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 ~  Abstract
 ~ Introduction
 ~ Material and Methods
 ~ Results
 ~ Discussion
 ~ Conclusions
 ~ Acknowledgement
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
BRIEF COMMUNICATION
Year : 2014  |  Volume : 32  |  Issue : 4  |  Page : 434-437
 

Clonal Diversity and Drug Resistance in Mycobacterium tuberculosis Isolated from Extra-pulmonary Samples in Central India - a Pilot Study


1 Department of Microbiology, Bhopal Memorial Hospital and Research Centre, Indian Council of Medical Research, Karond, Bhopal, Madhya Pradesh, India
2 Department of Microbiology and Molecular Biology, National JALMA Institute of Leprosy and other Mycobacterial Diseases Institute of Leprosy and Other Mycobacterial Diseases, Indian Council of Medical Research, Agra, Uttar Pradesh, India
3 State TB Officer, Directorate of Health Services, Satpura Bhavan, Madhya Pradesh, India

Date of Submission16-Jul-2013
Date of Acceptance24-Feb-2014
Date of Web Publication4-Oct-2014

Correspondence Address:
P Desikan
Department of Microbiology, Bhopal Memorial Hospital and Research Centre, Indian Council of Medical Research, Karond, Bhopal, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.142255

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 ~ Abstract 

In India, extrapulmonary tuberculosis (EPTB) accounts for 10 - 15% of all types of tuberculosis. To identify and compare predominant spoligotypes and drug-resistance patterns in strains of Mycobacterium tuberculosis isolated from extrapulmonary and pulmonary specimens in central India, drug susceptibility testing and spoligotyping were carried out. Spoligotyping data was analyzed using SITVIT2 database. ST11/EAI3_Ind with 33% isolates among extrapulmonary specimens and ST26/CAS1_DEL with 28% isolates among pulmonary specimens were the most predominant lineages. Multidrug resistance was found in 5.5% of the strains isolated from extrapulmonary specimens in contrast to 17% isolated from pulmonary specimens.


Keywords: Central India, extrapulmonary, M. tuberculosis, spoligotyping


How to cite this article:
Desikan P, Chauhan D S, Sharma P, Panwalkar N, Yadav P, Ohri B S. Clonal Diversity and Drug Resistance in Mycobacterium tuberculosis Isolated from Extra-pulmonary Samples in Central India - a Pilot Study . Indian J Med Microbiol 2014;32:434-7

How to cite this URL:
Desikan P, Chauhan D S, Sharma P, Panwalkar N, Yadav P, Ohri B S. Clonal Diversity and Drug Resistance in Mycobacterium tuberculosis Isolated from Extra-pulmonary Samples in Central India - a Pilot Study . Indian J Med Microbiol [serial online] 2014 [cited 2019 Sep 23];32:434-7. Available from: http://www.ijmm.org/text.asp?2014/32/4/434/142255



 ~ Introduction Top


Mycobacterium tuberculosis can affect virtually any part of the body. Extrapulmonary tuberculosis (EPTB) is a major problem worldwide. [1],[2] In India, EPTB accounts for 10 - 15% of all types of tuberculosis. [3] It is possible that certain patterns of genetic polymorphism may be present among strains of M. tuberculosis that cause EPTB in Central India. If so, it may be possible to predict EPTB in case of infection with specific strains of M. tuberculosis. There is no data available regarding genetic diversity among strains of M. tuberculosis that cause EPTB in Central India. There is also a paucity of data regarding multidrug resistance among these strains. We carried out a pilot study to identify predominant spoligotypes and drug-resistance patterns in strains of M. tuberculosis isolated from extrapulmonary specimens in Central India. Also, we compared clonal variations and drug-resistance patterns among both pulmonary and extrapulmonary M. tuberculosis isolates.


 ~ Material and Methods Top


A total of 36 M. tuberculosis strains were studied following approval from the Institutional Review Board, BMHRC, Bhopal. These included 18 strains consecutively isolated from extrapulmonary specimens, and 18 strains consecutively isolated from pulmonary specimens, all received between October 2010 and September 2011. Only 18 strains of M. tuberculosis were isolated from extrapulmonary samples during this period. Hence, the same number of consecutively isolated strains from pulmonary samples was included in the study. Nine extrapulmonary specimens were collected from females between the ages 30 and 58 years and 9 were collected from males with ages ranging from 33 to 65 years. Three pulmonary specimens were collected from females in the age range of 35 - 38 years and 15 were collected from males ranging in age from 21 to 75 years. All the patients included in the study were HIV negative and none of them had reported prior history of tuberculosis. None had received prior treatment with antitubercular therapy. M. tuberculosis strains were isolated from extrapulmonary samples that included colonic and duodenal biopsies, cervical lymph nodes, tissue from vertebral bodies, pleural fluid, pus from cold abscesses and urine. They were also isolated from pulmonary specimens that included sputa, bronchial aspirates and bronchial lavage samples.

