Email updates

Keep up to date with the latest news and content from BMC Infectious Diseases and BioMed Central.

Open Access Highly Accessed Research article

Molecular typing of mycobacterium tuberculosis isolates circulating in Jiangsu Province, China

Qiao Liu1, Dandan Yang2, Weiguo Xu2, Jianming Wang1, Bing LV3, Yan Shao2, Honghuan Song2, Guoli Li2, Haiyan Dong3, Kanglin Wan3 and Hua Wang12*

  • * Corresponding author: Hua Wang hua@jscdc.cn

  • † Equal contributors

Author Affiliations

1 Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, PR China

2 Department of Chronic Communicable Disease, Center for Disease Control and Prevention of Jiangsu Province, Nanjing, PR China

3 National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Beijing, PR China

For all author emails, please log on.

BMC Infectious Diseases 2011, 11:288  doi:10.1186/1471-2334-11-288


The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2334/11/288


Received:24 May 2011
Accepted:26 October 2011
Published:26 October 2011

© 2011 Liu et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Globally, China is the second place with high burden of tuberculosis (TB). To explore the characteristics of the pathogens of Mycobacterium tuberculosis (MTB) circulating in this area is helpful for understanding and controlling the spread of the strains. Recent developments in molecular biology have allowed prompt identification and tracking specific strains of MTB spreading through the population.

Methods

Spacer-oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) were performed in combination to yield specific genetic profiles of 260 MTB strains isolated from 30 counties of Jiangsu province in China between June and July 2010. The spoligotyping results were in comparison to the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4). Drug susceptibility test (DST) was performed on all strains by proportion method on Lowenstein-Jensen (LJ) culture media.

Results

Based on the spoligotyping method, 246 strains displayed known patterns and 14 were absent in the database. Predominant spoligotypes belonged to the Beijing family (80.4%). By using the 24-loci VNTR typing scheme, 224 different patterns were identified, including 20 clusters and 204 unique patterns. The largest clade comprised 195 strains belonging to the Beijing family. The combination of spoligotyping and 24-loci MIRU-VNTR demonstrated maximal discriminatory power. Furthermore, we observed a significant association between Beijing family strains and drug-resistant phenotypes. The Beijing family strains presented increased risks for developing multi-drug resistant TB, with the OR (95% CI) of 11.07(1.45-84.50).

Conclusions

The present study demonstrated that Beijing family isolates were the most prevalent strains circulating in Jiangsu province of China. The utility of spoligotyping in combination with 24-loci MIRU-VNTR might be a useful tool for epidemiological analysis of MTB transmission.

Background

Tuberculosis (TB) has been a global threat to human health and it remains a major public health burden in developing countries. As a public health dilemma, drug resistance has been an obstacle to achieve the goal of effective TB control. In 2009, it was reported that about 9.4 million incident cases (8.9 million-9.9 million) occurred globally and among them, 250 000 (230 000-270 000) were multi-drug resistant TB (MDR-TB) [1]. China is one of the five countries with the largest number of both incident cases (1.1-1.5 million) and MDR-TB cases (79 000-120 000) in absolute terms in 2008 [1]. The increased rate of drug-resistance and MDR strains of TB remain a serious problem of TB control in China.

MDR-TB may arise by inadequate antituberculosis treatment or by direct transmission of drug resistant strains from one individual to another [2]. The association with MDR-TB has led to concerns that Mycobacterium tuberculosis (MTB) strains may have a predilection for acquiring drug resistance. DNA fingerprinting of MTB isolates is a powerful tool to study the molecular epidemiological characteristics of MDR-TB. The traditional method used to genotype MTB complex (MTBC) is insertion sequence (IS) 6110 restriction fragment-length polymorphism (RFLP), which can be utilized in outbreak investigations, long-term surveillance and the detection of laboratory contamination. Despite the widespread use of IS6110-based RFLP analysis, it remains a labour intensive, technically demanding, and insufficiently discriminating strains with the low copy numbers (fewer than six) of IS6110 [3]. Following IS6110-based RFLP, spoligotyping has been the most widely used method for secondary typing of MTB strains and it has been particularly useful for identifying strains belonging to the Beijing/W family due to the absence of spacers 1-34 in the direct repeat (DR) region of the MTB genome [4]. Variable number tandem repeat (VNTR) is a newly developed typing method, which determines the number of repeated mycobacterial interspersed repetitive units (MIRU) by using small quantities of bacteria [5]. MIRU-VNTR has shown a higher discriminatory power when combining with spoligotyping. The advantages of such PCR-based genotyping techniques include their easy digitalization of the generated profiles and hence easy inter-laboratory comparison, as well as easy creation and maintenance of the database [6].

