VNTR typing studies of Mycobacterium leprae in China: assessment of methods and stability of markers during treatment.

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.

[Study on genotyping of Mycobacterium leprae and families with multi-cases].

OBJECTIVE: Multiple locus variable number-tandem repeat (VNTR) analysis (MLVA) had been proposed as a means of strain typing for tracking of source and studying the transmission chain of pathogens. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy. METHODS: MLVA on 7 VNTR loci was applied to the strain typing on prevalent Mycobacterium leprae isolates collected from Qiubei county, Yunnan province during 2002-2006 in the study on the relationship between geographic distribution and genotypes of M. leprae. The strain typing, combined with conventional epidemiological investigation was performed to trace the transmission chain. RESULTS: (1) Phylogenetic analyses through application of PAUP 4.0, The M. leprae were grouped into A, B, C, D and E strains according to the allelic range 9, 11-13, 15-26 and > 26 on the GTA9 locus. The strains with 9 copies on GTA9 locus, was named A. (2) Genotypes of strains from the five multi-case families located at North and North-West parts were similar and belonged to A strains. VNTR patterns of intra-family were identical or similar but not identical inter-family. (3) Not only A cluster appeared higher proportion in total isolates but also distributes cluster, indicating ongoing transmission from recent findings. CONCLUSION: VNTR strain typing was suitable to trace the short chain of transmission in both small area and intra-families. Multi-case families might constitute epidemic foci and source of M. leprae in villages, causing the predominant strain or cluster which tends to be those identified in multi-case families and resulted in the spreading of leprosy. A long-term study was underway to reveal whether A strain was predominant strain and to observe the evolution of M. leprae in this spatially and temporally defined endemic population.

Identification and distribution of Mycobacterium leprae genotypes in a region of high leprosy prevalence in China: a 3-year molecular epidemiological study.

Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been proposed as a means of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population are lacking. To this end, a study was initiated to assess the diversity and distribution of prevalent Mycobacterium leprae strains in Qiubei County, Yunnan Province, People's Republic of China, where the annual detection rate of leprosy is 10-fold higher than the national average rate. Sixty-eight newly diagnosed leprosy patients were included in the study. MLVA at eight M. leprae loci was applied using DNA extracts from skin biopsies. The number of alleles per locus ranged from 4 to 24, providing adequate strain discrimination. MLVA strain typing identified several clusters of patients whose M. leprae specimens shared similar VNTR profiles. Two of these clusters were comprised of patients who resided predominantly in the north and northwest parts of Qiubei County. Furthermore, it was found that multicase families are common in this county: 23 of the 68 patients were from 11 families. Intrafamilial VNTR profiles closely matched within six families, although they were different between the families. Moreover, VNTR patterns related to those found in some multicase families were also detected in patients in the same or adjacent townships, indicating the utility of VNTR strain typing to identify and detect short-range transmission events. Social contact through village markets is proposed as a means of transmission.