Mixed tuberculosis infections in rural South Vietnam

Tuberculosis patients may be infected with or have disease caused by more than one Mycobacterium tuberculosis strain, usually referred to as "mixed infections." These have mainly been observed in settings with a very high tuberculosis incidence and/or high HIV prevalence. We assessed the rate of mixed infections in a population-based study in rural Vietnam, where the prevalences of both HIV and tuberculosis are substantially lower than those in previous studies looking at mixed infections. In total, 1,248 M. tuberculosis isolates from the same number of patients were subjected to IS6110 restriction fragment length polymorphism (RFLP) typing, spoligotyping, and variable-number-tandem-repeat (VNTR) typing. We compared mixed infections identified by the presence of (i) discrepant RFLP and spoligotype patterns in isolates from the same patient and (ii) double alleles at ≥ 2 loci by VNTR typing and assessed epidemiological characteristics of these infections. RFLP/spoligotyping and VNTR typing identified 39 (3.1%) and 60 (4.8%) mixed infections, respectively (Cohen's kappa statistic, 0.57). The number of loci with double alleles in the VNTR pattern was strongly associated with the proportion of isolates with mixed infections according to RFLP/spoligotyping (P < 0.001). Mixed infections occurred more frequently in newly treated than in previously treated patients, were significantly associated with minor X-ray abnormalities, and were almost significantly associated with lower sputum smear grades. Although the infection pressure in our study area is lower than that in previously studied populations, mixed M. tuberculosis infections do occur in rural South Vietnam in at least 3.1% of cases.     

Genetic diversity of Mycobacterium tuberculosis in Sicily based on spoligotyping and variable number of tandem DNA repeats and comparison with a spoligotyping database for population-based analysis

In a previous study, we proposed to associate spoligotyping and typing with the variable number of tandem DNA repeats (VNTR) as an alternative strategy to IS6110-restriction fragment length polymorphism (RFLP) for molecular epidemiological studies on tuberculosis. The aim of the present study was to further evaluate this PCR-based typing strategy and to describe the population structure of Mycobacterium tuberculosis in another insular setting, Sicily. A collection of 106 DNA samples from M. tuberculosis patient isolates was characterized by spoligotyping and VNTR typing. All isolates were independently genotyped by the standard IS6110-RFLP method, and clustering results between the three methods were compared. The totals for the clustered isolates were, respectively, 15, 60, and 82% by IS6110-RFLP, spoligotyping, and VNTR typing. The most frequent spoligotype included type 42 that missed spacers 21 to 24 and spacers 33 to 36 and derived types 33, 213, and 273 that, together represented as much as 26% of all isolates, whereas the Haarlem clade of strains (types 47 and 50, VNTR allele 32333) accounted for 9% of the total strains. The combination of spoligotyping and VNTR typing results reduced the number of clusters to 43% but remained superior to the level of IS6110-RFLP clustering (ca. 15%). All but one IS6110-defined cluster were identified by the combination of spoligotyping and VNTR clustering results, whereas 9 of 15 spoligotyping-defined clusters could be further subdivided by IS6110-RFLP. Reinterpretation of previous IS6110-RFLP results in the light of spoligotyping-VNTR typing results allowed us to detect an additional cluster that was previously missed. Although less discriminative than IS6110-RFLP, our results suggest that the use of the combination of spoligotyping and VNTR typing is a good screening strategy for detecting epidemiological links for the study of tuberculosis epidemiology at the molecular level.     

Molecular analysis of clinical isolates of Mycobacterium bovis recovered from humans in Italy

In order to achieve a better knowledge of Mycobacterium bovis epidemiology in Italy, 42 clinical isolates from humans were genotyped. Predominant molecular patterns were found in one cluster of 15 isolates sharing spoligotype (ST482), variable-number tandem repeat (VNTR), and IS6110-based restriction fragment length polymorphism (one 1.9-kb band) profiles and in two clusters of 6 and 3 Mycobacterium bovis BCG isolates differing by one VNTR character. The remaining 18 isolates yielded unique profiles. Our results confirm the potential utility of spoligotyping and VNTR typing as a major typing system of M. bovis isolates.     

