Molecular epidemiology of Mycobacterium leprae: a solid beginning

Abstract

Until I was invited to present a workshop on phylogenetic methods at an IDEAL consortium meeting in March of 2007 I knew virtually nothing about Mycobacterium leprae. I was both astonished and awed as I learned about the many constraints that leprae investigators labour under; constraints that would drive most microbiologists and molecular biologists whimpering into a corner. The simple matter of growing a culture in order to prepare a sample of highly purified DNA for molecular strain typing purposes, something that most of us think of as a matter of taking a few hours, becomes a prodigious effort often requiring months to years – and the clonality of that culture is always uncertain. Likewise we take for granted the high level of genetic variability that permits the straight forward application of modern DNA technology to the problem of strain typing for epidemiological purposes. The apparent paucity of variability has forced leprae epidemiologists to turn to the variability that is inherent in short polynucleotide repeats (VNTR) as a basis of strain typing. At that March 2007 meeting it was evident that the field lacked a sufficient number of VNTR loci to reliably type strains or to estimate relationships among those strains. The variability in VNTR loci is with respect to the number of repeats of a short sequence, and the genotype of each allele is the repeat number. The papers in this special issue represent a concerted effort to provide a practical, reliable basis for molecular typing of M. leprae. The Gillis et al. paper identifies 16 potentially useful VNTR loci, provides a standard operating protocol (SOP) for determining the repeat number at each locus, and rigorously evaluates the reliability of each locus and its stability through time in a single infection. Six of the seven remaining papers employ that set of VNTR loci and apply the SOP in the field in six different countries as a practical trial of the value of those loci and the SOP in the field. Taken together, these papers represent both an amazing and an outstanding effort to create and validate the tools of a modern epidemiological system. The Gillis et al. paper sets rigorous standards for ‘certification’ of a locus and its SOP: the PCR conditions must permit reliable amplification, generating a full-length PCR product, from 10 cells, and the product must be able to be reliably sequenced. All 16 loci meet that standard. It turns out that determining the repeat number is not as simple as just reading the output sequence file. Slippage during the PCR amplification process can potentially result in