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  Table of Contents  
BRIEF COMMUNICATION
Year : 2017  |  Volume : 35  |  Issue : 1  |  Page : 129-133
 

Trends in spoligotype patterns of Mycobacterium tuberculosis strains in central India


1 Department of Microbiology, NRL, Bhopal Memorial Hospital and Research Centre, Bhopal, Madhya Pradesh, India
2 National JALMA Institute of Leprosy and Other Mycobacterial Diseases, Indian Council of Medical Research, Dr. Miyazaki Marg, Agra, Uttar Pradesh, India

Date of Web Publication16-Mar-2017

Correspondence Address:
Prabha Desikan
Department of Microbiology, NRL, Bhopal Memorial Hospital and Research Centre, Indian Council of Medical Research, Raisen Bypass Road, Karond, Bhopal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_16_12

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 ~ Abstract 


This study aims to understand trends in spoligotype patterns of Mycobacterium tuberculosis (MTB) in Central India. Elucidation of these trends may provide baseline information to understand the transmission dynamics of strains of MTB in the region. Spoligotyping was carried out on 340 MTB strains isolated from clinical samples received from 2007 to 2011. The prevalence of ST26/CAS1_Del, ST11/EAI3_IND, ST288/CAS2, ST25/CAS1_Del and Beijing lineages showed waxing and waning trends. ST26/CAS1_Del and ST11/EAI3_IND lineages were consistently present and were predominant. Well-established lineages showed a consistent presence in the community. New orphan lineages appeared to be less capable of establishing themselves.


Keywords: Central India, Mycobacterium tuberculosis, spoligotype patterns, trends


How to cite this article:
Desikan P, Chauhan DS, Panwalkar N, Sharma P, Yadav P. Trends in spoligotype patterns of Mycobacterium tuberculosis strains in central India. Indian J Med Microbiol 2017;35:129-33

How to cite this URL:
Desikan P, Chauhan DS, Panwalkar N, Sharma P, Yadav P. Trends in spoligotype patterns of Mycobacterium tuberculosis strains in central India. Indian J Med Microbiol [serial online] 2017 [cited 2017 Mar 25];35:129-33. Available from: http://www.ijmm.org/text.asp?2017/35/1/129/202331





 ~ Introduction Top


Tuberculosis, a disease of antiquity, continues to claim thousands of lives every year worldwide. The propagation success of Mycobacterium tuberculosis (MTB) probably depends on multiple factors which may be specific to each geographical region. This is underlined by the predominance of some lineages of MTB over others in a particular area. Studies show that certain spoligotype-defined lineages of MTB continue to be predominant while new spoligotype patterns are still emerging.[1],[2] Elucidation of these changes may provide important information to help understand transmission dynamics of strains of MTB in a region. The present study aims to understand the trends in spoligotype patterns of MTB over a period of 5 years in Central India.


 ~ Materials and Methods Top


Study samples

Three hundred and forty MTB strains isolated from clinical samples of tuberculosis patients from Bhopal and other districts of Madhya Pradesh from March 2007 to November 2011 were included in the study. There were 46 MTB strains isolated in 2007, 64 in 2008, 79 in 2009, 70 in 2010, and 81 in 2011.

Culture

Clinical samples were processed by the standard NaOH-NALC method for digestion and decontamination [3] and sediments were inoculated on two Lowenstein-Jensen slants (LJ slants). The organisms were identified as MTB by the absence of growth on LJ slants incorporated with P-Nitrobenzoic acid, a positive niacin accumulation test, a positive nitrate reduction test, negative catalase test, and rRNA-directed DNA hybridisation assay (Accuprobe ® Gen-probe, bioMérieux).

DNA extraction and spoligotyping

DNA from culture strains of MTB was extracted using QIAamp ® DNA Mini kit (QIAGEN ®, Dusseldorf, Germany) as per the prescribed manual. Spoligotyping was carried out by a commercial kit (Ocimum Biosolutions, Hyderabad) at the National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, as per the manufacturer's instructions. The extracted DNA was subjected to polymerase chain reaction using specific biotinylated primers directed towards 43 known spacers, interspersed between the DR-cluster of MTB genome. The amplicons were hybridised using streptavidin-conjugate on an activated membrane to which spacer oligonucleotides were covalently bound. For detection of hybridised DNA, an enhanced chemiluminescence detection system was used. The membrane was reversely blotted on an X-ray film to record the hybridisation signals. H37Rv was used as positive control and distilled water as negative control. The presence/absence of spacer oligonucleotides was documented in the form of binary code that was converted into octal code.

