www.elsevier.com/locate/meegid

Available online at www.sciencedirect.com

tion 8 (2008) 346–351

Infection, Genetics and Evolu

Role of spoligotyping and IS6110-RFLP in assessing genetic

diversity of Mycobacterium tuberculosis in India

Jitendra Prasad Mathuria a, Pragya Sharma c, Pradyot Prakash a,Jai Kumar Samaria b, Vishwa Mohan Katoch c, Shampa Anupurba a,*a Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India

b Respiratory Diseases, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, Indiac National Jalma Institute of Leprosy and other Mycobacterial Diseases, Tajganj, Agra 282001, India

Received 14 December 2007; received in revised form 12 February 2008; accepted 12 February 2008

Available online 17 February 2008

Abstract

In the present study, genetic diversity analysis of Mycobacterium tuberculosis isolated from patients attending a tertiary care hospital, North

India, has been attempted. Eighty three isolates of M. tuberculosis were subjected to DNA fingerprinting using spoligotyping and IS6110-RFLP

techniques. Spoligotype patterns showed that central Asian (32.5%), ill defined T (13.2%) and Beijing (10.8%) families were predominant in

ongoing transmission of the bacterium. Two STs; ST26 (CAS_Delhi) and ST1 (Beijing) represented 36.1% of the total M. tuberculosis population

in eastern Uttar Pradesh, North India. IS6110 RFLP analysis showed that isolates having low and zero copy number of the IS element were 15.6%

and 19.2%, respectively. Out of the 47 isolates clustered by spoligotyping, 40 could be further differentiated as unique strains by IS6110-RFLP.

Therefore, this study recommends that both the techniques be used simultaneously for DNA fingerprinting of M. tuberculosis in India.

# 2008 Elsevier B.V. All rights reserved.

Keywords: Tuberculosis; Spoligotyping; IS6110-RFLP; Genetic diversity

1. Introduction

Tuberculosis is still one of the major public health problems of

India. Approximately 3,22,322 deaths are caused each year by

tuberculosis (TB) with an estimated incidence and prevalence

rate of 168 and 299 per 1,00,000 population, respectively (http://

www.who.int/tb/publications/global_report/en/index.html).

Increasing prevalence of HIV infection, which makes people

more susceptible to TB, and drug resistant types of TB together

pose an increasingly serious public health hazard with a high

economic burden for India. The annual burden of TB to the

Indian economy is at least US$ 3 billion (http://www.tbcin-

dia.org/pdfs/TB%20India%202007.pdf).

Molecular typing, targeting different molecular markers, has

been utilized recently for the identification of potential sources

of infection and epidemiological investigations of tuberculosis,

both in the general population and in nosocomial settings (van

* Corresponding author. Tel.: +1 542 2368655.

E-mail address: shampa_anupurba@yahoo.co.in (S. Anupurba).

1567-1348/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.meegid.2008.02.005

Soolingen et al., 1993; van Embden et al., 1993; Behr and

Small, 1997). Restriction fragment length polymorphism

(RFLP) fingerprinting, with insertion element IS6110 as a

probe, has good discriminatory power and is frequently used as

a method of choice to differentiate strains of M. tuberculosis

isolates (Behr and Small, 1997). However, absence or low copy

number of IS6110 element in significant number of M.

tuberculosis strains, as reported elsewhere, further limit its

usefulness (Das et al., 1995; Radhakrishnan et al., 2001).

The introduction of new PCR-based typing methods,

spoligotyping, MIRU-VNTR and DRE-PCR typing has

allowed simultaneous detection and epidemiologic typing of

M. tuberculosis (Eisenach et al., 1990; Groenen et al., 1993;

Kamerbeek et al., 1997; Sola et al., 1998; Mazars et al., 2001).

Thus, molecular epidemiologic information can be combined in

the context of epidemic events and TB transmission.

Spoligotyping is basically a reverse hybridization technique

based on polymorphism in the direct repeat (DR) locus of the

mycobacterial chromosome (Kamerbeek et al., 1997). The

well-conserved 36-bp DRs are interspersed with unique spacer

sequences varying from 35 to 41 bp in size. Clinical isolates of

J.P. Mathuria et al. / Infection, Genetics and Evolution 8 (2008) 346–351 347

MTBC bacteria can be differentiated by the presence or

absence of one or more spacers. Although results can be

obtained rapidly from M. tuberculosis culture, it can also be

performed directly with clinical samples.

In the present study, we have attempted to assess genetic

diversity of M. tuberculosis strains isolated from patients

attending a tertiary care hospital in North India by spoligotyp-

ing and IS6110-RFLP fingerprinting.