Genotypic analysis of Mycobacterium tuberculosis in Bangladesh and prevalence of the Beijing strain

Genotypic analysis was performed on 48 Mycobacterium tuberculosis complex strains collected from a hospital in Dhaka city. Deletion analysis showed that the isolates were all M. tuberculosis; 13 of them were found to be of the "ancestral" type, while 35 were of the "modern" type, indicating that both endemic (ancestral type) and epidemic (modern type) strains cause tuberculosis in Bangladesh. Genotyping based on the spoligotype and variable-number tandem repeats (VNTR) of mycobacterial interspersed repetitive units (MIRU) was also done. A total of 34 strains (71%) were grouped by spoligotyping into nine different clusters; the largest comprised 15 isolates of the Beijing genotype, whereas the remaining eight clusters consisted of two to five isolates. MIRU-VNTR typing detected 32 different patterns among 44 tested strains, and the 15 Beijing strains were further discriminated by MIRU-VNTR typing (7 distinct patterns for the 15 isolates). These results indicate that MIRU-VNTR typing, along with spoligotyping and deletion analysis, can be used effectively for molecular epidemiological studies to determine ongoing transmission clusters; to our knowledge, this is the first report about the type of strains prevailing in Bangladesh.     

First molecular epidemiology study of Mycobacterium tuberculosis in Burkina Faso

We conducted a molecular epidemiology study on 120 Mycobacterium tuberculosis isolates from patients presenting pulmonary tuberculosis (TB) in Burkina Faso. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture. Molecular analysis of specific signatures showed that all TB cases reported in this study were caused by M. tuberculosis and identified no Mycobacterium bovis or Mycobacterium africanum isolates. This result is unexpected, as M. africanum strains were reportedly the etiologic agent in 20% of TB cases 2 decades ago. The comparison of spoligotypes from Burkina Faso with an international spoligotype database (SpolDB4) showed that the majority of isolates belong to major clades of M. tuberculosis (Haarlem, 9%; Latin American-Mediterranean, 30%; and T, 20%). The predominant group of isolates (30%) corresponds to spoligotype 61, described in Cameroon as the "Cameroon family." In Burkina Faso, as in Cameroon, this family could be associated with recent transmission of TB, suggesting a recent expansion in West Africa. Our data suggest a low level of primary drug resistance that may be a positive result of the Directly Observed Therapy Shortcourse program. Besides, based on spoligotyping plus MIRU-VNTR, data showed a high number of clusters in our sample, suggesting a high level of recent TB transmission in Burkina Faso. Nevertheless, an important genetic polymorphism was observed in this country, reflecting an endemicity situation where the control of TB would have less impact in the main towns.     

Epidemiological study of tuberculosis in the area of Angers,France, as studied by 3 PCR-based fingerprinting methods

The new genotyping methods efficiently complement classical epidemiological investigation in order to attempt a global approach to TB control. In the present work, we have studied the genomic diversity of Mycobacterium tuberculosis isolated during the year 1998 within the district of Angers, France (260,000 inhabitants distributed in 29 districts), in order to identify recent transmission events and any related risk factors. The methods used included "spacer oligonucleotide typing" or spoligotyping, "variable number of DNA tandem repeats" or VNTR, and "double repetitive element PCR" or DRE-PCR. The resulting spoligotyping and VNTR results were also feeded to international databases and compared with >10,000 isolates for spoligotyping and 500 isolates for VNTR, representative of about 60 countries. The results obtained underlined that most of the TB cases in our setting probably reflected reactivation cases, as clustered cases indicative of potential events of recent transmission were rare. Furthermore, interrogation of international databases showed that most of the isolates from the Angers region belonged to major conserved families of TB isolates representative of Europe, with only rare cases of Asian origin, or those previously reported in specific epidemies reported from elsewhere.     

Genetic diversity of Mycobacterium tuberculosis in Mbarara, South Western Uganda

Background

We determined the genetic diversity of mycobacteria isolated from tuberculosis patients in Mbarara Uganda, using region of difference (RD) analysis and spacer oligonucleotide typing (spoligotyping).

Methods

Sputum samples were cultured on Lowenstein Jensen media. The isolates were characterized using RD analysis and spoligotyping.

Results

The majority (92.8%) of the patients were new cases, 60% were males and 44% were HIV positive with a mean age of 33.7 years. All the 125 isolates were identified as M.tuberculosis sensu stricto. Most (92.8%) of the isolates were modern strains. Spoligotyping revealed 79 spoligotype patterns, with an overall diversity of 63.2%. Sixty (48%) isolates formed 16 clusters each consisting of 2–15 isolates. Mst (59.2 %) of the isolates were Uganda genotype strains. The major shared spoligotypes in our sample were SIT 135 (T2-Uganda) with 12 isolates and SIT 128 (T2) with 5 isolates. Sixty nine (87%) patterns had not yet been defined in the SpolDB4.0.database.