The Beijing family, first identified in 1995 in Beijing of China, almost omnipresent and significantly prevalent in certain world regions, e.g., East Asia and the former Union of Soviet Socialist Republics(USSR) [7]. Several studies have observed that the Beijing family MTB strains had exhibited important pathogenic features which might be associated with the drug resistant TB in China [6,8-10]. As the prevalence of drug resistant clones of MTB varies from one area to another, studies in geographical distribution of resistant clones are helpful for understanding the epidemiological characteristics of TB.

Jiangsu is a province locating along the eastern coast of China and it covers an area of 102.6 thousand square kilometers, with a total population of 77 million in 2009. The high prevalence of drug resistance among TB patients in Jiangsu province has been a major challenge for TB control. According to the most recently updated data, the proportion of primary and acquired MDR-TB were 7.63% and 33.7%, respectively [11].

The main goal of this study was to characterize the genotyping of MTB strains circulating in Jiangsu Province and to explore whether there is any evidence that the spreading of Beijing family of MTB strains was associated with the drug resistance.

Methods

Mycobacterial strains

The study was conducted in 30 counties of Jiangsu province, which were sampled according to the "Guidelines for surveillance of drug resistance in tuberculosis" developed by WHO/IUATLD [12]. Two hundred and sixty newly diagnosed sputum smear positive pulmonary TB patients from June 2010 to July 2010 in the study sites were recruited. Totally, there were 216 new cases and 44 previously treated ones. The average age of cases was 48.5 years old. All TB patients were diagnosed by referring to the national guidelines of China and reviewed by Jiangsu provincial Center for Disease Control and Prevention. Each subject present three sputum smear samples with labeled plastic bottles for sputum smear microscopy test. The two sputum samples with the highest bacterial counts were cultured, and one culture was submitted to the provincial reference laboratory for drug susceptibility test (DST). Sputum smear microscopy and culture were performed at the level of county (district) laboratory.

Strains isolation and drug susceptibility test

The sputum samples were cultured and isolated on Lowenstein-Jensen (LJ) culture media. LJ medium was impregnated with isoniazid (INH), rifampicin (RIF), streptomycin (SM), and ethambutol (EMB). The concentrations of anti-tuberculosis drugs were 0.2 μg/ml for INH, 40 μg/ml for RIF, 4 μg/ml for SM, and 2 μg/ml for EMB. The strain was declared resistant to the specific drug; or it was defined as sensitive when the growth rate was < 1% compared to the control. MDR-TB was defined as strains being resistant to at least RIF and INH [11]. Strains isolation, identification and drug susceptibility test (DST) were performed at the provincial reference laboratory.

Genomic DNA extraction

Mycobacterial genomic DNA was extracted from mycobacterial colonies growing on LJ medium by resuspending one loop of mycobacterial colonies in 200 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA) and was incubated at 85°C for 30 minutes to obtain genomic DNA. After centrifugation of the suspension, the supernatant fluid containing DNA was removed and stored at -20°C until further use [13]. Laboratory strain MTB H37Rv was used as a control for all microbiological and genetic procedures.

Genotyping

Spoligotyping of the isolates was performed as described by Kamerbeek et al [4]. In brief, genomic DNA of MTB isolates were amplified using polymerase chain reaction (PCR) with the primers of Dra (biotin labelled) and Drb, and then the PCR products were hybridized to a set of 43 oligonucleotide probes corresponding to each spacer, which were covalently bound to a membrane [4]. Spoligotypes in binary format were compared with the SpolDB4 database. In particular, strains with spoligotype patterns characterized by deletion of spacers 1-34 were defined as "typical" Beijing genotypes, whereas strains with additional deletion of one or more of the last nine spacers were defined as Beijing-like genotypes according to the criteria of the international database SpolDB4 [4,14-16].

