Evaluation of 24-locus MIRU-VNTR in extrapulmonary specimens: Study from a tertiary centre in Mumbai

doi:10.1016/j.tube.2012.01.002

Tuberculosis

Volume 92, Issue 3, May 2012, Pages 264-272

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Summary

Genotyping of Mycobacterium tuberculosis isolates is a useful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. There is a lack of information on the discriminatory power of standard 24-locus mycobacterial interspersed repetitive unit (MIRU) – variable number tandem repeats (VNTR) in India, which has the highest tuberculosis (TB) burden worldwide. Therefore, we assessed its utility on 69 M. tuberculosis (MTB) isolates from patients with extrapulmonary tuberculosis, in comparison to standard insertion sequence (IS) 6110-Restriction fragment length polymorphism (RFLP) fingerprinting and spoligotyping. IS6110-RFLP (HGDI, 0.9987) identified a single cluster of 3 (4.3%) single-copy IS6110 isolates. Spoligotyping showed 69.5% clustering (HGDI, 0.8857). In contrast, MIRU-VNTR analysis identified 69 (100%) unique strains (HGDI, 1.0000). Within the study limits, this observed high discriminatory power suggests that 24-locus MIRU-VNTR genotyping could potentially be used to study long-term transmission of MTB infection in Mumbai. Moreover, high congruence between the MIRU-VNTR-based and spoligotyping-based strain groupings suggests that CAS, EAI and Beijing are the predominant strain lineages in the Mumbai TB patient population. The Beijing lineage isolates were found to be more significantly associated with multi-drug resistance (p < 0.01) than CAS and EAI lineages.

Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB) is the leading cause of morbidity and mortality accounting for nearly 9.4 million global incident cases every year. India being a heavy TB burden country accounts for almost 21% of all forms of TB cases worldwide.¹ The situation has further worsened due to the increasing prevalence of HIV-TB co-infection; which in turn has led to an increase in cases of extrapulmonary TB (EPTB), now accounting for 10–15% of all forms of TB.² Thus, global epidemiological TB surveillance forms a vital aspect in understanding the transmission links and controlling of the spread of this disease.

IS6110-RFLP, the gold standard for genotyping MTB, has been heavily relied on for TB epidemiological studies because of its high level of discrimination, and correlation of IS6110-RFLP-based patient strain clusters with known or likely TB transmission risk factors.³ However, obvious limitations such as the need for large quantity of pure genomic DNA as the starting material, technical difficulties, differences in inter-laboratory results comparison and its limited capability in discriminating strains with low IS copy numbers have led to the development of PCR-based molecular epidemiological tools.⁴

Spoligotyping is a frequently used PCR-based genotyping method being used due to its technical simplicity and a portable data exchange format. However its application has been restricted by its low discriminatory power, especially in case of strains of the Beijing family.5, 6

In 2006, standardized genotyping based on interrogation by PCR of 24-loci containing variable numbers of tandem repeat DNA elements called mycobacterial interspersed repetitive units was proposed.⁴ This method can be performed on early positive cultures, reducing the turn-around-time as compared to IS6110-RFLP. Thus offering the following advantages: 1) TB diagnostics: detection of specimen cross-contamination during culture processing; evaluation of recurring TB; determination of mixed infection⁷; 2) Control of TB transmission: to determine whether increase in number of TB cases is due to an outbreak or a co-incidental occurrence. Further, when this information is coupled with contact tracing information, it aids in identification of high-risk groups susceptible to TB infection, improvement in case findings and contact investigations, and evaluation of TB control programmes⁷; and 3) The results provided are in an exchangeable format and are reproducible with minimal quality control and standard allele calling use, thus providing inter-laboratory data comparison. Twenty-four-locus MIRU-VNTR typing, especially when combined with spoligotyping, showed a comparable to slightly better predictive value than IS6110-RFLP to detect clusters of tuberculosis transmission in European population-based studies.⁸

