IS6110 Restriction Fragment Length Polymorphism Research Articles

Mycobacterium tuberculosis in Ontario, Canada: Insights from IS 6110 Restriction Fragment Length Polymorphism and Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Genotyping

A collection of 1,308 clinical Mycobacterium tuberculosis isolates from Ontario, Canada, was genotyped by IS6110 restriction fragment length polymorphism (RFLP) and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. RFLP or >12 MIRU-VNTR loci were necessary for resolution of Indo-Oceanic strains. The low clustering rate and high strain diversity indicate that, in Ontario, most tuberculosis results from reactivation of latent infections.

Molecular characterisation of Mycobacterium bovis isolates from cattle carcases at a city slaughterhouse in Uganda

During a period of eight months, the carcases of 16,800 slaughter cattle were inspected at a city abattoir in Uganda. Eighty-seven of them had tuberculosis-like lesions and tissue samples were...

Prospective Universal Application of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Genotyping To CharacterizeMycobacterium tuberculosisIsolates for Fast Identification of Clustered and Orphan Cases

The use of molecular tools for genotyping Mycobacterium tuberculosis isolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110 restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosis isolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110 RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.

Transmission permeability of tuberculosis involving immigrants, revealed by a multicentre analysis of clusters

In recent years, the number of cases of tuberculosis (TB) among immigrants in Spain has increased markedly, and led to this analysis of the recent transmission patterns of TB in the immigrant population in Madrid. The countries from which the highest number of immigrant cases have been reported were Ecuador (21%), Romania (16%), Morocco (12%), Peru (11%) and Bolivia (9%). Fifty-one per cent of the cases were from South America. In a multicentre study (2004–2006), IS6110 restriction fragment length polymorphism and spoligotyping were used to genotype the Mycobacterium tuberculosis isolates from 632 immigrant cases from 47 countries. A total of 183 cases (29%) were grouped into 59 clusters, which are markers of potential transmission events. Most of the clusters (81%) included patients living in different healthcare districts, and 54% of the clusters were multinational. When a sample of 478 autochthonous cases was included, 53% of the clusters involving immigrants also included autochthonous cases. This study revealed marked transmission permeability among nationalities and between the immigrant and the autochthonous populations.

Phenotypic and genotypic variant of MDR-Mycobacterium tuberculosis multiple isolates in the same tuberculosis episode, Rio de Janeiro, Brazil

Assuming that the IS6110-restriction fragment length polymorphism (RFLP) changes at a constant rate of 3.2 years, this methodology was applied to demonstrate, for the first time, variant patterns of Mycobacterium tuberculosis (MTB) in multiple isolates obtained at short time intervals from sputum and blood of an HIV+ patient with multiple admissions to the Emergency Room and to the multidrug-resistant tuberculosis (MDR-TB) Reference Center of a secondary-care hospital in Rio de Janeiro, Brazil. In sputum, the IS6110-RFLP appeared in isolates with two variant patterns with 10 and 13 IS6110 copies. However, blood presented only the pattern corresponding to 10 copies, suggesting compartmentalization. With regard to the exact match of 10 of 13 bands, this may be a subpopulation with the same clonal origin and this may be related to the IS6110 transposition. A susceptibility test demonstrated an MDR profile (INH(R), RIF(R), SM(R), and EMB(R), with the sputum isolate also exhibiting EMB(S) (R = resistant; S = sensitive). A gene mutation confirmed resistance only to streptomycin. There was agreement between the results of the phenotypic test and the clinical response to MDR-TB treatment, suggesting serious implications with regard to treatment administration based exclusively on molecular methods, and calling attention to the fact that more effective control strategies against the emergence of MDR strains must be implemented by the TB control program to prevent transmission of MDR-MTB strains at health facilities in areas highly endemic for TB.

