The document discusses rapid diagnosis of drug resistant tuberculosis. It provides an overview of conventional and newer diagnostic methods. Conventional methods like culture and drug susceptibility testing can take 8-12 weeks to identify resistance. Newer rapid phenotypic tests such as automated liquid cultures, thin layer agar cultures, TK medium and microscopic-observation drug susceptibility assay can reduce the time to 1-2 weeks but require specialized equipment. Molecular methods like real-time PCR and line probe assays that detect gene mutations associated with resistance have been commercialized and can provide results in 1-2 days, aiding early treatment decisions. Effective control of drug resistant tuberculosis will require scaling up rapid testing capacities and expanding use of novel molecular technologies.

1. Review Article
INDIAN JOURNAL OF MEDICAL SPECIALITIES 2012;3(2):159-164
Rapid diagnosis of drug resistant tuberculosis: current perspectives and challenges
Muktikesh Dash
Abstract
Tuberculosis (TB) caused by Mycobacterium tuberculosis complex remains one of the major public health
problems, especially in developing countries. The emergence of drug resistant tuberculosis (both multi-drug
resistant and extensively drug resistant tuberculosis) is widely considered a serious threat to global TB
control. Rapid diagnosis of drug resistant tuberculosis is one of the cornerstones for global TB control as it
allows early epidemiological and therapeutic interventions. The present article provides an overview of the
various diagnostic options available for drug resistant tuberculosis, including rapid conventional tools and
newer molecular methods. Newly developed rapid phenotypic tests include automated liquid based culture
and susceptibility tests, thin layer agar cultures, TK medium, microscopic-observation drug susceptibility
assay and phage-based assay. Among newly developed molecular methods, real-time polymerase chain
reaction (RT-PCR) and line probe assays (LPAs) have been commercialised and widely used in clinical
laboratories. To effectively address the threats of drug resistant tuberculosis, global initiatives are required
to scale-up culture and drug susceptibility testing capacities. In parallel efforts are needed to expand the
use of novel and emerging molecular technologies for rapid diagnosis of drug resistance.
Key words: Tuberculosis; diagnostic tests; drug resistance; PCR; mycobacterium.
Introduction
The impact of tuberculosis (TB) can be devastating
even today, especially in developing countries
suffering from high burdens of both TB and human
immunodeficiency virus (HIV) infections. In 2009
there were 9.4 million new cases of TB globally,
causing 1.7 million deaths [1]. Tuberculosis is a
major public health problem in India which accounts
for one-fifth of the global tuberculosis incident
cases. Each year nearly 2 million people in India
develop tuberculosis, of which around 0.87 million
are infectious cases. It is estimated that annually
around 330,000 Indians die due to tuberculosis
[2]. Drug resistance has enabled it to spread
with a vengeance. The prevalence of multi-drug
resistant tuberculosis (MDR-TB) and extensively-drug
resistant tuberculosis (XDR-TB) are increasing
throughout the world both among new tuberculosis
cases as well as among previously treated ones
[3]. Fortunately, the past few years have seen an
unprecedented level of funding and activity focused
on the development of new tools for diagnosis of
drug resistant tuberculosis. This should go a long
way in helping us arrest the spread of the disease.
Drug resistant tuberculosis
MDR-TB is a form of TB caused by a strain of M.
tuberculosis resistant to the most potent first line
anti-tuberculous drugs, i.e. isoniazid (INH) and
rifampicin (RIF). It has been estimated that India and
China account for nearly 50% of the global burden of
MDR-TB cases. Approximately 5% of all pulmonary
TB cases in India may be MDR. MDR rates are low in
new, untreated cases. The incidence in such cases
ranges from 1 to 5% (mostly <3%) in different parts
of India [4-6]. However, during the last decade,
there has been an increase in reported incidences
of drug resistance in Category II cases, particularly
among those treated irregularly, or with incorrect
regimens and doses. In such cases, the incidence of
Department of Microbiology, MKCG Medical College, Berhampur-760004, Odisha. India.
