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  Table of Contents  
Year : 2017  |  Volume : 35  |  Issue : 2  |  Page : 211-215

Genotypic characterisation of Mycobacterium tuberculosis isolates from tuberculous meningitis patients at a tertiary neurocare centre in Southern India

1 Department of Neuromicrobiology, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India
2 Public Health Research Institute TB Center, Newark, New Jersey 07103, USA

Date of Web Publication5-Jul-2017

Correspondence Address:
Manjunatha M Venkataswamy
Department of Neurovirology, 2nd Floor, Administrative Block, National Institute of Mental Health and Neurosciences, Bengaluru - - 560 078, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_16_166

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 ~ Abstract 

Aims: Specific genotypes of Mycobacterium tuberculosis (MTB) have been reported to cause outbreaks of pulmonary tuberculosis (TB) in geographical areas that are endemic to TB. However, since there is little epidemiological evidence on the association of particular genotypes that cause tuberculous meningitis (TBM), we sought to investigate the association of specific MTB strains with infection of the central nervous system (CNS). Materials and Methods: We carried out a genetic characterisation of 89 MTB isolates from TBM patients at a Southern Indian tertiary neurocare centre and compared the genotypes with strains of pulmonary TB isolated from Indian immigrants in New York City. We applied the standard methods of genotyping of MTB, namely, IS6110-based restriction fragment length polymorphism and spoligotyping for strain identification, along with principal genetic grouping and single-nucleotide polymorphism cluster analysis. Results: The analysis revealed a high-level of diversity amongst the strain population. The genotypes of the isolates from TBM patients paralleled the pulmonary TB strain population recovered from the Indian immigrants in NYC. Conclusions: We conclude that there is no apparent association between genotypes of MTB and propensity to infect CNS tissue.

Keywords: IS6110-RFLP, mycobacterium tuberculosis genotypes, spoligotyping, tuberculous meningitis

How to cite this article:
Chandramuki A, Khanna N, Shashkina E, Kurepina N, Mathema B, Kreiswirth BN, Venkataswamy MM. Genotypic characterisation of Mycobacterium tuberculosis isolates from tuberculous meningitis patients at a tertiary neurocare centre in Southern India. Indian J Med Microbiol 2017;35:211-5

How to cite this URL:
Chandramuki A, Khanna N, Shashkina E, Kurepina N, Mathema B, Kreiswirth BN, Venkataswamy MM. Genotypic characterisation of Mycobacterium tuberculosis isolates from tuberculous meningitis patients at a tertiary neurocare centre in Southern India. Indian J Med Microbiol [serial online] 2017 [cited 2018 Feb 20];35:211-5. Available from:

 ~ Introduction Top

Mycobacterium tuberculosis (MTB), the aetiologic agent of tuberculosis (TB), continues to be the second leading cause of mortality due to a single infectious pathogen worldwide after human immunodeficiency virus (HIV).[1] Our understanding of TB epidemiology and the efficacy of control activities have been complicated by an increase in drug resistance and the synergism of TB and HIV co-infection. Although commonly pulmonary, MTB can infect a variety of tissues including the meninges, lymph nodes, bone and spine tissues. As HIV infection increases the risk of extrapulmonary TB (EPTB), the rise in HIV infections has also increased the incidence of EPTB, including TB meningitis (TBM). TBM represents one of the most common extrapulmonary forms (1%–10%) and is the most severe form of TB. It is difficult to diagnose and treat, and is consequently associated with high morbidity and mortality.[2]

There is mounting evidence to suggest that different clinical strains of MTB can induce differential host immune response leading to variable pathogenesis and virulence in animal models.[3] However, a clear relationship between infecting MTB strain genotype and disease phenotype is currently unclear. In at least one report, the rabbit model of TBM showed the differential ability of infecting strains to cause disease consistent with TBM.[4] Here, the investigators found that infection with two W-Beijing strains led to higher bacillary loads in the cerebrospinal fluid (CSF) and more severe pathology when compared to another unrelated clinical strain CDC1551. This finding increased the possibility that certain bacterial traits, found in some strains of MTB (e.g., W-Beijing strains), have an increased ability to cause TBM and more severe disease. While there is some evidence for strain genotype-disease phenotype (i.e., TBM), the findings have not been generalised. For instance, a South African study investigating the relationship between extrapulmonary disease in children and strain types in a region where W-Beijing strains are prevalent showed no unique association as the genotypes distributed amongst meningitis cases paralleled the strains recovered from pulmonary cases.[5] In a recent study on MTB lineage causing HIV-associated TBM, patients infected with the 'modern' Beijing lineage strain showed lower mortality than patients infected with 'ancient' Indo-Oceanic lineage.[6] Despite being endemic to TB, there is a dearth of studies from India on genotyping of MTB isolates from CSF of TBM patients. In a report from northern India on EPTB isolates; although, the authors found a significant number of Beijing isolates causing bone and joint TB, there was no significant association of this genotype with TBM.[7] Another study from Puducherry, South India, on EPTB isolates reported novel spoligotypes; however, there were no isolates from TBM patients included in this study.[8] To that end, we examined the genetic diversity of MTB isolates recovered from the CSF of patients with TBM in a large tertiary neurologic institute in South India.

