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Drug resistant tuberculosis: A diagnostic challenge
Review Article 
Department of 
Microbiology, Maharaja 
Krishna Chandra Gajapati 
Medical College and 
Hospital, Berhampur, 
Odisha, India 
Address for correspondence: 
Dr. Muktikesh Dash, 
E‑mail: mukti_mic@yahoo. 
co.in 
Drug resistant tuberculosis: A diagnostic 
challenge 
Dash M 
ABSTRACT 
Tuberculosis (TB) is responsible for 1.4 million deaths annually. Wide‑spread misuse of anti‑tubercular drugs 
over three decades has resulted in emergence of drug resistant TB including multidrug‑resistant TB and 
extensively drug‑resistant TB globally. Accurate and rapid diagnosis of drug‑resistant TB is one of the paramount 
importance for instituting appropriate clinical management and infection control measures. The present article 
provides an overview of the various diagnostic options available for drug resistant TB, by searching PubMed for 
recent articles. Rapid phenotypic tests still requires days to weeks to obtain final results, requiring biosafety 
and quality control measures. For newly developed molecular methods, infrastructure, training and quality 
assurance should be followed. Successful control of drug resistant TB globally will depend upon strengthening 
TB control programs, wider access to rapid diagnosis and provision of effective treatment. Therefore, political 
and fund provider commitment is essential to curb the spread of drug resistant TB. 
KEY WORDS: Diagnosis, drug resistant, rapid, tuberculosis 
Received : 10‑10‑2012 
Review completed : 03‑12‑2012 
Accepted : 07‑06‑2013 
Introduction 
T he 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). In 2010, there were 8.8 million new cases of TB 
globally, causing 1.4 million deaths.[1] TB is a major public 
health problem in India, which accounts for one‑fifth of 
the global TB incident cases. Each year nearly 2 million 
people in India develop TB, of which around 0.87 million 
are infectious cases.[2] It is estimated that annually around 
2,80,000 (23/1,00,000 population) Indians die due to TB.[2] 
Drug resistance has enabled it to spread with a vengeance. 
The prevalence of multidrug‑resistant tuberculosis (MDR‑TB) 
and extensively‑drug resistant TB tuberculosis (XDR‑TB) are 
increasing throughout the world both among new TB cases 
as well as among previously treated ones.[3] Accurate and 
rapid diagnosis of drug‑resistant TB is one of the paramount 
importance for instituting appropriate clinical management 
and appropriate infection control measures.[4,5] 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 TB. This should go a long way in helping us 
arrest the spread of the disease. 
Sources and method included PubMed search for recent articles 
using MeSH terms “TB” and “resistance” and “diagnosis.” 
Furthermore, World Health Organization (WHO) reports and 
national guidelines were used. The inclusion criteria for selected 
articles were based on relevance to the purpose of the review. 
Drug Resistant TB 
MDR‑TB is a form of TB caused by a strain of Mycobacterium 
tuberculosis (MTB) resistant to the most potent first line anti‑TB 
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.[6] 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.[7,8] 
However, during the last decade, there has been an increase in 
reported incidences of drug resistance in category II TB cases, 
particularly among those treated irregularly or with incorrect 
regimens and doses. In such cases, the incidence of MDR‑TB 
varies from 11.8% to 47.1%.[9] 
XDR‑TB, is defined as TB caused by a strain of MTB 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‑TB 
Access this article online 
Quick Response Code: Website: 
www.jpgmonline.com 
DOI: 
10.4103/0022-3859.118038 
PubMed ID: 
*** 
 196 Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3
Dash: Diagnosis of drug resistant tuberculosis 
injectable drugs, i.e. kanamycin, capreomycin or amikacin. 
XDR‑TB was first described in 2006. Since then, there have 
been documented cases in 77 countries world‑wide by the 
end of 2011.[10] 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% and as high as 
21.1% in HIV infected persons suffering from MDR‑TB.[11,12] 
Treatment outcomes are significantly worse for patients with 
XDR‑TB, compared with patients with drug‑susceptible TB 
or MDR‑TB.[13,14] In the first recognized outbreak of XDR‑TB, 
53 patients in KwaZulu‑Natal, South Africa, who were 
co‑infected with XDR‑TB and HIV, survived for an average of 
16 days, with mortality of 98%.[15] XDR‑TB raises concerns of 
a future TB epidemic with restricted treatment options and 
jeopardizes the major gains made in TB control. 
Totally, drug resistant TB (TDR‑TB) or extremely drug resistant 
TB is resistant to all first line and second line anti‑tubercular 
drugs. The detection of four Mumbai cases, which were 
resistant to all first line and second line drugs.[16] 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 TB. 
Molecular basis of drug resistance 
RIF acts by binding to the beta‑subunit of the ribonucleic 
acid (RNA) polymerase (coded for by the rpoB gene), 
inhibiting RNA transcription. Subsequent deoxyribonucleic 
acid (DNA) sequencing studies have shown that more than 
95% of RIF resistant strains have mutations in an 81‑base 
pair region (codons 507‑533) of the rpoB gene. INH inhibits 
enoyl‑acyl carrier protein (ACP)‑reductase (coded by the inhA 
gene), which is involved in mycolic acid biosynthesis. INH is 
also a “pro‑drug,” which is converted to its active form by the 
catalase‑peroxidase enzyme (coded by katG gene). Resistant 
mutants can be due to different regions of several genes, 
including binding of activated INH to its inhA target, the 
activation of the pro‑drug by katG or by increased expression 
of the target inhA. Point mutations in codon 315 of the katG 
gene have been found in 50‑90% of high‑level INH resistant 
strains while 20‑35% of low‑level INH resistant strains have been 
reported mutations in inhA regulatory region and 10‑15% have 
mutations in the ahpC‑oxyR intergenic region (often together 
with katG mutations in other regions).[17] 
Conventional Phenotypic Methods for Diagnosis of Drug 
Resistant TB 
MTB is an extremely slow growing organism. Using the 
standardized drug susceptibility testing (DST) with conventional 
methods, 8‑12 weeks are required to identify drug resistant TB 
on solid media (i.e. Lowenstein‑Jensen [LJ] medium). In 
general, these methods assess inhibition of MTB growth in the 
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. The most common 
medium for the agar proportion method in resource limited 
countries is LJ medium; however, the Clinical and Laboratory 
Standards Institute, (CLSI) considers this medium to be 
unsuitable for susceptibility testing due to uncertainty about 
the potency of drugs following inspissation and also because 
components present in the eggs or the medium may negatively 
affect some drugs. Both Centers for Disease Control and 
Prevention and CLSI recommend that Middle brook 7H10 agar 
supplemented with oleic albumin dextrose catalase (OADC) 
be used as the standard medium for the agar proportion assay. 