Previously published reports from this tertiary care centre using spoligotyping as a genotyping tool has reported a high prevalence (35–62%) of Beijing genotype, considerably associated with both multi- and extensive-drug resistance9, 10; whereas a recent study from the same city region reported a predominance (26%) of Manu1 genotype with a smaller proportion (4%) of Beijing genotype.¹¹

In this study, we assessed the discriminatory power of 24-locus MIRU-VNTR typing in comparison against IS6110-RFLP and spoligotyping for clustering analysis of MTB complex isolates from patients with EPTB from Mumbai. These results would also help us to determine the most prevalent genotypes in one of the largest world megapoles.

Section snippets

Setting and patient specimen processing

This study was carried out from January–April, 2010 in a tertiary care hospital in Mumbai, with a referral bias towards the non-responders. The study was approved by the Institutional Review Board. A total of 476 consenting patients with a suspicion of EPTB were enrolled in the study. Specimens like pus, abscess, tissues, biopsies, lymph nodes and body fluids were processed by N-acetyl-l-cysteine and sodium hydroxide¹² followed with inoculation (specimens like CSF was inoculated directly) in

Results

The mean age (±Standard Deviation) of patients was 32 (±14.4) years, and the male:female sex ratio was 0.84. Of the 476 patients enrolled, 136 (pus/abscess, 27; body fluids, 15; lymph nodes, 12; tissues, 7; CSF, 1; biopsies, 7) were culture confirmed cases suffering from EPTB; with 94 patients (69%) being on anti-tubercular treatment for more than 6 months from the date of enrolment and 19 (13.9%) patients had a previous history of TB. IS6110-RFLP profiles and spoligotyping were available for

Discussion

The present study was designed with an objective to determine the diversity of MTB genotypes in patients suffering from EPTB and to explore the utility in Mumbai of 24-locus MIRU-VNTR as a fingerprinting tool in comparison with both, IS6110-RFLP and spoligotyping. Specimens representing various anatomical body sites were collected, one specimen per patient was considered for analysis in this study.

IS6110-RFLP genotyping (HGDI, 0.9987) detected only one cluster of three isolates with a single

Funding

The study was funded by the National Health and Education Society, P. D. Hinduja National Hospital and Medical Research Centre. The funding body did not have any role in the study design, in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication.

Competing interests

Dr. Philip Supply is a Consultant for Genoscreen.

Ethical approval

Not required.

Acknowledgements

We thank National Health and Education Society, P. D. Hinduja National Hospital and Medical Research Centre for their encouragement and support.

