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

Davis GL, Ray NA, Lahiri R, Gillis TP, Krahenbuhl JL, Williams DL, Adams LB - PLoS Negl Trop Dis (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.

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.

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Related in: MedlinePlus

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 3×107M. 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 3×103M. 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.
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pntd-0002404-g004: 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 3×107M. 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 3×103M. 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.

Mentions: In agreement with the conventional viability assays, neither rifampin nor rifapentine at a single dose decreased M. leprae viability when assessed by hsp18 (Figure 4A) or esxA (Figure 4B) qRT-PCR. Control FPs expressed ∼105hsp18 or esxA equivalents per 3×103M. leprae. Five doses of rifapentine reduced this to 1.88×103±1.37×103hsp18 (p = 0.001) and 3.04×103±1.33×103esxA (p = 0.001) equivalents, while 5× rifampin showed no significant decrease in expression in either assay. However, these molecular assays demonstrated that both rifampin and rifapentine were highly effective at 20 doses when measured by hsp18 expression (p = 0.016 and p = 0.019, respectively) or esxA expression (p<0.001 and p = 0.009, respectively).


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

Davis GL, Ray NA, Lahiri R, Gillis TP, Krahenbuhl JL, Williams DL, Adams LB - PLoS Negl Trop Dis (2013)

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 3×107M. 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 3×103M. 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.
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Related In: Results  -  Collection

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pntd-0002404-g004: 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 3×107M. 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 3×103M. 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.
Mentions: In agreement with the conventional viability assays, neither rifampin nor rifapentine at a single dose decreased M. leprae viability when assessed by hsp18 (Figure 4A) or esxA (Figure 4B) qRT-PCR. Control FPs expressed ∼105hsp18 or esxA equivalents per 3×103M. leprae. Five doses of rifapentine reduced this to 1.88×103±1.37×103hsp18 (p = 0.001) and 3.04×103±1.33×103esxA (p = 0.001) equivalents, while 5× rifampin showed no significant decrease in expression in either assay. However, these molecular assays demonstrated that both rifampin and rifapentine were highly effective at 20 doses when measured by hsp18 expression (p = 0.016 and p = 0.019, respectively) or esxA expression (p<0.001 and p = 0.009, respectively).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.

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.

Show MeSH
Related in: MedlinePlus