Evaluation of new variable-number tandem-repeat systems for typing Mycobacterium tuberculosis with Beijing genotype isolates from Beijing, China

The newly proposed variable-number tandem-repeat (VNTR) typing system, which includes a basic 15-locus set and a high-resolution 24-locus set (P. Supply et al., J. Clin. Microbiol. 44:4498-4510, 2006), demonstrated a high power for the discrimination of Mycobacterium tuberculosis isolates collected worldwide. To evaluate its ability to differentiate the Beijing genotype strains from the Beijing area in China, 72 isolates with typical Beijing or Beijing-like spacer oligonucleotide typing profiles were subjected to typing with the VNTR system (24 loci) and typing by restriction fragment polymorphism analysis with IS6110 (IS6110-RFLP). Compared to the “old” 12-locus VNTR typing method, use of the 15- and 24-locus systems had a dramatically improved power to discriminate the Beijing genotype strains. A subtle difference in the Hunter-Gaston discriminatory index (HGI) between the 15-locus and the 24-locus systems resulted from only one locus, Mtub29. However, the VNTR-based clusters could be further differentiated by IS6110-RFLP (HGI by IS6110 RFLP, 0.999), although in one case an IS6110 cluster was subdivided by the 15-locus VNTR system. In this sense, use of the newly proposed 15-locus VNTR system along with the Mtub29 locus can serve as a first-line typing method for the epidemiological study of M. tuberculosis isolates in Beijing, while secondary typing of clustered strains by IS6110-RFLP is still required.     

Phylogenetic reconstruction within Mycobacterium tuberculosis Beijing genotype in northwestern Russia

A selection of genetic markers was used to study the evolution of Mycobacterium tuberculosis Beijing family strains in northwestern Russia. A total of 221 of 434 epidemiologically unlinked isolates studied in 1996-2001 belonged to the Beijing family as determined by standard spoligotyping (signals 35-43). Ninety-six percent of these Beijing isolates ("typical") were closely related in IS6110-RFLP (D > 0.85) while 9 remaining isolates (2 different profiles, "atypical") were more distant from the rest (D = 0.6-0.7). Further analysis was performed on a selection of 12 typical and both atypical Beijing strains with different IS6110-RFLP profiles (2 isolates each). All 28 Beijing isolates studied had the KatG 463Leu allele, an intact mtp40 fragment of the mpcA gene, and an identical structure of the DR locus (15 DVRs) with an upstream IS6110 copy in opposite orientation. The IS6110-RFLP based neighbor-joining (distance) and quartet-puzzling (maximum-likelihood) trees showed that the branch lengths were considerably longer for atypical Beijing strains. Typical Beijing strains had the 1.02 kb Rv3135 PPE-family gene and two IS1547 copies (iplA and iplB) one of them (iplB) disrupted by IS6110 insertion. Atypical Beijing strains had the 1.97 kb Rv3135 gene and a single intact IS1547/iplA copy. We suggest that the M. tuberculosis Beijing family strains currently circulating in the northwest of Russia are relatively ancient and thus appear to be endemic in this region since evolutionarily distant time. The prevalent typical Beijing strains (96%) are likely to be of monophyletic origin and their ongoing dissemination has started recently: these strains differ in rapidly evolving IS6110-RFLP but have identical structure of other polymorphic genome regions studied. The atypical Beijing strains (4%) are evolutionary older; they probably had a common (unknown) ancestor with typical Beijing strains.     

First worldwide proficiency study on variable-number tandem-repeat typing of Mycobacterium tuberculosis complex strains

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.     

Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto

Leptospira interrogans sensu stricto is responsible for the most frequent and severe cases of human leptospirosis. The epidemiology and clinical features of leptospirosis are usually associated with the serovars and serogroups of Leptospira. Because of the difficulties associated with serological identification of Leptospira strains, we evaluated a novel PCR-based method for typing L. interrogans serovars. Based upon the genome sequence of L. interrogans serovar Lai type strain 5660, 44 loci were analyzed by PCR for their variability in size due to the presence of variable-number tandem repeats (VNTR). Seven VNTR loci were found to be powerful markers for serovar identification, epidemiology, and phylogenetic studies of L. interrogans. This rapid and easy method should greatly contribute to a better knowledge of the epidemiology of Leptospira.     