E-mail: mukti_mic@yahoo.co.in
Received: 21-06-2012 | Accepted: 05-08-2012 | Published Online: 20-08-2012
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (creativecommons.org/licenses/by/3.0)
Conflict of interest: None declared Source of funding: Nil
Indian Journal of Medical Specialities, Vol. 3, No. 2, Jul - Dec 2012 159

2. Muktikesh Dash
MDR-TB varies from 12-17% [6].
XDR-TB, is defined as TB caused by a strain of M.
tuberculosis that is resistant to RIF and INH, as well
as to any member of the quinolone family and at least
one of the second-line anti-tuberculous injectable
drugs i.e., Kanamycin, Capreomycin, or Amikacin.
XDR-TB was first described in 2006. Since then,
there have been documented cases in six continents
and 55 countries [7]. The global prevalence of XDR-TB
has been difficult to assess. The prevalence of
XDR-TB has been reported from India, which varies
between low i.e., 2.4% to as high as 33.3% among
HIV infected persons suffering from MDR-TB [8,9].
Treatment outcomes are significantly worse for
patients with XDR-TB, compared to patients with
drug-susceptible TB or MDR TB [10,11]. In the first
recognised outbreak of XDR-TB, 53 patients in
KwaZulu-Natal, South Africa, who were co-infected
with XDR TB and human immunodeficiency virus
(HIV), survived for an average of 16 days, with a
mortality of 98% [12]. XDR-TB raises concerns of
a future tuberculosis epidemic with restricted
treatment options, and jeopardises the major gains
made in tuberculosis control.
Totally drug resistant tuberculosis (TDR-TB) or
extremely drug resistant tuberculosis (XXDR TB)
is resistant to all first line and second line anti-tubercular
drugs. Four cases were detected in
Mumbai who were resistant to all first line and
second line drugs [13]. This kind of rapid progression
of drug resistance from MDR, to XDR and TDR-TB
underlines the need for rapid and accurate diagnosis
of drug resistant tuberculosis.
Conventional methods for diagnosis of drug
resistant tuberculosis
Mycobacterium tuberculosis is an extremely
slow growing organism. Using standardised drug
susceptibility testing (DST) with conventional
methods, 8 to 12 weeks are required to identify
drug resistant tuberculosis on solid media (i.e.,
Lowenstein-Jensen medium). In general, these
methods assess inhibition of M. tuberculosis
growth in presence of antibiotics to distinguish
between susceptible and resistant strains. As the
results usually take weeks, inappropriate choice
of treatment regimen may result in death such as
in case of XDR-TB (especially in HIV co-infected
patients). In addition, delayed diagnosis of drug
resistance results in inadequate treatment, which
may generate additional drug resistance and
continued transmission in community.
Rapid phenotypic methods for diagnosis of drug
resistant tuberculosis
Rapid automated liquid based culture and
sensitivity tests- Automated liquid culture
systems such as BACTEC radiometric system (Bactec
460TB;Becton Dickinson, USA), non radiometric
systems MB/BacT ALERT (BioMerieux, France),
Versa TREK (Trek Diagnostic System, USA) and
Mycobacteria Growth Indicator Tube (MGIT 960;
Becton Dickinson, USA) are more sensitive and
have a shorter turn-around time than solid media
cultures. These are also less labour intensive and
therefore, less vulnerable to manual errors. But
automated systems still require few weeks to obtain
final results [14]. Also, these instruments are costly,
require maintenance and can be extremely difficult
for most public health laboratories in developing
countries.
Thin layer agar (TLA) cultures and TK Medium-
Thin layers of 7H11 middle brook solid agar medium
are used to detect microcolonies by conventional
microscopy. As it can be adapted for the rapid
detection of drug resistance directly from sputum
samples, it has an average turn-around time of 11
days [15]. Newly developed test such as TK medium
(Salubris Inc., USA) is a colorimetric system that
indicates growth of mycobacteria by changing the
colour of the growth medium. Metabolic activity
of growing mycobacteria changes the colour of the
culture medium, and this allows for an early positive
identification before bacterial colonies appear.
Unfortunately, there is insufficient published
evidence on the field performance of these tests in
developing countries [16].