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Genetic diversity and characterization of M. tuberculosis isolates causing extrapulmonary tuberculosis in Bangladesh
2021, Infection, Genetics and Evolution
Citation Excerpt :
In our study, the second predominant lineage found among the EPTB cases was Beijing (20,14.2%). A study conducted in Mumbai, India reported 18.8% of Beijing lineage strains among EPTB isolates (Vadwai et al., 2012). In two independent studies from Thailand reported Beijing as the dominant lineage (56%) among isolates from TB meningitis patients (Faksri et al., 2011; Yorsangsukkamol et al., 2009).
Tuberculosis (TB) remains one of the leading causes of death and Bangladesh ranks 7th among the highest TB burden countries. Though molecular epidemiological data for pulmonary TB (PTB) have previously been described in Bangladesh, data on the molecular characterization and clinical association with different lineages among extrapulmonary TB (EPTB) is lacking. The aim of the study was to investigate the molecular characterization and lineage distribution of M. tuberculosis isolates obtained from patients with EPTB in Bangladesh. Between November 2015 and March 2017, a total of 1,340 EPTB specimens including lymph node, pus, tissue, ascitic fluid, cerebrospinal fluid, pleural fluid, abscess wall, urine etc. were collected from four tertiary care hospitals in Dhaka city, Bangladesh. Among the specimens, 141 were found positive on solid culture. Molecular characterization of the 141 isolates was done by deletion analysis, spoligotyping and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTR) analysis. Among the 141 isolates, 80 (56.7%) were found as ‘modern’ and the remaining 61 (43.3%) were ‘ancestral’ type. Spoligotyping results revealed 91 distinct patterns of which 74 isolates were unique and the remaining 67 were divided into 17 distinct clusters. East African- Indian (EAI) lineage was the most predominant, comprising 26 (18.4%) isolates, followed by the Beijing lineage (14.2%). 15-loci MIRU-VNTR analysis revealed that 132 isolates (93.5%) had unique patterns, whereas only 9 (6.5%) isolates were grouped into 4 distinct clusters. In conclusion, the study findings provide a first insight into genetic diversity of EPTB isolates in Bangladesh. The present study demonstrated that ‘modern’ strains were more prevalent among the EPTB cases, while EAI lineages were predominantly circulating in this region.
A review of published spoligotype data indicates the diversity of Mycobacterium tuberculosis from India is under-represented in global databases
2020, Infection, Genetics and Evolution
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A disproportionate number of isolates (42%) were from Southern India, which has 19% of the population. Studies from India have demonstrated that isolates forming spoligotype clusters are found to be unrelated when typed using IS6110 RFLP studies and/or MIRU-VNTR (Gutierrez et al., 2006; Mathuria et al., 2008; Sharma et al., 2008a; Stavrum et al., 2009; Narayanan et al., 2010; Purwar et al., 2011; Varma-Basil et al., 2011; Vadwai et al., 2012; Devi et al., 2015; Varma-Basil et al., 2016; Sharma et al., 2017; Sharma et al., 2008b). The diversity seen on spoligotyping and MIRU-VNTR studies is likely to be an underestimation of the diversity found in WGS studies (Gagneux, 2013).
Mycobacterium tuberculosis (MTBC) lineages differ in clinical presentation, virulence, transmission, drug resistance and immunological responses. Despite having the largest burden of tuberculosis (TB) in the world, strains from India are underrepresented in international databases. We reviewed published spoligotype data to determine the distribution and diversity of MTBC lineages in India.
A Pubmed/MEDLINE search identified 34 M. tuberculosis spoligotyping studies from India. Spoligotype patterns were extracted and the Spoligotype International Type (SIT) number, sub-lineage and lineage determined. Minimum Spanning Trees were used to determine relationships between patterns.
We identified 1528 spoligotype patterns distributed across 8300 isolates; 6733 isolates belonged to 472 SITs, with 53% of all isolates belonging to 12 SITs with at least 100 isolates each. Lineage 1 and Lineage 3 made up 67% of all isolates, although a lineage could not be assigned for 16% of isolates. Lineage 1 isolates were most common in Southern, Western and Eastern India, and Lineage 3 was most common in Northern and Central India. The RULE, CBN and KBBN lineage prediction algorithms from the TB-lineage tools performed variably, with the correct lineage predicted correctly for only 64% of patterns with known lineage. Using a consensus definition, 64% of the 1359 isolates with unknown lineage were assigned to Lineage 1, and 14% each were assigned to Lineages 3 and 4. With these lineage assignments, 80% of all isolates belonged to either Lineage 1 or Lineage 3.
Our findings indicate significant M. tuberculosis diversity in India. The documentation of 1056 orphan and unreported patterns indicate that this diversity is under-represented in global databases.
Molecular characterization and drug susceptibility profile of Mycobacterium tuberculosis isolates from Northeast Bangladesh
2018, Infection, Genetics and Evolution
Tuberculosis (TB) remains a major public health problem worldwide including in Bangladesh. Molecular epidemiological tools provide genotyping profiles of Mycobacterium tuberculosis (M. tuberculosis) strains that can give insight into the transmission of TB in a specific region. The objective of the study was to identify the genetic diversity and drug susceptibility profile of M. tuberculosis strains circulating in the northeast Bangladesh. A total of 244 smear-positive sputum specimens were collected from two referral hospitals in Mymensingh and Netrakona districts. The isolated strains were genotyped by deletion analysis, spoligotyping, and MIRU-VNTR typing. We also analyzed the distributions of drug susceptibility pattern and demographic data among different genotypes. All isolates were identified as M. tuberculosis and among them 167 strains (68.44%) were ‘ancestral’ and the remaining 77 (31.56%) were ‘modern’ type. Spoligotyping analysis yielded 119 distinct patterns, among them, 86 isolates had unique patterns and the remaining 158 were grouped into 33 distinct clusters containing 2 to 18 isolates. The predominant spoligotypes belong to the EAI lineage strains, comprising 66 (27.04%) isolates followed by Beijing (7.38%), T1 (6.15%), CAS1-Delhi (5.33), LAM9 (3.28%), MANU-2 and X2. MIRU-VNTR analysis revealed 167 isolates (68%) had unique patterns, whereas 77 (32%) were grouped into 26 clusters and the rate of recent transmission was 20.9%, suggesting that the majority of TB cases in this region are caused by the reactivation of previous TB infections rather than recent transmission. About 136 (55.7%) isolates were sensitive to four anti-TB drugs, 69 (28.3%) were resistant to one or more (except rifampicin and isoniazid combination) drugs and 39 (15.9%) were MDR. In conclusion, our study provides a first insight into molecular characterization and drug resistance profile of M. tuberculosis strains in northeast Bangladesh which will ultimately contribute to the national TB control program.
