|Year : 2015 | Volume
| Issue : 2 | Page : 267-270
Spoligotype patterns of Mycobacterium tuberculosis isolated from extra pulmonary tuberculosis patients in Puducherry, India
G Kandhakumari1, S Stephen1, S Sivakumar2, S Narayanan2
1 Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pondicherry, Tamil Nadu, India
2 Department of Immunology, National Institute for Research in Tuberculosis (NIRT - ICMR), Chennai, Tamil Nadu, India
|Date of Submission||26-Apr-2014|
|Date of Acceptance||02-Feb-2015|
|Date of Web Publication||10-Apr-2015|
Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pondicherry, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Purpose: Genotyping studies like spoligotyping are valuable tools in understanding the genetic diversity and epidemiology of Mycobacterium tuberculosis. Though there are reports of spoligotyping of M. tuberculosis isolates from pulmonary specimens from different parts of India, spoligotyping of extra pulmonary tuberculosis isolates are very few. Puducherry has not yet recorded spoligopatterns of M. tuberculosis from either pulmonary or extra pulmonary (EPTB) specimens. The aim of this study is to analyze the spoligotype patterns of EPTB strains circulating in Puducherry and neighboring districts of Tamil Nadu. Materials and Methods: During June 2011 to December 2013, 570 EPTB specimens were processed by culturing on to Lowenstein Jensen (LJ) medium and automated Mycobacterium Growth Indicator Tube system (MGIT960). Identification of M. tuberculosis was carried out as per standard procedures, and MPT 64 antigen positivity in a commercial immunochromatography kit. Spoligotyping was carried out at National Institute of Research in Tuberculosis (ICMR), Chennai. Results: M. tuberculosis was isolated from 67 single EPTB specimens (11.8%) like pus/cold abscess (34), TB spine (10), pleural fluid (10), urine (5), tissue bit (2), lymph nodes (2), ascitic fluid (2), synovial fluid (1) and endometrial curetting (1). Among 67 isolates with 41 spoligopatterns, EAI lineage with 28 isolates (41.8%) predominated followed by 18 orphans (26.9%), 10 Beijing (14.9%) and 8 U (11.9%). BOVIS1_BCG (ST482), T1-T2 (ST78) and H3 (ST50) were represented by one strain each (1.5%). C onclusions: Spoligotyping plays a significant role in the epidemiology of tuberculosis. Three spoligotypes, T1-T2 (ST78), EAI6 (ST292) and U (ST1429) are reported for the first time in India.
Keywords: Extrapulmonary tuberculosis, genotyping, Mycobacterium Growth Indicator Tube system 960, Mycobacterium tuberculosis, spoligotyping
|How to cite this article:|
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
|How to cite this URL:|
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 [serial online] 2015 [cited 2019 Jul 23];33:267-70. Available from: http://www.ijmm.org/text.asp?2015/33/2/267/154871
| ~ Introduction|| |
Genotyping methods like Spacer oligo nucleotide typing (Spoligotyping), Mycobacterial interspersed repetitive units-Variable number tandem repeat analysis (MIRU-VNTR) and IS6110 Restriction Fragment Length Polymorphism (IS6110-RFLP) are valuable tools in understanding the genetic diversity of M. tuberculosis isolates. This is borne out of the reports from different geographical areas of India. ,,,,,,,,,,,,, Spoligotyping is a hybridization assay which targets the deoxyribonucleic acid (DNA) polymorphism at the direct repeat locus (DR locus) of M. tuberculosis genome, which is unique for M. tuberculosis complex (MTBC). Reports of spoligotyping of M. tuberculosis isolates from patients with pulmonary specimens (PT) are available in Indian literature. ,,,,,,,,,,, However, spoligotype work on patients with Extra Pulmonary Tuberculosis (EPTB) from India is very few. ,, This is because, unlike pulmonary tuberculosis, EPTB reports are not many due to paucibacillary nature of clinical specimens, and detection and identification of M.tuberculosis takes several weeks. A polymerase chain reaction (PCR)-based technique developed by Kameerbeek et al.  requires only a small amount of DNA, can detect strains with low copy number of IS6110 elements and also simultaneously detects and types MTBC, thus resulting in the rapid confirmation of MTBC. Puducherry has not yet recorded spoligopatterns of M. tuberculosis isolates from patients with either PT or EPTB.