To identify a suitable MIRU-VNTR loci set for genotyping MTB in this area, the number of tandem repeats was determined in 24 MIRU-VNTR genetic loci: ten original MIRU-VNTR loci; six loci of exact tandem repeats (ETRs): ETR-A, -B, -C, -D, -E and -F; five Mtub loci: Mtub4, 21, 30, 38, and 39; and three Queen's University of Belfast (QUBs) loci: QUB-11b, -26, and 4156c. PCR amplification was performed for correspondent loci as described in the previous studies [17-20]. PCR products were analyzed on a 2% agarose gel against a 100-bp DNA ladder, and the copy number at each locus was calculated using the Quantity 1 gel imaging system [10]. Determination of the discriminatory power of the VNTR loci was calculated using the Hunter-Gaston discriminatory Index (HGDI) [21]. The HGDI was calculated using the following formula:

<a onClick="popup('http://www.biomedcentral.com/1471-2334/11/288/mathml/M1','MathML',630,470);return false;" target="_blank" href="http://www.biomedcentral.com/1471-2334/11/288/mathml/M1">View MathML</a>, where D is the numerical index of discrimination, N is the total number of strains in the typing scheme, s is the total number of different strain types, and nj is the number of strains belonging to the jth type.

Data analysis

Spoligotypes in binary format were entered in an Excel spreadsheet and compared with the spoligotyping database SpolDB4. The patterns were established clusters in the BioNumerics software version 5.0 (Applied Maths, Sint-Martens-Latem, Belgium). The strength of association between genotypes of MTB strains and drug resistance was estimated by odds ratio (OR) and 95% confidence interval (95% CI). All tests of significance were two sided and a significant threshold was set at 0.05. Statistical analyses were carried out by using SPSS software 13.0 (SPSS Inc., USA).

Ethical consideration

This project has been approved by Institutional Review Board of Nanjing Medical University. Written informed consent was obtained from all participants. Ethics has been respected throughout the whole study period.

Results

Spoligotyping analysis

Reproducible results were obtained for 260 strains and 34 distinct spoligotype clusters were obtained as referring to the SpolDB4.0 database. Twenty-seven spoligotypes represented the single isolate and the other 233 isolates were grouped into 7 clusters containing from 2 to 199 isolates (Figure 1). Among them, 14 (5.4%) strains were found to be undefined (absent in the SpolDB4.0 database) while 246(94.6%) were successfully clustered by spoligotyping and divided into 11 types (STs). Out of these, 5 STs had 2 or more strains whereas 6 STs had single isolate each. A determination of predominant STs were Beijing family (n = 209, 80.4%), T1 (n = 23, 8.8%) and T2 (n = 6, 2.3%), representing about 91.5% of total strains (Table 1).

thumbnailFigure 1. Samples with MIRU-VNTR and spoligotyping. Clustering was based upon an average of MIRU-VNTR and spoligotyping, clustered using the dice coefficient and the unweighted pair group method with arithmetic averages (UPGMA) in BioNumerics 5.0.

Table 1. Spoligotypes shared by Mycobacterium tuberculosis strains evaluated in this study

MIRU-VNTR

Among 260 MTB strains being genotyped, 224 different VNTR genotypes were detected. Two hundred and four (91.1%) were unique (i.e., observed for only one strain) and 56 strains could be grouped into 20 clusters, each including 2 to 8 strains (Additional file 1). Two main clusters which contained 8 (14.3%) and 7 (12.5%) strains showed 4 2 4 3 4 3 2 3 3 2 5 1 5 3 3 3 4 5 4 1 4 5 6 3 and 4 2 4 3 4 3 2 3 3 2 5 1 5 3 3 3 4 5 4 1 4 6 6 3 VNTR profiles, respectively (Figure 1). HGDI scores which were calculated for particular MIRU loci varied significantly from 0.717 of Qub11b to 0 of MIRU24. As suggested by Sola et al [22], the MIRU loci were further classified into highly (> 0.6), moderately (0.3 to 0.6), and poorly (< 0.3) discriminating based on the HGDI scores. Five loci (ETRB, ETRC, MIRU20, MIRU02, MIRU24) showed negligible diversity (HGDI < 0.1) whereas ETRE and Qub11b had highly discriminating ability, especially for Beijing family (Table 2).

Additional file 1. MIRU-VNTR genotypes of Mycobacterium tuberculosis isolates. Clustering was based upon an average of MIRU-VNTR, clustered using the categorical co-efficient and UPGMA in BioNumerics 5.0.