Polymorphism of variable-number tandem repeats at multiple loci in Mycobacterium tuberculosis

Genotyping based on variable-number tandem repeats (VNTR) is currently a very promising tool for studying the molecular epidemiology and phylogeny of Mycobacterium tuberculosis. Here we investigate the polymorphisms of 48 loci of direct or tandem repeats in M. tuberculosis previously identified by our group. Thirty-nine loci, including nine novel ones, were polymorphic. Ten VNTR loci had high allelic diversity (Nei's diversity indices >or= 0.6) and subsequently were used as the representative VNTR typing set for comparison to IS 6110-based restriction fragment length polymorphism (RFLP) typing. The 10-locus VNTR set, potentially providing >2 x 10(9) allele combinations, obviously showed discriminating capacity over the IS 6110 RFLP method for M. tuberculosis isolates with fewer than six IS 6110-hybridized bands, whereas it had a slightly better resolution than IS 6110 RFLP for the isolates having more than five IS 6110-hybridized bands. Allelic diversity of many VNTR loci varied in each IS 6110 RFLP type. Genetic relationships inferred from the 10-VNTR set supported the notion that M. tuberculosis may have evolved from two different lineages (high and low IS 6110 copy number). In addition, we found that the lengths of many VNTR loci had statistically significant relationships to each other. These relationships could cause a restriction of the VNTR typing discriminating capability to some extent. Our results suggest that VNTR-PCR typing is practically useful for application to molecular epidemiological and phylogenetic studies of M. tuberculosis. The discriminating power of the VNTR typing system can still be enhanced by the supplementation of more VNTR loci.     

Utility of new 24-locus variable-number tandem-repeat typing for discriminating Mycobacterium tuberculosis clinical isolates collected in Bulgaria

The present study evaluated new markers for molecular typing of Mycobacterium tuberculosis with a collection of strains circulating in Bulgaria. A study sample included 133 strains from epidemiologically unlinked patients from different regions of the country. Spoligotyping was used as a primary typing tool; it subdivided these strains into 37 types, including 15 clusters and 22 singletons. Traditional IS6110-restriction fragment length polymorphism (RFLP) typing and novel 24-locus variable number tandem-repeat (VNTR) typing methods were applied to the selection of 73 strains. Discriminatory power (Hunter-Gaston index [HGI]) of these methods was found to be 0.983 and 0.997, respectively. The 73 strains were subdivided into 66 types by a 24-locus mycobacterial interspersed repetitive unit (MIRU)-VNTR scheme, 62 types by a classical 12-locus MIRU-VNTR scheme, 51 types by IS6110-RFLP typing, and 31 types by spoligotyping. A combination of the five most polymorphic loci (MIRU40, Mtub04, Mtub21, QUB-11b, and QUB-26) was shown to achieve a high discrimination (HGI = 0.984). To conclude, a complete 24-locus scheme excellently differentiated strains in our study, whereas a reduced 5-locus set provided a sufficiently high differentiation and may be preliminarily suggested for the first-line typing of M. tuberculosis isolates in Bulgaria.     

Comparative nucleotide sequence analysis of polymorphic variable-number tandem-repeat Loci in Mycobacterium ulcerans

We analyzed a set of variable-number tandem-repeat (VNTR) loci to assess their nucleotide sequence diversity in isolates of three Mycobacterium ulcerans genotypes. Sequence variants in two loci resulted in intraspecies resolution of Southeast Asian and Asian genotypes in contrast to a homogenous sequence composition among African isolates. Nucleotide sequence polymorphism in repeat units can enhance discrimination of VNTR loci.     

