Molecular Assays for Determining Mycobacterium leprae Viability in Tissues of Experimentally Infected Mice

Abstract

BackgroundThe 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 FindingsTwo 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.

Dodecameric structure of a small heat shock protein from Mycobacterium marinum M

“…26,27 In pathogens like Mycobacteria, sHSPs are virulence factors and form biomarkers for identification of the disease. 28 Based on amino acid sequence similarity, three classes of sHSPs are known in Mycobacteria: acr1, acr2, and acr3 29 . Different mycobacterial species harbor varying number of sHSPs.…”

GSMN-ML- a genome scale metabolic network reconstruction of the obligate human pathogenMycobacterium leprae

“…Enzymes for catabolism of hexoses and glycerol have been detected in M. leprae isolated from armadillos [38, 39], prompting us to perform the DPA growth simulations with both (independently) glucose and glycerol. RNA was extracted and immediately processed from in vivo -grown viable M. leprae harvested and from mouse footpads [21] (see Methodology for details). The control condition was M. leprae from mouse footpads, as above, but then incubated in axenic culture for 48 hours under conditions (see Methodology) where the bacilli remain viable but do not replicate.…”

“…At 4-5 months post inoculation mice were euthanized and footpad tissues removed. These tissues were either fixed in 70% ethanol for at least 48 h or minced and homogenized immediately for purification of viable M. leprae [21]: in vivo M. leprae . Viable bacteria were then also added to axenic medium 7H9 containing Caesitone (0.1% w/v), BSA (0.5% w/v), dextrose (0.75% w/v) and ampicillin (50µg/ml), and incubated at 33°C, 5% O 2 for 48h.…”

Tuberculosis and Leprosy

“…69 ). Recent reports indicate that measurements of RNA from M leprae extracted directly from biopsies may be used to assess viability, 95,96 but these assays are still only available in research settings. MDT for leprosy was recommended by a WHO committee in 1981, but has not been studied in a randomized, controlled trial.…”