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Volume - 80, Issue - 3

Editorial
Pages 236 - 245
Commentary
Pages 246 - 249
Original Papers
Pages 250 - 260
  • Characterisation of Short tandem repeats for genotyping Mycobacterium leprae

    • Thomas Gillis
    • Varalakshmi Vissa
    • Masanori Matsuoka
    • Saroj Young
    • Jan Hendrik Richardus
    • Richard Truman
    • Barry Hall
    • Patrick Brennan
    • false The Ideal Consortium Partners
    Volume 80, Issue 3

    | Published on September 2009

    Objective

    Establish a typing system for Mycobacterium leprae based on polymorphic DNA structures known as short tandem repeats (STR).

    Design

    Assess 16 polymorphic STR for sensitivity, specificity and reproducibility in standard assays using reference strains of M. leprae.

    Results

    Primers for 16 STR loci were selected based on PCR product size and for their ability to sequence each STR locus from both directions. All primer pairs produced a visible PCR amplicon of appropriate size from PCR reactions containing 10 M. leprae cells. DNA sequences for each STR locus, except (AT)15, was correctly identified as M. leprae-specific in replicate samples containing 1000 M. leprae using either the forward or reverse PCR primers. Twelve of 13 M. leprae STR loci were stable during passage in heavily infected armadillo tissues over a 5 year and 7 month infection cycle.

    Conclusions

    Certain M. leprae STR provide suitable targets for strain typing with the potential for grouping M. leprae with shared genotypes that may prove useful for establishing linkages between leprosy cases within geographical regions.

Original Papers
Pages 261 - 271
  • VNTR typing studies of Mycobacterium leprae in China: Assessment of methods and stability of markers during treatment

    • Yan Xing
    • Jian Liu
    • Rama Murthy Sakamuri
    • Zheng Wang
    • Yan Wen
    • Varalakshmi Vissa
    • Xiaoman Weng
    Volume 80, Issue 3

    | Published on September 2009

    Objective

    To evaluate the reliability and feasibility of two methods of multilocus variable number of tandem repeat analysis (MLVA) for strain typing of M. leprae, and to study whether short tandem repeat loci are stable and suitable for epidemiological study of leprosy.

    Methods

    Total DNA was extracted from skin biopsies of 20 new multibacillary (MB) patients from China diagnosed in 2006. To determine the copy numbers of short tandem repeats (STRs) for 13 loci, we amplified each locus individually by PCR, followed by sequence analysis of the amplicons. Separately, the same loci, plus four others were amplified by Multiplex PCRs (MP) using fluorescent primers and the copy number was identified by fragment length analysis (MP-FLA). MLVA was also performed at different times during treatment for a subset of the patients.

    Results and conclusions

    Genetic variability of M. leprae in China can be assessed in microsatellite loci. (GTA)9 and (TTC)21 loci are hypervariable, with array sizes of 25 repeat units or more. The expansion of the (GTA)9 locus is a characteristic of some M. leprae isolates in China. A high level of allele concordance was observed between PCR-sequencing and MP-FLA methods. However, MP-FLA method was cost- effective, rapid, high throughput and suitable for strain typing. Five of the 20 isolates of M. leprae were from patients residing in the same township in Qiubei County, Yunnan, and matched closely by MLVA. Three of these patients are family contacts of previously diagnosed patients, with intra-familial strain types being similar, suggesting infections from common sources and transmission chain(s). The VNTR patterns were highly similar in biopsy and slit skin smears (SSS) before treatment, and in the SSS collected at various time points during treatment. Taken together, VNTR strain typing is a useful tool for study of short range transmission in leprosy.

Original Papers
Pages 272 - 279
  • A continuation: study and characterisation of Mycobacterium leprae short tandem repeat genotypes and transmission of leprosy in Cebu, Philippines

    • Rama Murthy Sakamuri
    • Jordan Harrison
    • Robert Gelber
    • Paul Saunderson
    • Patrick J. Brennan
    • Marivic Balagon
    • Varalakshmi Vissa
    Volume 80, Issue 3

    | Published on September 2009

    Objective

    To study the stability and allelic diversity of tandem repeat loci in M. leprae in leprosy patients of Cebu, Philippines, and the suitability of multilocus variable number of tandem repeat (VNTR) analysis (MLVA) typing for detecting transmission.

    Methods

    Seventy newly diagnosed leprosy patients consulting at the Leonard Wood Memorial, Cebu Skin Clinic Total DNA was extracted from slit skin smear (SSS) scrapings of each patient and used for amplification of 13 M. leprae VNTR loci by single locus or multiplex PCR. Number of repeats for each VNTR locus was obtained by DNA sequencing or fragment length analysis methods. Medical, social and geographic details were included in the molecular epidemiology database.

    Results and conclusions

    Multiplex PCR (MP) and fragment length analysis (FLA) methods were found to be more efficient and accurate compared to single short tandem repeat (STR) amplification and DNA sequencing. Intra-patient MLVA patterns from four different samples were conserved in the minisatellites, while differences in one or more of the polymorphic and stutter prone microsatellites was observed, in four of five patients. The 13 loci could differentiate M. leprae strains in Cebu, however, MLVA patterns were stable enough during incubation and transmission between individuals within multi-case families. Thus M. leprae MLVA has potential for strain typing and transmission studies in Cebu.

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