Rapid phenotypic methods for diagnosis of drug resistant TB 
Rapid automated liquid based culture and susceptibility 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 shorter turnaround time than solid media cultures. 
These are also less labor intensive and therefore, less vulnerable 
to manual errors. But, automated systems still requires few 
weeks to obtain final results.[18] Also these instruments are costly, 
require maintenance and can be extremely difficult for most 
public health laboratories in developing countries. 
Nitrate reductase assay (Griess method) 
The NRA is a liquid or solid medium technique that measures 
nitrate reduction by members of the MTB complex to indicate 
growth and to indicate resistance to INH and RIF. Since the 
NRA method uses nitrate reduction as a sign of growth, results 
are detected earlier than by examination of microcolonies on 
solid medium. However, the Griess reagent kills the organisms 
when added to the tubes, so multiple tubes must be inoculated 
if further testing is necessary. In addition, not all members of the 
MTB complex reduce nitrate, so the presence of nitrate‑negative 
acid fast bacilli (AFB) may require further testing. 
Thin layer agar cultures and TK medium 
Thin layers of middle brook 7H11 solid agar medium are used 
to detect microcolonies by conventional microscopy. It can 
be adapted for the rapid detection of drug resistance directly 
from sputum samples, but requires average turnaround time 
of 11 days.[19] 
Newly developed test such as TK medium (Salubris Inc., USA) 
is a colorimetric system that indicates growth of mycobacteria 
by changing the color of the growth medium. Metabolic activity 
of growing mycobacteria changes the color 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.[20] 
Microscopic observation drug susceptibility assay 
The MODS assay is based on characteristic cord formation 
of MTB that can be visualized microscopically (“strings 
and tangles” appearance) in liquid medium with or without 
antimicrobial drugs (for DST).[21] The test sensitivity is better 
Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3 197 
Dash: Diagnosis of drug resistant tuberculosis 
than traditional methods using LJ media with a turnaround time 
of 7 days for culture and drug susceptibility testing (C‑DST) for 
INH and RIF. It is cheap, simple and fairly accurate.[22] Biosafety 
level‑3 facilities are required if the plates are opened for further 
testing, such as for the confirmation of the identification of 
TB. Laboratories using MODS assay require a functioning 
biosafety cabinet, a safety centrifuge, an incubator, an inverted 
light microscope for observation of mycobacterial growth and 
supplemented liquid media (Middle brook 7H9 broth with 
OADC and PANTA). 
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 
MTB is detected in samples that contain the drug (i.e., RIF). 
When these assays performed on MTB culture isolates, they 
have shown high sensitivity and variable specificity, but the 
evidence is lacking about the accuracy when they are directly 
applied to sputum specimens.[23] It also requires high standards 
of biosafety and quality control. 
Luciferase reporter phages are genetically‑modified phages 
containing the flux gene encoding firefly luciferase. This 
catalyzes a reaction producing light in the presence of the 
luciferin substrate and adenosine triphosphate; light is only 
produced in the presence of viable mycobacteria. Detection 
of light released from viable mycobacteria can be achieved 
by a luminometer or photographic film within 2‑4 days from 
culture.[24] The luminometer readout is more sensitive and 
enables quantification of results while the use of Polaroid 
photographic film offers a lower‑tech approach with lower 
sensitivity. 
Rapid molecular methods for diagnosis of drug resistant TB 
Since the publication of genome details of MTB H37Rv strain 
in 1998, these have been utilized in development of nucleic 
acid amplification (NAA) tests for diagnosis of drug resistant 
TB. A number of NAA tests are now available, manual and 
automated, commercial and in the house, with varying 
performance characteristics. Real‑time polymerase chain 
reaction (RT‑PCR) and line probe assays (LPAs) have been 
commercialized and widely used in clinical laboratories merit 
special mention, detailed below. 
RT‑PCR 
Molecular tools are based on identification of specific mutations 
responsible for drug resistance, which are detected by the process 
of NAA in conjunction with electrophoresis, sequencing or 
hybridization. Direct sequencing techniques such as RT‑PCR 
that uses wild‑type primer sequences to amplify genes and 
enable the use of specific probes to identify mutations. Recently 
introduced semi‑quantitative nested RT‑PCR, which integrates 
and automates sample processing and simultaneously detects 
MTB and RIF resistance within the single‑use disposable 
cartridges. A study examined 1,730 patients with suspected 
drug‑sensitive or MDR pulmonary TB across Peru, Azerbaijan, 
South Africa and India. There was sensitive detection of MTB 
and RIF resistance directly from untreated sputum in < 2 h with 
minimal hands‑on time.[25] The WHO has recently supported 
the use of this system as an initial diagnostic test in respiratory 
specimens of patients with high clinical suspicion of having TB 
or who could be MDR.[26] These tests are very expensive require 
adequate maintenance and calibration of the equipment. 
Further, the specificity for RIF resistance in populations where 
MDR‑TB is rare requires confirmation by repeat testing and 
thus increases cost. 
LPAs 
LPAs are a family of novel DNA strip tests that use both PCR and 
reverse hybridization methods. In these assays, a specific target 
sequence is amplified and applied on nitrocellulose membranes. 