 ~ Materials and Methods Top

We evaluated 89 MTB isolates that were isolated from the CSF of 86 TBM patients between 1999 and 2000. Referrals to this tertiary care centre for neurologic, neurosurgical and psychiatric diseases are primarily from the Southern Indian population. The diagnosis of TBM was based on clinical findings including a headache, fever and vomiting for more than 3 weeks, pleocytosis, increased protein in the CSF and basal exudates with or without hydrocephalus, as revealed by cranial computed tomographic scan. All patients were tested for HIV infection.

The isolation of MTB from the CSF specimens and drug susceptibility testing of isoniazid, rifampin, ethambutol and streptomycin was carried out using the BACTEC 460 TB system (Becton Dickinson, MD, USA). Each isolate was genetically characterised using both IS6110-based restriction fragment length polymorphism (RFLP) and spoligotyping as per standardised methodologies.[9],[10] These genotypes were compared to the Public Health Research Institute (PHRI) TB Center IS6110 RFLP library that contains over 23,000 MTB isolates from diverse global sources. The majority of the PHRI collection was also genotyped by spoligotyping, and a large representative subgroup was further characterised by mycobacterial interspersed repetitive unit – variable number of tandem repeat analysis, principal genetic grouping (PGG) and genetic clusters by the sequence analysis of selected single-nucleotide polymorphism (SNP) nucleotides.[11],[12] The PHRI library includes isolates from all culture-positive New York City (NYC) cases since 2001. Country of origin was available for 2527 cases from 2001 to 2003, from which 101 isolates were recovered from Indian immigrants.[13] The genetic diversity of the MTB recovered from the Indian immigrants, who had primarily pulmonary disease, and the TBM isolates cultured in Bengaluru was compared to evaluate whether the genotypic makeup of the strains infecting lungs as against the central nervous system (CNS) differed.

 ~ Results Top

Eighty-nine isolates generated a total of 62 different IS6110 patterns including 34 unique patterns (denoted “001”) with no match to the PHRI database identified [Table 1]. Spoligotype profile divided the 89 isolates into 44 genotypes. The S00210 was the most common seen amongst 25% of the isolates followed by 18% each of S00002 and S00091, the S00115 accounted for 14% and the rest of the isolates were distributed amongst multiple spoligotypes [Table 1].
Table 1: Genotypes of Mycobacterium tuberculosis isolates from the cerebrospinal fluid of tuberculous meningitis patients from South India based on spoligotyping, IS6110 restriction fragment length polymorphism, principal genetic group and single-nucleotide polymorphism cluster analysis

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The majority of the strains (n = 56, 62%) were typed to PGG 1 reflective of the most ancestral TB isolates, and nearly all the rest were typed to PGG 2. A comparison of the TBM isolates with the 101 NYC isolates showed a similar strain distribution as 83% of the isolates were assigned to PGG 1, and genetic cluster IIA was overly represented in both pulmonary and extrapulmonary populations [Figure 1]. Of note, five TBM patients had the same strain, a single IS6110 copy strain 'BE' with spoligotype S00091 (octal code 477777777413071) – this also was the case in the NYC Indian population where four patients were diseased with this strain. Amongst the patients diagnosed with TBM, 16 (18%) were co-infected with HIV. There was no clear correlation between being HIV-positive and being infected with specific strain types. Further, there was no association between drug susceptibility profiles and genotype of the infecting isolate either in HIV or non-HIV associated TBM [Supplemental Table 1].
Figure 1: Comparison of the Mycobacterium tuberculosis strain populations between tuberculous meningitis isolates from Southern India and primarily pulmonary isolates recovered from Indian immigrants in New York City. The numbers represent the percentage of isolates in a specific genetic cluster. Genetic clusters I, II and IIA are members of principal genetic Group 1 (hatched boxes); clusters III, IV, V and VI are members of principal genetic Group 2 (dotted boxes); and clusters VII and VIII are members of principal genetic Group 3 (herringbone boxes).

