Mycobacterial Interspersed Repetitive Units Typing Research Articles

Extensive Homoplasy but No Evidence of Convergent Evolution of Repeat Numbers at MIRU Loci in Modern Mycobacterium tuberculosis Lineages.

More human deaths have been attributable to Mycobacterium tuberculosis than any other pathogen, and the epidemic is sustained by ongoing transmission. Various typing schemes have been developed to identify strain-specific differences and track transmission dynamics in affected communities, with recent introduction of whole genome sequencing providing the most accurate assessment. Mycobacterial interspersed repetitive unit (MIRU) typing is a family of variable number tandem repeat schemes that have been widely used to study the molecular epidemiology of M. tuberculosis. MIRU typing was used in most well-resourced settings to perform routine molecular epidemiology. Instances of MIRU homoplasy have been observed in comparison with sequence-based phylogenies, limiting its discriminatory value. A fundamental question is whether the observed homoplasy arises purely through stochastic processes, or whether there is evidence of natural selection. We compared repeat numbers at 24 MIRU loci with a whole genome sequence-based phylogeny of 245 isolates representing three modern M. tuberculosis lineages. This analysis demonstrated extensive homoplasy of repeat numbers, but did not detect any evidence of natural selection of repeat numbers, at least since the ancestral branching of the three modern lineages of M. tuberculosis. In addition, we observed good sensitivity but poor specificity and positive predictive values of MIRU-24 to detect clusters of recent transmission, as defined by whole-genome single nucleotide polymorphism analysis. These findings provide mechanistic insight, and support a transition away from VNTR-based typing toward sequence-based typing schemes for both research and public health purposes.

MOLECULAR TYPING AND DRUG RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS COMPLEX ISOLATES FROM JAMOT AND MBALMAYO DISTRICT HOSPITALS, CAMEROON

BackgroundCameroon is a country where tuberculosis still remains a major public health problem. The aim of the present research was to evaluate the potential of molecular markers in predicting first-line...

Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing.

BackgroundImproved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways.MethodsWe sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants.ResultsIsolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade.ConclusionWhole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster.

Genotypic diversity of multidrug-, quinolone- and extensively drug-resistant Mycobacterium tuberculosis isolates in Thailand

Drug-resistant tuberculosis (TB), which includes multidrug-resistant (MDR-TB), quinolone-resistant (QR-TB) and extensively drug-resistant tuberculosis (XDR-TB), is a serious threat to TB control. We aimed to characterize the genotypic diversity of drug-resistant TB clinical isolates collected in Thailand to establish whether the emergence of drug-resistant TB is attributable to transmitted resistance or acquired resistance. We constructed the first molecular phylogeny of MDR-TB (n=95), QR-TB (n=69) and XDR-TB (n=28) in Thailand based on spoligotyping and proposed 24-locus multilocus variable-number of tandem repeat analysis (MLVA). Clustering analysis was performed using the unweighted pair group method with arithmetic mean. Spoligotyping identified the Beijing strain (SIT1) as the most predominant genotype (n=139; 72.4%). The discriminatory power of 0.9235 Hunter–Gaston Discriminatory Index (HGDI) with the 15-locus variable-number tandem repeats of mycobacterial interspersed repetitive units typing was improved to a 0.9574 HGDI with proposed 24-locus MLVA, thereby resulting in the subdivision of a large cluster of Beijing strains (SIT1) into 17 subclusters. We identified the spread of drug-resistant TB clones caused by three different MLVA types in the Beijing strain (SIT1) and a specific clone of XDR-TB caused by a rare genotype, the Manu-ancestor strain (SIT523). Overall, 49.5% of all isolates were clustered. These findings suggest that a remarkable transmission of drug-resistant TB occurred in Thailand. The remaining 50% of drug-resistant TB isolates were unique genotypes, which may have arisen from the individual acquisition of drug resistance. Our results suggest that transmitted and acquired resistance have played an equal role in the emergence of drug-resistant TB. Further characterization of whole genome sequences of clonal strains could help to elucidate the mycobacterial genetic factors relevant for drug resistance, transmissibility and virulence.

