Infection, Genetics and Evolution 9 (2009) 832–839

Characterization of predominant Mycobacterium tuberculosis strains fromdifferent subpopulations of India

Jyoti Arora a, Urvashi Balbir Singh a,*, Naga Suresh a, Tanu Rana a, Chhavi Porwal a,Amit Kaushik a, Jitendra Nath Pande b

a Department of Microbiology, All India Institute of Medical Sciences, New Delhi, Indiab Sitaram Bharatiya Institute of Science and Research, New Delhi, India

A R T I C L E I N F O

Article history:

Received 31 October 2008

Received in revised form 27 April 2009

Accepted 3 May 2009

Available online 20 May 2009

Keywords:

Tuberculosis

Spoligotyping

Restriction fragment length polymorphism

Shared type

Clade

A B S T R A C T

The predominant strains from India belong to Central-Asian (CAS) and the East-African-Indian (EAI)

clade of Mycobacterium tuberculosis. The two clades have also been shown to be geographically

partitioned. The study of such strains may help to understand the characteristics that make M.

tuberculosis an effective pathogen and its overrepresentation in certain populations. M. tuberculosis

isolates characterized by spoligotyping under a population based tuberculosis study covering different

regions from the North and South India were further analyzed by restriction fragment length

polymorphism (RFLP) and by deletion analysis of M. tuberculosis specific deletion region 1 (TbD1). The

genetic relationship of the two clades inferred using different genetic markers showed good correlation.

In the North where the CAS clade predominates the isolates are characterized by presence of high IS6110

copy number and absence of TbD1 region whereas in the South where the EAI clade predominates the

isolates are characterized by low copy number of IS6110 and presence of TbD1 region. The ancestral EAI

strains were found to be less often associated with drug resistance or young age as compared to the CAS

clade. The study highlights that the EAI lineage is well established in India and that CAS may be emerging

or more recently introduced to India. The results depict a distinction in the lineage of strains from the

North versus South India indicating a need to study if the pathogen has adapted to specific human

populations.

� 2009 Elsevier B.V. All rights reserved.

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1. Introduction

The resurgence of tuberculosis (TB) has renewed interest inunderstanding the epidemiology and pathogenesis of the disease.Genotyping of clinical isolates in different parts of the world hasshown that global epidemiology of TB is propagated by thousands ofdifferent genotypes (van Soolingen et al., 1999; Warren et al., 1999).The strains occur at different frequencies, and the relativefrequencies in different areas vary between districts, cities, countriesand continents (Brudey et al., 2006; Filliol et al., 2002; Filliol et al.,2003). The dynamics of TB epidemic in a given area and time framemay therefore be a factor of the different strains circulating in thatregion. The knowledge of circulating strains can be used formolecular evolutionary and population genetics studies.

IS6110-based typing is the most widely applied genotypingmethod in the molecular epidemiology of Mycobacterium tubercu-

losis. The IS6110 insertion sequence is specific to the M. tuberculosis,

* Corresponding author. Tel.: +91 11 26588500/700x6436; fax: +91 11 26588641.

E-mail address: drurvashi@gmail.com (U.B. Singh).

1567-1348/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.meegid.2009.05.008

are present in different copy numbers (0–25) and their positions inthe genome are highly variable among different isolates (vanEmbden et al., 1993). Spoligotyping targeting the DNA polymorph-ism at the direct repeat locus (DR locus) of the genome of M.

tuberculosis complex, allows simultaneous detection and differ-entiation of M. tuberculosis complex strains (Kamerbeek et al., 1997).The DR locus is well conserved and stable enough rendering itspecific for detecting M. tuberculosis complex strains.

SNPs in the katG and gyrA genes classify M. tuberculosis isolatesinto three principal genetic groups (PGGs). TbD1 is specificallypresent in a subset of PGG1 strains, but absent in other strains ofPGG1, and in PGG2 and PGG3 strains. Based on the presence orabsence of an M. tuberculosis specific deletion (TbD1), M.

tuberculosis strains can be segregated into ‘‘ancestral’’ versus‘‘modern’’ lineages (Brosch et al., 2002). Though the known‘‘modern’’ M. tuberculosis families are very widely prevalentworldwide (Filliol et al., 2002; Filliol et al., 2003; Brudey et al.,2006), ancient principal genetic group 1 (PGG1) clones areresponsible for TB in India (Kulkarni et al., 2005; Gutierrezet al., 2006; Singh et al., 2007). Studies in India lack reference topreferential localization of certain strain families in different

Table 1Clinical and epidemiologic characteristics of patients harbouring isolates belonging

to EAI and CAS clade.

