Tuberculosis 91 (2011) 407e413

Contents lists available at ScienceDirect

Tuberculosis
journal homepage: http://intl.elsevierhealth.com/journals/tube

REVIEW

Molecular epidemiology of tuberculosis in India: Moving forward with a systems

biology approach
Niyaz Ahmed a, b, c, *, Seyed E. Hasnain b, d, *
a

Pathogen Biology Laboratory, School of Life Sciences, University of Hyderabad, Hyderabad, India
Institute of Life Sciences, University of Hyderabad, Hyderabad, India
c
Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia
d
Jawaharlal Nehru Centre for Advanced Scientic Research, Jakkur, Bengaluru, India
b

a r t i c l e i n f o

s u m m a r y

Article history:
Received 22 October 2010
Received in revised form
16 March 2011
Accepted 19 March 2011

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be the leading source of mortality
and morbidity across the world with India fast emerging as the TB capital of the world. In order to
develop effective intervention strategies it is equally important to focus not only on a system of information and efcient methods for localizing sources of infection, but also highlight tools that enable
enhanced understanding of the dynamics of spreading of disease. Accurate identication of the underlying strains in an epidemiological setting is therefore of paramount signicance. There is no scientic
evidence to explain that some strains of the TB bacilli spread faster and transmit more aggressively than
others although strains such as M. tuberculosis Beijing/W have been widely reported to cause large scale
and fatal outbreaks perhaps linked to their postulated propensity to transmit faster. We provide an
overview of the present scenario of molecular epidemiology and dissemination dynamics of M. tuberculosis and discuss how systematic, genome sequence based methods allow decipherment of the population genetic structure of M. tuberculosis in India which was not achievable with traditional
ngerprinting methods. We discuss the prevalence of ancestral genotypes in India which perhaps
represent less disseminating and more controllable lineages that infect a majority of TB patients in this
high burden country. Further, we suggest functional molecular infection epidemiology as a new discipline to guide investigation of the impact of pathogen diversity (as juxtaposed to the host response) on
the disease phenotype. We also propose systems biology to be a powerful new science to holistically
analyze the epidemic through integration of high-throughput multi-omics data to understand the
dynamic interactions that occur at the level of host-pathogen cross-talks and to identify potentially novel
drivers of the future control strategies.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Tuberculosis
Molecular epidemiology
Genomics
Systems biology
India

1. Tuberculosis incidence and transmission in India

According to World Health Organization (WHO),1 about 9
million new cases of tuberculosis (TB) and 1.7 million TB-related
deaths occur every year globally. Of the total number of TB cases
worldwide, 80% are concentrated in 22 nations, including India, the
TB capital of the world1 where it accounts for one fth of the global
burden of tuberculosis (TB), with 1.8 million new cases of active TB
each year - more new cases than any other country.2

* Corresponding authors. Institute of Life Sciences, University of Hyderabad

Campus, Prof. CR Rao Road, Gachibowli, Hyderabad 500046, India. Tel.: 91 40
23134585; fax: 91 40 66794585.
E-mail addresses: niyazSL@uohyd.ernet.in (N. Ahmed), seyedhasnain@gmail.
com (S.E. Hasnain).
1472-9792/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tube.2011.03.006

According to the estimates of the Revised National Tuberculosis

Control Programme (RNTCP) of India two in every ve Indians are
infected with latent TB.3 India also has a substantial burden of HIV
cases; recent estimates from the WHO and other agencies account
for about 2.5 million people living with HIV in India (an overall
population HIV prevalence of 0.36%).4,5 HIV co-infection greatly
magnies the risk of progression from latent TB infection to active
TB. TB has thus emerged as the leading cause of mortality in HIVinfected persons in India.2,5
Transmission dynamics of TB aerosols depend on crowding,
weather conditions and the extent of exposure. The chain of
transmission can therefore, be broken by isolating patients with
active disease and starting effective anti-tubercular therapy. After
two weeks of such treatment, people with non-resistant active TB
generally cease to be contagious.