Strain classification of Mycobacterium tuberculosis isolates in Brazil based on genotypes obtained by spoligotyping, mycobacterial interspersed repetitive unit typing and the presence of large sequence and single nucleotide polymorphism.

Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as “LAM-like”, 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.

Mycobacterial interspersed repetitive unit typing and mutational profile for multidrug-resistant and extensively drug-resistant tuberculosis surveillance in Portugal: a 3-year period overview

Multidrug tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) cases constitute a serious health problem in Portugal, of which the majority of isolates belong to the Lisboa family and the Q1 cluster, highly related to the Lisboa family. Here we sought to investigate the molecular basis of resistant TB as well as to determine the prevalence of specific drug resistance mutations and their association with MDR-TB and/or XDR-TB. In total, 74 Mycobacterium tuberculosis clinical isolates collected in Lisbon Health Region were genotyped by 24-loci mycobacterial interspersed repetitive units–variable number of tandem repeats (MIRU-VNTR), and the mutational profile associated with first- and second-line drug resistance was studied. Seven new mutations were found, whilst the remaining 28 mutations had been previously associated with drug resistance. None of the mutations was specifically associated with MDR-TB. The mutational patterns observed among isolates belonging to Lisboa3 and Q1 clusters were also observed in isolates with unique MIRU-VNTR patterns but closely related to these strains. Such data suggest that the genotyping technique employed discriminates isolates with the same mutational profile. To establish the most adequate genotyping technique, the discriminatory power of three different MIRU-VNTR sets was analysed. The 15-loci MIRU-VNTR set showed adequate discriminatory power, comparable with the 24-loci set, allowing clustering of 60% and 86% of the MDR-TB and XDR-TB isolates, respectively, the majority of which belonged to the Lisboa3 and Q1 clusters. From an epidemiological standpoint, this study suggests combined mutational and genotyping analysis as a valuable tool for drug resistance surveillance.

Temporal dynamics of Mycobacterium tuberculosis genotypes in New South Wales, Australia.

BackgroundMolecular epidemiology of Mycobacterium tuberculosis, its transmission dynamics and population structure have become important determinants of targeted tuberculosis control programs. Here we describe recent changes in the distribution of M. tuberculosis genotypes in New South Wales (NSW), Australia and compared strain types with drug resistance, site of disease and demographic data.MethodsWe evaluated all culture-confirmed newly identified tuberculosis cases in NSW, Australia, from 2010-2012. M. tuberculosis population structure and clustering rates were assessed using 24-loci Mycobacterial interspersed repetitive unit (MIRU) analysis and compared to MIRU data from 2006-2008.ResultsOf 1177 tuberculosis cases, 1128 (95.8%) were successfully typed. Beijing and East African Indian (EAI) lineage strains were most common (27.6% and 28.5%, respectively) with EAI strains increasing in relative abundance from 11.8% in 2006-2008 to 28.5% in 2010-2012. Few cases of multi-drug resistant tuberculosis were identified (18; 1.7%). Compared to 12-loci, 24-loci MIRU provided improved cluster resolution with 695 (61.6%) and 227 (20.1%) clustered cases identified, respectively. Detailed analysis of the largest cluster identified (an 11 member Beijing cluster) revealed wide geographic diversity in the absence of documented social contact.ConclusionsEAI strains of M. tuberculosis recently overtook Beijing family as a prevalent cause of tuberculosis in NSW, Australia. This lineage appeared to be less commonly related to multi-drug resistant tuberculosis as compared to Beijing strain lineage. The resolution provided by 24-loci MIRU typing was insufficient for reliable assessment of transmissions, especially of Beijing family strains.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-455) contains supplementary material, which is available to authorized users.