Specific DNA probes on the membrane hybridize with the 
amplified sequence applied on it. Color conjugates make the 
amplified target sequences appear as colored bands. These tests 
have been designed to identify MTB and simultaneously detect 
genetic mutations related to drug resistance both from clinical 
samples as well as culture isolates. These tests able to identify 
MTB complex and simultaneously detect genetic mutations in 
the rpoB gene region related to rifampin resistance. The LPA 
strip consists of 10 oligonucleotide probes: One is specific for 
the MTB complex, five are partially overlapping wild‑type probes 
that span the region at positions 509‑534 of the rpoB gene and 
four probes are specific for amplicons carrying the most common 
rpoB mutations (D516V, H526Y, H526D and S531L). According 
to a recent review, the sensitivities of the LPA are above 90% for 
clinical isolates with 99‑100% specificity [Table 1]. It identifies 
MTB complex and simultaneously detects mutations in the rpoB 
gene as well as mutations in the katG gene for high‑level INH 
resistance. These tests can also detect mutations in the inhA gene 
for low‑level INH resistance and mutations in the gyrA, rrs and 
embB genes for 2nd line anti‑tubercular drugs fluoroquinolones, 
aminoglycosides and ethambutol respectively [Table 2].[32] The 
newly developed assay may represent a reliable tool for detection 
of fluoroquinolones, amikacin, capreomycin and ethambutol 
resistance. LPA strip can use culture isolates and smear positive 
sputum as specimen and provide results in 1‑2 days. A recent 
laboratory evaluation study from South Africa estimated the 
accuracy of the GenoType MTBDRplus assay performed directly 
on AFB smear‑positive sputum specimens.[33] It showed high 
sensitivity, specificity, positive and negative predictive values 
for detection of RIF and INH resistance [Table 1]. However, 
a meta‑analysis on this assay found that sensitivity estimates 
for INH resistance were comparatively modest.[33,34] In general, 
LPAs are expensive and require dedicated equipments, reagents 
and facilities. Molecular drug resistance testing has technical 
limitations, i.e., LPAs can detect only well characterized 
drug resistance alleles, not every drug resistance allele can be 
discriminated via current tests and silent mutations (which do 
not confer drug resistance) can be detected by probes leading 
to misclassification of drug resistance. In addition, molecular 
tests cannot determine the proportion of drug resistant bacteria 
within a mixed population of cells (i.e., wild type and drug 
resistant). Cross contamination with amplicons generated from 
previous tests can be problematic especially when the tests have 
been employed in laboratories without appropriate staff training 
and quality control. 
 198 Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3
Dash: Diagnosis of drug resistant tuberculosis 
Table 1: Performance of methodologies used to diagnose drug resistant tuberculosis 
Drug resistance Methodology and products Sensitivity % (95% CI) Specificity % (95% CI) Reference number 
Phenotypic methods 
Automated liquid 
MB/BacT 
for rifampicin 
based CST systems 
ALERT 
resistance 
Versa TREK 100 100 
MGIT 960 99.8 99.2 
NRA Griess method 99 100 [28] 
MODS 98 (94.5‑99.3) 99.4 (95.7‑99.9) [29] 
Phage based assay FASTPlaque‑response 
(commercial) 
FASTPlaque‑Response 
(in‑house) 
Luciferase reporter 
phage 
Phenotypic methods 
for isoniazid 
resistance 
Automated liquid 
based CST systems 
MB/BacT ALERT 100 95 [27] 
Versa TREK 95.5 99 
MGIT 960 97.1 100 
NRA Griess method 94 100 [28] 
MODS 97.7 (94.4‑99.1) 95.8 (88.1‑98.6) [29] 
Molecular methods 
for rifampicin 
resistance 
RT‑PCR GeneXpert® MTB/RIF 98 (97‑99) 99 (98‑99) [31] 
Line probe assay INNO‑LiPA Rif. TB 93 (89‑96) 99 (99‑100) 
MTBDRplus 97 (92‑99) 98 (95‑99) 
Molecular methods 
for isoniazid 
resistance 
Line probe assay MTBDRplus 77 (69‑83) 99 (97‑100) 
Molecular methods 
for second‑line drug 
resistance 
Line probe assay MTBDRsl 
(fluoroquinolones)W 
Amikacin 90 (81‑96) 100 (98‑100) 
Kanamycin 83 (59‑96) 100 (96‑100) 
Capreomycin 87 (77‑94) 99 (96‑100) 
Ethambutol 60 (52‑68) 98 (94‑100) 
CST – Culture and susceptibility testing; NRA – Nitrate reductase assay; MODS – Microscopic observation drug susceptibility; RT‑PCR – Real time 
polymerase chain reaction; CI – Confidence interval; MTB – Mycobacterium tuberculosis; TB – Tuberculosis; RIF – Rifampicin; MGIT – Mycobacteria 
growth indicator tube 
Diagnostic testing algorithm for drug resistant TB 
Laboratory policies and testing of patients suspected of 
having drug‑resistant TB depend on the local epidemiology, 
local treatment policies, the existing laboratory capacity, 
specimen referral and transport mechanisms and human and 
financial resources. Mycobacterial culture (solid or liquid) 
and identification of MTB provide a definitive diagnosis of 
TB. Culture also provides necessary isolates for conventional 
DST. Thus LPAs, RT‑PCRs, MODS and NRA can be used in 
conjunction with culture (solid and liquid) and DST [Figure 1].[35] 
Revised national tuberculosis control program and diagnosis 
of drug resistant TB 
The RNTCP plans to strengthen laboratory capacity for MTB 
C‑DST and LPA across India. To date, 35 RNTCP accredited 
labs including 14 LPA and 4 liquid culture labs in public and 
private sectors are serving patients while another 30 labs 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.[36] In a policy statement released in June 
2008, the WHO endorsed the use of LPA for rapid screening 
of patients at risk of MDR‑TB and recommended the use 
99 98 [27] 
95.5 (92.2‑97.4) 95 (91.3‑97.2) [30] 
98.5 (96.1‑99.4) 97.9 (94.8‑99.2) 
99.3 (49.1‑100) 98.6 (92.5‑99.8) 
85 (78‑91) 100 (97‑100) 
of LPAs only on culture isolates and smear‑positive sputum 
specimens. It is not recommended as a complete replacement 
for conventional C‑DST.[37] As of January 2012, diagnosis of 
XDR‑TB can only be confirmed at three laboratories in India, 
which are quality assured for second line anti‑TB DST of 
flouroquinolones and injectable drugs. These are the National 
Reference Laboratories (NRL) of tuberculosis research centre/ 
national institute for research in tuberculosis (TRC/NIRT) 
Chennai, National Tuberculosis Institute (NTI) Bangalore 
and LRS Institute, New Delhi. Routine fluoroquinolone and 
injectable DST (i.e., XDR‑TB diagnosis) on all MDR‑TB 
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 
C‑DST laboratories used by RNTCP. Capacity building for 
second line DST is being undertaken through these NRLs.[26] 
Prevention and Control of Drug Resistant TB 
The principles of TB control are important for the prevention 
of drug resistant TB; these include prompt case detection, 
provision of curative treatment and prevention of transmission. 
Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3 199 
Dash: Diagnosis of drug resistant tuberculosis 
Table 2: Summary of technologies used to diagnose drug resistant tuberculosis 
Technologies Summary of technologies Estimated costs 
Description Product Training Infrastructure Equipment Consumable 
New solid 
culture 
methods 
Measures nitrate reduction 
to indicate RIF and INH 
resistance 
Non‑commercial (nitrate 
reductase assay) 
Simultaneously detects TB 
and indicate RIF and INH 
resistance 
Non‑commercial (thin layer 
agar culture) 
Liquid culture Broth based manual and 
automated culture system; 
can be configured for DST 
Commercial 
BacT/ALERT 
Versa TREK 
MGIT 960 
MODS Manual liquid culture 
technique uses inverted 
microscope to detect TB 
Non‑commercial Extensive BSL 2 to 
RT‑PCR Allows automated 
sample processing, DNA 
amplification detection of 
TB and screening for RIF 
resistance 
Commercial (GeneXpert® 
MTB/RIF) 
GeneXpert detect mutations 
in 81‑base pair core region 
of rpoB and in codon 315 
of katG genes respectively 
Molecular line 
probe assay 
Strip test simultaneously 
detects TB genetic 
mutations for RIF/INH 
resistance 
Commercial (INNO‑LiPA, 
MTBDR, MTBDRplus, 
MTBDRsl) 
INNO‑LiPA targets the 
16S‑23S DNA spacer 
region for mycobacteria 
and mutations in the rpoB 
gene region related to RIF 
resistance 
MTBDR detect the 
23S rRNA genes for 
mycobacteria and 
mutations in rpoB as well 
as katG genes for INH 
resistance 
MTBDRplus target in 
addition inhA for low level 
INH resistance 
MTBDRsl detects in 
addition gyrA, rrs and 
embB for fluoroquinolones, 
aminoglycosides and 
ethambutol respectively 
RIF – Rifampicin; INH – Isoniazid; MODS – Microscopic observation drug susceptibility; RT‑PCR – Real time polymerase chain reaction; 
TB – Tuberculosis; DST – Drug susceptibility testing. Basic laboratory (no specialized biosafety equipment). BSL 2 – Biosafety level 2 (specialized 
biosafety equipment required, such as biosafety cabinet); BSL 3 – Biosafety level 3 (biosafety cabinet and other safety equipment required. 
Controlled ventilation system that maintains a directional airflow into the laboratory also required); MGIT – Mycobacteria growth indicator tube; 
MTB – Mycobacterium tuberculosis; DNA – Deoxyribonucleic acid; rRNA – Ribosomal ribonucleic acid 
The WHO’s “Stop TB”[38] directly observed treatment short 
course (DOTS) strategy includes supervision and support 
of treatment, although there is little evidence that directly 
observed treatment alone improves cure rates.[39] Ineffective 
drug treatment is a strong risk factor for acquired drug resistance 
and proper administration of anti‑tubercular drugs is critical 
to reduce this risk. An enhanced DOTS program, DOTS‑plus 
has been developed for managing MDR‑TB in resource limited 
settings.[40] This program recommends additional facilities for 
C‑DST for detection of drug resistant TB and provision of 
appropriate second line anti‑tubercular drugs. 