Conclusion

The TB epidemic in Mbarara is caused mainly by modern M.tuberculosis strains of the Uganda genotype. The wide diversity of strains may indicate that the majority of the TB cases are reactivation rather than re-infection. However this needs to be ascertained with more discriminative finger printing techniques.     

Development of variable-number tandem repeat typing of Mycobacterium bovis: comparison of results with those obtained by using existing exact tandem repeats and spoligotyping

Various genetic markers have been exploited for fingerprinting the Mycobacterium tuberculosis complex (MTBC) in molecular epidemiological studies, mainly through identifying restriction fragment length polymorphisms (RFLP). In large-scale studies, RFLP typing has practical processing and analysis limitations; therefore, attempts have been made to move towards PCR-based typing techniques. Spoligotyping (spacer oligotyping) and, more recently, variable-number tandem repeat (VNTR) typing have provided PCR-derived typing techniques. This study describes the identification and characterization of novel VNTR loci, consisting of tandem repeats in the size range of 53 to 59 bp in the MTBC, and their assessment as typing tools in 47 Mycobacterium bovis field isolates and nine MTBC strains. Spoligotyping and the previously described set of exact tandem repeats (ETRs) (R. Frothingham and W. A. Meeker-O'Connell, Microbiology 144:1189-1196, 1998) were also applied to the same panel of isolates. The allelic diversity of the individual VNTR loci was calculated, and a comparison of the novel VNTRs was made against the results obtained by spoligotyping and the existing set of ETRs. Eleven unique spoligotypes were discriminated in the panel of 47 M. bovis isolates. Greater resolution was obtained through the combination of the most-discriminating VNTRs from both sets. Considerable discrimination was achieved, with the 47 M. bovis isolates resolved into 14 unique profiles, while all nine MTBC isolates were uniquely differentiated. The novel VNTR markers described increased the discrimination possible in strain typing of M. bovis, with the added benefit of an intuitive digital nomenclature, with the allele copy number of the individual VNTRs providing a profile. VNTR typing was shown to be a valuable technique with great potential for further development and application to epidemiological tracing of tuberculosis transmissions.     

Molecular characterization and drug resistance patterns of strains of Mycobacterium tuberculosis isolated from patients in an AIDS counseling center in Port-au-Prince,Haiti: a 1-year study

Tuberculosis (TB) is one of the most common opportunistic diseases that appear among human immunodeficiency virus (HIV)-positive patients in Haiti. In this context the probable emergence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis is of great epidemiological concern. However, as routine culture of M. tuberculosis and drug susceptibility testing are not performed in Haiti, it has not been possible so far to evaluate the rate of drug resistance among M. tuberculosis isolates from circulating TB cases. This report describes the first study on the molecular typing and drug resistance of M. tuberculosis isolates from patients with culture-positive pulmonary tuberculosis monitored at the GHESKIO Centers in Haiti during the year 2000. Clinical, epidemiological, and drug susceptibility testing results were available for 157 patients with confirmed cases of TB, with a total of 8.9% of patients harboring MDR M. tuberculosis. A significant association between the occurrence of resistance and previous TB treatment was observed (P < 0.001), suggesting that a previous history of TB treatment was a risk factor associated with MDR TB in Haiti. The DNAs of individual isolates from 106 samples were available and were typed by spoligotyping and determination of the variable number of tandem DNA repeats. Both typing methods provided interpretable results for 96 isolates, and the clusters observed were further confirmed by ligation-mediated PCR to define potential cases of active transmission. Thirty-three (34%) of the isolates were found to be grouped into 11 clusters with two or more identical patterns. However, an assessment of risk factors (sex, HIV positivity, previous treatment, drug resistance) showed that none was significantly associated with the active transmission of TB. These observations suggest that acquired MDR TB is prevalent in Haiti and may be associated with compliance issues during TB treatment since prior TB therapy is the strongest risk factor associated with MDR TB. Prevention of TB transmission in Haiti should target active case investigation, routine detection of drug resistance, and adequate treatment of patients. The use of directly observed short-course therapy should be enforced throughout the country; and relapses, reactivations, or newly acquired infections should be discriminated by genotyping methods.     