2. Material and methods

2.1. Specimen collection

Eighty three M. tuberculosis isolates, one isolate each from

non-related patients with pulmonary tuberculosis, during the

period September 2004–December 2005 in the Department of

Microbiology, Institute of Medical Sciences, Banaras Hindu

University, Varanasi, have been included in the study.

Representative isolates were selected from different locations

within eastern Uttar Pradesh. Biochemical identification was

performed using standard methods (Metchock et al., 1999).

2.2. DNA isolation

DNA was isolated as previously described (van Soolingen

et al., 1993) with slight modification.

2.3. Restriction fragment length polymorphism

RFLP was done using the protocol as described previously

(van Embden et al., 1993). Briefly, it included five major steps,

(i) restriction digestion of DNA by PvuII (Genei, Bangalore,

India) at 37 8C in a shaking water-bath for 4 h, (ii) separation of

restricted fragments by 1% agarose gel electrophoresis at 1.2 V/

cm, (iii) transfer of fragments onto positively charged nylon

membrane by southern blotting, (iv) hybridization with DIG

labeled 245 bp probe and (v) detection by recommended

procedure using DIG nucleic acid labeling and detection kit

(Roche Diagnostics, Germany). The fingerprints were com-

pared visually by two independent observers.

2.4. Spoligotyping

Spoligotyping was performed on genomic DNA to detect

presence or absence of 43 spacers by using the standard method

(Kamerbeek et al., 1997) with the help of commercially

available kit (Isogen Biosciences, BV, Maarsen, The Nether-

lands). Briefly, DR region was amplified with specific primers

and amplified DNA was hybridized with a set of spacer

oligonucleotide probes covalently linked to a membrane. The

hybridization pattern thus obtained was subsequently visua-

lized, after incubating with streptavidin peroxidase (Roche

Diagnostics, Germany) using Enhanced Chemiluminescent

detection system (Amersham Biosciences, Buckinghamshire,

United Kingdom). Proper controls (H37Rv, M. bovis BCG and

Negative control) were used with each experiment. The results

were entered into Microsoft excel sheet in binary format,

converted into octal code and compared with the international

spoligotyping database SpolDB4.0 (Brudey et al., 2006).

Further, those spoligo patterns not found in SpolDB4.0 were

analyzed with ‘‘Spotclust’’, which assigns families based on

SpolDB3.0, using a mixture model (Vitol et al., 2006).

3. Results

3.1. Patients characteristics

Of 83 cases included in the present study, 29 were on anti-

tubercular treatment. The mean age of the subjects was 33.9

years with a range of 6–82 years and majority of them (n = 61)

belonged to productive age group (15–45 years). The male to

female ratio was 3.36:1.

3.2. Spoligotyping

A total of 44 distinct spoligotype patterns were obtained

among 83 MTB isolates on comparing with SpolDB4.0, 19

isolates were found to be orphan, i.e. absent in the SpolDB4.0

database. The remaining 64 isolates could be allotted 26 shared

types (STs). Out of these 26 STs, 9 STs had 2 or more isolates

whereas 17 had single isolate each (Table 1).

Major STs found in the present study were ST26 (n = 22 and

26.5%) and ST1 (n = 8 and 9.6%) representing about 36.1% of

total M. tuberculosis population. ST53 and ST361 were also

present in this geographical area as minor shared types,

containing four and three isolates, respectively. Five clusters

(ST48, ST102, ST138, ST141 and ST298) had two isolates

each. At the phylogenetic level, majority of the isolates

belonged to Central Asian (CAS), ill defined T and Beijing

families with 27 (32.5%), 11 (13.2%) and 9 (10.8%) isolates

each. The minor families observed in this study were East

African Indian (EAI) (n = 8 and 9.6%) and Harleem (n = 6 and

7.2%). Similarly, while analyzing these 64 isolates with

‘‘Spotclust’’, the majority of isolates (n = 49) belonged to three

families CAS (n = 27), T1 (n = 12) and Beijing (n = 10)

(Table 1).

Further, orphan clades were analyzed with ‘‘Spotclust’’ to

assign families based on studies of SpolDB3.0. Of 19, 17

isolates had unique patterns and 2 isolates possessed similar

pattern. Seven of these belonged to EAI5 family. CAS,

Haarlem1 and T1 families had 3 strains each, whereas 33, 34

and EAI3 had 1 strain each (Table 2).