| ~ Materials and Methods|| |
This research work was approved by Institutional Human Ethical Committee. Patients' informed consent was obtained before collection of clinical samples. EPTB patients positive for pulmonary tuberculosis and those EPTB cases previously treated were excluded from our study. A total of 570 single clinical specimens collected between June 2011 to December 2013 from EPTB patients were processed. Direct Ziehl-Neelsen (ZN) smears were first examined. After decontamination, the specimens were inoculated onto two sets of culture media:
- Solid medium: Lowenstein-Jensen slant (LJ) and incubated at 37°C for a period of 8 weeks
- A liquid medium: Modified Middlebrook 7H9 broth and incubated at 37°C for a period of 6 weeks in the automated BACTEC MGIT 960 mycobacterial detection system (Becton Dickinson and company).
When growth was observed, ZN smears were made from LJ/MGIT 960 and those showing acid fast bacilli were subjected to conventional tests like niacin accumulation test, nitrate reduction test and catalase test.  Para nitro benzoic acid (PNB) susceptibility was performed in MGIT 960. MPT64 Ag detection test was done using SD Bioline TBAg MPT64 Rapid (Standard Diagnostics Seoul, South Korea) kit  to confirm the identity of MTBC.
M. tuberculosis strains which grew only in MGIT, but not on LJ, were sub-cultured onto LJ slants and incubated for spoligotyping and niacin tests. Growth on LJ media was scrapped and emulsified in sterile TE buffer in screw capped vials and were taken to Chennai. The isolates were heat killed at 80°C for 30 minutes and processed for DNA extraction. Spoligotyping was performed in National Institute of Research in Tuberculosis, Chennai as per the method of Kamerbeek et al.
| ~ Results|| |
Sixty-seven isolates of M. tuberculosis were recovered from various EPTB specimens like pus/cold abscess (34), TB spine (Pott's disease) (10), pleural fluid (10), urine (5), tissue bit (2) lymph nodes (2), ascitic fluid (2), synovial fluid (1) and endometrial curetting (1). These isolates were analyzed by spoligotyping which produced 41 distinct spoligopatterns [Table 1]. Out of these 41 patterns, only 15 (36.6%) clusters are available with Standard Type (ST) number, whereas the remaining 26 (63.4%) patterns do not possess ST numbers (unique patterns) in the Spoligotype International Types Variable number of DNA tandem repeats International Types database. The most predominant spoligotype among these 67 isolates was EAI lineage (28) (41.8%) which is the common type from Tamil Nadu, South India. The second most predominant are the orphans (18) (26.9%), followed by Beijing type (10) (14.9%) and U (8) (11.9%). T1-T2, H3 and BOVIS1_BCG spoligotypes were represented by a single isolate each (1.5% each).
|Table 1: Spoligopatterns of 67 extra pulmonary tuberculosis (EPTB) isolates of Mycobacterium tuberculosis from Puducherry |
Click here to view
Sub-lineages in our study
[Table 1] EAI sub lineages observed were EAI1, EAI3, EAI5 and EAI6. Eleven isolates belong to EAI3, comprising seven in EAI3_IND (ST11) and four new variants (Unique isolate). EAI1 shows two clusters ST48 and ST1389 containing three and one isolate respectively. EAI5 has three clusters: ST126 with two isolates, ST355 and ST474 with one isolate each. This EAI5 cluster additionally has three new variants. Two isolates belong to the sub lineage EAI5 or EAI3 (ST8). EAI6 has two clusters: ST292 and ST43 containing two isolates each. Spoligotypes which do not match any existing pattern in the database are called as orphans and 18 (26.9%) orphans were isolated in this study .
Spoligotypes of M. tuberculosis strains in India
The spoligopatterns of M. tuberculosis observed in different Indian states by several workers is presented in [Table 2].
|Table 2: Spoligopatterns of M. tuberculosis isolates from different parts of India |
Click here to view
Antibiotic susceptibility of M. tuberculosis from EPTB specimens
Fifty out of the 67 M. tuberculosis isolates in our study were sensitive to all the first line ATT drugs Streptomycin, Isoniazid, Rifampicin, Ethambutol and Pyrazinamide. Two isolates (Beijing and U) were resistant to both streptomycin and Isoniazid. Fifteen isolates were resistant to only one drug. Pyrazinamide-6 (EAI1_SOM (2), Beijing, U, EAI_5 and Bovis1_BCG one each); Streptomycin-3 (Orphan, EAI6_BD1 and EAI3_IND one each); Isoniazid-3 (Orphan-2 and U-one); Ethambutol-2 (Orphan); Rifampicin-1(U).
