Extraction and detection of Mycobacterium leprae DNA from ZNCF-stained skin smear slides for better identification of negative skin smears

Rr, Kamble; Vs, Shinde; Madhale, S. P.; Kamble, AA; Ravikumar, BP; Rs, Jadhav

doi:10.4103/0255-0857.58732

Cited by 12 publications

(6 citation statements)

References 9 publications

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“…In a preliminary trial of 9 independent 3+ positive slides, DNA extraction was attempted by 2 methods: an enzymatic method ( 7 ) and a classical thermic lysis in a Chelex suspension (InstaGene; Bio-Rad, Hercules, CA, USA) ( 8 ). In our study, only the Chelex method produced good results, i.e., enabled us to obtain DNA that was successfully PCR amplified and produced a full spoligotyping pattern (results not shown).…”

Section: The Studymentioning

confidence: 99%

Spoligotyping ofMycobacterium africanum, Burkina Faso

Gomgnimbou

Refrégier²,

Diagbouga³

et al. 2012

Emerg. Infect. Dis.

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Section: The Studymentioning

confidence: 99%

Spoligotyping ofMycobacterium africanum, Burkina Faso

Gomgnimbou

Refrégier²,

Diagbouga³

et al. 2012

Emerg. Infect. Dis.

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“…DNA was extracted from skin biopsies using the previously described lysis protocol 41 . Briefly, 25 mg of tissue was manually homogenized, mixed with 1 ml of 1X phosphate-buffered saline (PBS), centrifuged at 8600g for 10 min at 4 °C and the pellet was dried for 1 hr.…”

Section: Methodsmentioning

confidence: 99%

Structural Implications of Mutations Conferring Rifampin Resistance in Mycobacterium leprae

Vedithi

Malhotra

Das

et al. 2018

Sci Rep

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The rpoB gene encodes the β subunit of RNA polymerase holoenzyme in Mycobacterium leprae (M. leprae). Missense mutations in the rpoB gene were identified as etiological factors for rifampin resistance in leprosy. In the present study, we identified mutations corresponding to rifampin resistance in relapsed leprosy cases from three hospitals in southern India which treat leprosy patients. DNA was extracted from skin biopsies of 35 relapse/multidrug therapy non-respondent leprosy cases, and PCR was performed to amplify the 276 bp rifampin resistance-determining region of the rpoB gene. PCR products were sequenced, and mutations were identified in four out of the 35 cases at codon positions D441Y, D441V, S437L and H476R. The structural and functional effects of these mutations were assessed in the context of three-dimensional comparative models of wild-type and mutant M. leprae RNA polymerase holoenzyme (RNAP), based on the recently solved crystal structures of RNAP of Mycobacterium tuberculosis, containing a synthetic nucleic acid scaffold and rifampin. The resistance mutations were observed to alter the hydrogen-bonding and hydrophobic interactions of rifampin and the 5′ ribonucleotide of the growing RNA transcript. This study demonstrates that rifampin-resistant strains of M. leprae among leprosy patients in southern India are likely to arise from mutations that affect the drug-binding site and stability of RNAP.

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“…Several studies have reported successes to detect M . leprae by PCR in SSS showing its clear advantage over SSS microcopy [8,16]. Therefore, the present study was carried on 50 new patients comparing the routine conventional SSS microscopy with PCR on SSS in the leprosy diagnosis.…”

Section: Discussionmentioning

confidence: 99%

“…The tube containing lysate was centrifuged 30 sec at 10,000 rpm, then vortex for 10–15 sec and again centrifuged for 30 sec. And then it was incubated at 60°C for overnight, then temperature was set at 94°C and the reaction was terminated at 94°C for 15 minutes [16,17]. Then purification of DNA by extraction with Phenol:Choloroform method was done as per the method described elsewhere [18].…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Polymerase Chain Reaction (PCR) with Slit Skin Smear Examination (SSS) to Confirm Clinical Diagnosis of Leprosy in Eastern Nepal

et al. 2016

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BackgroundDetection of Mycobacterium leprae in slit skin smear (SSS) is a gold standard technique for the leprosy diagnosis. Over recent years, molecular diagnosis by using PCR has been increasingly used as an alternative for its diagnosis due to its higher sensitivity. This study was carried out for comparative evaluation of PCR and SSS microscopy in a cohort of new leprosy cases diagnosed in B. P. Koirala Institute of health Sciences, Dharan, Nepal.Methodology/Principal FindingsIn this prospective crossectional study, 50 new clinically diagnosed cases of leprosy were included. DNA was extracted from SSS and PCR was carried out to amplify 129 bp sequence of M. leprae repetitive element. Sensitivity of SSS and PCR was 18% and 72% respectively. Improvement of 54% case detection by PCR clearly showed its advantage over SSS. Furthermore, PCR could confirm the leprosy diagnosis in 66% of AFB negative cases indicating its superiority over SSS. In the paucibacillary (PB) patients, whose BI was zero; sensitivity of PCR was 44%, whereas it was 78% in the multibacillary patients.Conclusions/SignificanceOur study showed PCR to be more sensitive than SSS microscopy in diagnosing leprosy. Moreover, it explored the characteristic feature of PCR which detected higher level of early stage(PB) cases tested negative by SSS. Being an expensive technique, PCR may not be feasible in all the cases, however, it would be useful in diagnosis of early cases of leprosy as opposed to SSS.

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Extraction and detection of Mycobacterium leprae DNA from ZNCF-stained skin smear slides for better identification of negative skin smears

Cited by 12 publications

References 9 publications

Spoligotyping ofMycobacterium africanum, Burkina Faso

Spoligotyping ofMycobacterium africanum, Burkina Faso

Structural Implications of Mutations Conferring Rifampin Resistance in Mycobacterium leprae

Evaluation of Polymerase Chain Reaction (PCR) with Slit Skin Smear Examination (SSS) to Confirm Clinical Diagnosis of Leprosy in Eastern Nepal

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