Comparison of four-colour IS 6110-fAFLP with the classic IS 6110-RFLP on the ability to detect recent transmission in the city of Barcelona, Spain

The purpose of the study was to compare the IS6110-RFLP (RFLP) results obtained in a previous epidemiological study in the city of Barcelona, Spain [Borrell S, Espanol M, Orcau A, Tudo G, March F, Cayla JA, et al. Factors associated with differences between conventional contact tracing and molecular epidemiology in study of tuberculosis transmission and analysis in the city of Barcelona, Spain. J Clin Microbiol 2009 Jan;47(1):198-204.] with the results obtained with IS6110-fAFLP, [Thorne N, Evans JT, Smith EG, Hawkey PM, Gharbia S, Arnold C. An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110-restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting. Clin Microbiol Infect 2007 Oct;13(10):964-70.] on the ability to detect recent transmission. fAFLP was applied to DNA samples of RFLP clustered strains, with and without known epidemiological links, with the additional inclusion of four nucleotide-specific fluorophores to further increase the discrimination of the fragments obtained. Four-colour fAFLP was performed on 123 RFLP clustered strains with no epidemiological link (NELC) and on 28 epidemiologically linked RFLP clustered (ELC) strains grouped into 48 and 13 clusters respectively. Clustering results obtained by the two methods were highly congruent in ELC strains with fAFLP allocating 92.3% of the ELCs. For the NELCs, RFLP results were confirmed in 39/48 (81.2%) of fAFLP-clusters with 0-1 different fragments and 9/48 (18.8%) differed in 2-4 fragments, which are considered genetically related but not recently transmitted. In conclusion, overestimation of recent tuberculosis transmission can occur because of the inaccurate analysis of RFLP results. Four-colour fAFLP allows us to differentiate between recent transmission strains and epidemiologically unrelated but genetically related strains.

Comparison of a Variable-Number Tandem-Repeat (VNTR) Method for TypingMycobacterium aviumwith Mycobacterial Interspersed Repetitive-Unit-VNTR and IS1245Restriction Fragment Length Polymorphism Typing

Mycobacterium avium complex (MAC) infections are increasing annually in various countries, including Japan, but the route of transmission and pathophysiology of the infection remain unclear. Currently, a variable-number tandem-repeat (VNTR) typing method using the Mycobacterium avium tandem repeat (MATR) loci (MATR-VNTR) is employed in Japan for epidemiological studies using clinical isolates of M. avium. In this study, the usefulness of this MATR-VNTR typing method was compared with that of the IS1245-restriction fragment length polymorphism (IS1245-RFLP) typing method and a mycobacterial interspersed repetitive-unit (MIRU)-VNTR typing method reported previously (V. C. Thibault, M. Grayon, M. L. Boschiroli, C. Hubbans, P. Overduin, K. Stevenson, M. C. Gutierrez, P. Supply, and F. Biet, J. Clin. Microbiol. 45:2404-2410, 2007). Seventy clinical isolates identified as M. avium from human immunodeficiency virus-negative patients with MAC infections were used. MATR-VNTR typing using 15 loci distinguished 56 patterns of different allele profiles, yielding a Hunter-Gaston discriminatory index (HGDI) of 0.990. However, IS1245-RFLP and MIRU-VNTR typing yielded HGDIs of 0.960 and 0.949, respectively, indicating that MATR-VNTR has an excellent discriminatory power compared with MIRU-VNTR and IS1245-RFLP typing. Moreover, concomitant use of the MATR-VNTR method and IS1245-RFLP typing increased the HGDI to 0.999. MATR-VNTR typing is inexpensive and easy to perform and could thus be useful in establishing a digital multifacility database that will greatly contribute to the clarification of the transmission route and pathophysiology of M. avium infections.

Mycobacterium aviumsubsp.hominissuisInfection in a Pet Parrot

To the Editor: Tuberculosis is a chronic wasting disease in domestic birds (especially hens) and free-ranging birds worldwide (1). Most mycobacterial infections in birds are caused by Mycobacterium avium subsp. avium (mainly domestic birds) or by M. genavense (especially pet birds). Nontuberculous (potentially pathogenic) mycobacteria (i.e., M. fortuitum, M. gordonae, and M. nonchromogenicum) occasionally have been isolated from necropsied pet birds (2). Because potentially pathogenic mycobacteria also are increasingly problematic in immunocompromised human patients, they merit special attention. M. avium subsp. hominissuis can infect humans, especially immunocompromised persons. M. avium subsp. hominissuis infections have been documented in pigs and cattle (3) and rarely in dogs (4), birds (5), and other animals.

