ACTA SCIENTIFIC MICROBIOLOGY (ISSN: 2581-3226)

Volume 3 Issue 8 August 2020

Review Article

An Overview of the Drug Susceptibility Testing for Tuberculosis

Vikas Jha1*, BS Ajit Kumar2, Sampurna Panigrahi3, Gayatri Nair3 and

Nikitha Bangera3
1
Received: June 30, 2020
National Facility of Biopharmaceutical, Mumbai, India
2
Published: July 25, 2020
V.P.M's R. Z. Shah College Mulund, India
3 © All rights are reserved by Vikas Jha., et al.
School of Biotechnology and Bioinformatics, DY Patil deemed to be University, CBD
Belapur, Navi Mumbai, India
*Corresponding Author: Vikas Jha, National Facility for Biopharmaceuticals,
Mumbai, India.
DOI: 10.31080/ASMI.2020.03.0653

Abstract
There in an increase in demand for reliable, inexpensive and rapid drug susceptibility assay because of expanding anti-tuberculosis
drug-resistant Mycobacterium tuberculosis necessitating the need for appropriate treatment. One of the major challenges being
faced is the lack of resources and the limiting of reliable drug susceptibility test meeting acceptable levels only for isoniazid
and rifampicin. In this article, an overview of different drug susceptibility testing and assays is detailed and the advantages and
disadvantages highlighted. It discusses the perspective on conventional methods which have paved the way for modern DSTs along
with the advancements made in the conventional methods.
Keywords: Drug Susceptibility; Anti-tuberculosis; Mycobacterium tuberculosis; Conventional DST’s

Introduction
Tuberculosis is one of the top 10 causes of mortality world-
wide and was declared a global emergency nearly two and a half
decades ago [1,2]. The causal agent being Mycobacterium tubercu-
losis, is a rod-shaped, acid-fast bacterium, usually transmitted by
aerosol infection and is the leading cause of death from a single
infectious agent [2,3]. An estimation of nearly 10.0 million (range,
9.0 - 11.1 million) people have fallen sick globally due to tuber-
culosis in 2018 though the number has remained fairly stable in
recent years. There is a decline of 1.6% per year in the period 2000
- 2018 and 2.0% between 2017 and 2018 in the tuberculosis in-
cidence rate, the cumulative reduction being 6.3% between 2015
and 2018.

Many cases of active tuberculosis are still not detected and

are not treated in a timely manner, especially in developing coun- Figure 1: Global tuberculosis report 2019, WHO
tries. India and Indonesia are the two countries which rank first (Global report of cases).
and third worldwide in terms of estimated incident cases per year

Citation: Vikas Jha., et al. “An Overview of the Drug Susceptibility Testing for Tuberculosis". Acta Scientific Microbiology 3.8 (2020): 47-56.
An Overview of the Drug Susceptibility Testing for Tuberculosis

48

are primarily responsible for the increase in the global notifica- The expanding problem of resistance in Mycobacterium tuber-
tions of tuberculosis cases since 2013, observing an increase from culosis has driven a requirement for quick, cheap, and easy tech-
1.2 million cases to 2.0 million between 2013 and 2018 solely in niques to detect resistance. Various methods have been developed
India [2]. over the years to make susceptibility testing feasible. The deter-
mination of drug susceptibility of Mycobacterium tuberculosis can
be ascertained by the examination of a medium comprising an
anti-tuberculosis drug for growth or metabolic inhibition, or via
detection of the mutation in genes associated to drug action at the
molecular level. On the basis of technical standpoint, the detection
of drug susceptibility by growth or metabolic inhibition can be fol-
lowed out by means of observing drug-free and drug-containing
media consisting macroscopic growth or by metabolic activity or
products detection and measurement. It can also be carried out by
lysis with mycobacteriophage or genetic mutation detection avail-
ing molecular techniques [5]. Molecular tests promise a more rapid
drug resistance detection with a disadvantage of expenses.