Format: PDF Size: 21KB Download file

This file can be viewed with: Adobe Acrobat ReaderOpen Data

Table 2. Variability of 24-VNTR loci among Mycobacterium tuberculosis strains (n = 260)

Drug-susceptibility patterns of the MTB isolates

A total of 260 MTB strains isolated from the sputum samples of TB patients were used for drug susceptibility test. Among them, 13.1% (34/260) were MDR-TB strains, which were resistant to at least INH and RIF, the two most powerful antituberculosis drugs. Furthermore, we compared the distribution of drug resistance between Beijing and non-Beijing genotyping strains. As shown in table 3, among non-Beijing family strains, the proportion of mono-drug resistance was 7.8% (n = 4) to INH, 2.0% (n = 1) to RIF, 9.8% (n = 5) to SM, while 19.6% (n = 10) were resistant to at least one drug and 2.0% (n = 1) were MDR-TB. For Beijing family strains, the proportion of mono-drug resistance was 3.3% (n = 7) to INH, 1.4% (n = 3) to RIF, 5.7% (n = 12) to SM and 0.5% (n = 1) to EMB, respectively, while 11.0% (n = 23) were resistant to at least one drug and 15.8% (n = 33) were MDR-TB. A binary logistic regression model was applied to analyze the factors associated with MDR-TB. The Beijing family strains presented increased risks for developing MDR (15.8% vs.2.0%, OR: 11.07, 95%CI: 1.45-84.50) (Table 4).

Table 3. Differences of characteristics between Beijing and non-Beijing family

Table 4. Factors associated with multi-drug resistant tuberculosis

Discussion

This study demonstrates that the Beijing genotype is the most predominant lineage of TB strains in Jiangsu province, which is consistent to the findings from other areas of China. As described in table 5, Beijing family accounts for 85.12% to 92.59% of the MTB strains currently epidemic in the Beijing area [23,24] and it is also prevalent in Heilongjiang (89.50%) [10], Gansu (87.50%) [24], Jilin (89.88%) [24], Ningxia (67.12%) [25], Henan (80.00%) [24], Shandong (85.93%) [9], Shanxi (80.00%) [24], Tianjin(91.07%) [26], Tibet (90.38%) [24], Xinjiang (67.80%) [24], Anhui (85.35%) [24], Fujian (54.50%) [24], Hong Kong (68.45% and 70.00%) [27,28], Hunan (66.00%) [24], Jiangsu and Zhejiang (69.23%) [29], Shanghai (77.14%) [30], Zhejiang (64.84%) [24], Guangxi (55.29%) [24], Taipei (52.53%) [13], and Sichuan (57.89% to 63.64%) [24,31]. It was found that amongst all MTB strains studied, the "Beijing" genotype strains were highly prevalent in our geographic area. The proportion of Beijing family circulating in the north area (83.91%) is higher than that in the south area (66.40%) of China (P < 0.001).

Table 5. Distribution of Beijing family strains in China

Though world widely the Beijing genotype is apparently the second most prevalent genotype, following after AFR lineage [16], reasons for its successful global spread remain poorly understood. The infective success of this lineage seems to be associated with its effect on the immune response, in that it can control the release of the macrophage-derived cytokines that play a central role in directing the immune response towards a non-protective Th2 phenotype [32,33]. The widely distributed (but not universal) association of drug resistance and the Beijing genotype suggests that these strains may have a particular propensity for acquiring drug resistance [14]. Besides China, they are increasingly reported in other areas of the world and are frequently associated with outbreaks of TB or MDR-TB [14,29,34-37], but not all [9,10,27,38,39]. In the present study, we observed a significant difference of drug resistance between Beijing and non-Beijing strains, where the Beijing family was associated with an increased risk of MDR-TB. These data are accordant with the results from studies so far performed in China [29,40]. Definite conclusions on the extent of spread and associations with drug resistance, however, could not yet be drawn. This was due to the limited amount of information available from most areas of the world, the possible biases in many of the published reports, and the absence of standard definitions and study designs.