Characterization of IS6110 insertions in the dnaA–dnaN intergenic region of Mycobacterium tuberculosis clinical isolates

Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to be clonally related. The presence of IS6110 in the dnaA-dnaN intergenic region, one preferential locus for the integration of IS6110, was evaluated in 125 M. tuberculosis isolates. Five isolates had IS6110 inserted in this region, and two consisted of a mix of isogenic strains that putatively have evolved during a single infection. Strains from the same isolate had identical spoligo and mycobacterial interspersed repetitive unit-variable-number tandem repeat profiles, but had slight variations in IS6110 RFLP patterns, due to the presence of IS6110 in the dnaA-dnaN intergenic region. Duplication of the dnaA-dnaN intergenic region was found in one isogenic strain.

IS1245 RFLP analysis of strains of Mycobacterium avium subspecies hominissuis isolated from pigs with tuberculosis lymphadenitis in Portugal

From the end of 2004 increasing numbers of tuberculosis-like lesions were detected in regular meat inspections of pigs at regional abattoirs. Mycobacterium avium subspecies hominissuis was identified as the cause...

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

BackgroundMycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission.Results164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900 - restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property.ConclusionThe results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

Occupational tuberculosis following extremely short exposure

Transmission of Mycobacterium tuberculosis (MT) in most cases requires extended exposure. To document that MT transmission may occur even after very short exposure. All first-time culture-confirmed tuberculosis (TB) cases in Denmark have since 1992 been subjected to genotyping, using the IS6110-Restriction Fragment Length Polymorphism (RFLP) technique. A young nurse with no risk factors developed pulmonary TB: the DNA pattern of her MT strain was compared to The Danish TB Subtyping Database, comprising >6000 DNA patterns from TB patients nationwide. Only one single MT DNA pattern matched the DNA profile of the isolate from the nurse. The pattern originated from a patient shortly admitted to the department where she worked at the time. MT transmission had occurred in spite of very short exposure. By adding modern molecular epidemiological methods to traditional epidemiological surveys, a more detailed picture of MT-transmission pathways can be obtained, showing that MT transmission can occur even after extremely short exposure. This stresses the necessity for adequate respiratory protection among hospital staff taking care of patients with pulmonary symptoms suspected for TB.

Combined Multilocus Short-Sequence-Repeat and Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem-Repeat Typing ofMycobacterium aviumsubsp.paratuberculosisIsolates

Short-sequence-repeat (SSR) sequencing was applied to 127 Mycobacterium avium subsp. paratuberculosis isolates typed by mycobacterial interspersed repetitive unit-variable-number tandem repeats (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP). Combined MIRU-VNTR and SSR typing followed by secondary IS900 RFLP typing is an improved approach to high-resolution genotyping of this pathogen.

The Recent-Transmission of Mycobacterium tuberculosis Strains among Iranian and Afghan Relapse Cases: a DNA-fingerprinting using RFLP and spoligotyping