Mycobacterium tuberculosis isolates can take weeks to months

Figure 2: Global tuberculosis report 2019, WHO (India). to completely define the drug resistance pattern via conventional
phenotypic methods due to the very slow growth of this bacteri-
um. Drug susceptibility testing (DST) using the Lowenstein Jensen
Prompt identification of new incidents and rapid implementa-
(LJ) include proportion method, resistant ratio method and the
tion of effective treatment regiments to interpret transmission are
absolute concentration method which for major anti-tuberculosis
two critical components of tuberculosis transmission. The stan-
drugs is fairly well standardised with clinical samples. It is a time-
dard treatment combines isoniazid (INH), rifampin (RIF), pyrazin-
consuming phenotypic DST method, taking up to 6 - 8 weeks to re-
amide (PZA) and ethambutol (EMB) which are the four first-line
cover Mycobacteria or discriminate negative samples [6].
antibiotics, which when properly administered renders patients
suffering from tuberculosis noncontagious. Inefficient treatment Egg- or agar-based media for conventional culture methods are
can lead to drug resistance bacilli (acquired resistance) and those still frequently employed by many countries. Proportion method
resistant organisms can be transmitted to other individuals (pri- amid conventional methods is the most favoured option, but in
mary resistance). matters of frequency, absolute concentration method is also often
used due to its technical simplicity of inoculum preparation and re-
Multidrug-resistant (MDR) TB, i.e. bacilli resistant to the first-
sult interpretation.
line drugs isoniazid and rifampin (rifampicin) has garnered much
attention over the years along with the emergence of the more Numerous techniques have been devised for early detection of
deadly extensively drug-resistant tuberculosis (XDR) [4]. growth inhibition to shorten the turnaround time (TAT) and make
case management more convenient. The most often used systems
Drug-resistant tuberculosis continues to be a public health
are detection oare carbon dioxide production detection systems,
threat. In 2018, there were about half a million new cases of ri-
such a BACTEC 460 or MB/Bact, and consumption of oxygen, such
fampicin-resistant tuberculosis (of which 78% had multidrug-
as Mycobacteria Growth Indicator Tube (MGIT). BACTEC 460 TB
resistant tuberculosis). Extensively drug-resistant TB (XDR-TB) is
despite reducing TAT to 1 - 2 weeks it places higher demands on
defined as MDR-TB plus resistance to at least one of the fluoroqui-
equipment to be routinely used in poor-resource countries also
nolones and one of the injectable agents (second-line drugs) used
utilising substrates and expensive technology [8,9].
in MDR-TB treatment regimens [2].

Citation: Vikas Jha., et al. “An Overview of the Drug Susceptibility Testing for Tuberculosis". Acta Scientific Microbiology 3.8 (2020): 47-56.
An Overview of the Drug Susceptibility Testing for Tuberculosis