Strain classification of Mycobacterium tuberculosis isolates in Brazil based on genotypes obtained by spoligotyping, mycobacterial interspersed repetitive unit typing and the presence of large sequence and single nucleotide polymorphisms

Strain classification of Mycobacterium tuberculosis isolates in Brazil based on genotypes obtained by spoligotyping, mycobacterial interspersed repetitive unit typing and the presence of large sequence and single nucleotide polymorphisms

Recognizing Laboratory Cross-Contamination

Mycobacterium tuberculosis (MTB) isolation from clinical specimens is the standard for TB diagnosis. Positive MTB cultures are rarely questioned, but false-positive culture rates range from 2% to 4%. In December 2011, two smear-negative, culture-positive TB cases were reported to the Oklahoma State Department of Health (OSDH) in people without TB signs or symptoms. OSDH TB control officers interviewed physicians and laboratory personnel, reviewed patient charts, traced epidemiologic links, and performed microbiologic studies to determine if these were true TB cases. Both specimens were found to have been processed on the same day, at the same laboratory, under the same hood, and by the same technician sequentially after a strongly smear-positive TB specimen. No epidemiologic links were identified among the three patients. Spoligotyping and 24-locus mycobacterial interspersed repetitive unit typing of the three specimens were identical. Only liquid media grew MTB in the two questionable specimens. A laboratory splash incident was suspected, whereby all three liquid media sample lids were open during inoculation rather than being opened one at a time, causing cross-contamination. Also, the two specimens were incubated for 2-3 weeks longer than standard protocol before MTB growth was observed. Patient 1 was not treated for TB because her physician doubted the culture result. Patient 2, an organ transplant recipient, began four-drug anti-TB therapy, and an investigation was initiated for transplant-associated TB. Adherence to strict laboratory techniques and recognizing the possibility of false-positive MTB cultures, especially when inconsistent with clinical data, are essential in preventing erroneous TB diagnoses.

The use of mycobacterial interspersed repetitive unit typing and whole genome sequencing to inform tuberculosis prevention and control activities

Molecular strain typing of Mycobacterium tuberculosis has been possible for only about 20 years; it has significantly improved our understanding of the evolution and epidemiology of Mycobacterium tuberculosis and tuberculosis disease. Mycobacterial interspersed repetitive unit typing, based on 24 variable number tandem repeat unit loci, is highly discriminatory, relatively easy to perform and interpret and is currently the most widely used molecular typing system for tuberculosis surveillance. Nevertheless, clusters identified by mycobacterial interspersed repetitive unit typing sometimes cannot be confirmed or adequately defined by contact tracing and additional methods are needed. Recently, whole genome sequencing has been used to identify single nucleotide polymorphisms and other mutations, between genotypically indistinguishable isolates from the same cluster, to more accurately trace transmission pathways. Rapidly increasing speed and quality and reduced costs will soon make large scale whole genome sequencing feasible, combined with the use of sophisticated bioinformatics tools, for epidemiological surveillance of tuberculosis.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Three-year longitudinal study of genotypes of Mycobacterium tuberculosis in a low prevalence population

To investigate the molecular epidemiology of tuberculosis, temporal and spatial distribution of Mycobacterium tuberculosis isolates and associations between genotypes and clinical characteristics, in a low prevalence population. A total of 930 M. tuberculosis isolates referred to the New South Wales (NSW, Australia) Mycobacterium Reference Laboratory in 2004-2006 were characterised by mycobacterial interspersed repetitive unit (MIRU) and spacer oligonucleotide (spoligo) typing. Associations between genotypes, patient age, disease site and drug resistance were explored and the predictive power of molecular typing was analysed using Bayesian Belief Networks. Among isolates from 855 NSW residents, there were 287 spoligotypes, 494 MIRU types and 643 unique spoligotype-MIRU type combinations. They formed 73 spoligotype, 104 MIRU type and 76 spoligo-MIRU clusters, most of which contained only two isolates. The majority (87.7%) of spoligotype clusters contained several MIRU profiles and 64.4% of MIRU clusters contained several spoligotypes. The three most common M. tuberculosis clades were Beijing (24.1%), East African Indian (11.8%) and Central Asian (6.5%); 6.9% and 0.7% isolates were resistant to isoniazid and rifampicin, respectively. There was no proof of association between genotype and drug resistance but isoniazid resistance increased independently over time. Given the low rates of genotype clustering, statistical analysis of genotype-phenotype associations was limited. Potential associations were not confirmed by Bayesian classifiers. Spoligo and MIRU typing demonstrated low levels of M. tuberculosis clustering in NSW; temporal and spatial changes in M. tuberculosis genotypes reflected migration patterns to Australia. No analytically significant associations between M. tuberculosis genotypes and clinical phenotypes were detected.

