Enumeration of Mycobacterium leprae Using Real-Time PCR

James L Krahenbuhl,P Kyle Andrews,Richard W Truman,Thomas P Gillis,Pamela L C Small,Linda B Adams,Naoko Y Robbins

doi:10.1371/journal.pntd.0000328

Enumeration of Mycobacterium leprae Using Real-Time PCR

James L Krahenbuhl, P Kyle Andrews + Show 5 more

Open Access

https://doi.org/10.1371/journal.pntd.0000328

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Abstract

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae–infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.

Highlights

Because M. leprae can not be grown on synthetic media, the bacilli must be enumerated by direct microscopic counting
We describe the use of real-time PCR to provide a rapid, objective and consistent enumeration procedure for M. leprae
M. leprae were harvested from nude mouse foot pad tissues after infection for approximately 6 months

Summary

Introduction

Because M. leprae can not be grown on synthetic media, the bacilli must be enumerated by direct microscopic counting. Developed by Shepard [1] in the 1960’s, this technique has survived as the ‘‘gold standard’’ for enumerating M. leprae for almost 50 years It is a highly specialized procedure, cumbersome to perform and limited in terms of sensitivity and specificity. Various methods have been described to minimize error in direct microscopic counting of M. leprae, including the use of special slide coatings, staining procedures, and methods to calibrate microscopes [2,4]. These steps add to the complexity of the technique and the inherent insensitivity of the method requires that multiple samples be processed in large group sizes in order to reduce error. M. leprae cannot be differentiated from other acid-fast bacteria by microscopic examination alone, and clinical assessment of suspect biopsies requires that additional tests be applied when a mixed infection is suspected [5,6,7,8,9,10]

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