Pathogen genotyping and cluster analysis are means of identifying both recent and unsuspected transmission, which can then be used to inform control programs on disease trends and aid in the implementation of corrective measures (Scott et al., 2004
). Longitudinal VNTR strain typing of M. leprae
in an endemic population, at first during a 43 month period (69 patients, though only 68 were previously reported) and extended by an additional 51 months (95 patients), has been informative and instructive at multiple levels. Though overall genetic diversity of M. leprae
in Qiubei is minimal, by using an appropriate selection of seven polymorphic VNTR loci we were able to identify clustered genotypes; track the temporal, spatial, and ethnogeographic distribution of leprosy; observe VNTR genotype evolution; and identify at-risk populations.
Within the sample size available for population level analysis, genotypes were not found to significantly differ based on clinical form or patient gender. Geographical clustering was evident, indicating that transmission continued to occur among people living within short distances of each other (in the same or neighboring township). Certain regional strains of M. leprae continued to present, suggesting a lack of adequate control in the infected populations. No major regional shifts in dominant alleles were seen between the two study periods. The majority of genotype clusters (19/21) had samples from both study periods, with multiple clusters (6/21) showing histories of longer than 2 years (). In particular, the B group in the North continued to expand from 2004 through 2010. In one patient, it was demonstrated that relapse, not reinfection, was the cause of disease recurrence after 5 years.
Strain typing was performed using a single skin biopsy from each patient as the source of M. leprae
. As the majority of MCFs demonstrated shared genotypes, this implies that these skin derived specimens represent the dominantly transmitted strain type, and perhaps the source of infection. Job et al. have shown that untreated multibacillary patients shed M. leprae
via their skin and nasal secretions, and that close contacts are at risk for exposure to the pathogen (Job et al., 2008
). Allelic differences have been seen across bacterial samples taken from dermal and nervous tissue (Young et al., 2004
; Young et al., 2008
), however the transmissibility of M. leprae
residing in nervous tissue remains to be clarified. Subtle genotypic shifts were seen in some related strains, such as the (AC)8a allele in members of MCFs F13 and F17 (). Of the seven loci mapped, the stutter prone locus (AT)15 (, ) had the greatest variability within MCFs and clusters.
The combination of epidemiological and molecular data was able to provide depth beyond what either data set could provide alone. The epidemiological data revealed a disparity in the leprosy burden across ethnic populations within Qiubei, with case detection rates of the Zhuang and Miao significantly higher than that of the Han majority (). We were able to capture a similar story with the molecular data; where, as an estimate of active transmission, the percent of clustered genotypes for both the Zhuang and Miao also surpassed the value for the Han majority (). Further, there were marked quantitative distinctions between the case detection rates and percent of clustered genotypes that extended the story. We found that although the detection rate among the Miao was higher than that of the Zhuang, the percent of clustered genotypes among the Miao was lower; thus implying a more fragmented transmission pattern among the Miao. Analogous to the molecular clusters identified by VNTR strain typing, MCFs represent contact linked clusters of infection. We found that the percent of MCFs among the Zhuang was similarly higher than for the Miao, reinforcing the conclusion of a more fragmented transmission pattern among the Miao. The underlying reason for the difference in transmission patterns is unclear, but it may be rooted in geographic, socioeconomic and cultural factors.
Genotypic clustering serves as a method to measure the temporal concomitance of transmission events. When overall clustering is high and individual clusters are large, a recent pathogen invasion may be implied (Tanaka and Francis, 2006
). In the townships of the North, where the Zhuang form the majority, genotypic clustering was found to be highest, and the largest observed supercluster, the A/B group, was found almost exclusively in the North. In the South, clusters were smaller and genotypes were more diverse, implying a more historical endemicity. Thus, the difference between the Zhuang and Miao transmission patterns may be a product of geography, with the underlying cause being a more recent introduction of leprosy in the Zhuang communities of the North. “Recent” must be interpreted cautiously, as it may refer to a time long before the beginning of this study.
Chen et al. found that within endemic regions of China the median delay between self-reported onset of symptoms and diagnosis is 23 months (Chen et al., 2000
). The long delay before treatment of symptomatic leprosy presents substantial opportunity for secondary transmission of M. leprae
. When socioeconomic development is slower, the delay in treatment is significantly longer, likely due to less accessible health care systems (Chen et al., 2000
). In the South, the pace of economic development is faster. As a consequence, communication and transportation infrastructure is superior to that of the North, and more employment opportunities outside farming exist, such as in the construction, service and administrative fields (personal communication). Therefore, a difference in treatment delay may also explain the difference in transmission patterns across ethnicities and regions (Shen et al., 2010
). Where health care accessibility may affect entire villages, as in the North, community clusters may dominate. Shen et al. compared the case finding methods in China and found that passive methods, such as skin clinics and referrals, contribute to fewer cases in Southwestern endemic provinces than in the less endemic Eastern provinces (52.2% vs
83.7%) (Shen et al., 2010
). In contrast, methods such as contact, clue and group surveys resulted in more detected cases. Just as with patient access to care, active case detection is dependent on adequate communication and transportation infrastructure. As such, regional differences in the efficiency of case finding methods may also exist in Qiubei. In the North, where cluster sizes are large, active case finding methods may need to be strengthened.
Another contributing factor may be local customs. Among the Zhuang in the North it is common for extended families to live in adjoined community housing structures, while for the Miao it is common for people to leave one place and move to another in search of suitable farm land (personal communication). Families that live in closely connected communities may share community strains, leading to higher percent clustering. Those families that choose to relocate may carry local strains with them, and promote a higher number of unclustered cases. This study has been based only in Qiubei and leprosy in adjacent counties has not been explored. In a few family histories there is knowledge of recent migration of patients or family members from Guangnan and Luxi counties. Women traditionally relocate to the spouse’s family after marriage, which may also contribute to the migration of people from one county or township to another, and promote the dispersal of M. leprae genotypes. Detailed family histories may have been illuminating, but were not undertaken, or feasible, for all patients. Further contributing culture factors may include language barriers between ethnicities and amenability to BCG vaccination (personal communication).
As seen by the overall concordance between molecular and epidemiologically derived conclusions, VNTR strain typing can estimate transmission patterns when patient history and demographic data are unavailable, though the greatest degree of resolution is found by combining molecular and patient history data. The findings in this study provide adequate validation for continued application of VNTRs, in contrast to the conclusions by Monot et al. that VNTRs may be too dynamic for use as epidemiological markers in leprosy (Monot et al., 2008
). Recognizing that homoplasy and convergent evolution of VNTR are certainly possible, this study shows that valuable information can be gained from longitudinal VNTR strain typing. When closely and continuously monitored, VNTR strain typing may be used to estimate the half-life of both strain types and individual alleles, and provide a means for surveillance of transmission. Other candidate M. leprae
microsatellite loci exist and exploration of these additional loci may identify more distant relationships among the currently unclustered strains (Zhang et al, 2005
). Furthermore, universally adopting FLA as the typing system would yield more rapid and economic typing of multiple samples, provide a consistent framework for comparing the results of different studies, permit multiplexing of loci, and allow for the detection of mixed allelic signatures (Kimura et al., 2009
). We conclude that VNTRs, when appropriately used, are sensitive tools for stain typing that have sufficient resolution at the regional and community level where SNP types may be invariant.