The specimens were digested and decontaminated by the standard NaOH-NALC method [4] and then 0.5 ml of sediment was inoculated on two Lowenstein-Jensen slants (LJ slants) as well as in a MB/BacT process bottle as per the manufacturer's protocol. All the inoculated bottles were incubated at 37°C. Growth of acid-fast bacilli in either of the media was confirmed by smear microscopy using Ziehl-Neelsen stain. The isolate was confirmed as M. tuberculosis by the absence of growth on LJ slants incorporated with P-Nitrobenzoic acid, a positive niacin accumulation test, a positive nitrate reduction test, a negative catalase test and a positive rRNA directed DNA hybridisation assay for M. tuberculosis (GenProbe, bioMérieux, France).

Drug Susceptibility Test (DST) on LJ medium to first-line anti-TB drugs was performed by 1% proportion method as per the RNTCP guidelines. [5] The critical concentrations used were 4 μg/ml for streptomycin (SM), 0.2 μg/ml for Isoniazid (INH), 40 μg/ml for Rifampicin (RIF) and 2 μg/ml for Ethambutol (ETM). An isolate was defined as resistant if the proportion of growth on drug-containing medium to drug-free medium was found to be equal or higher than 1. If a strain was found to be resistant to Isoniazid and Rifampicin with or without resistance to other drugs, it was defined as Multi-drug resistant (MDR).

Genomic DNA from M. tuberculosis strains was extracted using QiAmp DNA Mini kit (Dusseldorf, Germany) following manufacturer's guidelines. PCR was carried out at the National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, using specific primers to amplify the direct repeat variable regions. The amplicons were subjected to spoligotyping by using a commercial kit (Ocimum Biosolutions, Hyderbad) as per the manufacturer's procedural directives in order to identify Spoligotype International types (SIT). SITs were assigned to each spoligotype pattern according to the international SITVIT2 database. [6] SPOTCLUST database was consulted to assign most probable lineage to the orphan isolates. [7]


 ~ Results Top


Out of the 18 M. tuberculosis isolates from extrapulmonary specimens, 10 (55%) could be clustered in three lineages [Figure 1]. On the other hand, out of the 18 M. tuberculosis strains isolated from pulmonary specimens, 14 (78%) could be clustered in five lineages [Figure 2]. Five isolates from extrapulmonary specimens (28%) had unique spoligotype patterns. However, only one isolate from pulmonary specimens (5.5%) had a unique spoligotype pattern. The predominant circulating lineage among extrapulmonary specimens was found to be SIT11/EAI3_Ind with six (33%) isolates. Conversely, SIT26/CAS1_DEL was found to be the most predominant lineage among pulmonary specimens, with five (28%) isolates. The Beijing genotype was found in only one (5%) of the isolates from extrapulmonary specimens whereas it was found in two (11%) isolates from pulmonary specimens. Other lineages found among strains isolated from both, extrapulmonary and pulmonary specimens, were SIT381/CAS, SIT48/EAI_SOM and SIT25/CAS1_DEL. Two lineages, SIT52/T2, and SIT1391/U, were found exclusively in strains isolated from extrapulmonary specimens. Orphan patterns were found in 3 (17%) strains among strains isolated from both, pulmonary and extrapulmonary specimens, respectively. However, all these orphan strains belonged to different lineages [Table 1].
Figure 1: Distinct spoligotypes of Mycobacterium tuberculosis isolates from extrapulmonary patients. The dendogram was generated using the dice coefficient and the unweighted pair group method with arithmetic averages (UPGMA) using the MIRU-VNTRplus program. R - Rifampicin, I - Isoniazid, E - Ethambutol, S - Streptomycin

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Figure 2: Distinct spoligotypes of Mycobacterium tuberculosis isolates from pulmonary TB patients. The dendrogram was generated using the dice coefficient and the unweighted pair group method with arithmetic averages (UPGMA) using the MIRU-VNTRplus program. S-Streptomycin, I-Isoniazid, R-Rifampicin, E-Ethambutol

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Table 1: Details of spoligotypes and incidence of multidrug resistance in the study sample

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Multidrug resistance was found in only one (5.5%) of the strains isolated from extrapulmonary specimens in contrast to three strains (17%) isolated from pulmonary specimens. The MDR strain isolated from an extrapulmonary specimen was of the Beijing genotype. Of the three MDR strains isolated from pulmonary specimens, one belonged to the Beijing genotype, one belonged to the shared type SIT26/CAS1_DEL and one belonged to an orphan lineage. No Rifampicin or Isoniazid monoresistance was observed in any of the strains studied. Using Fisher's Exact test, however, a statistically significant association could not be demonstrated between isolates from pulmonary and extrapulmonary samples; and multidrug resistance; as well as spoligotypes.