Molecular and conventional epidemiology of Mycobacterium tuberculosis in Botswana: a population-based prospective study of 301 pulmonary tuberculosis patients

Little is known about patterns of tuberculosis (TB) transmission among populations in developing countries with high rates of TB and human immunodeficiency virus (HIV) infection. To examine patterns of TB transmission in such a setting, we performed a population-based DNA fingerprinting study among TB patients in Botswana. Between January 1997 and July 1998, TB patients from four communities in Botswana were interviewed and offered HIV testing. Their Mycobacterium tuberculosis isolates underwent DNA fingerprinting using IS6110 restriction fragment length polymorphism, and those with matching fingerprints were reinterviewed. DNA fingerprints with >5 bands were considered clustered if they were either identical or differed by at most one band, while DNA fingerprints with < or =5 bands were considered clustered only if they were identical. TB isolates of 125 (42%) of the 301 patients with completed interviews and DNA fingerprints fell into 20 different clusters of 2 to 16 patients. HIV status was not associated with clustering. Prior imprisonment was the only statistically significant risk factor for clustering (risk ratio, 1.5; 95% confidence interval, 1.1 to 2.0). In three communities where the majority of eligible patients were enrolled, 26 (11%) of 243 patients overall and 26 (25%) of 104 clustered patients shared both a DNA fingerprint and strong antecedent epidemiologic link. Most of the increasing TB burden in Botswana may be attributable to reactivation of latent infection, but steps should be taken to control ongoing transmission in congregate settings. DNA fingerprinting helps determine loci of TB transmission in the community.     

Evaluation and genotyping of multidrug-resistant cases of tuberculosis in southern Brazil

Sixty isolates of Mycobacterium tuberculosis identified as multidrug-resistant (MDR) at a reference laboratory in Rio Grande do Sul State during the years 1999 and 2000 were analyzed using the IS6110-restriction fragment length polymorphism (RFLP) technique. We also genotyped 202 susceptible strains to compare the genotyping results, as well as the clinical and demographic data. Spacer oligotyping (spoligotyping) analysis was performed for isolates presenting low IS6110 copy number. Patients with identical DNA pattern strains were considered clustered. From 262 isolates, 94 (36%) belonged to 20 distinct RFLP clusters, and after spoligotyping analysis, 89 of the isolates (34%) remained in cluster. MDR isolates did not differ statistically in clustering proportion from susceptible strains. A significant association between the occurrence of MDR and previous tuberculosis (TB) treatment was observed (p < 0.001), as well as failure on TB treatment (p < 0.001). Human immunodeficiency virus (HIV)-positive patients were associated with susceptible tuberculosis (p = 0.024). We also identified that unmarried patients were more likely to develop TB due to recent transmission than married patients (p < 0.005). The introduction of directly observed therapy short-course (DOTS) strategy will be important in decreasing default and failure rates and avoiding the development of new MDR strains.     

Use of variable-number tandem-repeat typing to differentiate Mycobacterium tuberculosis Beijing family isolates from Hong Kong and comparison with IS6110 restriction fragment length polymorphism typing and spoligotyping

The Mycobacterium tuberculosis Beijing family isolates may cause more than a quarter of all tuberculosis cases worldwide, are emerging in some areas, and are often associated with drug resistance. Early recognition of transmission of this genotype is therefore important. To evaluate the usefulness of variable-number tandem-repeat (VNTR) typing to discriminate and recognize strains of the Beijing family, M. tuberculosis isolates from Hong Kong were subjected to VNTR analysis, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. The allelic diversity of the 14 VNTR loci included in the analysis varied from 0 to 0.618 among Beijing strains. The discriminatory power of VNTR analysis was slightly lower than that of IS6110 RFLP. Our analysis shows that VNTR typing, which has many practical advantages over RFLP typing, can be used for epidemiological studies of Beijing strains. However, VNTR-defined clusters should be subtyped with IS6110 RFLP for maximal resolution.     

Molecular typing of Mycobacterium tuberculosis based on variable number of tandem DNA repeats used alone and in association with spoligotyping

Fingerprinting based on variable numbers of tandem DNA repeats (VNTR), a recently described methodology, was evaluated for molecular typing of Mycobacterium tuberculosis in an insular setting. In this study, VNTR fingerprinting was used alone or as a second-line test in association with spoligotyping, double-repetitive-element PCR (DRE-PCR), and IS6110 restriction fragment length polymorphism (RFLP) analysis, and the discriminatory power for each method or the combination of methods was compared by calculating the Hunter-Gaston discriminative index (HGI). The results obtained showed that in 6 out of 12 (50%) cases, VNTR-defined clusters were further subdivided by spoligotyping, compared to 7 out of 18 (39%) cases where spoligotyping-defined clusters were further subdivided by VNTR. When used alone, VNTR was the least discriminatory method (HGI = 0.863). Although VNTR was significantly more discriminatory when used in association with spoligotyping (HGI = 0.982), the combination of spoligotyping and DRE-PCR (HGI = 0.992) was still the most efficient among rapid, PCR-based methodologies, giving results comparable to IS6110 RFLP analysis. Nonetheless, VNTR typing may provide additional phylogenetical information that may be helpful to trace the molecular evolution of tubercle bacilli.     