A conformal Bayesian network for classification of Mycobacterium tuberculosis complex lineages

BackgroundWe present a novel conformal Bayesian network (CBN) to classify strains of Mycobacterium tuberculosis Complex (MTBC) into six major genetic lineages based on two high-throuput biomarkers: mycobacterial interspersed repetitive units (MIRU) and spacer oligonucleotide typing (spoligotyping). MTBC is the causative agent of tuberculosis (TB), which remains one of the leading causes of disease and morbidity world-wide. DNA fingerprinting methods such as MIRU and spoligotyping are key components in the control and tracking of modern TB. ResultsCBN is designed to exploit background knowledge about MTBC biomarkers. It can be trained on large historical TB databases of various subsets of MTBC biomarkers. During TB control efforts not all biomarkers may be available. So, CBN is designed to predict the major lineage of isolates genotyped by any combination of the PCR-based typing methods: spoligotyping and MIRU typing. CBN achieves high accuracy on three large MTBC collections consisting of over 34,737 isolates genotyped by different combinations of spoligotypes, 12 loci of MIRU, and 24 loci of MIRU. CBN captures distinct MIRU and spoligotype signatures associated with each lineage, explaining its excellent performance. Visualization of MIRU and spoligotype signatures yields insight into both how the model works and the genetic diversity of MTBC.ConclusionsCBN conforms to the available PCR-based biological markers and achieves high performance in identifying major lineages of MTBC. The method can be readily extended as new biomarkers are introduced for TB tracking and control. An online tool (http://www.cs.rpi.edu/~bennek/tbinsight/tblineage) makes the CBN model available for TB control and research efforts.

Human Tuberculosis due toMycobacterium bovisin the United States, 1995–2005

Understanding the epidemiology of human Mycobacterium bovis tuberculosis (TB) in the United States is imperative; this disease can be foodborne or airborne, and current US control strategies are focused on TB due to Mycobacterium tuberculosis and airborne transmission. The National TB Genotyping Service's work has allowed systematic identification of M. tuberculosis-complex isolates and enabled the first US-wide study of M. bovis TB. Results of spacer oligonucleotide and mycobacterial interspersed repetitive units typing were linked to corresponding national surveillance data for TB cases reported for the period 2004-2005 and select cases for the period 1995-2003. We also used National TB Genotyping Service data to evaluate the traditional antituberculous drug resistance-based case definition of M. bovis TB. Isolates from 165 (1.4%) of 11,860 linked cases were identified as M. bovis. Patients who were not born in the United States, Hispanic patients, patients <15 years of age, patients reported to be HIV infected, and patients with extrapulmonary disease each had increased adjusted odds of having M. bovis versus M. tuberculosis TB. Most US-born, Hispanic patients with TB due to M. bovis (29 [90.6%] of 32) had extrapulmonary disease, and their overall median age was 9.5 years. The National TB Genotyping Service's data indicated that the pyrazinamide-based case definition's sensitivity was 82.5% (95% confidence interval; 75.3%-87.9%) and that data identified 14 errors in pyrazinamide-susceptibility testing or reporting. The prevalence of extrapulmonary disease in the young, US-born Hispanic population suggests recent transmission of M. bovis, possibly related to foodborne exposure. Because of its significantly different epidemiologic profile, compared with that of M. tuberculosis TB, we recommend routine surveillance of M. bovis TB. Routine surveillance and an improved understanding of M. bovis TB transmission dynamics would help direct the development of additional control measures.