Moderate BSL 2 to 
BSL 3 
Low Medium 
Extensive BSL 2 to 
BSL 3 
Low Medium 
Extensive 
(3 weeks) 
BSL 3 High High 
BSL 3 
Medium Medium 
Minimal Basic 
laboratory 
High High 
Moderate 
(3 days) 
BSL 2 to 
BSL 3 
High High 
As most of the resistance arises from either inadequate or 
inappropriate treatment of active disease, prevention of 
active disease indirectly prevents drug resistance. Contact 
tracing including family members and health‑care workers, 
those who are at risk of acquiring drug resistant TB[41] is one 
of the fundamental measures to detect active disease, must 
be aligned to all health services. Prevention of transmission 
in healthcare settings is difficult in places where resources 
are limited with no isolation facilities; one approach is to 
manage cases with similar resistance profiles in segregated 
groups. However, simple, low‑cost interventions, such as 
 200 Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3
Dash: Diagnosis of drug resistant tuberculosis 
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Drug resistant tuberculosis: A diagnostic challenge

  • 2. Review Article Department of Microbiology, Maharaja Krishna Chandra Gajapati Medical College and Hospital, Berhampur, Odisha, India Address for correspondence: Dr. Muktikesh Dash, E‑mail: mukti_mic@yahoo. co.in Drug resistant tuberculosis: A diagnostic challenge Dash M ABSTRACT Tuberculosis (TB) is responsible for 1.4 million deaths annually. Wide‑spread misuse of anti‑tubercular drugs over three decades has resulted in emergence of drug resistant TB including multidrug‑resistant TB and extensively drug‑resistant TB globally. Accurate and rapid diagnosis of drug‑resistant TB is one of the paramount importance for instituting appropriate clinical management and infection control measures. The present article provides an overview of the various diagnostic options available for drug resistant TB, by searching PubMed for recent articles. Rapid phenotypic tests still requires days to weeks to obtain final results, requiring biosafety and quality control measures. For newly developed molecular methods, infrastructure, training and quality assurance should be followed. Successful control of drug resistant TB globally will depend upon strengthening TB control programs, wider access to rapid diagnosis and provision of effective treatment. Therefore, political and fund provider commitment is essential to curb the spread of drug resistant TB. KEY WORDS: Diagnosis, drug resistant, rapid, tuberculosis Received : 10‑10‑2012 Review completed : 03‑12‑2012 Accepted : 07‑06‑2013 Introduction T he 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). In 2010, there were 8.8 million new cases of TB globally, causing 1.4 million deaths.[1] TB is a major public health problem in India, which accounts for one‑fifth of the global TB incident cases. Each year nearly 2 million people in India develop TB, of which around 0.87 million are infectious cases.[2] It is estimated that annually around 2,80,000 (23/1,00,000 population) Indians die due to TB.[2] Drug resistance has enabled it to spread with a vengeance. The prevalence of multidrug‑resistant tuberculosis (MDR‑TB) and extensively‑drug resistant TB tuberculosis (XDR‑TB) are increasing throughout the world both among new TB cases as well as among previously treated ones.[3] Accurate and rapid diagnosis of drug‑resistant TB is one of the paramount importance for instituting appropriate clinical management and appropriate infection control measures.[4,5] 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 TB. This should go a long way in helping us arrest the spread of the disease. Sources and method included PubMed search for recent articles using MeSH terms “TB” and “resistance” and “diagnosis.” Furthermore, World Health Organization (WHO) reports and national guidelines were used. The inclusion criteria for selected articles were based on relevance to the purpose of the review. Drug Resistant TB MDR‑TB is a form of TB caused by a strain of Mycobacterium tuberculosis (MTB) resistant to the most potent first line anti‑TB 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.[6] 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.[7,8] However, during the last decade, there has been an increase in reported incidences of drug resistance in category II TB cases, particularly among those treated irregularly or with incorrect regimens and doses. In such cases, the incidence of MDR‑TB varies from 11.8% to 47.1%.[9] XDR‑TB, is defined as TB caused by a strain of MTB 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‑TB Access this article online Quick Response Code: Website: www.jpgmonline.com DOI: 10.4103/0022-3859.118038 PubMed ID: ***  196 Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3
  • 3. Dash: Diagnosis of drug resistant tuberculosis injectable drugs, i.e. kanamycin, capreomycin or amikacin. XDR‑TB was first described in 2006. Since then, there have been documented cases in 77 countries world‑wide by the end of 2011.[10] 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% and as high as 21.1% in HIV infected persons suffering from MDR‑TB.[11,12] Treatment outcomes are significantly worse for patients with XDR‑TB, compared with patients with drug‑susceptible TB or MDR‑TB.[13,14] In the first recognized outbreak of XDR‑TB, 53 patients in KwaZulu‑Natal, South Africa, who were co‑infected with XDR‑TB and HIV, survived for an average of 16 days, with mortality of 98%.[15] XDR‑TB raises concerns of a future TB epidemic with restricted treatment options and jeopardizes the major gains made in TB control. Totally, drug resistant TB (TDR‑TB) or extremely drug resistant TB is resistant to all first line and second line anti‑tubercular drugs. The detection of four Mumbai cases, which were resistant to all first line and second line drugs.[16] 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 TB. Molecular basis of drug resistance RIF acts by binding to the beta‑subunit of the ribonucleic acid (RNA) polymerase (coded for by the rpoB gene), inhibiting RNA transcription. Subsequent deoxyribonucleic acid (DNA) sequencing studies have shown that more than 95% of RIF resistant strains have mutations in an 81‑base pair region (codons 507‑533) of the rpoB gene. INH inhibits enoyl‑acyl carrier protein (ACP)‑reductase (coded by the inhA gene), which is involved in mycolic acid biosynthesis. INH is also a “pro‑drug,” which is converted to its active form by the catalase‑peroxidase enzyme (coded by katG gene). Resistant mutants can be due to different regions of several genes, including binding of activated INH to its inhA target, the activation of the pro‑drug by katG or by increased expression of the target inhA. Point mutations in codon 315 of the katG gene have been found in 50‑90% of high‑level INH resistant strains while 20‑35% of low‑level INH resistant strains have been reported mutations in inhA regulatory region and 10‑15% have mutations in the ahpC‑oxyR intergenic region (often together with katG mutations in other regions).