02562nas a2200229   4500000000100000008004100001260001000042653001900052653003100071653002500102100002100127700001200148700001100160700001300171700001300184245015100197856005200348300000900400490000700409520190200416022001402318       2023                            d  bLepra10aGeneral Energy10a molecular viability assay10aMycobacterium leprae1 aHunter Collins J1 aLenz SM1 aRay NA1 aLahiri R1 aAdams LB00aAssessment of esxA, hsp18, and 16S transcript expression as a measure of Mycobacterium leprae viability: A comparison with the mouse footpad assay  uhttps://leprosyreview.org/article/94/1/20-22078  a7-180 v943 a<p>Objectives:&nbsp;Shepard’s mouse footpad (MFP) assay is the definitive method for ascertaining&nbsp;Mycobacterium leprae&nbsp;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&nbsp;hsp18&nbsp;and&nbsp;esxA&nbsp;transcript expression, with growth in the MFP as a determinant of&nbsp;M. leprae&nbsp;viability.</p>

<p>Methods:&nbsp;Receiver operating characteristic curve analyses were performed to determine viability cut-off values for&nbsp;M. leprae&nbsp;expression of&nbsp;hsp18,&nbsp;esxA, and 16S transcripts. To verify these values, immunocompetent BALB/c mice were inoculated in the footpads with a high dose of viable&nbsp;M. leprae. Footpads were harvested on Day 1 and at various timepoints up to 12 months post infection.&nbsp;M. leprae&nbsp;viability was determined by MVA or by sub-inoculating bacilli into passage mice per Shepard’s MFP assay.</p>

<p>Results:&nbsp;Cut-off levels for expression of each transcript for&nbsp;M. leprae&nbsp;viability were established with high confidence (CI = 99%,&nbsp;P&nbsp;&lt;&nbsp;0.0001). In the high-dose infection assay,&nbsp;M. leprae&nbsp;exhibited a significant decrease in transcript detection by three months post-infection. Expression of&nbsp;hsp18&nbsp;and&nbsp;esxA&nbsp;transcripts by&nbsp;M. leprae&nbsp;correlated with bacterial growth in the MFP assay. In contrast, 16S transcripts were detected in&nbsp;M.&nbsp;leprae&nbsp;populations confirmed dead by the MFP assay.</p>

<p>Conclusion:&nbsp;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&nbsp;M. leprae&nbsp;viability.</p>
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