Spread of drug-resistant Mycobacterium tuberculosis strains of the Beijing genotype in the Archangel Oblast,Russia

A collection of 119 strains of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in the Archangel Oblast, Russia, in 1998 and 1999 were studied by using restriction fragment length polymorphism (RFLP) analysis with the IS6110 probe and spoligotyping. Resistance of the strains to antituberculosis drugs was analyzed by the BACTEC method, and mutations associated with rifampin resistance were detected by using the Inno-LiPA Rif. TB test. RFLP analysis and spoligotyping demonstrated that 53 (44.5%) of the strains belonged to the Beijing genotype. These strains showed a significantly higher rate of resistance than M. tuberculosis strains of other genotypes circulating in the region. In particular, 43.4% of the strains of the Beijing genotype were multidrug resistant; in contrast, only 10.6% of the other strains were. Of the strains of the Beijing genotype, 92.5% were part of a cluster, while only 33.3% of the remaining strains were clustered. Analysis of the medical records of the patients demonstrated that individuals infected with a strain of the Beijing genotype were significantly more likely to be alcohol abusers and to have chronic obstructive pulmonary disease prior to the tuberculosis diagnosis. Multivariate analysis showed that both variables were independently associated with infection by strains belonging to the Beijing genotype. Our study demonstrated that strains of the Beijing genotype are an important cause of tuberculosis in the Archangel Oblast and that dissemination of these strains is associated with the high incidence of drug resistance.     

Use of multilocus variable-number tandem-repeat analysis for typing Mycobacterium avium subsp. paratuberculosis

The etiology of Crohn's disease in humans is largely unknown. Clinical signs of Crohn's disease partly resemble the clinical picture of Johne's disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis. Because of the high prevalence of these bacteria in (products of) ruminants and their remarkable thermostability, concern has been raised about the possible role of these bacteria in the pathogenesis of Crohn's disease. In an attempt to develop a molecular typing method to facilitate meaningful comparative DNA fingerprinting of M. avium subsp. paratuberculosis isolates from the human and animal reservoirs, multilocus variable-number tandem-repeat analysis (MLVA) was explored and compared to IS900 restriction fragment length polymorphism (RFLP) typing. MLVA typing subdivided the most predominant RFLP type, R01, into six subtypes and thus provides a promising molecular subtyping approach to study the diversity of M. avium subsp. paratuberculosis.     

Differential activation of dendritic cells by Mycobacterium tuberculosis Beijing genotype

Mycobacterium tuberculosis (Mtb) inhibits dendritric cells (DC) function in order to delay T cell response. Furthermore, there is increasing evidence that genetic diversity of Mtb strains can affect their interaction with the immune system. Beijing genotype has attracted attention because of its high prevalence and multi-drug resistance. Although it is known that this genotype is hypervirulent and differentially activates macrophages when compared to other genotypes, little is known about its interaction with DC. In order to address this issue, murine bone marrow derived DC (BMDC) were stimulated with soluble extracts (SE) from BCG, H37Rv, Canetti and Beijing genotypes. We observed that unlike other mycobacteria strains, SE-Beijing was unable to induce maturation of DC as assessed by cell surface MHC-II expression. DC stimulated with SE-Beijing failed to produce IL-12 and TNF-α, but did secrete IL-10. Interestingly, SE-Beijing induced CCR7 and PDL-1 on BMDC, but did not induce the expression of CD86. When BMDC stimulated with SE-Beijing were used to activate CD4+ cells they were unable to induce a Th1 response when compared with less virulent genotypes. These results indicate that Beijing is able to modulate DC activation and function, which may be related to the pathogenesis induced by this genotype.     

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.     