Both spoligotyping and MIRU-VNTR have different abilities to discriminate MTB strains and can be performed in combination. For example, among 260 strains included in this analysis, 209 (80.4%) were classified as the Beijing genotype. The majority of these Beijing genotype isolates showed the typical spoligotype pattern (hybridization to all of spacers 35-43 and no hybridization to spacers 1-34). Four strains showed the non-Beijing genotype spoligotype pattern, but had specific Beijing genotype signatures of MIRU-VNTR. Furthermore, another four strains showing the typical Beijing genotype spoligotype pattern, but can't be distinguished by MIRU-VNTR (Figure 1). One possible explanation might be the multiple infections of strains in these eight samples [41-43], however, it is rarely found when using the DNA fingerprinting method. Till now, no definition of the Beijing genotype on the basis of genetic markers will be 100% perfect, because there will always be exceptional strains [14]. As shown in our study, when 209 strains in the Beijing clade identified by spoligotyping were further discriminated by MIRU-VNTR, nearly all (205/209, 98.1%) Beijing strains were parts of a clade (Figure 1). This study further demonstrated that MTB strains grouped into the Beijing family by spoligotyping had similar patterns of MIRU-VNTR. This was borne out by the fact that all MIRU-VNTR patterns of Beijing family strains were highly similar.

Spoligotyping has been proved to be the most useful method to recognize Beijing lineage strains as it is the most rapid and easiest method to apply and it correlates well with the other methods [14]. But this method had a poor discriminability when it was used independently, thus should be performed in combination with another independent method (for example MIRU-VNTR typing) to allow effective epidemiological investigation [22]. As shown in the present study, spoligotyping alone yielded 34 genotypes, 7 clusters, with 27 unique genotypes and a HGDI of 0.409. MIRU-VNTR clustered 21.5% of strains with an HGDI of 0.998. The combination of spoligotyping and MIRU-VNTR typing in our population clustered 19 of all 260 strains. Different VNTR typing sets showed various efficiencies in different Beijing genotype strains (Table 6). The discriminatory power of the traditional 12 loci MIRU-VNTR has been found to be insufficient [5], which is often failed to differentiate clonal strains in areas where the Beijing family genotype is predominant [27]. Other sets of MIRU-VNTR loci, such as an optimized set of 24 loci, have also been defined. However, all 24 loci are not required for MTB genotyping in any given situation [5], as the number of loci required depends on the lineage known to be prevalent in the investigated area. We have evaluated MIRUs loci, QUB loci and ETR loci individually for their abilities to differentiate the Beijing and non-Beijing genotype families of MTB. When the top 12 loci (MIRU26,40,10, 39; QUB11b,26; Mtub21,04,39,30; ETR E,D) were selected for typing, the set produced a HGDI value of 0.996 in all stains and 0.994 in Beijing family stains, which was almost equal to that obtained in 24 loci MIRU-VNTR (0.998 and 0.997). This optimized 12 loci VNTR typing set could be an important tool for tracking MTB of the Beijing family in this area.

Table 6. Comparative analysis of genotyping methods applied to Mycobacterium tuberculosis strains (n = 260)

As observed by 24-MIRU-VNTR analysis, it appeared that individuals infected with strains belonging to the Beijing genotype were more likely to be part of a cluster. Beijing strains were isolated from patients from different counties of Jiangsu Province, suggesting that these virulent strains are spreading throughout the province. In addition to Beijing family strains, we also detected strains belonging to other families such as T1, T2, H3, H4, CAS, LAM, U, and MANU2. The CAS family was previously found primarily in India [44], and also detected in Tibet and Xinjiang [24]. The LAM family was predominantly prevalent in South America and West-Africa, and also found in Taiwan of China [13]. It can be suggested that strains of these families have started spreading across Jiangsu province; of course, this requires further testing using more extended typing of more clinical strains.

Future studies are needed to clarify the characteristics of Beijing family, one important genotype of MTB. Possible variations in host-pathogen interactions among strains of various genotypes needs to be identified and the role of Beijing genotype infection as a possible risk factor for drug resistance and/or treatment failure must urgently be addressed in longitudinal studies in the affected high incident regions. In short, the effect of this genotype on TB control efforts need to be further investigated.

Conclusion

This is the first report on the genotypes of MTB stains using spoligotyping in combination with 24-loci MIRU-VNTR technology in Jiangsu province of China. Based on our preliminary data, Beijing family strains have predominantly prevalent in Jiangsu and shown a high number of clusters in the study population. Furthermore, we also observed that the high prevalence of Beijing genotype may be associated with the risk of MDR-TB. Further genome level studies will be needed to investigate the molecular specificities of the major subgroup.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

QL, HW, WX conceived the study, carried out the molecular genetic studies, analyzed the data and drafted the manuscript; DY, YS, HS, GL participated in the study design, implemented the field investigation and performed DST tests; BL and YD participated in the genotyping analysis. KW and JW participated in the study design and helped draft the manuscript. All authors contributed to the study and have read and approved the final manuscript.