 ~ Discussion Top


A higher number of clustered isolates (33.33%) of the SIT11/EAI3_Ind lineage among strains isolated from extrapulmonary samples indicate an ongoing transmission of circulating M. tuberculosis strains involving extrapulmonary sites within this community. Only 16.66% of the strains isolated from pulmonary specimens had this lineage. The presence of 28% unique patterns in strains isolated from extrapulmonary specimens could indicate reactivation of existing lineages in this region. Only 5.55% of the strains isolated from pulmonary specimens had a unique pattern. This might indicate that pulmonary tuberculosis is associated more commonly with strains belonging to the more frequently isolated lineages in this community. SIT11/EAI3_Ind with 6 (33.33%) isolates was found to be the most predominant lineage among strains isolated from extrapulmonary samples in this region. However, a study from north India reported that CAS1_DEL was the most predominant lineage among strains isolated from extrapulmonary samples. [8] CAS, EAI and Beijing have been reported as the predominant strain lineages among isolates from patients with EPTB in Mumbai. [9]

Only one strain (5.55%) among those isolated from extrapulmonary samples was multidrug resistant. This strain belonged to the Beijing genotype. In contrast, 33.33% of strains isolated from pulmonary samples were multidrug resistant.

These results are in contrast to previous studies from other geographical areas which document higher percentages of drug resistance in M. tuberculosis strains isolated from extrapulmonary samples. [10],[11],[12] Further studies on large number of samples would provide a greater insight into the pattern in our geographical area.


 ~ Conclusions Top


The predominant circulating lineage among extrapulmonary specimens was found to be SIT11/EAI3_Ind with 6 (33%) isolates whereas SIT26/CAS1_DEL was found to be predominant spoligotype among the strains of M. tuberculosis isolated from pulmonary sites with 5 (28%) isolates. A relatively lower incidence of drug resistance was observed among the strains of M. tuberculosis isolated from EPTB when compared to the strains isolated from pulmonary specimens.

While our pilot study shows clustering of a specific lineage of M. tuberculosis strains isolated from extrapulmonary samples, a larger number of strains isolated from extrapulmonary samples need to be studied in order to confirm the predilection, if any, of a specific lineage of M. tuberculosis to extrapulmonary sites in this geographical area. If such a predilection is demonstrated, diagnostic modalities could be tailored to detect these lineages with greater specificity and sensitivity.


 ~ Acknowledgement Top


Indian Council of Medical Research, for Financial Support.

 
 ~ References Top

1.Sudre P, Hirschel BJ, Gatell JM, Schwander S, Vella S, Katlama C, et al. Tuberculosis among European patients with the acquired immune deficiency syndrome. The AIDS in Europe Study Group. Tuber Lung Dis 1996;77:322-8.  Back to cited text no. 1
    
2.Stelianides S, Belmatoug N, Fantin B. Manifestations and diagnosis of extrapulmonary tuberculosis. Rev Mal Respir 1997;14:S72-87.  Back to cited text no. 2
    
3.Wares F, Balasubramanian R, Mohan A, Sharma SK. Extrapulmonary Tuberculosis: Management and Control. In: Tuberculosis Control in India. Agarwal SP, Chauhan LS, editors. Directorate General of Health Services, New Delhi; India. Reed Elsevier India Private Ltd; 2005.p. 95-114.  Back to cited text no. 3
    
4.World Health Organization. Anti-tuberculosis Drug Resistance in the World Report No. 4. Geneva, Switzerland: World Health Organization; 2008. Publication No. WHO/HTM/TB/2008.394.  Back to cited text no. 4
    
5.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; 2009.  Back to cited text no. 5
    
6.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. 6
    
7.Vitol I, Driscoll J, Kreiswirth B, Kurepina N, Bennett KP. Identifying Mycobacterium tuberculosis complex strain families using spoligotypes. Infect Genet Evol 2000;6:491-504.  Back to cited text no. 7
    
8.Sankar MM, Selvaraj JS, Diana CA, 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. 8
    
9.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.  Back to cited text no. 9
    
10.Maurya AK, Kant S, Nag VL, Kushwaha RA, Dhole TN. Trends of anti-tuberculosis drug resistance pattern in new cases and previously treated cases of extrapulmonary tuberculosis cases in referral hospitals in northern India. J Postgrad Med 2012;58:185-9.  Back to cited text no. 10
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11.Butt T, Kazmi SY, Ahmad RN, Mahmood A, Karamat KA, Anwar M. Frequency and antibiotic susceptibility pattern of mycobacterial isolates from extra-pulmonary tuberculosiscases. J Pak Med Assoc 2003;53:328-32.  Back to cited text no. 11
    
12.Vadwai V, Boehme C, Nabeta P, Shetty A, Alland D, Rodrigues C. Xpert MTB/RIF: A new pillar in diagnosis of extrapulmonary tuberculosis? J Clin Microbiol 2011;49:2540-5.  Back to cited text no. 12
    


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