Reconstruction of the epidemic history of the Beijing genotype of Mycobacterium tuberculosis in Russia and former soviet countries using spoligotyping

All known genotypes (3925 spoligoprofiles of M. tuberculosis) were selected using the SITVIT database in nine countries, including countries of the former Soviet Union: Russia, Latvia, Estonia, Poland, Finland, Italy, Portugal, Japan, and Vietnam. The programs SpolTools and DESTUS were used to construct consensus networks to identify the epidemic genotypes of M. tuberculosis in the studied countries. In Russia, Latvia, and Estonia, the Beijing strain was determined as the main epidemic genotype. In Finland, Poland, Portugal, and Italy, all epidemic genotypes belonged to other families. The hypothesis on the explosive nature of the spreading of the Beijing genotype of M. tuberculosis was put forward for Russia and other former Soviet countries in the 20th century. The basic idea of the hypothesis was that the pattern of dissemination of the Beijing genotype occurred at the CER (Chinese Eastern Railway), in the Gulag, and in the civilian society of Soviet Union. The Beijing genotype of M. tuberculosis affected Russian builders of the CER during the end the 19th and early 20th centuries. With the repression of CER builders in the Soviet Union, the Beijing genotype was spread among Gulag prisoners; after 1953, it had spread throughout the civilian society of the entire country. The distribution of epidemic genotypes of M. tuberculosis in the studied countries was interpreted as evidence of the suggested hypothesis.     

Rapid detection of Mycobacterium tuberculosis Beijing genotype strains by real-time PCR

Mycobacterium tuberculosis strains of the Beijing genotype were first identified in China and neighboring countries and have attracted special attention due to their global emergence and association with drug resistance. To further analyze the spread and special characteristics of Beijing genotype strains, accurate, rapid and sensitive methods that overcome the drawbacks of the classical methods such as IS6110 DNA fingerprinting or spoligotyping for the identification of strains of this genotype are needed. Based on the nucleotide sequences of M. tuberculosis SAWC0780 and H37Rv, primers and fluorogenic 5' nuclease (TaqMan) probes for real-time PCR assays specific for Beijing and non-Beijing strains, respectively, were designed. The detection limits for the real-time PCR assays were about 5 and 10 copies of chromosomal DNA, respectively. In mixtures of Beijing and non-Beijing DNA, a multiplex assay was able to detect (i) one copy of Beijing DNA in approximately 1,000 copies of non-Beijing DNA and (ii) one copy of non-Beijing DNA in approximately 2,000 copies of Beijing DNA. In a blinded analysis of a collection of 103 multidrug-resistant strains isolated in Germany in 2001, all 62 Beijing and all 41 non-Beijing strains were correctly identified. In conclusion, the real-time assay allows for the rapid and specific detection of Beijing and non-Beijing strains. The major advantages of this test in comparison to other methods used for the identification of Beijing strains are its simplicity and sensitivity and the fact that amplification and detection occur within one reaction tube.     

Limitations of spoligotyping and variable-number tandem-repeat typing for molecular tracing of Mycobacterium bovis in a high-diversity setting

This study describes the attempt to trace the first Mycobacterium bovis outbreak in alpacas (Lama pacos) in Spain by spoligotyping and variable-number tandem-repeat (VNTR) analysis. Due to high genotype diversity, no matching source was identified, but local expansion of a clonal group was found and its significance for molecular tracing is discussed.     

Stability of variable-number tandem repeats of mycobacterial interspersed repetitive units from 12 loci in serial isolates of Mycobacterium tuberculosis

Variable number tandem repeats (VNTRs) of elements named mycobacterial interspersed repetitive units (MIRUs) have previously been identified in 12 minisatellite loci of the Mycobacterium tuberculosis genome. These markers allow reliable high-throughput genotyping of M. tuberculosis and represent a portable approach to global molecular epidemiology of M. tuberculosis. To assess their temporal stability, we genotyped 123 serial isolates, separated by up to 6 years and belonging to a variety of distinct IS6110 restriction fragment length polymorphism (RFLP) families, from 56 patients who had positive sputum cultures. All 12 MIRU VNTR loci were completely identical within the groups of serial isolates in 55 out of 56 groups (98.2%), although 11 pairs of isolates from the same patients with conserved MIRU VNTRs displayed slightly different IS6110 RFLP profiles. In a single case, serial isolates with an unchanged IS6110 RFLP profile showed a change in 1 out of 12 MIRU VNTR loci. These results indicate that MIRU VNTRs are stable over time and therefore are suitable for reliable follow-up of patients chronically infected with tuberculosis over long periods. Moreover, they support MIRU VNTR genotyping as a powerful first-line method followed by subtyping by IS6110 RFLP to define ongoing transmission clusters.     