[17] Conventional Phenotypic Methods for Diagnosis of Drug Resistant TB MTB is an extremely slow growing organism. Using the standardized drug susceptibility testing (DST) with conventional methods, 8‑12 weeks are required to identify drug resistant TB on solid media (i.e. Lowenstein‑Jensen [LJ] medium). In general, these methods assess inhibition of MTB growth in the 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. The most common medium for the agar proportion method in resource limited countries is LJ medium; however, the Clinical and Laboratory Standards Institute, (CLSI) considers this medium to be unsuitable for susceptibility testing due to uncertainty about the potency of drugs following inspissation and also because components present in the eggs or the medium may negatively affect some drugs. Both Centers for Disease Control and Prevention and CLSI recommend that Middle brook 7H10 agar supplemented with oleic albumin dextrose catalase (OADC) be used as the standard medium for the agar proportion assay. Rapid phenotypic methods for diagnosis of drug resistant TB Rapid automated liquid based culture and susceptibility 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 shorter turnaround time than solid media cultures. These are also less labor intensive and therefore, less vulnerable to manual errors. But, automated systems still requires few weeks to obtain final results.[18] Also these instruments are costly, require maintenance and can be extremely difficult for most public health laboratories in developing countries. Nitrate reductase assay (Griess method) The NRA is a liquid or solid medium technique that measures nitrate reduction by members of the MTB complex to indicate growth and to indicate resistance to INH and RIF. Since the NRA method uses nitrate reduction as a sign of growth, results are detected earlier than by examination of microcolonies on solid medium. However, the Griess reagent kills the organisms when added to the tubes, so multiple tubes must be inoculated if further testing is necessary. In addition, not all members of the MTB complex reduce nitrate, so the presence of nitrate‑negative acid fast bacilli (AFB) may require further testing. Thin layer agar cultures and TK medium Thin layers of middle brook 7H11 solid agar medium are used to detect microcolonies by conventional microscopy. It can be adapted for the rapid detection of drug resistance directly from sputum samples, but requires average turnaround time of 11 days.[19] Newly developed test such as TK medium (Salubris Inc., USA) is a colorimetric system that indicates growth of mycobacteria by changing the color of the growth medium. Metabolic activity of growing mycobacteria changes the color 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.[20] Microscopic observation drug susceptibility assay The MODS assay is based on characteristic cord formation of MTB that can be visualized microscopically (“strings and tangles” appearance) in liquid medium with or without antimicrobial drugs (for DST).[21] The test sensitivity is better Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3 197 
  • 4. Dash: Diagnosis of drug resistant tuberculosis than traditional methods using LJ media with a turnaround time of 7 days for culture and drug susceptibility testing (C‑DST) for INH and RIF. It is cheap, simple and fairly accurate.[22] Biosafety level‑3 facilities are required if the plates are opened for further testing, such as for the confirmation of the identification of TB. Laboratories using MODS assay require a functioning biosafety cabinet, a safety centrifuge, an incubator, an inverted light microscope for observation of mycobacterial growth and supplemented liquid media (Middle brook 7H9 broth with OADC and PANTA). 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 MTB is detected in samples that contain the drug (i.e., RIF). When these assays performed on MTB culture isolates, they have shown high sensitivity and variable specificity, but the evidence is lacking about the accuracy when they are directly applied to sputum specimens.[23] It also requires high standards of biosafety and quality control. Luciferase reporter phages are genetically‑modified phages containing the flux gene encoding firefly luciferase. This catalyzes a reaction producing light in the presence of the luciferin substrate and adenosine triphosphate; light is only produced in the presence of viable mycobacteria. Detection of light released from viable mycobacteria can be achieved by a luminometer or photographic film within 2‑4 days from culture.[24] The luminometer readout is more sensitive and enables quantification of results while the use of Polaroid photographic film offers a lower‑tech approach with lower sensitivity. Rapid molecular methods for diagnosis of drug resistant TB Since the publication of genome details of MTB H37Rv strain in 1998, these have been utilized in development of nucleic acid amplification (NAA) tests for diagnosis of drug resistant TB. A number of NAA tests are now available, manual and automated, commercial and in the house, with varying performance characteristics. Real‑time polymerase chain reaction (RT‑PCR) and line probe assays (LPAs) have been commercialized and widely used in clinical laboratories merit special mention, detailed below. RT‑PCR Molecular tools are based on identification of specific mutations responsible for drug resistance, which are detected by the process of NAA in conjunction with electrophoresis, sequencing or hybridization. Direct sequencing techniques such as RT‑PCR that uses wild‑type primer sequences to amplify genes and enable the use of specific probes to identify mutations. Recently introduced semi‑quantitative nested RT‑PCR, which integrates and automates sample processing and simultaneously detects MTB and RIF resistance within the single‑use disposable cartridges. A study examined 1,730 patients with suspected drug‑sensitive or MDR pulmonary TB across Peru, Azerbaijan, South Africa and India. There was sensitive detection of MTB and RIF resistance directly from untreated sputum in < 2 h with minimal hands‑on time.[25] The WHO has recently supported the use of this system as an initial diagnostic test in respiratory specimens of patients with high clinical suspicion of having TB or who could be MDR.