Spoligotype patterns analysis

The spoligotype patterns were analysed using the SITVIT2 database (Institut Pasteur de la Guadeloupe) to assign spoligotype lineages.[4] A strain was defined as 'cluster' when two or more strains were found to have same spoligotype international type (SIT); as 'unique' when a single strain was found to have specific SIT and as 'orphan' when no match for SIT for a strain was found in the database.[5],[6]


 ~ Results Top


Out of a total 46 strains in 2007, 24 (52.17%) could be clustered in 3 SITs; 10 (21.73%) were unique and 12 (26.08%) were orphan strains. ST11/EAI3_IND, ST26/CAS1_Del and ST288/CAS2 with 13 (28.26%), 7 (15.21%) and 4 (8.69%) strains, respectively, were the three predominant lineages in 2007. Out of a total 64 strains in 2008, 40 (62.5%) could be clustered in 5 SITs, 06 (9.37%) were unique and 18 (28.12%) were orphan strains. ST26/CAS1_Del, ST11/EAI3_IND, and ST25/CAS1_Del with 17 (26.56%), 13 (20.31%) and 06 (9.37%) strains, respectively, were the three predominant lineages in 2008. Out of a total 79 strains in 2009, 51 (64.55%) could be clustered in 11 SITs, 18 (22.78%) were unique and 10 (12.65%) were orphan strains. ST26/CAS1_Del, ST11/EAI3_IND and ST25/CAS1_Del with 18 (22.78%), 8 (10.12%) and 4 (5.06%) strains, respectively, were the three predominant lineages in 2009. Out of a total 70 strains in 2010, 48 (68.57%) could be clustered in 7 SITs, 14 (20%) were unique and 8 (11.42%) were orphan strains. ST26/CAS1_Del, ST11/EAI3_IND and ST288/CAS2 with 18 (22.78%), 8 (10.12%) and 4 (5.06%) strains, respectively, were the three predominant lineages in 2010. Out of a total 81 strains in 2011, 48 (59.25%) could be clustered in 8 SITs, 20 (24.69%) were unique and 13 (16.04%) were orphan strains. ST26/CAS1_Del with 18 (22.22%), ST11/EAI3_IND with 11 (13.58%), ST288/CAS2 and ST25/CAS1_Del with 5 (6.17%) strains each were the predominant lineages in 2011. The prevalence of ST11/EAI3_IND decreased over the initial 3 years from 28.26% strains in 2007 to 10.12% strains in 2009, increased to 24.28% strains in 2010 and again decreased to 13.58% in 2011. The prevalence ST26/CAS1_Del lineage increased from 15.21% of the strains in 2007 to 26.56% in 2008, reduced to 22.78% in 2009, again increased to 25.71% in 2010 and again decreased to 22.22% in 2011. The prevalence of Beijing strain increased from 2.17% strains in 2007 to 5.06% in 2009 and then, reduced over the next 2 years to 3.7% of strains in 2011 [Figure 1]. The prevalence of orphan strains increased from 26.08% of the strains in 2007 to 28.12% in 2008, reduced over the next 2 years to 11.42% in 2010 and increased to 16.04% in 2011.
Figure 1: Trends in spoligotype patterns.

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 ~ Discussion Top


The long-standing association of MTB and humans since ancient times indicates some adaptive genetic changes in the pathogen. The emerging multidrug resistance [7] among the strains of MTB is the classic paradigm of adaptation. MTB can exist in diverse metabolic states, following genetic alterations that help its escape through the conventional anti-tuberculosis agents.[8] Study of mobile genetic elements or repetitive DNA segments has enhanced our knowledge on 'ancestral' and 'modern' strains of MTB which may be related to a possible adaptive mechanism in the pathogen.[9],[10] In our study, the two foremost lineages, the 'ancient' ST11/EAI3_IND and the 'modern' ST26/CAS1_Del were found to be well established over 5 years. However, the prevalence of these lineages alternately increased and decreased over each consecutive year till 2011. Continuous clustering of EAI and CAS lineages indicates that these lineages are well adapted to this area, and therefore, show a successful transmission. The CAS and EAI lineages have shown consistent predominance and clustering patterns in the Indian subcontinent for many years.[5],[11] The EAI lineage has always been found to be prevalent in South India.[11] In the present study, ST11/EAI3_IND and ST26/CAS1_Del showed waxing and waning trends of prevalence over the 5 years of study.