408

N. Ahmed, S.E. Hasnain / Tuberculosis 91 (2011) 407e413

People with prolonged, frequent, or intense contact such as

those sharing a long haul ight with infected persons on-board are
at highest risk of becoming infected, with an estimated infection
rate of 22%. Others at risk include patients immuno-compromised
by conditions such as HIV/AIDS, people residing in TB endemic
areas, drug abusers, residents and employees of high-risk congregate settings, medically under-served and low-income populations,
high-risk racial or ethnic minority populations, children exposed to
adults in high-risk categories, patients on immunosuppressant
therapy, and often health care workers serving these high-risk
clients.6 Diabetes is also emerging as a risk factor for TB as reviewed
by us previously.7 The situation in India unfortunately qualies for
most of the above predispositions and risk factors.
In India, specic data on transmission dynamics of TB are not
available. Also, there are no reported occurrences of institutionalized TB outbreaks due to droplet infection. Systematic contact
studies have not been carried out/reported as on today whereby the
transmission potential of individual strains of the causal organisms
can be determined.
About 3.3 Million people are living with tuberculosis in India
with new case nding rates of 168/100, 000 people. Despite a 91%
DOTS coverage with 86% treatment success, 322,322 people are
dying with TB every year (http://www.globalhealthfacts.org). India
noticed a steep rise in the number of HIV cases, exceeding South
Africa in prevalence, with an estimated 5.7 million cases (statistics
from: globalhealthfacts.org) and therefore, the country may be at
the edge of a TB catastrophe due to the HIV-TB alliance, aggravated
by the more aggressive Beijing strains, where the threat of a series
of outbreaks in several years to come looms large. With India sitting
on a diabetic volcano,8 the synergy between TB and diabetes will
prove perhaps the most serious public health challenge ever with
grave implications. Thankfully enough, India has not witnessed
large institutionalized outbreaks until now. The high cure rates
under DOTS and much restrained MDR rates compare very favorably and are in contrast with the former USSR countries and the
Africa. Based on these statistics it will be wise to investigate if
Indian strains have some protective advantage.9

2. The genetic makeup of tubercle bacilli relevant in

epidemiology
Downsizing of the genomic content, occurring through serial
deletions, is the major underlying force in the emergence of ttest
and successful strain variants in the Mycobacterium tuberculosis
complex.10 Another minor contribution to genome evolution is
through in situ duplication events, most notably in Mycobacterium
bovis BCG strains. A few predominant genotypes circulating
throughout the world are responsible for the major outbreaks of
the recent past and these belong to the so-called Beijing, Haarlem
and African clusters.11,12 These major strain groups have been
classied based on the repetitive element IS6110 genotyping and
spoligotyping patterns and have been described as the predominant genotypes in the world. Although the M. tuberculosis genome
contains several repetitive elements, only a few are polymorphic
and these have not been rigorously studied.13 The rarity of polymorphism related to the mobility of repetitive elements coupled
with the restricted number of single nucleotide polymorphisms
indicate that transposition and homologous recombination are
(obscurant) events at the base of negligible genetic heterogeneity
thus reinforcing the importance of clonality of the M. tuberculosis
gene pool.14 In addition, polymorphisms seen with different
molecular markers reveal high degree of mutual association. This
supports the hypothesis that M. tuberculosis has a strong clonal
population structure.14,15