[26] These tests are very expensive require adequate maintenance and calibration of the equipment. Further, the specificity for RIF resistance in populations where MDR‑TB is rare requires confirmation by repeat testing and thus increases cost. LPAs LPAs are a family of novel DNA strip tests that use both PCR and reverse hybridization methods. In these assays, a specific target sequence is amplified and applied on nitrocellulose membranes. Specific DNA probes on the membrane hybridize with the amplified sequence applied on it. Color conjugates make the amplified target sequences appear as colored bands. These tests have been designed to identify MTB and simultaneously detect genetic mutations related to drug resistance both from clinical samples as well as culture isolates. These tests able to identify MTB complex and simultaneously detect genetic mutations in the rpoB gene region related to rifampin resistance. The LPA strip consists of 10 oligonucleotide probes: One is specific for the MTB complex, five are partially overlapping wild‑type probes that span the region at positions 509‑534 of the rpoB gene and four probes are specific for amplicons carrying the most common rpoB mutations (D516V, H526Y, H526D and S531L). According to a recent review, the sensitivities of the LPA are above 90% for clinical isolates with 99‑100% specificity [Table 1]. It identifies MTB complex and simultaneously detects mutations in the rpoB gene as well as mutations in the katG gene for high‑level INH resistance. These tests can also detect mutations in the inhA gene for low‑level INH resistance and mutations in the gyrA, rrs and embB genes for 2nd line anti‑tubercular drugs fluoroquinolones, aminoglycosides and ethambutol respectively [Table 2].[32] The newly developed assay may represent a reliable tool for detection of fluoroquinolones, amikacin, capreomycin and ethambutol resistance. LPA strip can use culture isolates and smear positive sputum as specimen and provide results in 1‑2 days. A recent laboratory evaluation study from South Africa estimated the accuracy of the GenoType MTBDRplus assay performed directly on AFB smear‑positive sputum specimens.[33] It showed high sensitivity, specificity, positive and negative predictive values for detection of RIF and INH resistance [Table 1]. However, a meta‑analysis on this assay found that sensitivity estimates for INH resistance were comparatively modest.[33,34] In general, LPAs are expensive and require dedicated equipments, reagents and facilities. Molecular drug resistance testing has technical limitations, i.e., LPAs can detect only well characterized drug resistance alleles, not every drug resistance allele can be discriminated via current tests and silent mutations (which do not confer drug resistance) can be detected by probes leading to misclassification of drug resistance. In addition, molecular tests cannot determine the proportion of drug resistant bacteria within a mixed population of cells (i.e., wild type and drug resistant). Cross contamination with amplicons generated from previous tests can be problematic especially when the tests have been employed in laboratories without appropriate staff training and quality control.  198 Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3
  • 5. Dash: Diagnosis of drug resistant tuberculosis Table 1: Performance of methodologies used to diagnose drug resistant tuberculosis Drug resistance Methodology and products Sensitivity % (95% CI) Specificity % (95% CI) Reference number Phenotypic methods Automated liquid MB/BacT for rifampicin based CST systems ALERT resistance Versa TREK 100 100 MGIT 960 99.8 99.2 NRA Griess method 99 100 [28] MODS 98 (94.5‑99.3) 99.4 (95.7‑99.9) [29] Phage based assay FASTPlaque‑response (commercial) FASTPlaque‑Response (in‑house) Luciferase reporter phage Phenotypic methods for isoniazid resistance Automated liquid based CST systems MB/BacT ALERT 100 95 [27] Versa TREK 95.5 99 MGIT 960 97.1 100 NRA Griess method 94 100 [28] MODS 97.7 (94.4‑99.1) 95.8 (88.1‑98.6) [29] Molecular methods for rifampicin resistance RT‑PCR GeneXpert® MTB/RIF 98 (97‑99) 99 (98‑99) [31] Line probe assay INNO‑LiPA Rif. TB 93 (89‑96) 99 (99‑100) MTBDRplus 97 (92‑99) 98 (95‑99) Molecular methods for isoniazid resistance Line probe assay MTBDRplus 77 (69‑83) 99 (97‑100) Molecular methods for second‑line drug resistance Line probe assay MTBDRsl (fluoroquinolones)W Amikacin 90 (81‑96) 100 (98‑100) Kanamycin 83 (59‑96) 100 (96‑100) Capreomycin 87 (77‑94) 99 (96‑100) Ethambutol 60 (52‑68) 98 (94‑100) CST – Culture and susceptibility testing; NRA – Nitrate reductase assay; MODS – Microscopic observation drug susceptibility; RT‑PCR – Real time polymerase chain reaction; CI – Confidence interval; MTB – Mycobacterium tuberculosis; TB – Tuberculosis; RIF – Rifampicin; MGIT – Mycobacteria growth indicator tube Diagnostic testing algorithm for drug resistant TB Laboratory policies and testing of patients suspected of having drug‑resistant TB depend on the local epidemiology, local treatment policies, the existing laboratory capacity, specimen referral and transport mechanisms and human and financial resources. Mycobacterial culture (solid or liquid) and identification of MTB provide a definitive diagnosis of TB. Culture also provides necessary isolates for conventional DST. Thus LPAs, RT‑PCRs, MODS and NRA can be used in conjunction with culture (solid and liquid) and DST [Figure 1].[35] Revised national tuberculosis control program and diagnosis of drug resistant TB The RNTCP plans to strengthen laboratory capacity for MTB C‑DST and LPA across India. To date, 35 RNTCP accredited labs including 14 LPA and 4 liquid culture labs in public and private sectors are serving patients while another 30 labs 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.[36] In a policy statement released in June 2008, the WHO endorsed the use of LPA for rapid screening of patients at risk of MDR‑TB and recommended the use 99 98 [27] 95.5 (92.2‑97.4) 95 (91.3‑97.2) [30] 98.5 (96.1‑99.4) 97.9 (94.8‑99.2) 99.3 (49.1‑100) 98.6 (92.5‑99.8) 85 (78‑91) 100 (97‑100) of LPAs only on culture isolates and smear‑positive sputum specimens. It is not recommended as a complete replacement for conventional C‑DST.[37] As of January 2012, diagnosis of XDR‑TB can only be confirmed at three laboratories in India, which are quality assured for second line anti‑TB DST of flouroquinolones and injectable drugs. These are the National Reference Laboratories (NRL) of tuberculosis research centre/ national institute for research in tuberculosis (TRC/NIRT) Chennai, National Tuberculosis Institute (NTI) Bangalore and LRS Institute, New Delhi. Routine fluoroquinolone and injectable DST (i.e., XDR‑TB diagnosis) on all MDR‑TB 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 C‑DST laboratories used by RNTCP. Capacity building for second line DST is being undertaken through these NRLs.[26] Prevention and Control of Drug Resistant TB The principles of TB control are important for the prevention of drug resistant TB; these include prompt case detection, provision of curative treatment and prevention of transmission. Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3 199 