Acknowledgements and funding

This study is supported by "Transmission Mode of Tuberculosis Project of the National Key Programme of Mega Infectious" (2008ZX10003-010) and "National S&T Major Project Foundation of China" (2009ZX10004-904).

References

  1. Global tuberculosis control - surveillance,planning,financing.WHO Report

    2010.

  2. Toungoussova OS, Bjune G, Caugant DA: Epidemic of tuberculosis in the former Soviet Union: social and biological reasons.

    Tuberculosis (Edinb) 2006, 86(1):1-10. Publisher Full Text OpenURL

  3. Christianson S, Wolfe J, Orr P, Karlowsky J, Levett PN, Horsman GB, Thibert L, Tang P, Sharma MK: Evaluation of 24 locus MIRU-VNTR genotyping of Mycobacterium tuberculosis isolates in Canada.

    Tuberculosis (Edinb) 2010, 90(1):31-38. Publisher Full Text OpenURL

  4. Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, Bunschoten A, Molhuizen H, Shaw R, Goyal M, et al.: Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.

    Journal of Clinical Microbiology 1997, 35(4):907-914. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  5. Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rusch-Gerdes S, Willery E, Savine E, de Haas P, van Deutekom H, Roring S, et al.: Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis.

    Journal of Clinical Microbiology 2006, 44(12):4498-4510. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  6. Mokrousov I, Narvskaya O, Limeschenko E, Vyazovaya A, Otten T, Vyshnevskiy B: Analysis of the allelic diversity of the mycobacterial interspersed repetitive units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations.

    Journal of Clinical Microbiology 2004, 42(6):2438-2444. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  7. van Soolingen D, Qian L, de Haas PE, Douglas JT, Traore H, Portaels F, Qing HZ, Enkhsaikan D, Nymadawa P, van Embden JD: Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia.

    Journal of Clinical Microbiology 1995, 33(12):3234-3238. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  8. Hu Y, Hoffner S, Jiang W, Wang W, Xu B: Extensive transmission of isoniazid resistant M. tuberculosis and its association with increased multidrug-resistant TB in two rural counties of eastern China: a molecular epidemiological study.

    BMC Infect Dis 2010, 10:43. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  9. Ma X, Wang H, Deng Y, Liu Z, Xu Y, Pan X, Musser JM, Graviss EA: rpoB Gene mutations and molecular characterization of rifampin-resistant Mycobacterium tuberculosis isolates from Shandong Province, China.

    Journal of Clinical Microbiology 2006, 44(9):3409-3412. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  10. Wang J, Liu Y, Zhang CL, Ji BY, Zhang LZ, Shao YZ, Jiang SL, Suzuki Y, Nakajima C, Fan CL, et al.: Genotypes and characteristics of clustering and drug-susceptibility of Mycobacterium tuberculosis isolates in Heilongjiang Province, China.

    Journal of Clinical Microbiology 2011. OpenURL

  11. Shao Y, Yang D, Xu W, Lu W, Song H, Dai Y, Shen H, Wang J: Epidemiology of anti-tuberculosis drug resistance in a chinese population: current situation and challenges ahead.

    BMC Public Health 2011, 11:110. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  12. Guidelines for surveillance of drug resistance in tuberculosis (WHO/HTM/TB/2009.422) [http://www.who.int/tb/publications/mdr_surveillance/en/index.html] webcite

  13. Dou HY, Tseng FC, Lin CW, Chang JR, Sun JR, Tsai WS, Lee SY, Su IJ, Lu JJ: Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosis in Taipei.

    BMC Infect Dis 2008, 8:170. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  14. Kremer K, Glynn JR, Lillebaek T, Niemann S, Kurepina NE, Kreiswirth BN, Bifani PJ, van Soolingen D: Definition of the Beijing/W lineage of Mycobacterium tuberculosis on the basis of genetic markers.

    Journal of Clinical Microbiology 2004, 42(9):4040-4049. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  15. Gori A, Bandera A, Marchetti G, Degli Esposti A, Catozzi L, Nardi GP, Gazzola L, Ferrario G, van Embden JD, van Soolingen D, et al.: Spoligotyping and Mycobacterium tuberculosis.