Multispacer Sequence Typing for Mycobacterium tuberculosis Genotyping

BackgroundGenotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected after complete genome sequence analysis. It has been applied to various pathogens, but not to M. tuberculosis.Methods and FindingsIn M. tuberculosis, the MST approach yielded eight variable intergenic spacers which included four previously described variable number tandem repeat loci, one single nucleotide polymorphism locus and three newly evaluated spacers. Spacer sequence stability was evaluated by serial subculture. The eight spacers were sequenced in a collection of 101 M. tuberculosis strains from five phylogeographical lineages, and yielded 29 genetic events including 13 tandem repeat number variations (44.82%), 11 single nucleotide mutations (37.93%) and 5 deletions (17.24%). These 29 genetic events yielded 32 spacer alleles or spacer-types (ST) with an index of discrimination of 0.95. The distribution of M. tuberculosis isolates into ST profiles correlated with their assignment into phylogeographical lineages. Blind comparison of a further 93 M. tuberculosis strains by MST and restriction fragment length polymorphism-IS6110 fingerprinting and mycobacterial interspersed repetitive units typing, yielded an index of discrimination of 0.961 and 0.992, respectively. MST yielded 41 different profiles delineating 16 related groups and proved to be more discriminatory than IS6110-based typing for isolates containing <8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts ≤ 42 cycles in internal transcribed spacer-real time PCR.ConclusionsThese results support MST as an alternative, sequencing-based method for genotyping low IS6110 copy-number M. tuberculosis strains. The M. tuberculosis MST database is freely available (http://ifr48.timone.univ-mrs.fr/MST_MTuberculosis/mst).

Molecular Characterization ofMycobacterium tuberculosisIsolates from Different Regions of Bulgaria

Mycobacterium tuberculosis isolates from different regions of Bulgaria were studied by a variety of molecular typing tools. Based on spacer oligonucleotide typing (spoligotyping), the 113 strains were subdivided into 35 spoligotypes: 5 unique profiles and 15 profiles shared by two to 29 strains; the Hunter-Gaston diversity index (HGI) was 0.9. Comparison with the international database SITVIT2 at the Institut Pasteur de Guadeloupe showed the presence of two globally distributed shared types, ST53 (25.7%) and ST47 (6.2%). Nineteen (16.8%) and six (5.3%) strains belonged to the ST125 (LAM/S subfamily) and ST41 (LAM7_TUR subfamily) types described in SITVIT2 as ubiquitous/rare and ubiquitous/common types, respectively. Seven spoligoprofiles (12 strains) were not found in the database; two of them constituted new shared types. The Beijing genotype strains were not found in the studied collection in spite of close contacts with Russia in the recent and historical past. Additional subtyping by IS6110-restriction fragment length polymorphism (RFLP) and 12-locus mycobacterial interspersed repetitive unit (MIRU)-variable number of tandem repeat analyses were performed within selected spoligotypes. In particular, MIRU typing showed better discrimination within ST125 than IS6110-RFLP typing (HGI = 0.83 versus 0.39). A high gradient for ST125 in Bulgaria compared to its negligible presence in the global database and neighboring countries leads us to suggest a Bulgarian phylogeographic specificity of this spoligotype. To conclude, this first study of the Bulgarian M. tuberculosis population demonstrated its heterogeneity and predominance of several worldwide-distributed and Balkan-specific spoligotypes.

Utility of mycobacterial interspersed repetitive unit typing for differentiating Mycobacterium tuberculosis isolates in Wuhan, China

Mycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping of Mycobacterium tuberculosis, and may represent a feasible approach to study M. tuberculosis molecular epidemiology. To evaluate the use of MIRU typing in discriminating M. tuberculosis strains, isolates from 105 patients in Wuhan City, China, were genotyped by this method as compared to spoligotyping. MIRU typing identified 55 types that defined 21 clusters and 34 unique isolates. The discriminatory power was high [Hunter-Gaston discriminatory index (HGDI), 0.97]. Spoligotyping showed that 86 (81.9 %) of 105 isolates belonged to the Beijing family genotype. For Beijing family and non-Beijing strains, the discriminatory power of MIRU was high (HGDI, 0.95 and 0.98, respectively). Among the alleles of the MIRU loci for the Beijing family, only locus 26 was highly discriminative, but for non-Beijing strains, loci 10, 16 and 26 were highly discriminative. MIRU typing is a simple and fast method which may be used for preliminary screening of M. tuberculosis isolates in China.