  • 6. Dash: Diagnosis of drug resistant tuberculosis Table 2: Summary of technologies used to diagnose drug resistant tuberculosis Technologies Summary of technologies Estimated costs Description Product Training Infrastructure Equipment Consumable New solid culture methods Measures nitrate reduction to indicate RIF and INH resistance Non‑commercial (nitrate reductase assay) Simultaneously detects TB and indicate RIF and INH resistance Non‑commercial (thin layer agar culture) Liquid culture Broth based manual and automated culture system; can be configured for DST Commercial BacT/ALERT Versa TREK MGIT 960 MODS Manual liquid culture technique uses inverted microscope to detect TB Non‑commercial Extensive BSL 2 to RT‑PCR Allows automated sample processing, DNA amplification detection of TB and screening for RIF resistance Commercial (GeneXpert® MTB/RIF) GeneXpert detect mutations in 81‑base pair core region of rpoB and in codon 315 of katG genes respectively Molecular line probe assay Strip test simultaneously detects TB genetic mutations for RIF/INH resistance Commercial (INNO‑LiPA, MTBDR, MTBDRplus, MTBDRsl) INNO‑LiPA targets the 16S‑23S DNA spacer region for mycobacteria and mutations in the rpoB gene region related to RIF resistance MTBDR detect the 23S rRNA genes for mycobacteria and mutations in rpoB as well as katG genes for INH resistance MTBDRplus target in addition inhA for low level INH resistance MTBDRsl detects in addition gyrA, rrs and embB for fluoroquinolones, aminoglycosides and ethambutol respectively RIF – Rifampicin; INH – Isoniazid; MODS – Microscopic observation drug susceptibility; RT‑PCR – Real time polymerase chain reaction; TB – Tuberculosis; DST – Drug susceptibility testing. Basic laboratory (no specialized biosafety equipment). BSL 2 – Biosafety level 2 (specialized biosafety equipment required, such as biosafety cabinet); BSL 3 – Biosafety level 3 (biosafety cabinet and other safety equipment required. Controlled ventilation system that maintains a directional airflow into the laboratory also required); MGIT – Mycobacteria growth indicator tube; MTB – Mycobacterium tuberculosis; DNA – Deoxyribonucleic acid; rRNA – Ribosomal ribonucleic acid The WHO’s “Stop TB”[38] directly observed treatment short course (DOTS) strategy includes supervision and support of treatment, although there is little evidence that directly observed treatment alone improves cure rates.[39] Ineffective drug treatment is a strong risk factor for acquired drug resistance and proper administration of anti‑tubercular drugs is critical to reduce this risk. An enhanced DOTS program, DOTS‑plus has been developed for managing MDR‑TB in resource limited settings.[40] This program recommends additional facilities for C‑DST for detection of drug resistant TB and provision of appropriate second line anti‑tubercular drugs. Moderate BSL 2 to BSL 3 Low Medium Extensive BSL 2 to BSL 3 Low Medium Extensive (3 weeks) BSL 3 High High BSL 3 Medium Medium Minimal Basic laboratory High High Moderate (3 days) BSL 2 to BSL 3 High High As most of the resistance arises from either inadequate or inappropriate treatment of active disease, prevention of active disease indirectly prevents drug resistance. Contact tracing including family members and health‑care workers, those who are at risk of acquiring drug resistant TB[41] is one of the fundamental measures to detect active disease, must be aligned to all health services. Prevention of transmission in healthcare settings is difficult in places where resources are limited with no isolation facilities; one approach is to manage cases with similar resistance profiles in segregated groups. However, simple, low‑cost interventions, such as  200 Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3
  • 7. Dash: Diagnosis of drug resistant tuberculosis $)%VPHDU0,52623@ 1HJDWLYH 8/785( 6ROLGRU/LTXLG
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  • 12. ;'5 6XVFHSWLEOH 1RW0'5UHVLVWDQWRWKHUGUXJV 6XVFHSWLEOH Figure 1: Diagnostic algorithm for use of conventional microscopy, culture (solid or liquid), molecular assays and drug susceptibility testing methods. AFB – Acid fast bacilli, LPA – Line probe assay, PCR – Polymerase chain reaction, NRA – Nitrate reductase assay, MODS – Microscopic observation drug susceptibility assay, MDR – Multidrug‑resistant, XDR – Extensively drug‑resistant opening windows and doors for adequate ventilation, can reduce transmission of TB.[42] Conclusion Successful control of drug resistant TB globally will depend on strengthening TB control programs, wider access to rapid C‑DST along with emerging molecular diagnostic technologies and provision of effective treatment. Rapid and accurate diagnosis of drug resistant TB will require massive scaling‑up of C‑DST capacity and simultaneous use of 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 TB 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. Therefore, political and fund provider commitment is essential to curb the spread of drug resistant TB. References 1. World Health Organization. Tuberculosis global facts 2011/2012, 2011. Available from: http://www.who.int/tb/publications/2011/ factsheet_tb_2011.pdf. [Cited on 2012 Jun 26]. 2. Government of India. TB India 2011 RNTCP: Annual Status Report, 2011. Available from: http://planningcommission.nic.in/reports/ genrep/health/RNTCP_2011.pdf. [Cited on 2012 Jun 26]. 3. World Health Organization. Towards universal access to diagnosis and treatment of multi‑drug resistant and extensively resistant tuberculosis by 2015: WHO progress report 2011, 2011. Available from: http://whqlibdoc.who.int/publications/2011/9789241501330_ eng.pdf. [Cited on 2012 Jun 26]. 4. Schaaf HS, Moll AP, Dheda K. Multidrug‑and extensively drug‑resistant tuberculosis in Africa and South America: Epidemiology, diagnosis and management in adults and children. Clin Chest Med 2009;30:667‑83, vii. 5. Wallis RS, Pai M, Menzies D, Doherty TM, Walzl G, Perkins MD, et al. Biomarkers and diagnostics for tuberculosis: Progress, needs, and translation into practice. Lancet 2010;375:1920‑37. 6. Nathanson E, Nunn P, Uplekar M, Floyd K, Jaramillo E, Lönnroth K, et al. MDR tuberculosis – Critical steps for prevention and control. N Engl J Med 2010;363:1050‑8. 7. Prasad R. Management of multi‑drug resistant tuberculosis: Practitioner’s view point. Indian J Tuberc 2007;54:3‑11. 8. World Health Organization. Multidrug and extensively drug-resistant TB (M/XDR‑TB): 2010 global report on surveillance Journal of Postgraduate Medicine July 2013 Vol 59 Issue 3 201 
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