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Genotypic Variation and Stability of Four Variable-Number Tandem Repeats and Their Suitability for Discriminating Strains of Mycobacterium leprae

Laboratory Research Branch, National Hansen's Disease Program at Louisiana State University, HRSA/BPHC, Baton Rouge, Louisiana 70894,¹ Leprosy Laboratory, Department of Tropical Medicine,² Laboratory of Molecular Biology and Diagnosis of Infectious Diseases, Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Fiocruz, Manguinhos 21045-900, Rio de Janeiro, Brazil³

Received 11 December 2003/ Returned for modification 17 February 2004/ Accepted 16 March 2004

It has not been possible to distinguish different strains of Mycobacterium leprae according to their genetic sequence. However, the genome contains several variable-number tandem repeats (VNTR), which have been used effectively in strain typing of other bacteria. To determine their suitability for differentiating M. leprae, we developed PCR systems to amplify 5 different VNTR loci and examined a battery of 12 M. leprae strains derived from patients in different regions of the United States, Brazil, Mexico, and the Philippines, as well as from wild armadillos and a sooty mangabey monkey. We found diversity at four VNTR (D = 0.74), but one system (C₁₆G₈) failed to yield reproducible results. Alleles for the GAA VNTR varied in length from 10 to 16 copies, those for AT₁₇ varied in length from 10 to 15 copies, those for GTA varied in length from 9 to 12 copies, and those for TA₁₈ varied in length from 13 to 20 copies. Relatively little variation was seen with interspecies transfer of bacilli or during short-term passage of strains in nude mice or armadillos. The TA₁₈ locus was more polymorphic than other VNTR, and genotypic variation was more common after long-term expansion in armadillos. Most strain genotypes remained fairly stable in passage, but strain Thai-53 showed remarkable variability. Statistical cluster analysis segregated strains and passage samples appropriately but did not reveal any particular genotype associable with different regions or hosts of origin. VNTR polymorphisms can be used effectively to discriminate M. leprae strains. Inclusion of additional loci and other elements will likely lead to a robust typing system that can be used in community-based epidemiological studies and select clinical applications.

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