Implementation of standardised multidrug regimens in the 1980s and 1990s has had a major impact on global leprosy prevalence, through shortening the duration of treatment. While it was reasoned that effective treatment of individual patients would reduce the spread of Mycobacterium leprae within communities, there is little evidence that the elimination programme has had a significant impact on disease incidence [1]. Continued controversies over global trends in the epidemiology of leprosy highlight gaps in our knowledge of the basic mechanisms of infection transmission and pathogenesis of this poorly understood disease [2]. While direct spread by aerosol or contact with infected individuals is thought to be the major route for dissemination of M. leprae, a role for zoonotic or environmental reservoirs cannot be excluded.

Publication of the genome sequence of an M. leprae isolate in 2001 revealed an organism which has undergone an extensive evolutionary process of gene decay [3]. Subsequent genetic studies have revealed that current human isolates of M. leprae from around the world show very little variation [4]. Identification of a limited number of informative single nucleotide polymorphisms (SNPs) allowed the elucidation of a high-level global phylogeny for M. leprae, but the extraordinary degree of sequence conservation has so far precluded application of SNP-based typing in the analysis of local epidemiology and mapping of transmission chains.

In contrast to the extreme conservation uncovered by SNP analysis, several researchers have reported a highly dynamic pattern of variation in copy number of short tandem repeat sequences in the M. leprae genome [5]–[8]. Using these loci, considerable variability was observed amongst panels of isolates within restricted geographical areas [5], [8]–[11]. In most, though not all, cases, the pattern of polymorphisms was conserved during passage in experimentally infected nude mice [12]. Inability to grow M. leprae in axenic culture has prevented quantitative measurement of the frequency in which changes in copy number are generated.

The aim of the present study was to assess whether differences in copy number of short tandem repeats provides information that is informative about the transmission of M. leprae. To evaluate this, we have applied molecular typing to leprosy patient biopsies collected in the course of epidemiological studies carried out in northern Malawi and in Hyderabad, India. This has allowed two different evaluations: firstly to assess the stability of the genotype within individuals; and secondly to assess transmission within leprosy multi-case households.

Our findings in the Malawi patients are consistent with previous publications demonstrating extensive polymorphism in the copy number of short tandem repeat sequences of M. leprae [5]–[8]. Comparison with the relative paucity of SNP polymorphisms suggests that M. leprae may have acquired a specific lesion in the mechanisms required for maintenance of fidelity during replication of repeat sequences, possibly as one of the consequences of the overall pattern of gene decay in this organism [18]. Comparison of different repeat loci indicates that changes are most extensive in the case of very short repeat motifs comprising only 2 or 3 base pairs. This is consistent with the reported absence of polymorphism in longer repeat sequences resembling the mycobacterial interspersed repeat units (MIRU) that have proved useful in typing of M. tuberculosis [18].

While we cannot exclude the possibility that changes in repeat copy number have potentially selectable phenotypic consequences, inspection of the predicted changes does not indicate any obvious biological significance. Three of the repeats are located outside of coding regions. The 6bp ML1505 locus introduces a variable number of Pro-Ala repeats within a conserved hypothetical protein; the 12bp ML1182 locus encodes a Glu-Val-Val-Glu repeat in a member of the PPE protein family; the ML0058 21bp repeat, ML2469 23bp repeat and the ML2418 25bp repeat are located in pseudogenes.

Analysis of multiple Malawi biopsies collected at the same time reproduced our previous finding among Indian patients [5], with significantly greater genotypic differences between M. leprae samples collected from skin and nerve than between multiple skin samples from the same patient (7/7 skin-nerve versus 1/7 skin-skin patients; p<0.01). This could indicate that the patients with more than one M. leprae genotype had been infected by more than one isolate, and that the isolates were tissue specific, or that progression of the infection results in expansion of different bacterial populations in different anatomical sites. To explore further the possibility that the genotype may change during disease progression, we analysed serial samples from four patients from Malawi. In no patient were the serial isolates identical. While again we cannot totally exclude the possibility of multiple infections, the variation in copy number observed over time suggests that the dominant genotype can undergo changes within a single individual.

The potential occurrence of genotypic variation within individual patients points to a need for considerable caution in any application of this type of analysis to tracking of transmission between individuals. On the other hand, when we analysed repeat copy numbers at three loci for individuals with a high probability of sharing a transmission link as a result of living in the same household, in Hyderabad, India, we observed a strong concordance in bacterial genotype. This is consistent with our earlier observation in this same population [5].

The homogeneity in genotypes between individuals within households in the Hyderabad series contrasts with the differences observed between individuals in Malawi, and also with the differences observed within individuals over time and between tissues in the Malawi series. We offer three comments on these patterns. First, as only three loci were examined in the Hyderabad series, versus five in the Malawi patients, our ability to detect differences was lower for the Indian than for the Malawian series. This may have increased the apparent homogeneity of the Hyderabad household sets. Second, the Indian series included more individuals towards the lepromatous pole than did the Malawi series. Perhaps the relatively unrestrained growth of M. leprae in lepromatous patients allows selection of dominant clones, and/or these patients were infected with large numbers of genetically identical bacilli within their household environments. Third, the Malawi patients were appreciably older than the Indian patients. Though tuberculoid disease is thought to have a shorter incubation period than lepromatous disease, this age difference, and the fact that leprosy has declined rapidly in Malawi in recent years [13],[14], means that the Malawian patients are likely to have been infected for longer than the Indian patients, which could explain the genetic divergence of the M. leprae populations between individuals. The fact that the two oldest patients in the Hyderabad series had isolates which differed from those of their household contacts is consistent with this.

Taken together, our findings suggest that genotyping of M. leprae on the basis of short tandem repeat copy numbers may provide insights into disease progression within individual patients and, when used with care, may assist in analysis of short and recent transmission chains. Current evidence indicates that it does not provide a robust assay to distinguish recent transmission from relapse, or reinfection from reactivation, in the way that molecular tools have proved useful for study of the epidemiology of tuberculosis. It remains possible that further repeat loci will be identified as having an intermediate stability suitable for wider transmission tracking.