| ~ Discussion|| |
Research works conducted in Kerala and Chennai reported the high prevalence of EAI strains compared to North India. ,,, Singh et al. in 2007 reported the highest prevalence of 89% EAI strains from Kerala. Similarly in 2013, Joseph and co-workers  reported 64.3% of these strains in Kerala. In 2007 and 2011 Singh et al., and Shanmugam and coworkers  reported 67.5% and 59.3% of these EAI strains, respectively, from Chennai. Our study conducted in Puducherry identified 41.8% of EAI strains, which is the predominant spoligotype. Research works from different parts of North India reports only 3.8% EAI strains from Delhi,  4.44% from Maharashtra,  9.6% in U.P and Varanasi  and 10% from the whole of North India. 
With reference to the Central and Middle Eastern Asia (CAS) family, this is the predominant lineage seen in North India. The highest prevalence of 57.2% was reported from extra pulmonary specimens by Sankar et al. in North India. Singh et al.  reported 56.5%, 53.6 and 44.6% from Delhi, Pune and Lucknow, respectively. Varma-Basil et al. reported CAS strains at a percentage of 43.6% in Delhi. It is also reported at a rate of 45.7% from Madhya Pradesh, central India.  Contrary to this high prevalence of CAS in North India, South India recorded a very low prevalence of 1.5% in Trivandrum.  3.5% in Chennai  and 5.3% in Kerala.  A total absence of CAS strains from extra pulmonary specimens was observed in our study. This supports the concept of clear demarcation between the strains prevalent in North and South India. This common finding is slightly different from the experience of Thomas et al. from Andhra Pradesh, who reported a higher prevalence rate of 40% for CAS type. This could be perhaps due to the presence of Non-Resident Indians (NRI) from Hyderabad who work in Middle East countries and migrant population from North Indian states who come and work in Hyderabad, which is the commercial capital of Andhra Pradesh state.
Although Beijing isolates were reported in different areas with prevalence ranging from 2.2%  to 11.5%  from pulmonary specimens, their prevalence rate was higher in EPTB cases like 10% and 18.8%. , It is also reported that 63.6% of the Beijing strains from EPTB specimens were multidrug resistant (MDR) and from treatment failure cases. , In our study, Beijing was isolated at a rate of 14.9%, but only one isolate showed resistance to streptomycin and isoniazid. None of our 67 isolates were of MDR type. It was also pointed out from earlier studies that Beijing strains were absent in rural areas of Andhra Pradesh and rural TB units of Mumbai, though it was prevalent at a rate 6.8% in urban population of Andhra Pradesh, 10.4% among treatment failure cases and 3.4% from new cases of urban Mumbai. ,
As far as Manu strains are concerned, studies conducted in North India by various researchers have reported varying prevalence of these strains at a frequency of 1.2%,  0.55%  and even 0%  in particular areas. Central India  has not recorded these strains. Chittoor district of South Andhra Pradesh in South India  reported 6.93% of Manu strains. A study conducted in North India by Sankar and co-workers  in 2013 on EPTB specimens reported 6.4% of Manu strains, all of which are pansensitive. Also they have reported the isolation of Manu strains from all extra pulmonary sites except bone. Studies conducted by Chatterjee et al. recorded the highest percentage (26.4%) of Manu1 strains (ST100) from pulmonary isolates in Maharashtra, thus indicating the prevalence of circulating local strains. But our analysis of EPTB isolates did not identify any Manu strains.
Desikan and his colleagues from Madhya Pradesh  reported that their three EPTB isolates were orphans. However, this is only a small number. Twenty-six (38.8%) unique patterns were available in our study, four isolates belong to EAI3, three to EAI5, one to Beijing type and the remaining 18 are orphans. Bovis_1 BCG strain was identified in our study from a case of lymphadenitis after BCG vaccination in a child. BCG, primarily used as a vaccine may cause complications which include regional lymphadenitis, osteomyelitis and disseminated infections. 