49

Colorimetric methods is another in the developmental stage of parallel sets and one drop of bacillary suspension is spread on
DST relying on oxidation-reduction indicators like resazurin or tet- the surface of each drug-containing slopes of media of a series of
razolium bromide [10]. Phage based technique is a method devel- concentration, with similar procedure followed with H37Rv strain.
oped combining phenotypic assays and deploying bacteriophage All tubes are incubated for 4 weeks at 370C and weekly observed.
to introduce into any viable isolate of M. tuberculosis the firefly The growth is defined as the presence of 20 or more colonies in the
luciferase gene (Fflux) [8]. Detection of a low-level multiplication drug-containing media. The isolates are resistant when the growth
of M. tuberculosis by particle-counting immunoassay can also cur- appears on the media containing a given drug concentration in
tail TAT. These techniques are quite difficult to be implemented in which control strain is susceptible. DST critical concentrations for
developing countries and countries where these are required in- second-line drugs have not yet been adequately validated for the
dispensably due to the drawbacks of being expensive, technically resistance ratio and absolute concentration methods.
complex and absence of appropriately trained human resources.
Among the three methods, the proportion method is the most
Detection of gene mutations related to resistance using mo- commonly used method worldwide [13]. In the proportional meth-
lecular techniques including hybridization of amplified gene seg- od, Löwenstein-Jensen slants with critical drug concentrations are
ments or other PCR-based methods can be utilized but as primary prepared for different anti-TB drugs. Parallel preparation of drug-
amplification is required for molecular techniques when followed free control media is carried out. The standardised culture suspen-
on a routinely basis for long periods of time false results can be sion is diluted in sterile distilled water in different dilutions. From
generated due to contaminating amplicons and/or chromosomal each dilution a drug-containing media is inoculated with one loop-
DNA [5]. full of bacillary suspension as well as controls of plain LJ media are
inoculated with the respective diluted bacillary suspension. The
Phenotypic method slopes are observed after 28 days of incubation in 370C. Any colo-
Solid culture nies growing on drug-containing medium inoculated with the 10-1
dilution that equal or more the number of colonies growing on the
In the 1960s, Cannetti., et al. described the first DST method for
control medium inoculated with the 10-3 dilution represents 1% or
M. tuberculosis, classifying the test as direct and indirect where the
more of the test population. If the calculation was 1% or more then
direct test involves the sputum homogenate or other pathological
it is interpreted as resistant [14].
material cultured directly on drug-containing medium, though the
results obtained are not considered reliable. In indirect test the Liquid culture
primary diagnostic culture was inoculated in a drug-containing In comparison to solid culture liquid culture reduces the turn-
medium. This was further classified into three main categories: around time significantly for results and are around 10% more sen-
(a) the absolute-concentration method; (b) the resistance-ratio sitive than solid media culture. The confirmation can be obtained
method; and (c) the proportion method [11]. within two weeks and can be used for susceptibility testing for both
first-line and second-line drugs [13].
According to the method developed at the National Institute for
Public Health and the Environment (RIVM), Bilthoven, The Nether- BACTEC 460
lands in the absolute concentration method, a concentration series
BACTEC 460 is a semi-automated, well-established broth-based
of anti-tuberculosis drugs are added to 7H10 medium distributed
method providing rapid detection of mycobacterium in a closed
in 25-well plates. Furthermore, to check the sizes of the inocula
system. The introduction of the BACTEC 460 TB System revolution-
and to compare the mycobacterial growth levels control is includ-
ized laboratory testing for mycobacteria and has established itself
ed in the presence of different concentrations of anti-tuberculosis
as the gold standard for culture and susceptibility testing [15].
drugs in a proportional manner. The determination of MIC can also
be ascertained by this method [12]. The major drawback harboured by this system is the radiomet-
ric method used to detect the mycobacterial growth. BACTEC 460
In the resistance-ratio method, media containing two-fold di-
generates radioactive waste due to usage of radioisotopes making
lution of the primary anti-tuberculosis drugs are prepared as

Citation: Vikas Jha., et al. “An Overview of the Drug Susceptibility Testing for Tuberculosis". Acta Scientific Microbiology 3.8 (2020): 47-56.
An Overview of the Drug Susceptibility Testing for Tuberculosis