3. Shortcomings of the gold-standard typing method and

evolution of secondary genotyping approaches
The presently available typing systems designed for molecular
epidemiology16 are not capable of classifying strains on the whole
genome basis including various evolutionary changes and random
base substitutions.17 Because of these limitations, knowledge about
the mycobacterial population structure in India remained largely
unexplored and sketchy. There is a need for a genome sequence
based classication of predominant lineages and to nd out their
preponderances etc. so as to assist global efforts aimed at controlling this deadly disease. IS6110 RFLP based ngerprinting18 has
been used to study the mycobacterial population structure from
Southern India, Northern India and the Delhi region.19e22 However,
the usefulness of IS6110 ngerprinting is limited because of the
high proportion of M. tuberculosis strains with low copy numbers or
devoid of IS6110 in several regions of India.19,21 IS6110 typing also
suffers from a relative lack of portability, which hinders comparison
with other studies.23 Fingerprinting methods targeting polymorphic spacer sequences in the direct repeat (DR) region,
including spoligotyping, have been used in some of these regions
and in Bombay.24,25 However, these methods considerably underestimate the clonal diversity when used alone.26
The face of molecular epidemiology of tubercle bacilli has
changed after the availability27 of genome sequence data in the
public domain. Subsequent to this revolution, DNA microarrays have
been used for comparative genomics of different M. tuberculosis
clones for which clinical and epidemiological information was
available.28,29
Because of the clonal structure of M. tuberculosis,14,30 comparative genotypic analyses from widespread geographic areas, such as
the Indian sub-continent, or from different human populations can
give unique insights into dissemination dynamics and evolutionary
genetics of the pathogen.31,32
3.1. Fluorescent Amplied Fragment Length Polymorphism (FAFLP)
Fluorescent amplied fragment length polymorphism (FAFLP)
analysis provides a means of examining DNA segments distributed
over the entire genome of an organism. This information can be used
to dissect the routes of infection, study evolutionary genetics and
identify novel genes involved in resistance and virulence of pathogens in an epidemiological setting. It is a modication of Amplied
Fragment Length Polymorphism (AFLP) technique, commonly used
to type infectious agents.33 This technique offers more discriminatory powers than other ngerprinting techniques. The information
generated by this technique can be digitized offering quicker
dissemination and inter-laboratory comparisons. Other advantages
of this technique are the requirement of less amount of starting DNA
and the range of markers generated which can be used for diagnosis
or may possibly reect the resistance and virulence loci in the
genome of M. tuberculosis strains and also provide information about
the functional biology of this microorganism.
The technique is based on the digestion of known/unknown
DNA with two restriction enzymes (REs). Generally, one enzyme is
a frequent cutter while the other is a rare cutter. The digested
fragments are ligated to adaptors (with RE recognition sequences
identical to that used in digestion). Primers based on the adaptor
sequences are then used for PCR amplication. Sequences to which
both adaptors are attached are exponentially amplied while
fragments with one attached adaptor amplify linearly and are thus
in negligible quantity. A second optional amplication can be
carried out by primers, which extend into the unknown sequence
by attaching one to three bases at the 30 end of the primers. This
leads to selective amplication of sequences having bases