Genetic diversity of Mycobacterium tuberculosis complex isolates circulating in an area with high tuberculosis incidence: Using 24-locus MIRU-VNTR method
2018, Tuberculosis
Citation Excerpt :
We noted a good discriminatory power for MIRU-VNTR typing method used in this study. However, this discriminatory power is consistent with higher values that reported in previous studies from different provinces of Iran ranged from 0.972 to 0.9932, as well as previous studies in other countries ranged from 0.920 to 1.000 [6,23,25,33,45,46,53–57]. Among 24 loci, 7 loci had high allelic diversity in this study.
We aimed to determine the genetic diversity, phylogenetic relationship and transmission dynamics of Mycobacterium tuberculosis complex (MTBC) genotypes in an area with high tuberculosis (TB) incidence. A set of 164 MTBC isolates from new TB patients of Golestan province, Iran, were subjected to genotyping using the standard 24-locus MIRU-VNTR method. Recent TB transmission was evaluated and phylogenetic relationships were analysed by minimum spanning tree and cluster-graph methods. Among the 164 isolates, 132 distinct patterns were detected. The 48 clustered isolates (29.3%) were distributed into 16 clusters ranging in size from 2 to 12 isolates. The most frequent genotype was Central Asian Strain/Delhi (CAS/Delhi) (n = 67, 40.8%), followed by NEW-1 (n = 53, 32.3%) and Beijing (n = 19, 11.6%) genotypes. Thirty five (72.9%) of NEW-1 isolates were recovered from immigrant patients and 84.2% (n = 16) of Beijing genotypes recovered from native cases. Statistically significant association was found between clustering and smoking (p = 0.047), drug addiction (p = 0.01) and prison history (p = 0.003). The estimated proportion of recent transmission was 19.5%. Presence of highly diverse MTBC isolates circulating in this province without a dominant genotype might be a consequence of importation of various genotypes in this area.
Whole genome sequencing of clinical strains of Mycobacterium tuberculosis from Mumbai, India: A potential tool for determining drug-resistance and strain lineage
2017, Tuberculosis
Citation Excerpt :
This extends our earlier reports of M.tb drug profiles using spoligotyping [20] and MIRU-VNTR [19] profiles of M.tb in India. While WGS enabled maximum strain discrimination, it reiterated the high amount of strain diversity in an endemic locale such as Mumbai [19,20,39]. The sensitivities for predicting resistance to the drugs in this study were comparable to those reported by Walker et al. [40].
Amplification of drug resistance in Mycobacterium tuberculosis (M.tb) and its transmission are significant barriers in controlling tuberculosis (TB) globally. Diagnostic inaccuracies and delays impede appropriate drug administration, which exacerbates primary and secondary drug resistance. Increasing affordability of whole genome sequencing (WGS) and exhaustive cataloguing of drug resistance mutations is poised to revolutionise TB diagnostics and facilitate personalized drug therapy. However, application of WGS for diagnostics in high endemic areas is yet to be demonstrated. We report WGS of 74 clinical TB isolates from Mumbai, India, characterising genotypic drug resistance to first- and second-line anti-TB drugs. A concordance analysis between phenotypic and genotypic drug susceptibility of a subset of 29 isolates and the sensitivity of resistance prediction to the 4 drugs was calculated, viz. isoniazid-100%, rifampicin-100%, ethambutol-100% and streptomycin-85%. The whole genome based phylogeny showed almost equal proportion of East Asian (27/74) and Central Asian (25/74) strains. Interestingly we also found a clonal group of 9 isolates, of which 7 patients were found to be from the same geographical location and accessed the same health post. This provides the first evidence of epidemiological linkage for tracking TB transmission in India, an approach which has the potential to significantly improve chances of End-TB goals. Finally, the use of Mykrobe Predictor, as a standalone drug resistance and strain typing tool, requiring just few minutes to analyse raw WGS data into tabulated results, implies the rapid clinical applicability of WGS based TB diagnosis.
Spoligotype defined lineages of Mycobacterium tuberculosis and drug resistance: Merely a casual correlation?
2017, Indian Journal of Medical Microbiology
Drug-resistant tuberculosis (TB) is a major challenge to TB control strategy worldwide. Analysis of genetic polymorphism among drug resistant Mycobacterium tuberculosis (MTB) strains may help provide some insight into the transmission dynamics of these strains. Spoligotyping is a widely used technique to identify genetic polymorphism, based on 43 known spacers interspersed between direct repeat regions. Considerable work has been done in various parts of the world using this technique to identify and analyse the polymorphic nature of MTB. Many studies have been carried out to determine the association of drug resistance with spoligotype defined lineages, and much data has been produced over the years. New information continues to be generated. This review aims to put together the findings of relevant studies in an attempt to understand the correlation of drug resistance with spoligotype defined lineages of MTB. This would help provide a perspective of the available data that can be used as a starting point to understand the molecular epidemiology of drug resistant TB.