Parameters Number (%) of patients in p Value

CAS clade EAI clade

Age (y)

15–45 126 (84) 119 (70) 0.0048

>46 24 (16) 51 (30)

Gender

Male 114 (76) 122 (72) NS

Female 36 (24) 48 (28)

Birth place

Village 129 (86) 140 (82) NS

City 21 (14) 30 (18)

Previous history of TB

No previous therapy 67 (45) 119 (70) 0.0003

Failed/defaulter/relapsed 70 (46) 42 (25)

Cured 13 (9) 9 (5)

Smoking

Yes 69 (46) 81 (45) NS

No 81 (54) 89 (55)

Alcohol

Yes 72 (48) 73 (44) NS

No 78 (52) 97 (56)

Family TB

Yes 39 (26) 41 (24) NS

No 111 (74) 129 (76)

TB death

Yes 24 (16) 27 (16) NS

No 126 (84) 143 (84)

Drug resistance

Resistance to one or more drug 69 (46) 56 (33) 0.023

Susceptible to all drugs 81 (54) 114 (66)

X-ray

Ext 102 (68) 107 (63) NS

Lim 48 (32) 63 (37)

Radiologic findings

Cav 105 (70) 119 (70) NS

Noncav 45 (30) 51 (30)

Sputum smear results

Negative 12 (8) 12 (7) NS

1+ 52 (40) 56 (33)

2+ or 3+ 78 (52) 102 (60)

NS, not significant statistically; y, year.

J. Arora et al. / Infection, Genetics and Evolution 9 (2009) 832–839 833

subpopulations among different regions of the country. Despite thehigh burden of TB and various epidemiological studies done in theNorth and the South regions of India (Das et al., 1995; Narayananet al., 1997; Radhakrishnan et al., 2001; Siddiqi et al., 2001; Bhanuet al., 2002; Mistry et al., 2002; Kulkarni et al., 2005; Gutierrezet al., 2006; Suresh et al., 2006; Singh et al., 2007; Sharma et al.,2008; Ahmed et al., 2009; Stavrum et al., 2009) there is limited dataavailable pertaining to strains circulating in the country.

Epidemiological data from different studies suggests thatdifference in transmissibility and virulence among M. tuberculosis

strains are related to genetic makeup of the organisms (Valwayet al., 1998; Caminero et al., 2001; Lopez et al., 2003). Over-representation of clades suggests that they possess biologicaladvantage in specific populations and study of such strains mayhelp to understand the characteristics that make M. tuberculosis aneffective pathogen.

Based upon our earlier results of spoligotyping on 540 M.

tuberculosis isolates from five different geographical regions ofIndia, we concluded that different parts of India also differed inregard to predominant spoligotypes, underlining the differences inthe introduction and evolution of TB in India, the East-African-Indian (EAI) clade being more commonly found in Southern partsand the (Central-Asian) CAS clade being more predominant in theNorth (Singh et al., 2007). The present study was undertaken tostudy genetic composition of the EAI and CAS isolates usingdifferent genetic markers.

The study also reflects on the associations between lineages andpatient demographic variables.

2. Materials and methods

2.1. Study population

Clinical isolates of M. tuberculosis were obtained during thestudy period of January 2001–September 2003 under a multi-centric (eight centres over the country) project supported byINDIA-CLEN (INCLEN, USA). Samples were processed on the day ofarrival by modified Petroff’s method and cultured on LJ slants at37 8C for 6–8 weeks in the respective laboratories. Testing forsusceptibility to Rifampin (RIF, 40.0 mg/ml), Ethambutol (EMB,2.0 mg/ml), Isoniazid (INH, 0.2 mg/ml) and Streptomycin (SM,4.0 mg/ml) was performed according to the Proportion method(Laszlo et al., 1997). Randomly selected culture isolates (n – 540)from three North Indian cities (Delhi, Pune and Lucknow) and twoSouth Indian cities (Chennai and Trivandrum) were collected at theAll India Institute of Medical Sciences (AIIMS), New Delhi,laboratory and stored at �70 8C for further workup. These werewell characterized by spoligotyping (Singh et al., 2007) using thestandard protocol (Kamerbeek et al., 1997). Of the total isolatesspoligotyped, 324 isolates belonged to the two predominantclades, CAS and EAI (153 isolates belonged to CAS clade and 171isolates belonging to EAI clade). These isolates were furtherworked up.

