Rapid Variable-Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens

Mycobacterium leprae is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of M. leprae has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of M. leprae isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (n = 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the M. leprae genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (n = 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of M. leprae in different countries where leprosy is endemic.The causative pathogen of leprosy is Mycobacterium leprae. A continued incidence, defying global campaigns to eliminate leprosy even after years of rigorous case finding and the availability of multidrug therapy regimens (28, 29, 30, 31), is attributed to subclinical human and environmental reservoirs of the pathogen (1, 8, 13). In recent years, molecular strain-typing methodologies have complemented conventional infectious disease epidemiology. With the publication in 2001 of the complete genome sequence of an isolate from Tamil Nadu, India, called the TN strain (4), selection of potential polymorphic genomic markers for strain typing was feasible. The first genetic markers that showed polymorphism were short tandem repeats (STRs) in the M. leprae genome. One was a 6-bp intragenic sequence in the rpoT gene, and the second, a trinucleotide (TTC) repeat element upstream of a pseudogene (17, 23). These sequences exhibit variable numbers of tandem repeats (VNTRs) when sequenced in different isolates. Based on these observations, we short-listed 44 loci (including the rpoT and TTC loci) by in silico analyses of the M. leprae genome and accomplished the screening of 11 STR loci, of which 9 were polymorphic when tested in a small panel of four human isolates derived from passage through armadillos (6). Five were minisatellites (6- to 50-bp repeat units), and four were microsatellites (1- to 5-bp repeat units). Since then, others have also shown that VNTR loci exist in M. leprae isolates (25, 33, 34). Three single-nucleotide polymorphisms have also been discovered by comparing sequences of a limited number of strains (20).The goal of our work has been to discover and apply DNA variation among M. leprae isolates to identify sources and chains of transmission of leprosy in regions of endemicity. There are, however, physiological and practical issues relevant to strain typing of M. leprae in the clinical setting, such as the long incubation period and low transmissibility of leprosy and the requirement for clinical specimens such as slit skin smears and skin biopsy specimens from leprosy patients due to the inability of M. leprae to grow in culture. During the course of the last 4 years, field studies in which STR mapping was implemented have been reported. Matsuoka et al. (16) applied the microsatellite locus (TTC)21 to type M. leprae strains obtained from nasal swabs and slit skin smears from patients grouped by village, dwelling, or household in Indonesia, while Young et al. (33) combined (AT)15, (GTA)9, and (TTC)21 VNTR loci for the identification of short- and long-range M. leprae transmission chains in areas within and surrounding the city of Hyderabad, India. Monot et al. mapped five M. leprae STR loci in patients from Mali, Africa (19). The results from those studies demonstrated heterogeneity in prevalent haplotypes, indicating that genotype mapping with a small panel of one to five microsatellite VNTR loci was insufficient to discern strain relatedness. However, within an intrafamilial case, three markers were congruent (33). The authors of those studies concluded that in these areas of endemicity, multiple rather than single dominant isolates are found and that additional genomic markers are necessary for strain typing.For these reasons, assays for the amplification and differentiation of multiple genomic loci are needed. When these requirements have been met, it becomes possible to undertake systematic strain-typing studies that include suitable sampling strategies and conventional epidemiology methods for monitoring transmission and detecting clusters of cases. In light of these laboratory, field, and clinical issues, we further explored multiple-locus VNTR analysis (MLVA) techniques. In this paper, we report the development and testing of multiplex-PCR methods for MLVA for reference armadillo-derived M. leprae isolates and clinical materials and address allelic properties of individual loci and the reproducibility and feasibility of the techniques. In a future paper, we will apply and extend these methods to the data from population-based studies in Cebu, the Philippines (R. M. Sakamuri, M. Kimura, W. Li, K. Madanahally, H.-C. Kim, H. Lee, M. Balagon, R. Gelber, W. C. Black, S.-N. Cho, P. J. Brennan, and V. Vissa, submitted for publication).