BackgroundRelapse of tuberculosis (TB) may develop as the result of reactivation of the endogenous primary infection, or as a result of a exogenous reinfection. This survey evaluated the rate of reactivation versus recent transmission among Iranian and Afghan relapse cases.MethodsThe sputum specimens were digested, examined microscopically for acid-fast bacilli, and inoculated into Löwenstein-Jensen slants by standard procedures. Thereafter, the susceptibility and identification tests were performed on culture positive specimens. Subsequently, the strains that were identified as Mycobacterium tuberculosis (258 isolates) were subjected to IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. Additional patient's information was collected for further epidemiological analysis. Patients whose isolates had identical genotyping patterns were considered a cluster with recent transmission episode.ResultsOut of 258 available isolates, 72(28%) had multi-drug resistant (MDR-TB) in ratio and 42 (16.2%) had other resistant. Notably, 38 of MDR-TB cases (52%) were isolated from Afghan patients. By IS6110-RFLP typing method, 65 patients (25%) were clustered in 29 clusters. In cluster cases, the intra-community transmissions between Iranian and Afghan patients were 41%. All MDR-TB patients in clusters had either Haarlem I or Beijing characteristic. The risk factors like sex, family history, close contact, living condition, PPD test result and site of TB infection were not associated with clustering. Although, the MDR-TB strains were more frequent in non-cluster cases (31%) than cluster one(18%) (P < 0.05). Majority of M. tuberculosis strains isolated from non-cluster cases were belong to EAI3 (51; 30%) and CASI(32;18.6%) superfamilies.ConclusionDuring the studied period, reactivation of a previous infection remain the more probable cause of recurrence. Although, the evidence of intra- community transmission between Iranian and Afghan TB cases, highlighted the impact of afghan immigrants in national tuberculosis control program (NTP) of Iran.

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.

Determinants of Cluster Size in Large, Population-Based Molecular Epidemiology Study of Tuberculosis, Northern Malawi

Tuberculosis patients with identical strains of Mycobacterium tuberculosis are described as clustered. Cluster size may depend on patient or strain characteristics. In a 7-year population-based study of tuberculosis in Karonga District, Malawi, clusters were defined by using IS6110 restriction fragment length polymorphism, excluding patterns with <5 bands. Spoligotyping was used to compare strains with an international database. Among 682 clustered patients, cluster size ranged from 2 to 37. Male patients, young adults, and town residents were over-represented in large clusters. Cluster size was not associated with HIV status or death from tuberculosis. Spoligotypes from 9 (90%) of 10 large cluster strains were identical or very similar (1 spacer different) to common spoligotypes found elsewhere, compared with 37 (66%) of 56 of those from nonclustered patients (p = 0.3). Large clusters were associated with factors likely to be related to social mixing, but spoligotypes of common strains in this setting were also common types elsewhere, consistent with strain differences in transmissibility.

Evaluation and Strategy for Use of MIRU-VNTRplus, a Multifunctional Database for Online Analysis of Genotyping Data and Phylogenetic Identification ofMycobacterium tuberculosisComplex Isolates

Because of its portable data, discriminatory power, and recently proposed standardization, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing has become a major method for the epidemiological tracking of Mycobacterium tuberculosis complex (MTBC) clones. However, no public MIRU-VNTR database based on well-characterized reference strains has been available hitherto for easy strain identification. Therefore, a collection of 186 reference strains representing the primary MTBC lineages was used to build a database, which is freely accessible at http://www.MIRU-VNTRplus.org. The geographical origin and the drug susceptibility profile of each strain were stored together with comprehensive genetic lineage information, including the 24-locus MIRU-VNTR profile, the spoligotyping pattern, the single-nucleotide- and large-sequence-polymorphism profiles, and the IS6110 restriction fragment length polymorphism fingerprint. Thanks to flexible import functions, a single or multiple user strains can be analyzed, e.g., for lineage identification with or without the use of reference strains, by best-match or tree-based analyses with single or combined marker data sets. The results can easily be exported. In the present study, we evaluated the database consistency and various analysis parameters both by testing the reference collection against itself and by using an external population-based data set comprising 629 different strains. Under the optimal conditions found, lineage predictions based on typing by 24-locus MIRU-VNTR analysis optionally combined with spoligotyping were verified in >99% of the cases. On the basis of this evaluation, a user strategy was defined, which consisted of best-match analysis followed, if necessary, by tree-based analysis. The MIRU-VNTRplus database is a powerful tool for high-resolution clonal identification and has little equivalent in terms of functionalities among the bacterial genotyping databases available so far.