Microscopic observation drug susceptibility assay
(MODS)- The MODS assay is based on characteristic
cord formation of M. tuberculosis that can be
visualised microscopically (‘strings and tangles’
appearance) in liquid medium with or without
antimicrobial drugs (for DST) [17]. The test
sensitivity is better than traditional methods using
LJ media with a turnaround time of 7 days for culture
and drug sensitivity. Besides, it is cheap, simple
160 Indian Journal of Medical Specialities, Vol. 3, No. 2, Jul - Dec 2012

3. Drug resistant tuberculosis
and fairly accurate [18]. One minor disadvantage
of MODS assay is the requirement for an inverted
microscope for observation of the mycobacterial
growth.
Phage based assay- Phage amplification-based test
(FAST Plaque-Response, Biotech Laboratories Ltd.,
UK), has been developed for direct use on sputum
specimens. Drug resistance is diagnosed when M.
tuberculosis is detected in samples that contain the
drug (i.e., RIF). When these assays were performed
on M. tuberculosis culture isolates, they have shown
high sensitivity and variable specificity, but evidence
is lacking about the accuracy when they are directly
applied to sputum specimens [19]. It also requires
high standards of bio-safety and quality control.
Rapid molecular methods for diagnosis of drug
resistant tuberculosis
Since publication of genome details (M. tuberculosis
H37Rv strain) in 1998, these have been utilised in
development of nucleic acid amplification (NAA)
tests for diagnosis of drug resistant tuberculosis. A
number of NAA tests are now available, manual and
automated, commercial and in-house, with varying
performance characteristics. Real-time polymerase
chain reaction (RT-PCR) and line probe assays (LPA)
have been commercialised and widely used in
clinical laboratories.
Real-time polymerase chain reaction (RT-PCR)-
Molecular tools are based on identification of specific
mutations responsible for drug resistance, which are
detected by the process of nucleic acid amplification
in conjunction with electrophoresis, sequencing or
hybridisation. Direct sequencing techniques such
as real-time polymerase chain reaction (RT-PCR)
uses wild-type primer sequences to amplify genes
and enables the use of specific probes to identify
mutations. Among these is a recently introduced
semi-quantitative nested RT-PCR, i.e., GeneXpert
MTB/RIF (Cepheid, USA and FIND Diagnostics,
Geneva, Switzerland). It integrates and automates
sample processing and simultaneously detects
M. tuberculosis and rifampicin resistance within
single-use disposable cartridges. A study examined
1730 patients with suspected drug-sensitive or
multidrug-resistant pulmonary tuberculosis across
Peru, Azerbaijan, South Africa and India. There was
sensitive detection of M. tuberculosis and rifampicin
resistance directly from untreated sputum in less
than two hours with minimal hands-on time [20].
The W.H.O. has recently supported the use of this
system as an initial diagnostic test in respiratory
specimens of patients with high clinical suspicion
of having tuberculosis or who could be multidrug
resistant [21]. These tests are expensive and
complicated, even if highly sensitive and specific.
Line probe assays (LPAs)- Line probe assays are a
family of novel DNA strip tests that use both PCR
and reverse hybridisation methods. In these assays,
a specific target sequence is amplified, and applied
on nitrocellulose membranes. Specific DNA probes
on the membrane hybridise with the amplified
sequence applied on it. Colour conjugates make
the amplified target sequences appear as coloured
bands. These tests have been designed to identify
M. tuberculosis and simultaneously detect genetic
mutations related to drug resistance both from
clinical samples as well as culture isolates.
Commercially available kits include the INNO-LiPA
Rif.TB (Innogenetics, Belgium), the GenoType
MTBDR, MTBDRplus, MTBDRsl assay (Hain Lifescience,
Germany). INNO-LiPA Rif.TB test is able to identify
M. tuberculosis complex and simultaneously
detect genetic mutations in the rpoB gene region
related to rifampicin resistance. The GenoType
MTBDR assay, introduced in 2004, identifies M.
tuberculosis complex and simultaneously detects
mutations in the rpoB gene as well as mutations in
the katG gene for high-level isoniazid resistance.