The more recently evolved Beijing genotype was not a predominant lineage in this region though it had a continuous representation. Beijing lineage is well known for its virulent nature and its association with drug resistance.[12]

The prevalence of other predominant lineages, ST288/CAS2 and ST25/CAS1_Del lineages also showed waxing and waning trends over the period of study [Figure 1]. Poorly represented lineages appeared and disappeared intermittently over 5 years [Table 1]. These lineages may have not been able to gain a foothold in this region. This is similar to the findings from a study from New Delhi, India.[13] On the other hand, MANU, a poorly represented lineage in our area, was found to be the most predominant lineage among the drug-susceptible strains of MTB in Mumbai, India.[14]
Table 1: Year wise occurrence of spoligotype lineages over the period of five years

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Well-established lineages show a consistent presence in the community. ST26/CAS1_Del and ST11/EAI3_IND lineages were consistently present and were the most predominant lineages over 5 years. Beijing lineage had a continuous, though less predominant presence. Other poorly represented lineages appeared and disappeared intermittently. It is possible that a mix of host, environmental and bacterial factors is involved in the propagation success of some lineages and poor propagation of others. These factors may vary from region to region. Identification and mapping of these factors may help plan strategies for targeted and successful control of tuberculosis.

Acknowledgment

The authors acknowledge the help provided by laboratory staff of Bhopal Memorial Hospital and Research Centre, Bhopal and National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra.

Financial support and sponsorship

The authors acknowledge the Indian Council of Medical Research for financial support for the study.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Mokrousov I, Vyazovaya A, Narvskaya O. Mycobacterium tuberculosis Latin American-Mediterranean family and its sublineages in the light of robust evolutionary markers. J Bacteriol 2014;196:1833-41.  Back to cited text no. 1
    
2.
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.  Back to cited text no. 2
    
3.
Revised National TB Control Programme, Training Manual for Mycobacterium tuberculosis Culture and Drug Susceptibility Testing, Central TB Division, Directorate General of Health Services, Ministry of Health and Family Welfare, Nirman Bhawan; April, 2009.  Back to cited text no. 3
    
4.
Demaya C, Liensa B, Burguièrea T, Hilla V, Couvina D, Milleta J, et al. SITVITWEB – A publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology. Infect Genet Evol 2012;12:755-66.  Back to cited text no. 4
    
5.
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.  Back to cited text no. 5
    
6.
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.  Back to cited text no. 6
    
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Manjula S, Sritharan V. Microbial pathogenesis: An insight into Mycobacterium tuberculosis. Indian J Med Microbiol 2002;20:61-8.  Back to cited text no. 7
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Negi SS, Singh U, Gupta S, Khare S, Rai A, Lal S. Characterization of RPO B gene for detection of rifampicin drug resistance by SSCP and sequence analysis. Indian J Med Microbiol 2009;27:226-30.  Back to cited text no. 8
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Gagneux S, DeRiemer K, Van T, Kato-Maeda M, de Jong BC, Narayanan S, et al. Variable host-pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 2006;103:2869-73.  Back to cited text no. 9
    
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Reiling N, Homolka S, Walter K, Brandenburg J, Niwinski L, Ernst M, et al. Clade-specific virulence patterns of Mycobacterium tuberculosis complex strains in human primary macrophages and aerogenically infected mice. MBio 2013;4. pii: E00250-13.  Back to cited text no. 10
    
11.
Das S, Paramasivan CN, Lowrie DB, Prabhakar R, Narayanan PR. IS6110 restriction fragment length polymorphism typing of clinical isolates of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Madras, South India. Tuber Lung Dis 1995;76:550-4.  Back to cited text no. 11
    
12.
Huyen MN, Buu TN, Tiemersma E, Lan NT, Dung NH, Kremer K, et al. Tuberculosis relapse in Vietnam is significantly associated with Mycobacterium tuberculosis Beijing genotype infections. J Infect Dis 2013;207:1516-24.  Back to cited text no. 12
    
13.
Stavrum R, Myneedu VP, Arora VK, Ahmed N, Grewal HM. In-depth molecular characterization of Mycobacterium tuberculosis from New Delhi – Predominance of drug resistant isolates of the 'modern' (TbD1) type. PLoS One 2009;4:e4540.  Back to cited text no. 13
    
14.
Bhatter P, Chatterjee A, D'souza D, Tolani M, Mistry N. Estimating fitness by competition assays between drug susceptible and resistant Mycobacterium tuberculosis of predominant lineages in Mumbai, India. PLoS One 2012;7:e33507.  Back to cited text no. 14
    


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