2.2. Data analysis and statistics

Demographic data from patients including age, sex, previoushistory of TB, history of smoking, alcohol and family TB, andhometown were collected at admission in standard question-naires. The clinical data were analyzed from 320/324 patients withthe EAI or CAS clade isolate (the data from four patients wasincomplete). Data were analyzed using the EpiInfo v.6.0 programand observed frequencies were compared by means of two-by-twocontingency tables. A p value of <0.05 was considered statisticallysignificant. The data and demographic parameters are summarizedin Table 1.

2.3. Restriction fragment length polymorphism (RFLP)

46/153 clinical isolates belonging to CAS clade and 44/171isolates belonging to EAI clade were selected at random (usingrandom table) from a total of 324 isolates, for RFLP. Thedistribution of isolates from different cities/geographical regions,their shared-types according to SpolDB4 (Brudey et al., 2006) andthe IS6110 copy number are shown in Table 2.

DNA extraction and IS6110 RFLP were performed using therecommended international standard protocol (van Embden et al.,1993). The IS6110 RFLP patterns were analyzed by visualexamination as well as computer assisted analysis by use ofGelCompar version 3.5 software. (Applied Maths, Belgium).Similarities between RFLP patterns were calculated by using theDice coefficient, and the dendrogram was produced with theunweighted pair group method using arithmetic averages algo-rithm (UPGMA).

2.4. TbD1 PCR analysis

The TbD1 region was analyzed using PCR primers designed forTbD1 regions according to the method of Brosch et al. (2002).Primers complementary to the internal sequence as well as thoseflanking the deleted regions were used. Product was obtained withthe internal primers or with the flanking primers depending on thepresence or absence of the TbD1 region.

J. Arora et al. / Infection, Genetics and Evolution 9 (2009) 832–839834

3. Results

3.1. Patient demographics

The statistical analysis was done for 320 patients (harbouringisolates belonging to EAI and CAS clade). 73.8% were male and26.2% were female. 77% of the patients were in the age groups 15–45, whereas the remaining 23% were above 45 years. Approxi-mately, half the patients had a prior history of smoking andalcohol. History of TB in the family was present in 25% of patients.58% of the cases were new while 35% belonged to the category offailure/relapsed/treatment completed and 7% were cured.

39% of patients harboured strains that were resistant to one ormore drugs.

The distribution of the two clades was different among differentage groups. The age group of 46 and older was significantly moreoften infected by EAI clade (30% of the isolates) while CAS clade hadonly 16% isolates from this age group (p = 0.0048). The two cladesalso showed difference in proportion of resistance to at least onedrug. Patient isolates belonging to CAS clade showed a higher levelof drug resistance as compared to EAI clade (p = 0.0003).

Similar results were also obtained when the two clades werecompared with reference to new cases vis-a-vis patients withhistory of treatment (defaulters/relapsed). CAS clade was moreoften found in patients who had already taken anti-tuberculartreatment (ATT) as compared to EAI which was more often found inpatients who had never been exposed to ATT drugs. Most of the

Table 2The geographical partitioning as evidenced by Spoligotyes, TbD1 deletion and IS6110 R

other parameters showed nearly similar results among the twoclades (Table 1, Fig. 1).

In the multivariate logistic regression analysis age and previoushistory of TB were found to be significant. Patients with age morethan 46 are 53% less likely to have CAS group as compared to EAIgroup [odds ratio (OR) �0.47 (95% confidential interval (CI) 0.27,0.83)]. Patients who had previous history of TB were 2.7 timesmore likely to have CAS group as compared to EAI group afteradjusting for other covariates [odds ratio (OR) �2.71 (95%confidential interval 1.69, 4.35)].