We are reporting for the first time in India three spoligotypes: T1-T2 (ST78), EAI6 (ST292) and U (ST1429), which are not reported by other Indian researchers so far. The predominance of 26.9% orphans and presence of 63.4% spoligopatterns without ST numbers in our study highlights the circulating genetically diverse M. tuberculosis and emphasize the need for more molecular epidemiological studies. MIRU typing could not be carried out by us at that point of time due to resource constraints.
To conclude, Spoligotyping of M. tuberculosis is an important tool for studying the epidemiology of this pathogen causing PT and EPTB. The high costs of instruments/reagents and sophisticated technology are perhaps the main hindering factors faced by resource poor laboratories in the developing countries.
| ~ References|| |
Joseph BV, Soman S, Radhakrishnan I, Hill V, Dhanasooraj D, Kumar RA, et al.
Molecular epidemiology of Mycobacterium tuberculosis
isolates from Kerala, India using IS6110-RFLP, spoligotyping and MIRU-VNTRs. Infect Genet Evol 2013;16:157-64.
Vadwai V, Shetty A, Supply P, Rodrigues C. Evaluation of 24-locus MIRU-VNTR in extrapulmonary specimens: Study from a tertiary centre in Mumbai. Tuberculosis (Edinb) 2012;92:264-72.
Varma-Basil M, Kumar S, Arora J, Angrup A, Zozio T, Banavaliker JN, et al
. Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis
in Delhi, North India. Mem Inst Oswaldo Cruz 2011;106:524-35.
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.
Sharma P, Chauhan DS, Upadhyay P, Faujdar J, Lavania M, Sachan S, et al
. Molecular typing of Mycobacterium tuberculosis
isolates from a rural area of Kanpur by spoligotyping and mycobacterial interspersed repetitive units (MIRUs) typing. Infect Genet Evol 2008;8:621-6.
Shanmugam S, Selvakumar N, Narayanan S. Drug resistance among different genotypes of Mycobacterium tuberculosis
isolated from patients from Tiruvallur, South India. Infect Genet Evol 2011;11:980-6.
Mathuria JP, Sharma P, Prakash P, Samaria JK, Katoch VM, Anupurba S. Role of spoligotyping and IS6110-RFLP in assessing genetic diversity of Mycobacterium tuberculosis
in India. Infect Genet Evol 2008;8:346-51.
Desikan P, Chauhan DS, Sharma P, Panwalkar N, Gautam S, Katoch VM. A pilot study to determine genetic polymorphism in Mycobacterium tuberculosis
isolates in Central India. Indian J Med Microbiol 2012;30:470-3.
Thomas SK, Iravatham CC, Moni BH, Kumar A, Archana BV, Majid M, et al.
Modern and ancestral genotypes of Mycobacterium tuberculosis
from Andhra Pradesh, India. PLoS One 2011;6:e27584.
Chatterjee A, D'Souza D, Vira T, Bamne A, Ambe GT, Nicol MP, et al.
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 2010;48:3593-9.
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.
Singh UB, Suresh N, Bhanu NV, Arora J, Pant H, Sinha S, et al
. Predominant tuberculosis spoligotypes, Delhi, India. Emerg Infect Dis 2004;10:1138-42.
Kulkarni S, Sola C, Filliol I, Rastogi N, Kadival G. Spoligotyping of Mycobacterium tuberculosis
isolates from patients with pulmonary tuberculosis in Mumbai. India. Res Microbiol 2005;156:588-96.
Gutierrez MC, Ahmed N, Willery E, Narayanan S, Hasnain SE, Chauhan DS, et al.
Predominance of ancestral lineages of Mycobacterium tuberculosis
in India. Emerg Infect Dis 2006;12:1367-74.
Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, et al.
Simultaneous detection and strain differentiation of Mycobacterium tuberculosis
for diagnosis and epidemiology. J Clin Microbiol 1997;35:907-14.
Forbes BA, Sahm DF, Weissfeld AS, editors. Mycobacteria. In: Bailey and Scott's Diagnostic Microbiology. 12 th
ed. St. Louis: Mosby Elsevier; 2002. p. 478-509.
Abe C, Hirano K, Tomiyama T. Simple and rapid identification of the Mycobacterium tuberculosis
complex by immunochromatographic assay using anti-MPB64 monoclonal antibodies. J Clin Microbiol 1999;37:3693-7.
Perumal AK, Singh D. Extensive osteomyelitis of humerus following Bacille Calmette-Guérin vaccination. Int J Med Sci Public Health 2013;2:465-7.
[Table 1], [Table 2]