Outbreak of Tuberculosis Caused by Mycobacterium caprae in a Zoological Garden

In the autumn of 2004, tuberculosis caused by Mycobacterium caprae occurred in a zoo in Slovenia. A dromedary camel (Camelus dromedarius) was killed after a history of progressive emaciation. Necropsy findings indicated disseminated tuberculosis, which was confirmed by cultivation of M. caprae. Consequently, a tuberculin skin test was performed in all epidemiologically linked animals and another dromedary camel and six bison (Bison bison) were positive and killed. Mycobacterium caprae was isolated from two bison while M. scrofulaceum and Mycobacterium spp. were found in two other bison, respectively. The second dromedary camel was found to be negative for mycobacteria under both microscopic and culture tests. The isolates were investigated with commercial identification kits, IS6110 PCR, IS6110 restriction fragment length polymorphism analysis, spoligotyping and mycobacterial interspersed repetitive units typing. Genotyping results revealed that the dromedary camel and the two bison were infected by the same M. caprae.

Use of Mycobacterial Interspersed Repetitive Unit Locus 26 for Rapid Identification of Beijing Genotype Mycobacterium tuberculosis Strains

In a recent paper by Rao et al. (3), the easy and rapid mycobacterial interspersed repetitive unit (MIRU) locus 26 PCR assay was found to be 100% specific and sensitive for the discrimination of the Beijing genotype Mycobacterium tuberculosis strains from the non-Beijing strains based on the assumption that all Beijing strains have seven copies and non-Beijing strains have fewer or more than seven MIRU copies at locus 26. Unfortunately, however, it is known that not all Beijing isolates carry seven MIRU copies at locus 26 (2, 4). Particularly, in some geographic regions, such as the countries of the former USSR, Beijing genotype strains with fewer than seven MIRU copies at locus 26 are in striking predominance over strains with seven copies (2). Recently, we showed that Beijing genotype strains recovered in Istanbul were carried over from the countries of the former USSR, and of the 46 Beijing genotype strains, 37 strains were found to have fewer than seven MIRU copies at locus 26 (1). After having performed spoligotyping and MIRU typing for more than 2,000 M. tuberculosis strains in Istanbul, we also encountered some non-Beijing strains which had seven repeats at MIRU locus 26. These non-Beijing strains with seven repeats at locus 26 were of the T1, LAM-9, Haarlem 1, CAS 1, 703740004000760, 703777740003371, and 777777774760771 spoligotypes. In conclusion, the sensitivity and specificity of the MIRU locus 26 PCR assay may not be 100% as was found in this study (3), and this assay seems to be far from reliable as a screening tool to identify and discriminate Beijing genotype strains from non-Beijing strains, since there are Beijing genotype strains with fewer or more than seven repeats at locus 26 and, conversely, non-Beijing strains with seven repeats.

Characterization ofMycobacterium capraeIsolates from Europe by Mycobacterial Interspersed Repetitive Unit Genotyping

Mycobacterium caprae, a recently defined member of the Mycobacterium tuberculosis complex, causes tuberculosis among animals and, to a limited extent, in humans in several European countries. To characterize M. caprae in comparison with other Mycobacterium tuberculosis complex members and to evaluate genotyping methods for this species, we analyzed 232 M. caprae isolates by mycobacterial interspersed repetitive unit (MIRU) genotyping and by spoligotyping. The isolates originated from 128 distinct epidemiological settings in 10 countries, spanning a period of 25 years. We found 78 different MIRU patterns (53 unique types and 25 clusters with group sizes from 2 to 9) but only 17 spoligotypes, giving Hunter-Gaston discriminatory indices of 0.941 (MIRU typing) and 0.665 (spoligotyping). For a subset of 103 M. caprae isolates derived from outbreaks or endemic foci, MIRU genotyping and IS 6110 restriction fragment length polymorphism were compared and shown to provide similar results. MIRU loci 4, 26, and 31 were most discriminant in M. caprae, followed by loci 10 and 16, a combination which is different than those reported to discriminate M. bovis best. M. caprae MIRU patterns together with published data were used for phylogenetic inference analysis employing the neighbor-joining method. M. caprae isolates were grouped together, closely related to the branches of classical M. bovis, M. pinnipedii, M. microti, and ancestral M. tuberculosis, but apart from modern M. tuberculosis. The analysis did not reflect geographic patterns indicative of origin or spread of M. caprae. Altogether, our data confirm M. caprae as a distinct phylogenetic lineage within the Mycobacterium tuberculosis complex.