Mycobacterium tuberculosis transmission in a country with low tuberculosis incidence: role of immigration and HIV infection

Immigrants from high-burden countries and HIV-coinfected individuals are risk groups for tuberculosis (TB) in countries with low TB incidence. Therefore, we studied their role in transmission of Mycobacterium tuberculosis in Switzerland. We included all TB patients from the Swiss HIV Cohort and a sample of patients from the national TB registry. We identified molecular clusters by spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis and used weighted logistic regression adjusted for age and sex to identify risk factors for clustering, taking sampling proportions into account. In total, we analyzed 520 TB cases diagnosed between 2000 and 2008; 401 were foreign born, and 113 were HIV coinfected. The Euro-American M. tuberculosis lineage dominated throughout the study period (378 strains; 72.7%), with no evidence for another lineage, such as the Beijing genotype, emerging. We identified 35 molecular clusters with 90 patients, indicating recent transmission; 31 clusters involved foreign-born patients, and 15 involved HIV-infected patients. Birth origin was not associated with clustering (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 0.73 to 3.43; P = 0.25, comparing Swiss-born with foreign-born patients), but clustering was reduced in HIV-infected patients (aOR, 0.49; 95% CI, 0.26 to 0.93; P = 0.030). Cavitary disease, male sex, and younger age were all associated with molecular clustering. In conclusion, most TB patients in Switzerland were foreign born, but transmission of M. tuberculosis was not more common among immigrants and was reduced in HIV-infected patients followed up in the national HIV cohort study. Continued access to health services and clinical follow-up will be essential to control TB in this population.

"Pseudo-Beijing": evidence for convergent evolution in the direct repeat region of Mycobacterium tuberculosis

Background Mycobacterium tuberculosis has a global population structure consisting of six main phylogenetic lineages associated with specific geographic regions and human populations. One particular M. tuberculosis genotype known as “Beijing” has repeatedly been associated with drug resistance and has been emerging in some parts of the world. “Beijing” strains are traditionally defined based on a characteristic spoligotyping pattern. We used three alternative genotyping techniques to revisit the phylogenetic classification of M. tuberculosis complex (MTBC) strains exhibiting the typical “Beijing” spoligotyping pattern. Methods and Findings MTBC strains were obtained from an ongoing molecular epidemiological study in Switzerland and Nepal. MTBC genotyping was performed based on SNPs, genomic deletions, and 24-loci MIRU-VNTR. We identified three MTBC strains from patients originating from Tibet, Portugal and Nepal which exhibited a spoligotyping patterns identical to the classical Beijing signature. However, based on three alternative molecular markers, these strains were assigned to Lineage 3 (also known as Delhi/CAS) rather than to Lineage 2 (also known as East-Asian lineage). Sequencing of the RD207 in one of these strains showed that the deletion responsible for this “Pseudo-Beijing” spoligotype was about 1,000 base pairs smaller than the usual deletion of RD207 in classical “Beijing” strains, which is consistent with an evolutionarily independent deletion event in the direct repeat (DR) region of MTBC. Conclusions We provide an example of convergent evolution in the DR locus of MTBC, and highlight the limitation of using spoligotypes for strain classification. Our results indicate that a proportion of “Beijing” strains may have been misclassified in the past. Markers that are more phylogenetically robust should be used when exploring strain-specific differences in experimental or clinical phenotypes.

Bacillus anthracis: Molecular taxonomy, population genetics, phylogeny and patho-evolution

Bacillus anthracis, the etiological agent of anthrax, manifests a particular bimodal lifestyle. This bacterial species alternates between short replication phases of 20-40 generations that strictly require infection of the host, normally causing death, interrupted by relatively long, mostly dormant phases as spores in the environment. Hence, the B. anthracis genome is highly homogeneous. This feature and the fact that strains from nearly all parts of the world have been analysed for canonical single nucleotide polymorphisms (canSNPs) and variable number tandem repeats (VNTRs) has allowed the development of molecular epidemiological and molecular clock models to estimate the age of major diversifications in the evolution of B. anthracis and to trace the global spread of this pathogen, which was mostly promoted by movement of domestic cattle with settlers and by international trade of contaminated animal products. From a taxonomic and phylogenetic point of view, B. anthracis is a member of the Bacillus cereus group. The differentiation of B. anthracis from B. cereus sensu strict, solely based on chromosomal markers, is difficult. However, differences in pathogenicity clearly differentiate B. anthracis from B. cereus and are marked by the strict presence of virulence genes located on the two virulence plasmids pXO1 and pXO2, which both are required by the bacterium to cause anthrax. Conversely, anthrax-like symptoms can also be caused by organisms with chromosomal features that are more closely related to B. cereus, but which carry these virulence genes on two plasmids that largely resemble the B. anthracis virulence plasmids. (C) 2011 Elsevier B.V. All rights reserved.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

Standard genotyping overestimates transmission of Mycobacterium tuberculosis among immigrants in a low incidence country.

Immigrants from high tuberculosis (TB) incidence regions are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520 Mycobacterium tuberculosis isolates from 2000-2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-loci MIRU-VNTR and spoligotyping). Here, we used whole genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of clusters involving only foreign-born patients. The overall clustering proportion using standard genotyping was 17% (90 patients, 95% confidence interval [CI]: 14-21%), but only 8% (43 patients, 95% CI: 6-11%) using WGS. The clustering proportion was 17% (67/401, 95% CI: 13-21%) using standard genotyping and 7% (26/401, 95% CI: 4-9%) using WGS among foreign-born patients, and 19% (23/119, 95% CI: 13-28%) and 14% (17/119, 95% CI: 9-22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence for an association between birth origin and transmission (aOR 2.2, 95% CI: 0.9-5.5, comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland when compared to WGS, particularly among immigrants from high TB incidence regions, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in low TB incidence settings.