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[Additional file 1]

 ~ Discussion Top

The ability to accurately genotype bacteria and subspeciate MTB clinical isolates into unique phylogenetic lineages provides the opportunity to examine and correlate the genetic diversity of particular bacillary population with specific disease phenotypes (e.g., TBM).[3] The genotyping of MTB using a combination of complementing genotyping methods, IS6110-based RFLP, spoligotyping and SNP analysis, reveal that specific genotypes are often associated with patients from different ethnic backgrounds and geographic regions.[11] In this study, we had the opportunity to evaluate a collection of MTB isolates recovered from CSF amongst a large referral population in South India. The primary focus of this study was to investigate whether MTB isolated from TBM cases are genetically restricted group of strains, as suggested in previous studies. In addition, we compared the distribution of genotypes from our South Indian TBM population to a primarily pulmonary TB group of Indian immigrants in NYC.

Arvanitakis et al. evaluated TBM amongst all TB cases in Manitoba, Canada, from 1979 to 1996.[14] Amongst 2334 TB patients, 33 (1.4%) of the cases were diagnosed with TBM and 13 (39%) of these cases identified in individuals of First Nations origin. Amongst the 33 cases, 19 (58%) MTB isolates recovered between 1979 and 1996 were available for IS6110 analysis. Genotyping identified one common IS6110 hybridisation pattern, type 1, amongst six patients who were allFirst Nation individuals living in disparate regions of Manitoba. During the same period, 372 isolates from non-TBM cases were genotyped, and 77 (21%) had type 1 and 83% of these cases were cultured from the patients of theFirst Nations origin. The authors suggested that while several strains can cause CNS infection, the occurrence of TBM may be strain-dependent (e.g., type 1). Alternatively, the strains that cause TBM may simply reflect the genotypic profile of the general bacillary population in the same region.

Caws et al. evaluated the prevalence of the two predominant strain types in Vietnam amongst patients with TBM.[15] Of the 222 TBM patients, 35 were HIV-positive and 187 were HIV-negative; 69% of the HIV-positive cases and 42% of the HIV-negative TBM cases were infected with W-Beijing genotype. In contrast, the Vietnam (non-W-Beijing) genotype was recovered from 9% of the HIV-positive patients and 19% of HIV-negative TBM cases. The authors pointed out that the W-Beijing strains were comparable in causing TBM (42%) and pulmonary TB (37%) amongst HIV-negative TB patients; however, they noted an association with TBM amongst HIV-positive patients.

A recent study from a paediatric referral centre in the Western Cape, South Africa, between 1992 and 2003 identified 59 culture-confirmed TBM cases.[16] Each MTB isolate was divided into one of the three PGGs and then further segregated into strain families on the basis of IS6110 DNA fingerprints. Analysis of the overall strains showed that nearly 60% of the strains were from PGG 2, 27% typed to genetic Group 1 and the rest were from Group 3. Amongst the 59 paediatric TBM cases, 25% of the cases were caused by the W-Beijing family of strains. The distribution of the IS6110 strain families in meningitis isolates mirrored the most prevalent strains in the Western Cape adult pulmonary population, the presumed reservoir for these paediatric TBM cases. The authors noted that there was no association between genotypes (PGG or W-Beijing strains) and clinical presentation and outcome. A similar study from the Western Cape examined MTB isolates from 285 children with pulmonary and extrapulmonary disease. Amongst the total sample, two strain families, LAM3/F11 and W-Beijing, predominated with 31% and 23%, respectively.[5] The authors noted no significant differences in the propensity of a particular strain family to cause extrapulmonary disease and more specifically TBM. A study from Egypt attests the findings from South Africa where 66 MTB isolates recovered from TBM patients were genetically heterogeneous (53 distinct genotypes), and the one cluster of six patient isolates was a noted common strain previously identified amongst pulmonary TB patients.[17]

In this study, we found that the genetic diversity of MTB causing TBM was similar to the NYC Indian, primarily pulmonary, TB population. The finding from 87 TBM cases at the National Institute of Mental Health and Neurosciences (NIMHANS) in South India parallels the experience in other studies. Collectively, in the present study and other studies share one common observation; the genetic diversity of MTB strains from a pulmonary TB population in a given region approximates the MTB population cultured from the CSF. In contrast to other studies which focus on W-Beijing strains and meningitis, we had only one case in our TBM sample as these strains are not commonly seen in Indian TB populations.[18] In this study, which included both HIV-positive and HIV-negative meningitis patients and where two genotyping methods were used to differentiate strains, we found minimal strain clustering and no clear association between the severity of disease and a given strain type, family or PGG. Most significantly, the findings in this study strongly support the hypothesis that all MTB strains are able to cause TBM and no strain-specific tropism exists for causing infections in the meninges.


