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tion 8 (2008) 346–351
Infection, Genetics and EvoluRole 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: [email protected] (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
J.P.
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1E
AI3
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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