3.2. IS6110 RFLP

3.2.1. DNA polymorphism of M. tuberculosis strains

In all, 60 different IS6110 RFLP patterns were found amongninety isolates belonging to 28 spoligotypes from EAI and CASclade. 59 patterns were unique and observed only once while onepattern having a single IS6110 element at 1.5 kb position wasshared by 31 isolates. The number of IS6110 copies ranged from 1to 17. Majority of isolates (n = 35) had IS6110 copies in the range of11 to 14. No isolate lacking IS6110 element was found. All isolateswith greater than one IS6110 element were unique (Fig. 2).

3.2.2. Differences in IS6110 RFLP patterns among isolates belonging to

CAS and EAI clade

44 isolates belonging to EAI clade were subjected to IS6110 RFLP(Table 2). Majority of isolates from EAI clade harboured few copies

FLP copy nos.

Table 2 (Continued )

J. Arora et al. / Infection, Genetics and Evolution 9 (2009) 832–839 835

of IS6110 (1–5 bands). 86.4% (38/44) of isolates had five or lessbands and 13.6% (6/44) isolates harboured �6 bands (Fig. 2).

46 isolates belonging to CAS clade were subjected to RFLP.Isolates belonging to CAS clade harboured multiple IS6110 copiesranging in number from 9 to 17 except a single isolate (P501) thathad three bands (Fig. 2). 87% (40/46) of isolates from CAS cladewere ‘‘Delhi type’’ (Bhanu et al., 2002). The CAS strains studiedshowed an overall homology of >60%. Isolates among CAS cladehad high copy number of IS6110 whereas EAI clade had isolateswith low copy number of IS6110 element.

Fig. 1. Histogram depicting the clinical and epidemiologic characteristics of patients

found significant among EAI and CAS clade. Note: NPT – no previous treatment, PT –

previously treated, R – resistant to one or more drugs and S – sensitive to all drugs.

3.2.3. TbD1 analysis

TbD1 PCR analysis was performed on all isolates. TbD1 regionwas present in all isolates constituting EAI clade but the region wasabsent from all isolates belonging to CAS clade consistent with theearlier findings (Brosch et al., 2002).

4. Discussion

EAI and CAS are the two predominant clades found in India(Singh et al., 2004; Kulkarni et al., 2005; Suresh et al., 2006; Singhet al., 2007). Further distribution of these clades and prevalenceof genetically different strains in different regions of the countryhas also been highlighted before. A clear difference among thestrains circulating in the North and the South regions wasobserved in our previous study which suggests that M.

tuberculosis strains have originated and evolved differently inNorth and South. Though ST26 was the most predominant type, itwas completely absent in Trivandrum. A decrease in this sharedtype as we moved from North to South. The reverse was true forST11. ST11was found to be present in all five regions but itshigher percentage in South suggests that probably this was thepoint of entry from where it was transmitted to the rest of thecountry (Singh et al., 2007).

Little work is available on genetic diversity of these two cladesusing additional genetic markers. The present study was under-taken to evaluate the geographically partitioned strains (Singhet al., 2007) further and to understand their phylogeny usingdifferent molecular markers among these two lineages. The study

Fig. 2. IS6110 RFLP profiles of isolates belonging to EAI and CAS clade. The figure illustrates the analysis of IS6110-RFLP of 90 Mycobacterium tuberculosis strains using

GelCompar version 3.5 software (Applied Maths, Belgium). Note: isolates preceded with D are from Delhi, P from Pune, T from Trivandrum, C from Chennai, and L (followed by

H, P or D) from Lucknow. ST – shared-type, EAI – East-African Indian and CAS – Cental Asian.

J. Arora et al. / Infection, Genetics and Evolution 9 (2009) 832–839836

J. Arora et al. / Infection, Genetics and Evolution 9 (2009) 832–839 837

shed light on some novel observations of associations betweenlineages and patient demographic variables.

In the present study the spoligopatterns of IS6110 high copynumber strains differed completely from spoligopatterns ofisolates with single copies, as majority of the multiple copystrains belonged to CAS clade. CAS strains have been shown to havemultiple copies of IS6110 (Gutacker et al., 2006) except for onerecent study in which 2/29 strains had zero copy of IS6110 elementthough the rest revealed multiple IS6110 copies (Ali et al., 2007).Contrary to this, strains in the EAI family have low-copy-number(or zero-copy-number) of IS6110 element (Brosch et al., 2002; Sunet al., 2004).