Molecular epidemiology and prevalence of mutations conferring rifampicin and isoniazid resistance in Mycobacterium tuberculosis strains from the southern Ukraine

Understanding the molecular epidemiology of tuberculosis (TB) and mutations in genes associated with drug resistance may contribute to the development of appropriate interventions to improve tuberculosis control. A structured questionnaire was used to collect basic epidemiological data from 589 patients with radiologically confirmed TB in the Odessa and Nikolaev regions of the Ukraine in 2003-2004. A non-commercial reverse hybridisation assay and DNA sequencing were used to detect mutations associated with rifampicin and isoniazid resistance. Genotyping was performed using multilocus variable number tandem repeat (VNTR) typing and spoligotyping. Mutations conferring rifampicin and isoniazid resistance were detected in 32.9% and 44.0%, respectively, of 225 Mycobacterium tuberculosis isolates from individual consecutive patients. Mutations in codon 531 and codon 315 of the rpoB and katG genes, respectively, were predominant among drug-resistant isolates. Multidrug (MDR) resistance rates were significantly higher among former prison inmates compared with non-prisoners (54.8% vs. 27.3%; RR 2.01; 95% CI 1.35-2.97) and the prevalence of mutations was higher in Beijing strains sharing the VNTR signature 223325173533424 than in other Beijing strains (71.4% vs. 45.7%; RR 1.74; 95% CI 1.17-2.57), suggesting that this group may be responsible for rapid transmission of MDR TB in the southern Ukraine.     

Sensitivities and specificities of spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing methods for studying molecular epidemiology of tuberculosis

The development of PCR-based genotyping modalities (spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat [MIRU-VNTR] typing) offers promise for real-time molecular epidemiological studies of tuberculosis (TB). However, the utility of these methods depends on their capacity to appropriately classify isolates. To determine the operating parameters of spoligotyping and MIRU-VNTR typing, we have compared results generated by these newer tests to the standard typing method, IS6110 restriction fragment length polymorphism, in analyses restricted to high-copy-number IS6110 isolates. Sensitivities of the newer tests were estimated as the percentages of isolates with identical IS6110 fingerprints that had identical spoligotypes and MIRU-VNTR types. The specificities of these tests were estimated as the percentages of isolates with unique IS6110 fingerprints that had unique spoligotypes and MIRU-VNTR types. The sensitivity of MIRU-VNTR typing was 52% (95% confidence interval [CI], 31 to 72%), and the sensitivity of spoligotyping was 83% (95% CI, 63 to 95%). The specificity of MIRU-VNTR typing was 56% (95% CI, 51 to 62%), and the specificity of spoligotyping was 40% (95% CI, 35 to 46%). The proportion of isolates estimated to be due to recent transmission was 4% by identical IS6110 patterns, 19% by near-identical IS6110 patterns, 33% by MIRU-VNTR typing, and 53% by spoligotyping. The low calculated specificities of spoligotyping and MIRU-VNTR typing led to misclassification of cases, inflated estimates of TB transmission, and low positive predictive values, suggesting that these techniques have unsuitable operating parameters for population-based molecular epidemiology studies.     

Characterization of Finnish Mycobacterium tuberculosis isolates by spoligotyping

The molecular epidemiology of tuberculosis (TB) in Finland was studied by spoligotyping 380 Mycobacterium tuberculosis isolates. The isolates were obtained during a 1-year study period from July 2000 to June 2001 and represented 90% of new M. tuberculosis findings by culture in the whole country during the study period. The spoligotyping results were compared to the World Spoligotyping Database of the Institut Pasteur de Guadeloupe, which contains data from >14,000 M. tuberculosis isolates obtained worldwide. A total of 138 different spoligotypes were identified among the 380 M. tuberculosis isolates. Thirty-eight (10%) isolates had unique spoligotypes, while 342 (90%) isolates belonged to 100 shared types. The four most common spoligotypes caused approximately one-third of the Finnish TB cases. Forty-seven of the 138 (34.1%) spoligotypes and 61 (16.1%) of the 380 M. tuberculosis isolates had spoligotypes that had not been previously reported. Only four (1.1%) patients were infected with an isolate belonging to the Beijing genotype. The characterization of Finnish M. tuberculosis isolates by spoligotyping shows that ubiquitous spoligotypes were common, but many spoligotypes specific to Finland were also found. However, Beijing family isolates were rarely encountered, although this spoligotype is predominant in our eastern and southern neighbors.     