Multiple-locus variable-number tandem-repeat analysis for rapid typing of Candida glabrata

A multiple-locus variable-number tandem-repeat analysis (MLVA) using six microsatellite markers was assessed in 127 Candida glabrata isolates. Thirty-seven different genotypes, stable both in vitro and in vivo, were observed. The highest discriminatory power (D = 0.902) was reached by using only four markers. MLVA seems to be relevant for C. glabrata typing.     

Origin and primary dispersal of the Mycobacterium tuberculosis Beijing genotype: clues from human phylogeography

We suggest that the evolution of the population structure of microbial pathogens is influenced by that of modern humans. Consequently, the timing of hallmark changes in bacterial genomes within the last 100,000 yr may be attempted by comparison with relevant human migrations. Here, we used a lineage within Mycobacterium tuberculosis, a Beijing genotype, as a model and compared its phylogeography with human demography and Y chromosome-based phylogeography. We hypothesize that two key events shaped the early history of the Beijing genotype: (1) its Upper Palaeolithic origin in the Homo sapiens sapiens K-M9 cluster in Central Asia, and (2) primary Neolithic dispersal of the secondary Beijing NTF::IS6110 lineage by Proto-Sino-Tibetan farmers within east Asia (human O-M214/M122 haplogroup). The independent introductions of the Beijing strains from east Asia to northern Eurasia and South Africa were likely historically recent, whereas their differential dissemination within these areas has been influenced by demographic and climatic factors.     

Mycobacterium tuberculosis Beijing genotype strains and unfavourable treatment outcomes: a systematic review and meta-analysis

ObjectivesThe Mycobacterium tuberculosis Beijing genotype was first described in 1995 and is now the predominant strain among patients with tuberculosis in many Asian countries. The rapid global spread of the Beijing genotype is receiving increasing attention because it can cause a higher risk of treatment failures. Our objective was to assess the association between the Beijing genotype and unfavourable treatment outcomes of tuberculosis.MethodsWe searched for eligible studies through PubMed, Web of Science, Chinese National Knowledge Infrastructure and Wanfang Data. We included cohort studies that evaluated treatment outcomes and Beijing genotype strains. Participants were individuals with active pulmonary tuberculosis. The association between Beijing genotype and the risk of unfavourable treatment outcomes was assessed using the pooled odds ratios (ORs) with corresponding confidence intervals (CIs).ResultsIn total, 7489 tuberculosis patients were involved in the analysis. Patients infected with the Beijing genotype were more likely to have unfavourable treatment outcomes, with the OR of 2.04 (95% CI 1.52–2.75). The pooled OR was 2.33 (95% CI 1.71–3.16) for recurrence, 2.36 (95% CI 1.69–3.30) for relapse and 2.62 (95% CI 1.90–3.61) for treatment failure, respectively. Subgroup analysis revealed that Beijing genotype was a significant risk factor for unfavourable treatment outcomes in Asians (OR 2.28, 95% CI 1.82–2.86) or in drug-susceptible TB patients (OR 2.11, 95% CI 1.31–3.39). No significant association was observed among non-Asian populations (OR 1.17, 95% CI 0.73–1.86) or patients with multidrug-resistant (MDR) tuberculosis (OR 0.97, 95% CI 0.48–1.94).ConclusionsOur results suggest that Mycobacterium tuberculosis Beijing genotype is associated with an increased risk of unfavourable treatment outcomes, including treatment failure and relapse.