M. tuberculosis strains with low copy numbers of IS6110 havebeen more frequently isolated from Asian patients than frompatients of European origin (Das et al., 1995; Gutierrez et al., 1998;Bauer et al., 1999; Park et al., 2000). Earlier it was thought that theSouth Indian strains have descended from the M. bovis pool,characterized by lesser copy numbers of the insertion element, butBrosch et al. (2002) have changed the previous belief andconcluded that the common ancestor of the tubercle bacilliresembled M. tuberculosis or M. canetti.

SNP analysis by katG463-gyrA95 classified ancestral tuberclebacilli as PGG1 organisms, while ‘‘modern’’ M. tuberculosis strains falleither into PGG1, PGG2, or PGG3 (Sreevatsan et al., 1997). Based onthe presence or absence of an M. tuberculosis specific deletion(TbD1), M. tuberculosis strains can be divided into ‘‘ancestral’’ and‘‘modern’’ strains. TbD1 is specifically present in a subset of PGG1strains, but absent in other strains of PGG1, and in PGG2 and PGG3strains. According to Brosch et al. (2002), TbD1 deletion occurred inPGG1 M. tuberculosis clone which then established the ‘‘modern’’PGG1, PGG2, and PGG3 M. tuberculosis lineages.

W-Beijing, Haarlem, CAS and M. tuberculosis H37Rv belong to‘‘modern’’ M. tuberculosis families, whereas the EAI clade repre-sents an ‘‘ancestral’’ M. tuberculosis lineage (Sola et al., 2001;Brosch et al., 2002). Apart from the two predominant clades, CASand EAI other sub-lineages observed were Beijing/Beijing like(5.9%), T (6.6%), H (1.8%)), X (1%)), Manu (0.5%) and LAM (0.5%)clade (Singh et al., 2007).

Based on the TbD1 results on our strains, we conclude thatalthough ancient PGG1 clones are prevalent in the whole of India,‘‘ancestral strains’’ characterized by presence of TbD1 region areprevalent in the South whereas ‘‘modern strains’’ characterized byabsence of TbD1 region are prevalent in the North and Central India.

The difference of the predominant strains between North andSouth India can be attributed to their historical contacts andmigration histories. India is now broadly characterized by Indo-European populations found in the central and northern regionsand by Dravidian populations in the southern and southeasternregions. The significant difference in allele and genotype frequen-cies between the two populations has been highlighted in differentstudies (Soya et al., 2005; Prasad and Thelma, 2007).

India’s contacts with Central Asia are ancient ones. Immigrationby the people from Central Asia into India is a well-known fact ofhistory. Ancient India and Central Asia had common andcontiguous borders, climatic continuity, similar geographicalfeatures and geo-cultural affinity. There has always been sharingof material and the ideas between the two. The Indian and foreignliterary sources attest to the fact.

Various studies provide insights into prehistoric and earlyhistoric patterns of migration into India. The results of thesestudies reveal that a substantial part of today’s North Indianpaternal gene pool was contributed by Central-Asian lineages.West Asia and Central Asia have been the two major geographicalsources of genes in the contemporary Indian gene pool. NorthIndians are genetically placed between the West Asian andCentral-Asian populations. This is consistent with gene flow from

West Asia and Central Asia into India. (Mukherjee et al., 2001; Zhaoet al., 2009). It may be possible that ST26 belonging to CAS cladewas introduced from Central Asia to the North India in the past andwith time spread throughout the North India. Cruciani et al. (2002)hypothesized that the EAI ancestral strains spread back from Asiato Africa through India associated with human migrations and thatevolution gave rise to the CAS lineage, and possibly to all ‘‘modern’’TB lineages (Filliol et al., 2003). If this is true then the question thatremains unanswered is that why in South India the proportion ofisolates belonging to CAS clade is negligible.

The preservation of certain banding pattern of RFLP andspoligotypes in different parts of India suggests that these markersremained stable over a long period of time and that thesegenotypes represent clones that evolved in the distant past andhave become disseminated in predominance.