N. Ahmed, S.E. Hasnain / Tuberculosis 91 (2011) 407e413

complementary to the ones attached to the primer(s). The primer

(forward or reverse) is tagged with uorescent dyes, which when
excited, emit light of a particular wavelength, which forms the
basis of visualizing the amplied DNA. These signals represent
a particular length of the DNA fragment amplied and form the
basis of typing. The strains with the identical set of amplied
products represent a single strain. This technique offers the
advantage of typing the strains based on the polymorphism
present over the whole genome and thus has added phylogenetic
value.34
3.2. VNTRs
More recently, molecular typing methods based on Variable
Number Tandem Repeats (VNTRs), of genetic elements named
Mycobacterial Interspersed Repetitive Units (MIRUs)35 have been
developed.36,37 MIRU-VNTR typing shows a discriminatory power
close to that of IS6110 ngerprinting, and is particularly efcient in
distinguishing M. tuberculosis isolates with few or no IS6110 elements.37e39 MIRU-VNTRs are sufciently stable to track epidemic
strains.37,38,40 There is diversity in the M. tuberculosis strain in 12
different regions covering Northern, Central and Southern India on
a total sample size of 91 isolates by using a set of 21 VNTR loci,
including the 12 MIRU-VNTR loci described previously35,36 and 9
additional loci containing VNTRs of other interspersed genetic
elements.41e43 All these loci are collectively designated as MIRUVNTR loci.
3.3. Region of Difference (RD) analysis and the new lineage
nomenclatures
In addition to the above markers, single nucleotide polymorphism (SNP) genotyping related to certain candidate genes was
used to assess consistency of the genetic relationships obtained by
VNTR typing at a broader evolutionary level.9 According to Sreevatsan et al. (1997), the M. tuberculosis strains can be classied into
three genetic groups based on two polymorphisms that occur with
high frequency in the genes encoding catalase-peroxidase (katG)
and the A subunit of DNA gyrase (gyrA). Group 1 has the katG codon
463 CTG (Leu) and gyrA codon 95 ACC (Thr); group 2 has the katG
463 CGG (Arg) and the gyrA codon 95 ACC (Thr), and group 3 has
the katG codon 463 CGG (Arg) and the gyrA codon 95 AGC (Ser). In
terms of evolutionary hierarchy, Group 1 represents the older
isolates followed by Group 2 and Group 3. However, Principal
Genetic Group (PGG) alone is not a reliable approach and many
groups have used spoligotyping in addition to PGG as the latter
being less discriminatory and almost redundant in view of the new
ndings (see later). It is therefore useful only in broadly identifying
genotype families.26,44,45 In fact, classication of lineages based on
PGG has always been confusing and was proved wrong in the
aftermath of large scale genome sequencing studies revealing an
important SNP discovery bias that identied only two informative
SNPs, when in reality there are many more SNPs stratifying
different M. tuberculosis strains. Accordingly the classications/
nomenclatures were revised mainly based on the deletion patterns
reective of ancestral deletion events.
Based on the presence or absence of a M. tuberculosis specic
deletion (TbD1)46 a new holistic evolutionary scenario for the
evolution of the M. tuberculosis complex46 and other mycobacteria47 has been proposed. TbD1 is specically present in ancestral
lineages of M. tuberculosis.46 Population wide deletion analysis
described recently48 suggests that there could be substantial
genomic variability among different M. tuberculosis genotypes in
the world. It is likely that new genotypes indeed exist but they go
unnoticed largely because of non-availability of high-resolution

409

genomic tools. Currently, the nomenclature of M. tuberculosis

lineages is based on genome wide deletion analysis and comparative genomics. Firstly, the regions of difference,46 then the
sequencing of 89 genes49 and lately the whole genome sequences50
have shown that there are six main lineages which are dispersed in
a geographically compartmentalized manner. TbD1 (deletion
event) most probably occurred in the common ancestor of the socalled modern strains including Euro-American, Beijing strains
and the North Indian strains (CAS in spoligotype notation). These
studies have provided a viable framework in the form of different
RD markers, and SNPs which could be successfully exploited in
lineage identication, in particular for accurately determining the
modern and ancestral types.
4. Strain typing in India: from convenient sampling to more
systematic analyses
M. tuberculosis genotypes from the Indian sub-continent have
largely been described in the context of locally available isolates
and in most cases single techniques have been used to dene
a conveniently sampled bacterial diversity.19,20,51e54 Such studies,
although important, have probably skewed the interpretation of
the diversity of M. tuberculosis because of their small-scale, localized, random sampling and or the choice of a single genotyping
method. IS6110 restriction-fragment length polymorphism (RFLP)
has been the age old standard for population-based molecular
epidemiological analyses of the tubercle bacilli with the purpose of
identifying circuits and predictors of ongoing TB transmission.
However, some M. tuberculosis isolates, especially those belonging
to ancestral lineages, have few or no IS6110 copies. Moreover,
systematic ngerprinting of all M. tuberculosis isolates by IS6110
RFLP is time-consuming, cumbersome and the results have poor
inter-laboratory portability, apart from the fact that mobile
elements have their own limitations in terms of instability and
molecular clock rates. Thus, a combination of two rapid PCR-based
molecular typing methods; spoligotyping and variable number
tandem repeats of mycobacterial interspersed repetitive unit
(MIRU-VNTR) has become an attractive alternative to IS6110 RFLP.9
Of late, few workers have systematically analyzed the tubercle
bacilli from India using multiple markers on rigorously sampled
isolates,9,51,55,56 such analyses revealed a clear predominance of
two important genogroups, each representing modern and ancestral group of strains respectively conned to the North and the
South of India. While the geno-family of East African Indian (EAI)
strains predominates in the southern part of the country, the CAS
types are overwhelmingly represented in Delhi and its adjoining
states.54,55,57,58 Also, in our experience, in some major cosmopolitan
cities, a blend of the two major strain families together with their
sublineages could be readily discerned whenever a modest,
random sample is genotyped; recently, Manu type isolates representing a sublineage of the ancestral EAI genogroup was identied in Andhra Pradesh (SK Thomas, N Ahmed, unpublished)
(Figure 1).
5. Ancestral lineages and adaptive advantage: do docile
strains offer any promise to the TB control programs?
Some of the early observations showing that the South Indian
strains caused low grade pathology,59 lend support to the potential
old is gold hypothesis.7 However, since M. tuberculosis gene pool
diversity was not completely understood at that time, it is not
possible to convey if these observations were really destined to
explain specic strain advantages. However, in the absence of in
vivo experiments to independently conrm such ndings, we
cannot be 100% sure that the EAI type strains are less virulent. Many