3.3. Restriction fragment length polymorphism

In IS6110 RFLP analysis, 19.2% (16/83) of these isolates in

which IS6110 element was not found were characterized as

zero copy number strains. Further, 15.6% (13/83) isolates were

found to be low copy number strains having <6 copies of

IS6110 element and the rest 65.0% (54/83) were multiple copy

number strains with �6 copies of the element. None of the

isolates showed presence of more than 16 copies of the IS

element (Fig. 1).

Table 1

Distribution of spoligotype patterns

Binary spoligotype Shared type No. and % in database No. and % in present study Family

SpolDB4.0 Spotclust

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST1 3758 (9.48%) 8 (9.64%) Beijing Beijing

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST11 237 (0.60%) 1 (1.20%) EAI3_IND EAI3

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST26 404 (1.02%) 22 (26.51%) CAS1_Delhi CAS

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST37 155 (0.39%) 1 (1.20%) T3 T1

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST47 721 (1.82%) 1 (1.20%) H1 H1

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST48 243 (0.61%) 2 (2.41%) EAI1_SOM EAI5

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST50 1504 (3.78%) 1 (1.20%) H3 T1

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST53 2497 (6.30%) 4 (4.82%) T1 T1

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST73 114 (0.28%) 1 (1.20%) T2–T3 T1

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST100 39 (0.0984%) 1 (1.20%) MANU1 33

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST102 46 (0.12%) 2 (2.41%) T1 T1

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST138 69 (0.17%) 2 (2.41%) EAI5 EAI5

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST141 10 (0.025%) 2 (2.41%) CAS1_Delhi CAS

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST236 91 (0.23%) 1 (2.41%) EAI5 EAI5

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST242 5 (0.012%) 1 (1.20%) T1 T1

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST283 30 (0.075%) 1(1.20%) H1 LAM8

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST288 40 (0.10%) 1 (1.20%) CAS2 CAS

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST298 26(.065%) 2 (2.41%) EAI3_IND EAI3

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST361 4 (0.01%) 3 (3.61%) H4 H3

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST616 3 (0.007%) 1 (1.20%) U Beijing

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST777 24 (0.06%) 1 (1.20%) H4 H3

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST1060 2 (0.005%) 1 (1.20%) T1 T1

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST1344 2 (0.005%) 1 (1.20%) CAS1_Delhi CAS

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST1426 2 (0.005%) 1 (1.20%) T3 T1

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST1591 2 (0.005%) 1 (1.20%) CAS2 CAS

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&& ST1651 2 (0.005%) 1 (1.20%) Beijing Beijing

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Fig. 1. Distribution of IS6110 copy number in M. tuberculosis isolates.

J.P. Mathuria et al. / Infection, Genetics and Evolution 8 (2008) 346–351 349

The 67 isolates, who had the IS element, showed 61 different

RFLP patterns. Whereas 56 isolates showed unique patterns,

rest 11 were distributed among 5 clusters with a maximum of 3

isolates in a cluster having 9 IS6110 elements in each of them.

4. Discussion

Although tuberculosis is one of the major health problems in

India, our knowledge regarding the circulating strains of M.

tuberculosis is scarce. Spoligotype patterns showed that CAS

family was the largest family (32.5%), represented by ST26,

ST141, ST1344, ST288 and ST1591. The most predominant

spoligotype corresponded to ST26 (CAS_Delhi type) which

was formed by 26.5% (n = 22) isolates. The occurrence of this

spoligotype has also been reported earlier from various other

Indian studies in the range of 7–24% (Singh et al., 2004, 2007;

Kulkarni et al., 2005; Gutierrez et al., 2006). A study from

Pakistan also showed CAS_Delhi type (39%) as the dominant

strain that was responsible for TB transmission (Hasan et al.,

2006). ST26 represents 1.02% of isolates in SpolDB4.0

database, and has been reported from 34 countries in varying

numbers, with maximum number of isolates from Asian

countries.

The W-Beijing family has worldwide distribution with

varying prevalence in different continents. Various studies

conducted in South East Asia, found this family was a

predominant genotype, varying from 34% to 72% of the

circulating strains (Glynn et al., 2002). In the present study, ST1

is the second largest cluster and included eight (9.6%) isolates

belonging to Beijing family. There was one isolate ST1651

which also belonged to this family. Studies from other

adjoining countries like Pakistan (6%) and Bangladesh

(31.2%) have reflected the prevalence of Beijing clade in their

areas (Banu et al., 2004; Hasan et al., 2006).