The ‘‘modern’’ M. tuberculosis families (W-Beijing, Haarlem, andCentral-Asian1) are widely prevalent worldwide; though their‘‘ancestral’’ counterparts (EAI) contribute to the global diseaseburden substantially (Filliol et al., 2002, 2003; Brudey et al., 2006;Gutierrez et al., 2006; Gagneux and Small, 2007). On the contrary, inIndia, EAI strains are present throughout the country though withvarying proportions. In the present study, EAI isolates were moreoften found in older patients probably reflecting that EAI areendemic isolates and have been circulating in the country for long. Avery significant observation was that EAI were less often associatedwith resistance and patients who had already taken ATT even once.Various studies have highlighted that the ancestral South Indianstrains (EAI) are less virulent and may not be lethal (Mitchison, 1964;Naganathan et al., 1986; Ahmed and Leblebicioglu (2006);Narayanan et al., 2008; Ahmed et al., 2009). It is also speculatedthat such strains disseminate less rapidly than the modern typespossibly due to a ‘genomic load’ of TbD1 and might be comparativelyless prone to acquisition of resistance to antimicrobials (Ahmed andLeblebicioglu, 2006; Ahmed et al., 2009). This would probably partlyexplain how this family has become endemic.

CAS isolates on the contrary were found more often in patientsin the age group of 15–45 years. This reflects on its ongoingtransmission in the community. This family was more oftenassociated with drug resistance and was found in patients havingtaken ATT treatment in the past. It has been suggested that CASclade is evolutionarily younger and since it is more prevalentworldwide it is hypothesized that ‘‘modern’’ M. tuberculosis

lineages are better human pathogens with enhanced virulencewhich facilitates their near-worldwide penetration (Goh et al.,2005).

Though there is intermixing of populations from the North andthe South, the strong geographic structuring of M. tuberculosis

population is striking. There is a need to know why particularstrain types are causing a large burden of disease in particularareas. Hirsh et al. (2004) suggested that the adaptation of strains ofM. tuberculosis to different host populations is responsible for thespread of the organism within defined ethnic groups. Their studysuggested that M. tuberculosis is organized into several large,genetically differentiated populations, which are stably associatedwith geographically defined host populations. They have alsosuggested that the strong association between host’s place of birthand parasite genotype is not due to the movement of individual M.

tuberculosis lineages through distinct human populations but thestrong associations that are centuries old. Significant variationsobserved in the protective efficacy of Bacille Calmette-Guerin(BCG) in different parts of India (Mitchison, 1964; Fine, 2000), mayin part be attributed to the fact that different strains aregeographically structured.

Using comparative genomics and molecular epidemiologicaltools Gagneux et al. (2006) demonstrated that global populationstructure of M. tuberculosis is defined by six phylogeographical

J. Arora et al. / Infection, Genetics and Evolution 9 (2009) 832–839838

lineages and that these are adapted to particular humanpopulations. Our results agree with the previous observation thatgeographic subdivision exists for M. tuberculosis and suggest thathuman migrations leading to human population bottleneck mightbe responsible for this area specific distribution of the pathogen.With time, EAI and CAS might have adapted to transmit and causesecondary cases specifically in South and North India.

We conclude that the association between host and pathogenpopulations remains strong despite social exchange and travel. Thestudy depicts that the EAI lineage is well established in India andthat CAS appears to be emerging or more recently introduced toIndia. The study also reflects on the association of these two cladesto ongoing transmission. The ancestral EAI strains seemed to beless often associated with drug resistance as compared to theevolutionarily younger CAS clade. The association of drugresistance with this prevalent clade will have implications on TBcontrol in North India. The association between the CAS isolatesand drug-resistance observed in the present study is a majorconcern and careful characterization of the clade with regards totheir virulence and transmissibility is required. Although this ispreliminary data, these findings are important for making publichealth strategies, control programs and evolutionary biology.

Acknowledgements

The authors Jyoti Arora, Tanu Rana, Amit Kaushik and theauthor Naga Suresh gratefully acknowledge the research fellow-ship from the Council for Scientific and Industrial Research andUniversity Grants Commission (Govt. of India) respectively. GovindPal is acknowledged for technical assistance. This work wassupported in part by India Clinical Epidemiology Network (INDIA-CLEN). We thank Dr. Anju Kagal, and Dr. D. R. Joshi, BJ MedicalCollege, Pune; Dr. Amita Jain and Dr. R. C. Ahuja, KG Medial College,Lucknow; Dr. K Jagannath, Institute of Thoracic Medicine, Chennaiand Dr. Ghosh and Dr. Resmi Rajan, CERTC Medical College,Trivandrum for providing isolates for the study.

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