Transmission dynamics and molecular characterization of Mycobacterium tuberculosis isolates with low copy numbers of IS6110

Population-based analysis of Mycobacterium tuberculosis transmission in Houston, Tex., over 5 years identified 377 patients infected with an isolate containing one to four copies of IS6110. The isolates were analyzed by spoligotyping and assigned to one of three major genetic groups based on nucleotide polymorphisms in codons katG 463 and gyrA 95. Prospectively obtained patient interviews were reviewed to assess epidemiologic links between apparently clustered patients. A total of 13 groups of isolates with the same IS6110 profile were identified, representing 326 of the 377 patients (86.5%; range 2 to 113 patients). In contrast, 28 groups of isolates containing 334 patients (88.6%) had the same spoligotype (range, 2 to 143 patients). Combination of IS6110 profile and spoligotype data identified 31 clusters with 300 patients (79.6%; range, 2 to 82 patients). All 377 isolates belonged to major genetic group 1 (77 patients) or genetic group 2 (300 patients); no major genetic group 3 isolates were identified. Among the 228 patients interviewed, 33 patients (14.5%) were directly linked to another patient in the same cluster. Possible epidemiologic links were also found among 11 patients. Moreover, many clusters consisted of individuals with the same ethnicity. In conclusion, we confirmed that IS6110 profiling and spoligotyping together provide enhanced molecular discrimination of M. tuberculosis isolates with low copy numbers of IS6110. Identification of epidemiologic links among some of the patients verified that the combination of these two methods reliably indexes tuberculosis transmission.     

Study of Restriction Fragment Length Polymorphism Analysis and Spoligotyping for Epidemiological Investigation of Mycobacterium bovis Infection

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS6110, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type Mycobacterium bovis isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS6110 or DR probes. The majority of isolates from all species carried a single IS6110 copy. In four RFLP types IS6110 polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of M. bovis infection in Ireland.     

Molecular epidemiology of disease due to Mycobacterium bovis in humans in the United Kingdom

Mycobacterium bovis is the causative agent of bovine tuberculosis, with a wide host range. Fifty human M. bovis isolates were typed using spoligotyping and variable number tandem repeats (VNTR). Fifteen of these spoligotypes have not yet been recorded in cattle. The predominant spoligotype in humans and cattle was subdivided by VNTR.     

Evaluation of Mycobacterium tuberculosis typing methods in a 4-year study in Schleswig-Holstein, Northern Germany

In order to evaluate the discriminatory power of different methods for genotyping of Mycobacterium tuberculosis complex (MTBC) isolates, we compared the performance of (i) IS6110 DNA fingerprint typing, (ii) spoligotyping, and (iii) 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in a long-term study on the epidemiology of tuberculosis (TB) in Schleswig-Holstein, the northernmost federal state of Germany. In total, we analyzed 277 MTBC isolates collected from patients between the years 2006 and 2010. The collection comprised a broad spectrum of 13 different genotypes, among which strains of the Haarlem genotype (31%) were most prominent, followed by strains belonging to the Delhi and Beijing lineages (7% and 6%, respectively). On the basis of IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping analyses, 211 isolates had unique patterns (76%) and 66 isolates (24%) were in 20 clusters. MIRU-VNTR combined with spoligotyping analyses revealed 202 isolates with unique patterns (73%) and 75 isolates in 18 clusters (27%). Overall, there was 93.1% concordance between the typing results obtained; 198 strains were identified as unique, and 60 isolates were clustered by both typing combinations (including all 31 isolates with confirmed epidemiological links). Of the remaining 19 isolates with discrepant results, 15 were falsely clustered by MIRU-VNTR (six Beijing genotype strains) and four were clustered by IS6110 RFLP (low IS6110 copy number) only. In conclusion, in the study population investigated, a minority of isolates, especially of the Beijing genotype, clustered by standard 24-loci MIRU-VNTR and without an obvious epidemiological link may require second-line typing by IS6110 RFLP or hypervariable MIRU-VNTR loci.