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EpidemiologyEvaluation of 24-locus MIRU-VNTR in extrapulmonary specimens: Study from a tertiary centre in Mumbai

Summary

Introduction

Section snippets

Setting and patient specimen processing

Results

Discussion

Funding

Competing interests

Ethical approval

Acknowledgements

Tuberculosis

Clin Microbiol Infect

Clin Microbiol Infect

Res Microbiol

Tuberculosis control in India

Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility

J Clin Microbiol

Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis

J Clin Microbiol

Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review

Emerg Infect Dis

Definition of the Beijing/W lineage of Mycobacterium tuberculosis on the basis of genetic markers

J Clin Microbiol

Assessment of an optimized mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing system combined with spoligotyping for population-based molecular epidemiology studies of tuberculosis

J Clin Microbiol

Mutation detection and accurate diagnosis of extensively drug-resistant tuberculosis: report from a tertiary care center in India

J Clin Microbiol

High incidence of the Beijing genotype among multidrug-resistant isolates of Mycobacterium tuberculosis in a tertiary care center in Mumbai, India

Clin Infect Dis

Strains of Mycobacterium tuberculosis from Western Maharashtra, India, exhibit a high degree of diversity and strain-specific associations with drug resistance, Cavitary disease, and treatment failure

J Clin Microbiol

Epidemiology
Evaluation of 24-locus MIRU-VNTR in extrapulmonary specimens: Study from a tertiary centre in Mumbai