Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice

Abstract

Background: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues.

Methodology/principle findings: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations.

Conclusions: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.

Figure 1. Enumeration and quantification of M. leprae in mouse FPs using conventional assays.

BALB/c (gray bars) and athymic nu/nu (black bars) mice were infected in the FPs with 3×10⁷ M. leprae. Bacilli were harvested on day 1 and at 4, 8, 12, and 17 weeks post infection and enumerated by direct counting (A). Viability of M. leprae was determined by BacLight viability staining (B) and RR (C). Dotted line represents the lower detection limit of the assay. Bars represent mean and standard deviation for each group. The value at each time point was compared to its respective value at 1 day. * = probability of statistical significance (p)<0.05, ** = probability of statistical significance (p)<0.01, and *** = probability of statistical significance (p)<0.001.

Figure 2. Enumeration and quantification of M. leprae in mouse FPs by molecular assays.

BALB/c (gray bars) and athymic nu/nu (black bars) mice were infected in with 3×10⁷ M. leprae. FP tissues were fixed in 70% ethanol on day 1 and at 4, 8, 12, and 17 weeks post infection. DNA and RNA were purified using a FastPrep protocol. M. leprae were enumerated by RLEP PCR on the DNA fraction (A). cDNA was prepared from an RNA equivalent of 3×10³ M. leprae for determination of viability by hsp18 (B) and esxA (C) qRT-PCR. Bars represent mean and standard deviation for each group. The value at each time point was compared to its respective value at 1 day. * = probability of statistical significance (p)<0.05, ** = probability of statistical significance (p)<0.01, and *** = probability of statistical significance (p)<0.001.

Figure 3. Enumeration and quantification using conventional assays of M. leprae from RMP and RPT treated mice.

Athymic nu/nu (black bars) mice were infected in the FPs with 3×10⁷ M. leprae. At 18 weeks post infection, groups of mice were treated with 1 dose (1×), 5 daily doses (5×), or 20 daily doses (20×) of RMP (striped bars) or RPT (crosshatched bars), each at 10 mg/kg. Control mice received vehicle alone (gray bars). One month after the last dose of each regimen, bacilli were harvested and viability of M. leprae was determined by BacLight viability staining (A) and RR (B). Dotted line represents the lower detection limit of the assay. Bars represent mean and standard deviation for each group. * = probability of statistical significance (p)<0.05, and *** = probability of statistical significance (p)<0.001.

Figure 4. Enumeration and quantification via molecular assays of M. leprae from RMP and RPT treated mice.

Athymic nu/nu (black bars) mice were infected in the FPs with 3×10⁷ M. leprae. At 18 weeks post infection, groups of mice were treated with 1 dose (1×), 5 daily doses (5×), or 20 daily doses (20×) of RMP (striped bars) or RPT (crosshatched bars), each at 10 mg/kg. Control mice received vehicle alone (gray bars). One month after the last dose of each regimen, FP tissues were fixed in 70% ethanol. DNA and RNA were purified using a FastPrep protocol. M. leprae were enumerated by RLEP PCR on the DNA fraction, and cDNA was prepared from an RNA equivalent of 3×10³ M. leprae for determination of viability by hsp18 (A) and esxA (B) qRT-PCR. Bars represent mean and standard deviation for each group. * = probability of statistical significance (p)<0.05, ** = probability of statistical significance (p)<0.01, and *** = probability of statistical significance (p)<0.001. 5× RPT vs. 20× RPT: (p) = 0.396 for hsp18; (p) = 0.569 for esxA.