    Emerging Infectious Diseases 2005, 11(8):1242-1248. PubMed Abstract | Publisher Full Text OpenURL

  16. Brudey K, Driscoll JR, Rigouts L, Prodinger WM, Gori A, Al-Hajoj SA, Allix C, Aristimuno L, Arora J, Baumanis V, et al.: Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology.

    BMC Microbiol 2006, 6:23. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  17. Le Fleche P, Fabre M, Denoeud F, Koeck JL, Vergnaud G: High resolution, on-line identification of strains from the Mycobacterium tuberculosis complex based on tandem repeat typing.

    BMC Microbiol 2002, 2:37. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  18. Meeker-O'Connell RFaWA: Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats.

    Microbiology 1998, 144:1189-1196. PubMed Abstract | Publisher Full Text OpenURL

  19. Skuce RA, McCorry TP, McCarroll JF, Roring SM, Scott AN, Brittain D, Hughes SL, Hewinson RG, Neill SD: Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets.

    Microbiology 2002, 148(Pt 2):519-528. PubMed Abstract | Publisher Full Text OpenURL

  20. Supply P, Lesjean S, Savine E, Kremer K, van Soolingen D, Locht C: Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units.

    Journal of Clinical Microbiology 2001, 39(10):3563-3571. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  21. Hunter PR: Reproducibility and indices of discriminatory power of microbial typing methods.

    Journal of Clinical Microbiology 1990, 28(9):1903-1905. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  22. Sola C, Ferdinand S, Mammina C, Nastasi A, Rastogi N: 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.

    Journal of Clinical Microbiology 2001, 39(4):1559-1565. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  23. Jiao WW, Mokrousov I, Sun GZ, Guo YJ, Vyazovaya A, Narvskaya O, Shen AD: Evaluation of new variable-number tandem-repeat systems for typing Mycobacterium tuberculosis with Beijing genotype isolates from Beijing, China.

    Journal of Clinical Microbiology 2008, 46(3):1045-1049. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  24. Dong H, Liu Z, Lv B, Zhang Y, Liu J, Zhao X, Wan K: Spoligotypes of Mycobacterium tuberculosis from different Provinces of China.

    Journal of Clinical Microbiology 2010, 48(11):4102-4106. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  25. Li WM, W S, Pei XY, Liu ZQ, Zhong Q, Qian M, Zhao B, Duan Mu HJ: Mycobacterium tuberculosis strains from Beijing, Guangdong and Ningxia.

    Chin J Epidemiol 2003, 24(5):381-384. OpenURL

  26. Xie T, JH , Zhao DF, Mu C, Zhao H, Wu ZP, Wang XX: Polymorphism of Variable-Number Tandem Repeat in Mycobacterium Tuberculosis Strains Isolated from Tianjin.

    Tianjin Med J 2010, 38:940-943. OpenURL

  27. Kam KM, Yip CW, Tse LW, Wong KL, Lam TK, Kremer K, Au BK, van Soolingen D: Utility of mycobacterial interspersed repetitive unit typing for differentiating multidrug-resistant Mycobacterium tuberculosis isolates of the Beijing family.

    Journal of Clinical Microbiology 2005, 43(1):306-313. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  28. Chan MY, Borgdorff M, Yip CW, de Haas PE, Wong WS, Kam KM, Van Soolingen D: Seventy percent of the Mycobacterium tuberculosis isolates in Hong Kong represent the Beijing genotype.

    Epidemiol Infect 2001, 127(1):169-171. PubMed Abstract | PubMed Central Full Text OpenURL

  29. Hu Y, Ma X, Graviss EA, Wang W, Jiang W, Xu B: A major subgroup of Beijing family Mycobacterium tuberculosis is associated with multidrug resistance and increased transmissibility.

    Epidemiol Infect 2011, 139(1):130-138. PubMed Abstract | Publisher Full Text OpenURL

  30. Qiao KWH, Yang CG, Luo T, Mei J, Gao Q: The application of variable number of tandem repeats in the microevolution study of Mycobacterium tuberculosis strain of Beijing genotype in Chongming Island, Shanghai.

    Journal of Microbes and Infections 2010, 5(4):208-213. OpenURL

  31. Peng ZZC, X LL, L RX: Correlation between Beijing genotype strains of Mycobacterium tuberculosis and drug resistant phenotypes in Chongqing children with tuberculosis.