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N. Ahmed, S.E. Hasnain / Tuberculosis 91 (2011) 407e413

Figure 1. Distribution of M. tuberculosis lineages in Andhra Pradesh. Very clearly visible are the two distinct geno-families, modern (led by CAS in blue followed by Beijing in pink)
and ancestral (led by EAI in green and its sublineages in light blue, yellow and brown followed by a very distinct Manu cluster in red) (SK Thomas, Ahmed N et al., unpublished
data). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article).

studies using animal models have nonetheless revealed that

differential clinical morbidity or resolution of the infection could be
ascribed to different genotypes as also recently reviewed by Nicol
and Wilkinson (2008).60 In consideration of this situation, one can
espouse that Mitchison possibly looked at the strains of EAI type
given their large-scale presence (nearly 80%) in Madras area. Also,
historically, no epidemic or an institutionalized outbreak took place
in this region. Given this scenario it appears reasonable to consider
these strains as less rapidly disseminating and thereby less prone to
the acquisition of drug resistance.61 An indirect conrmation of this
hypothesis comes from the reported lack of association of drug
resistance with ancestral isolates.62
By contrast, the modern M. tuberculosis (Beijing) genotypes
were described as hyper virulent in laboratory animals, culminating in higher bacillary load thereby augmenting dissemination63
and leading to early death.64 However, no such evidence is forthcoming from the studies involving human subjects. The Gambian
house-hold contact studies reveal that escalation to clinical stage
TB was signicantly reduced in individuals infected by Mycobacterium africanum, a relatively ancestral strain as compared to the
ones exposed to M. tuberculosis; and within M. tuberculosis lineage,
those infected with a Beijing family strain were more prone to
active disease.65 Also, one recent study62 revealed signicant
association of multiple drug resistance (MDR) with the Beijing
strain in contrast to the non-Beijing types.
Given the above, predominance of EAI, in proportion to the
Beijing-W and other minority types, could be one of the strong
reasons why India has so far not witnessed any institutionalized
outbreaks despite a historical burden of TB. This ancestral strain

advantage perhaps translates to the lack of direct correlation

between M. tuberculosis infection rate and TB disease burden
in India.
6. Toward functional molecular infection epidemiology of TB
Todays India presents a genetic playground with its races,
ethnic distributions, cultures, and languages66 with the populations largely categorized as urban and rural dwellers with tribal
or mainstream backgrounds. Sixteen hundred different dialects
emanating from the four main language families [the largest being
Indo European, which is prevalent in North, and the second largest
Dravidian group represents languages spoken in the South] are
spoken. Such a complex cultural diversity might account for the
presence of many different populations and sub-populations of
M. tuberculosis especially when it is now clear that this pathogen
has coevolved with the humans.49 But, until now, only two major
genetic lineages of the tubercle bacillus have been reported in
India. The predominance of a single M. tuberculosis population in
the South (EAI) and another one in the North (CAS) might therefore, point to a distinct adaptive evolution of these strains. The
extent and mechanistic aspects of this adaptation are to be
dissected and its signicance related to the control of TB has to be
unraveled.
Since whole genome sequencing on next generation platforms
offers possibilities to reduce time and cost in a considerable
manner, study of a single mycobacterial genome has become
almost pedestrian while the genome sequence based phylogenetics of multiple strains and species is within the relatively easy