Knowledge of tuberculosis transmission among recently infected patients in Glasgow

To examine perceptions and understanding of disease causation in tuberculosis patients with few epidemiological links detected by contact tracing. An exploratory qualitative study using semi-structured interviews was undertaken. Patients' beliefs were explored against a background of medical thinking about tuberculosis transmission and the current approach to contact tracing. Interviews were undertaken with patients (n=23) or suitable next of kin (n=3). Study patients were diagnosed with tuberculosis in Glasgow, an urban area of Scotland, between 1997 and 2004. All had a genetically indistinguishable 15-banded IS6110 restriction fragment length polymorphism pattern of the Beijing family of Mycobacterium tuberculosis, suggestive of recently transmitted infection, yet few had epidemiological links detectable as a result of contact tracing (30.8%). Interviewees had varying levels of knowledge, but most believed that tuberculosis was caused by a pathogen, spread by person-to-person contact. Modes of transmission were thought to include airborne transmission, sharing utensils with an infected individual, consumption of contaminated foods/liquids, and exchange of bodily fluids. Prolonged contact was not thought to be required for transmission to occur. Impaired immunity, social factors and environmental factors were believed to enhance the potential for transmission. Patients have complex beliefs about tuberculosis transmission and causation, which do not always mirror those of health professionals. Adopting and implementing an approach to contact identification that is aligned with lay beliefs may result in improved contact tracing outcomes.

Spoligotypes of Mycobacterium tuberculosis isolates of a high tuberculosis burden aboriginal township in Taiwan

To understand the genotype of Mycobacterium tuberculosis isolates circulating in the aboriginal Sioulin Township, the highest tuberculosis (TB) endemic area in Taiwan, a total of 138 isolates were collected between January 2003 and December 2004 for genotyping. Genotyping consisted of spacer oligonucleotide typing (spoligotyping), IS 6110-restriction fragment length polymorphism (RFLP) and mgtC and ogt single nucleotide polymorphisms (SNPs) characterizations. Spoligotyping data were compared with those from the fourth international spoligotyping database, SpolDB4. Of 27 resolved spoligotypes, 14 spoligotype patterns matched those found in SpolDB4 and 13 (TW1–13) were identified as novel. The most common among the 14 defined spoligotypes was Beijing ST1 (35.5%, 49/138), followed by Haarlem ST742 (10.9%, 15/138), Latin-American-Mediterranean (LAM) ST33 (5.8%, 8/138) and Haarlem ST50 (3.6%, 5/138). Of the 13 novel spoligotypes, 5 (TW 6–8, 12 and 13) were identified as “Haarlem-like” lineages according to clade analyses of spoligotyping and RFLP dendrograms. Overall, major spoligotypes found in Sioulin Township were Haarlem and Haarlem-like (39.1%), Beijing (38.4%), and LAM (5.8%) lineages. Interestingly, the results did not indicate any East-African-Indian lineages, which are highly prevalent in Far-East Asia. Our data also contained the first evidence of ST33 (LAM3 lineage) in Asia. This study provides first depiction of molecular epidemiology of M. tuberculosis in this isolated aboriginal population and further elucidation of the global historical expansion of the isolates.

Highly polymorphic variable-number tandem repeats loci for differentiating Beijing genotype strains ofMycobacterium tuberculosisin Shanghai, China

The PCR-based variable-number tandem repeats (VNTR) typing method is a very promising tool for the molecular epidemiological study of Mycobacterium tuberculosis. The discriminatory power of the VNTR loci that were optimized in many previous studies has not been evaluated in Shanghai, an area where Beijing genotype strains dominate. In the present study, we first performed a literature search to identify VNTR loci that were at least 45 bp in length. Second, we determined the Hunter-Gaston discriminatory index (HGI) values of each of the 45 VNTR loci that we identified, using Beijing genotype strains from a 'test set' of isolates from a population with low migration in Chongming Island, Shanghai, China. Third, we optimized two sets of VNTR loci, which we named VNTR-7 and VNTR-16. The HGI value of VNTR-7 was slightly lower than that of IS6110 restriction fragment length polymorphisms (RFLP), and the HGI values of VNTR-16 and IS6110 RFLP were comparable. Our results suggest that VNTR-7, followed by VNTR-16 and IS6110 RFLP, can be used routinely as a tool to discriminate between M. tuberculosis isolates in population-based epidemiologic studies of M. tuberculosis transmission in Shanghai, China.