    Journal of Third Military Medical University 2010, 32(23):2525-2528. OpenURL

  32. Reed MB, Domenech P, Manca C, Su H, Barczak AK, Kreiswirth BN, Kaplan G, Barry CE: A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response.

    Nature 2004, 431(7004):84-87. PubMed Abstract | Publisher Full Text OpenURL

  33. Manca C, Reed MB, Freeman S, Mathema B, Kreiswirth B, Barry CE, Kaplan G: Differential monocyte activation underlies strain-specific Mycobacterium tuberculosis pathogenesis.

    Infection and Immunity 2004, 72(9):5511-5514. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  34. Purwar S, Chaudhari S, Katoch VM, Sampath A, Sharma P, Upadhyay P, Chauhan DS: Determination of drug susceptibility patterns and genotypes of Mycobacterium tuberculosis isolates from Kanpur district, North India.

    Infect Genet Evol 2011, 11(2):469-475. PubMed Abstract | Publisher Full Text OpenURL

  35. Caws M, Thwaites G, Stepniewska K, Nguyen TN, Nguyen TH, Nguyen TP, Mai NT, Phan MD, Tran HL, Tran TH, et al.: Beijing genotype of Mycobacterium tuberculosis is significantly associated with human immunodeficiency virus infection and multidrug resistance in cases of tuberculous meningitis.

    Journal of Clinical Microbiology 2006, 44(11):3934-3939. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  36. Al Hajoj S, Rastogi N: The emergence of Beijing genotype of Mycobacterium tuberculosis in the Kingdom of Saudi Arabia.

    Ann Thorac Med 2010, 5(3):149-152. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  37. Brown T, Nikolayevskyy V, Velji P, Drobniewski F: Associations between Mycobacterium tuberculosis strains and phenotypes.

    Emerging Infectious Diseases 2010, 16(2):272-280. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  38. Ani A, Bruvik T, Okoh Y, Agaba P, Agbaji O, Idoko J, Dahle UR: Genetic diversity of Mycobacterium tuberculosis Complex in Jos, Nigeria.

    BMC Infect Dis 2010, 10:189. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  39. Lai CC, Tan CK, Lin SH, Liao CH, Huang YT, Chou CH, Hsu HL, Wang CY, Lin HI, Hsueh PR: Clinical and genotypic characteristics of extensively drug-resistant and multidrug-resistant tuberculosis. European journal of clinical microbiology & infectious diseases.

    official publication of the European Society of Clinical Microbiology 2010, 29(5):597-600. Publisher Full Text OpenURL

  40. Mokrousov I, Jiao WW, Sun GZ, Liu JW, Valcheva V, Li M, Narvskaya O, Shen AD: Evolution of drug resistance in different sublineages of Mycobacterium tuberculosis Beijing genotype.

    Antimicrob Agents Chemother 2006, 50(8):2820-2823. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  41. Warren RM, Victor TC, Streicher EM, Richardson M, Beyers N, Gey van Pittius NC, van Helden PD: Patients with active tuberculosis often have different strains in the same sputum specimen.

    American journal of respiratory and critical care medicine 2004, 169(5):610-614. PubMed Abstract | Publisher Full Text OpenURL

  42. Cox HS, Kubica T, Doshetov D, Kebede Y, Rusch-Gerdess S, Niemann S: The Beijing genotype and drug resistant tuberculosis in the Aral Sea region of Central Asia.

    Respiratory Research 2005, 6:134. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  43. Mallard K, McNerney R, Crampin AC, Houben R, Ndlovu R, Munthali L, Warren RM, French N, Glynn JR: Molecular detection of mixed infections of Mycobacterium tuberculosis strains in sputum samples from patients in Karonga District, Malawi.

    Journal of Clinical Microbiology 2010, 48(12):4512-4518. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  44. Singh UB, Suresh N, Bhanu NV, Arora J, Pant H, Sinha S, Aggarwal RC, Singh S, Pande JN, Sola C, et al.: Predominant tuberculosis spoligotypes, Delhi, India.

    Emerging Infectious Diseases 2004, 10(6):1138-1142. PubMed Abstract | Publisher Full Text OpenURL

Pre-publication history

The pre-publication history for this paper can be accessed here:

http://www.biomedcentral.com/1471-2334/11/288/prepub