months post infection. M. leprae viability was determined by MVA or by sub-inoculating bacilli into passage mice per Shepard’s MFP assay. Results Cut-off levels for expression of each transcript for M. leprae viability were established with high confidence (CI = 99%, P < 0.0001). In the high-dose infection assay, M. leprae exhibited a significant decrease in transcript detection by three months post-infection. Expression of hsp18 and esxA transcripts by M. leprae correlated with bacterial growth in the MFP assay. In contrast, 16S transcripts were detected in M. leprae populations confirmed dead by the MFP assay. Conclusion The MVA, which is a sensitive, specific, and rapid viability indicator, was directly validated by the MFP assay. This study confirms the accuracy of the MVA for determining M. leprae viability.