02760nas a2200385   4500000000100000008004100001260001300042653001200055653002300067653001600090653001900106653001100125653001200136653001600148653000900164653002400173653002500197653003000222653002400252653002500276100001600301700001300317700001500330700001500345700001400360700001300374700001600387245008700403856007200490300001200562490000700574050001800581520176100599022001402360       2009                            d  c2009 Jul10aAnimals10aBacterial Proteins10aDNA Primers10aDNA, Bacterial10aHumans10aleprosy10aMacrophages10aMice10aMicrobial Viability10aMycobacterium leprae10apolymerase chain reaction10aRNA, Ribosomal, 16S10aSuperoxide Dismutase1 aMartinez AN1 aLahiri R1 aPittman TL1 aScollard D1 aTruman RW1 aMoraes M1 aWilliams DL00aMolecular determination of Mycobacterium leprae viability by use of real-time PCR.  uhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708532/pdf/0512-09.pdf  a2124-300 v47  aMARTINEZ 20093 a<p>Mycobacterium leprae, the etiological agent of leprosy, is noncultivable on axenic media. Therefore, the viability of M. leprae for clinical or experimental applications is often unknown. To provide new tools for M. leprae viability determination, two quantitative reverse transcriptase PCR (RT-PCR) assays were developed and characterized. M. leprae sodA mRNA and 16S rRNA were used as RNA targets, and M. leprae repetitive element (RLEP) DNA was used to determine relative bacterial numbers in the same purified bacterial preparations or from crude biological specimens. Results demonstrated that both assays were good predictors of M. leprae viability during short-term experiments (48 h) involving rifampin (rifampicin) treatment in axenic medium, within rifampin-treated murine macrophages (MPhi), or within immune-activated MPhi. Moreover, these results strongly correlated those of other M. leprae viability assays, including radiorespirometry-based and Live/Dead BacLight viability assays. The 16S rRNA/RLEP assay consistently identified the presence of M. leprae in eight multibacillary leprosy patient biopsy specimens prior to multidrug therapy (MDT) and demonstrated a decline in viability during the course of MDT. In contrast, the sodA/RLEP assay was able to detect the presence of M. leprae in only 25% of pretreatment biopsy specimens. In conclusion, new tools for M. leprae viability determination were developed. The 16S rRNA/RLEP RT-PCR M. leprae viability assay should be useful both for short-term experimental purposes and for predicting M. leprae viability in biopsy specimens to monitor treatment efficacy, whereas the sodA/RLEP RT-PCR M. leprae viability assay should be limited to short-term experimental research purposes.</p>  a1098-660X