We acknowledge Dr. Jeffrey R. Driscoll (formerly at Wadsworth Center, New York) for his support with the analysis of spoligotyping data. We would like to thank the Department of Neurovirology, NIMHANS for the HIV testing of study patients.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 ~ References Top

World Health Organization. Global Tuberculosis Report 2014. Geneva: World Health Organization; 2014.  Back to cited text no. 1
Zuger A, Lowy F. Tuberculosis of the brain, meninges and spinal cord. In: Rom W, Garay S, editors. Tuberculosis. Toronto: Little Brown and Company; 1996. p. 541-56.  Back to cited text no. 2
Mathema B, Kurepina NE, Bifani PJ, Kreiswirth BN. Molecular epidemiology of tuberculosis: Current insights. Clin Microbiol Rev 2006;19:658-85.  Back to cited text no. 3
Tsenova L, Ellison E, Harbacheuski R, Moreira AL, Kurepina N, Reed MB, et al. Virulence of selected Mycobacterium tuberculosis clinical isolates in the rabbit model of meningitis is dependent on phenolic glycolipid produced by the bacilli. J Infect Dis 2005;192:98-106.  Back to cited text no. 4
Nicol MP, Sola C, February B, Rastogi N, Steyn L, Wilkinson RJ. Distribution of strain families of Mycobacterium tuberculosis causing pulmonary and extrapulmonary disease in hospitalized children in Cape Town, South Africa. J Clin Microbiol 2005;43:5779-81.  Back to cited text no. 5
Tho DQ, Török ME, Yen NT, Bang ND, Lan NT, Kiet VS, et al. Influence of antituberculosis drug resistance and Mycobacterium tuberculosis lineage on outcome in HIV-associated tuberculous meningitis. Antimicrob Agents Chemother 2012;56:3074-9.  Back to cited text no. 6
Sankar MM, Singh J, Diana SC, Singh S. Molecular characterization of Mycobacterium tuberculosis isolates from North Indian patients with extrapulmonary tuberculosis. Tuberculosis (Edinb) 2013;93:75-83.  Back to cited text no. 7
Kandhakumari G, Stephen S, Sivakumar S, Narayanan S. Spoligotype patterns of Mycobacterium tuberculosis isolated from extra pulmonary tuberculosis patients in Puducherry, India. Indian J Med Microbiol 2015;33:267-70.  Back to cited text no. 8
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van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: Recommendations for a standardized methodology. J Clin Microbiol 1993;31:406-9.  Back to cited text no. 10
Gutacker MM, Mathema B, Soini H, Shashkina E, Kreiswirth BN, Graviss EA, et al. Single-nucleotide polymorphism-based population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites. J Infect Dis 2006;193:121-8.  Back to cited text no. 11
Mazars E, Lesjean S, Banuls AL, Gilbert M, Vincent V, Gicquel B, et al. High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc Natl Acad Sci U S A 2001;98:1901-6.  Back to cited text no. 12
Clark CM, Driver CR, Munsiff SS, Driscoll JR, Kreiswirth BN, Zhao B, et al. Universal genotyping in tuberculosis control program, New York City, 2001-2003. Emerg Infect Dis 2006;12:719-24.  Back to cited text no. 13
Arvanitakis Z, Long RL, Hershfield ES, Manfreda J, Kabani A, Kunimoto D, et al. M. tuberculosis molecular variation in CNS infection: Evidence for strain-dependent neurovirulence. Neurology 1998;50:1827-32.  Back to cited text no. 14
Caws M, Thwaites G, Stepniewska K, Nguyen TN, Nguyen TH, Nguyen TP, et al. Beijing genotype of Mycobacterium tuberculosis is significantly associated with human immunodeficiency virus infection and multidrug resistance in cases of tuberculous meningitis. J Clin Microbiol 2006;44:3934-9.  Back to cited text no. 15
Maree F, Hesseling AC, Schaaf HS, Marais BJ, Beyers N, van Helden P, et al. Absence of an association between Mycobacterium tuberculosis genotype and clinical features in children with tuberculous meningitis. Pediatr Infect Dis J 2007;26:13-8.  Back to cited text no. 16
Cooksey RC, Abbadi SH, Woodley CL, Sikes D, Wasfy M, Crawford JT, et al. Characterization of Mycobacterium tuberculosis complex isolates from the cerebrospinal fluid of meningitis patients at six fever hospitals in Egypt. J Clin Microbiol 2002;40:1651-5.  Back to cited text no. 17
Singh UB, Arora J, Suresh N, Pant H, Rana T, Sola C, et al. Genetic biodiversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in India. Infect Genet Evol 2007;7:441-8.  Back to cited text no. 18


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