N. Ahmed, S.E. Hasnain / Tuberculosis 91 (2011) 407e413

411

Figure 2. Ontology of a proposed genomic and systems biology approach guiding the discovery efforts targeted at the control of tuberculosis. A central role for molecular
epidemiology has been proposed which is likely to provide vital, eld level/clinical feedback to genomics and systems level processes.

reach.67 This holds tremendous potential for genome sequence

based epidemiology of TB in India. Such an approach will not only
reveal within-lineage diversity of the major strain groups (EAI
and CAS), but also strengthen the cause of functional molecular
infection epidemiology, an emerging area of medical microbiology that entails correlation of genetic variations such as SNPs in
a pathogen, with a unique host function related to disease
severity, disease progression, or host susceptibility to a particular
genotype. Such a functional epidemiology should encompass not
only the descriptive host-pathogen (genomic level) associations,68 but also the global juxtaposition of pathogen and host
genomic variations with a prospective role in pathogen/infection
biology.
7. Combining systems biology with systems epidemiology e
the path forward
Large scale, federated availability of the genome sequences
from both the pathogen and host sides is likely to usher the
discipline of tuberculosis research into a more predictive, interdisciplinary and data intensive discipline called systems biology.
This new science is perhaps capable of systematically unraveling
the differential attributes of this dreaded disease, leading to
measurable, biological outcomes of perturbations (system properties) within a system comprising of the pathogen with all its
diverse gene pool repertoire as juxtaposed to the host diversity
and the environment. Such a systems approach in its fullest form
will require integration of high-throughput multi-omics data to
develop predictive models of the gene regulatory and functional

level networks explaining the dynamic interactions that occur at

the level of the complex host-pathogen cross-talks (Figure 2). This
highly complex proposal requires state of the art technology
platforms and computational infrastructure. Fortunately, India has
an established information technology industry and highly trained
man power in computation. It has huge collection of isolates
archived in national repositories and a highly successful DOTS
program reaching out to millions of patients and possibly to
contacts. Emergence of multiple drug resistant isolates and high
prevalence of HIV/AIDS make the case further interesting. Moreover, the costs of next generation sequencing (NGS) have gone
down substantially and almost all major NGS platforms are
available in the market. Given this, it appears that India will
become a global hub for conducting systems biology inspired
studies in TB. Such approaches are likely to give rise to very highly
sought-after deliverables: systems level understanding of the
host-pathogen interactions, testable models of virulence/
dormancy, vaccine candidates tailored to the community host
immune response, accurate diagnostic markers and novel drug
targets (Figure 2). However, improved knowhow of the complex
interactions occurring between genetically distinct hosts and their
coevolved pathogens (or exotic strains thereof) amidst the
changing bio-geo-socio-economic backgrounds and the environment will need even newer multidisciplinary approaches. In
particular, as suggested,69,70 the marriage of systems biology with
social anthropology and ecology, resulting in what might be
described as systems epidemiology, appears to be a very
reasonable future ramication of tuberculosis epidemiology in the
post genomic, systems era.

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N. Ahmed, S.E. Hasnain / Tuberculosis 91 (2011) 407e413

Funding:
Research in our laboratories was supported by
a Centre of Excellence Grant of the Department of Biotechnology of
the Indian Government entitled Multidisciplinary approaches
aimed at interventions against Mycobacterium tuberculosis [BT/
01/C0E/07/02]. We would like to also acknowledge a (matching)
grant by the University of Hyderabad/UGC (India) under the aegis of
a German Research Foundation (DFG) sponsored international
research training group entitled Internationales graduiertenkollegfunctional molecular infection epidemiology-GRK1673 (BerlinHyderabad) of which Niyaz Ahmed is a speaker. SEH is a JC Bose
National Fellow.
Competing interests:
Ethical approval:

None Declared.
Not required.

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