Systematic Survey of Clonal Complexity in Tuberculosis at a Populational Level and Detailed Characterization of the Isolates Involved

Abstract

Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R؉E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R؉E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R؉E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS6110-based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.In recent years, the application of molecular tools such as IS6110-based restriction fragment length polymorphism (RFLP), spoligotyping, and mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) analysis has revealed that infection by Mycobacterium tuberculosis could be more complex than initially assumed. Several clonally complex phenomena have been observed, including mixed infections with more than one strain (29,30,35), simultaneous presence of clonal variants (microevolution phenomena) (1, 2), or even different distributions of strains/clonal variants infecting independent anatomical sites in the same patient (6,15).Most reports of clonal complexity phenomena are descriptions of anecdotal cases and/or studies of clonal complexity are often restricted to contexts where overexposure to tuberculosis (TB) can be expected. This makes it difficult to evaluate the frequen...

Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates

“…The rates of mixed infections ranged from 2.1 to 57.9% by 7 to 24 loci in MIRU-VNTR-based analysis in different regions (Table 5), and the rates of clonal heterogeneity were 1.3 to 9.3% (9,(25)(26)(27). One may speculate that the differences in the proportions of mixed infections are caused not only by differences in the methodology of detection, but they also might vary by geographic region.…”

Methodological and Clinical Aspects of the Molecular Epidemiology of Mycobacterium tuberculosis and Other Mycobacteria

“…Microevolution is not restricted to SNPs and may occur at any other mutation site, resulting in a change of the typing pattern. In M. tuberculosis, a difference of one band in the IS6110-RFLP pattern or one repeat unit in a single MIRU-VNTR locus does not necessarily mean that the individuals represent different strains (423,425). Importantly, even identical genotyping patterns may not reflect an actual transmission link.…”

Mixed-Strain Mycobacterium tuberculosis Infections and the Implications for Tuberculosis Treatment and Control

“…The timing of sputum collection relative to the initiation of treatment is also likely to affect the sensitivity of tests for mixed infections (59,78); ideally, pretreatment specimens would be collected, as heterogeneity should be more frequently detected in these unselected bacterial populations. In several cases, mixed-strain M. tuberculosis infections have been identified by using specimens sampled from multiple sites (e.g., lung, spleen, blood, and lymph nodes) (9,16,74), including specimens collected during autopsy (22,33). It is reasonable to assume that as the numbers and types of specimens included in studies are expanded, especially considering that some lineages may exhibit organ tropism (55), the sensitivity for the detection of mixed infections will increase.…”