In this study, ST53 and ST50 which were major STs reported

from Finland (Puustinen et al., 2003), constituted only 4.8%

(n = 4) and 1.2% (n = 1) of the isolates, respectively. Only one

isolate was assigned to ST11 belonging to EAI_3 family which

was observed to form the second largest cluster in a recent study

from New Delhi (Singh et al., 2007).

The presence of a large number of circulating strains in this

region is contrary to the hypothesis that an endemic area might

have relatively few circulating strains but is similar to a study

J.P. Mathuria et al. / Infection, Genetics and Evolution 8 (2008) 346–351350

from New Delhi, India (Singh et al., 2007). The present study

reported six shared types for the first time from India. ST361

had three isolates and five shared types, namely ST236, ST242,

ST616, ST777 and ST1060, had one isolate each but these STs

have been infrequently reported elsewhere (Brudey et al.,

2006). Further, eight (9.6%) strains belonged to T1 family, a

widespread yet poorly defined super family, needing better

markers for proper characterizations (Singh et al., 2004). We

also found one isolate of a poorly characterized group of strains

baptized ‘‘Manu’’ which has been reported from Delhi and

Bombay in low frequency. It is presumed to be the probable

ancestor of both the CAS and EAI (East African Indian) (Singh

et al., 2004; Kulkarni et al., 2005). Presence of 19 orphan

isolates indicates the need of conducting multicentric studies to

know all the prevalent spoligotypes in Indian subcontinent.

While comparing the spoligotype patterns with spotclust as

well as SpolDB4.0, it was seen that the ‘‘Spotclust’’ model

more or less confirmed families, which were assigned with help

of SpolDB4.0 database. However, some STs (ST50, ST100,

ST283 and ST616) representing T1, 33, LAM8 and Beijing

families by ‘‘Spotclust’’ were found to be H3, Manu1, H1 and U

with spolDB4.0, respectively.

RFLP-IS6110 pattern, as reported from southern part of

India, have shown that significant number of the strains were

either of low (40–67%) or zero copy number (1–24%) (Das

et al., 1995; Radhakrishnan et al., 2001; Narayanan et al.,

2002). In the present study, only 15.6% of the strains had low

copy number of IS6110 element. Further, 19.2% isolates had

zero copy number of the insertion sequence which is similar to

studies conducted elsewhere in India but significantly higher

than those reported from San Francisco and Vietnam which

reported <1 and 2%, respectively (Agasino et al., 1998; Park

et al., 2000). Further, we found greater polymorphism among

isolates by IS6110 RFLP. Strains possessing IS6110 element

showed 61 different patterns; 56 were unique and the remaining

5 were clustered. There were 11 isolates within these 5 clusters.

Isolates with zero copy number of IS6110 showed 13

spoligotype patterns, whereas amongst the low copy number

isolates, there were 11 patterns by RFLP and 10 patterns by

spoligotyping. The high copy number isolates showed greater

polymorphism by RFLP with 50 patterns. By spoligotyping,

they showed only 27 patterns.

In 47 isolates clustered by spoligotyping, 40 isolates were

subdivided as unique strains by IS6110-RFLP typing. The

remaining seven isolates were clustered in three groups by both

the methods. On comparison with spoligotyping, two (n = 5)

groups belonged to ST26 (CAS_Delhi) and one (n = 2) group

ST138 (EAI5). It was observed that a number of IS positive

isolates, clustered by spoligotyping without any epidemiolo-

gical link, were well differentiated by IS6110 RFLP into

distinct genotypes.

Hence, although spoligotyping is a fast, simple, robust

method and useful in categorizing M. tuberculosis isolates

(including those with low and zero copy number) into different

families, the possibility of false clustering with this method

cannot be ruled out. Further, the limited discriminatory power

of spoligotyping is primarily because it targets a single locus

that accounts for less than 0.1% of the M. tuberculosis genome,

unlike IS6110-based RFLP analysis, which examines the

distribution of IS6110 throughout the entire genome.

In conclusion, the present study demonstrates the need for

both spoligotyping and IS6110- RFLP for better discrimination

between individual strains; rather than using these methods

alone. Further, the knowledge of different clades prevalent in

the communities endemic for tuberculosis is important and by

executing multi centric studies at regular intervals, we can

assess the success of tuberculosis control programme.

Acknowledgements

The authors acknowledge the Head, Department of

Microbiology, IMS, BHU, Varanasi, Director, National

JALMA Institute for Leprosy and Other Mycobacterial

Diseases, Tajganj, Agra and University Grants Commission,

India.

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