The second generation MTBDRplus and MTBDRsl
assays also detect mutations in the inhA gene for
low-level isoniazid resistance and mutations in the
gyrA, rrs and embB genes, respectively. The new
MTBDRsl assay may represent a reliable tool for
detection of floroquinolone, amikacin, capreomycin
and ethambutol resistance. A recent laboratory
evaluation study from South Africa estimated
the accuracy of the GenoType MTBDRplus assay
performed directly on AFB smear-positive sputum
specimens. It showed high sensitivity, specificity,
positive and negative predictive values for detection
of rifampicin and INH resistance. However, a
meta-analysis on this assay found that sensitivity
estimates for INH resistance were comparatively
modest [22,23].
Indian Journal of Medical Specialities, Vol. 3, No. 2, Jul - Dec 2012 161

4. Revised National Tuberculosis Control Programme
(RNTCP) and drug resistant tuberculosis
The Revised National Tuberculosis Control
Programme (RNTCP) plans to strengthen laboratory
capacity for M. tuberculosis culture, drug sensitivity
testing (C-DST) and Line probe assay (LPA) across
India. To date, 35 RNTCP accredited laboratories
including 14 LPA and 4 liquid culture laboratories
in public and private sectors are serving patients
while another 30 laboratories are under the process
of up-gradation and accreditation under RNTCP,
most of them include LPA and Liquid Culture for first
and second line drugs [24]. In a policy statement
released in June 2008, the WHO endorsed the
use of LPA for rapid screening of patients at risk
of MDRTB and recommended the use of line probe
assays only on culture isolates and smear-positive
sputum specimens. It is not recommended as a
complete replacement for conventional culture and
drug susceptibility testing [25]. As of January 2012,
diagnosis of XDRTB can only be confirmed at three
laboratories in India, which are quality assured for
second-line anti-tuberculosis drug susceptibility
testing of flouroquinolones and injectables. These
are the National Reference Laboratories (NRL) of
TRC/NIRT Chennai, NTI Bangalore and LRS Institute,
New Delhi. Routine fluoroquinolone and injectable
DST (i.e. XDR TB diagnosis) on all MDRTB patients at
the beginning of treatment has been recommended
by the RNTCP National Laboratory Committee in
2011, but the capacity to conduct that testing is
not yet present in most culture and DST laboratories
used by RNTCP. Capacity building for second line
DST is being undertaken through these NRLs [24].
Conclusion
Effective control of drug resistant tuberculosis
will require massive scaling-up of culture and DST
capacity, and simultaneous use of rapid molecular
assays. Furthermore, all molecular tests require
DNA extraction, gene amplification and detection of
mutations and are, therefore, relatively expensive,
demand resources and skills. These are usually
unavailable in developing countries where rates of
drug resistant tuberculosis are high. The challenge,
therefore, is to not only develop new tools, but to
also make sure that benefits of promising new tools
actually reach the populations that need it most,
but can least afford them.
Key Points
• The emergence of drug resistant
tuberculosis is a serious threat to control of
tuberculosis. For initiating early treatment
and prevention, rapid diagnosis of drug
resistant tuberculosis is essential.
• Conventional methods take weeks for
detection and result in delayed diagnosis
resulting in deterioration of patient’s
condition and inadequate treatment, which
may generate additional drug resistance
and continued transmission in community.
• Newly developed rapid phenotypic tests
including automated liquid based culture,
thin layer agar cultures, TK medium,
microscopic-observation drug susceptibility
assay and phage-based assay are expensive
and not suitable for field testing in
developing countries.
• The WHO has supported the use of RT-PCR
system as an initial diagnostic test in
respiratory specimens of patients with high
clinical suspicion of having tuberculosis or
who could be multidrug resistant.
• WHO endorsed the use of LPA for rapid
screening of patients at risk of MDRTB
and recommended its use only on culture
isolates and smear-positive sputum
specimens. It is not recommended as a
complete replacement for conventional
culture and drug susceptibility testing.
• Global initiatives are required to scale-up
culture and drug susceptibility testing
capacities and expand the use of novel and
emerging molecular technologies for rapid
diagnosis of drug resistance.
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