50

disposal pose a considerable logistical problem as well an increase Numerous low-cost colorimetric assays described are mainly
in expenses [16]. Another disadvantage of this method is its re- based on the reduction of a coloured indicator added to the cul-
quirement for plating thus increasing the duration of incubation ture medium after the exposure of M. tuberculosis in in-vitro to dif-
thereby elevating the risk of cross contamination [15]. Another ferent antibiotics. The detection of resistance is indicated by the
disadvantage of this method is its requirement for plating thus in- change in colour of the indicator, which is directly proportional to
creasing the duration of incubation thereby elevating the risk of the number of viable Mycobacteria in the medium. Different growth
cross contamination. indicator have been used for the assay such as tetrazolium salts:
XTT [2,3-bis- (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-
BACTEC MGIT 960 5-carboxanilide] and MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphe-
It is a fully automated, nonradiometric instrument which de- nyltetrazoliumbromide] and the redox indicators Alamar blue and
tects the growth of mycobacterium by exploiting the fluorescence resazurin [19].
of an oxygen sensor. In the study conducted by Enrico Tortoli., et
al. 1999 the BACTEC MGIT 960 performance was compared to Colorimetric methods of drug susceptibility testing produce
those of the radiometric BACTEC 460 instrument and egg-based results more quickly than standard culture methods and are less
Lowenstein-Jensen medium. The shortest time for detection was costly than molecular methods. The average time to have first re-
obtained with the BACTEC MGIT 960 system though the contami- sults was between 7 and 14 days compared with the reference
nation rate observed was intermediate (10.0%) to that of radio- standard method which takes 3 - 6 weeks. An example being the
metric system (3.7%) and the egg-based medium (17.0%). resazurin microtiter assay (REMA), which is based on a redox reac-
tion which induces a blue to pink colour change in the presence of
This system is comparable to BACTEC 460 eliminating two core live bacteria [20].
problems posed by the system one being reducing needle punc-
ture risk and overcoming the risk of disposal of radioactive waste. Colorimetric nitrate reductase-based antibiotic susceptibility or
Further the advancements in automation of the MGIT 960 has led CONRAS is a nitrate reductase-based test (NRA) for M. tuberculosis
to easy and continuous monitoring of the positive fluorescence in Middlebrook 7H9 broth cultures. It is an indirect assay carried
depending on the bacterial growth. It is a non-invasive and elimi- out on a solid media, with the media being supplemented by potas-
nates the requirement of labour thus dismissing the possibility of sium and sodium nitrate at 1000 mg/l concentration to function as
reading difficulties during the visual assessment of the tubes. The a growth indicator. This method employs the reduction of nitrate to
susceptibility can be automatically determined with the help of the nitrite by M. tuberculosis using the nitrate reductase enzyme which
threshold algorithms [17]. Although this system is faster than LJ is detected by a reagent (Griess reagent) which turns a pink-purple
culture the expenses are relatively high restricting their usage in colour [21].
low-income, heavy burden regions. Although this system is faster
These tests, however, have limitations such as Mycobacteria
than LJ culture the expenses are relatively high restricting their us-
other than M. tuberculosis can produce cord factor, in MTT assay
age in low-income, heavy burden regions.
isoniazid can interfere with formazan production giving rise to
Colorimetric method false-resistant results. In these tests the use of liquid medium in
It is a quantitative measurement of the susceptibility of M. tu- a micro-titre plate format could also prove to be disadvantageous
berculosis against anti-tuberculosis agents. Diagnostic test for tu- due to possible contamination between wells as well as for being a
berculosis is either expensive (molecular methods and automated biohazard [22].
liquid-based culture systems) or slow (culture on solid media and
Microscopic observation drug susceptibility
biochemical tests). Therefore, an alternative method was devel-
Developed by a research team in Lima, Peru microscopic ob-
oped which is rapid, quantitative, and nonradiometric and does
servation drug susceptibility (MODS) is a highly sensitive/specific
not require the use of instrumentation [18].
assay for rapid and economic detection of Mycobacterium tubercu-

Citation: Vikas Jha., et al. “An Overview of the Drug Susceptibility Testing for Tuberculosis". Acta Scientific Microbiology 3.8 (2020): 47-56.
An Overview of the Drug Susceptibility Testing for Tuberculosis

51

losis and DST directly from sputum. This is a test reserved to two The primary principle of LPA is based on the reverse hybridiza-
drugs isoniazid and rifampicin in regards to drug susceptibility tion of the DNA on a strip. The detection is based on the binding
testing. The workings of this test as explained by Linwei Wang., of amplicons (DNA amplification products) to probes targeting the
et al. is as follows, a 24-well plate with 4 wells allotted for each first and second-line agents affiliated to most common resistance
patient specimen is used, two of the wells contain RIF and INF and mutation and to probes targeting the complementary wild-type
the other two are drug-free. The plates are sealed after inoculation DNA sequence. The detection of mutation is carried out by observ-
and incubated M. tuberculosis is grown rapidly in liquid medium. ing the pattern of binding of amplicon and the lack of hybridization
The morphological characterization patterns specific to M. tuber- of the amplicons to the corresponding wild type probes.
culosis is employed for diagnosis following inoculation under an
inverted microscope. In case of the drug rifampicin, the mode of action of rifampicin is
by binding to the beta-subunit of the RNA polymerase (coded for by
MODS has the advantage of having the ability to be used as an rpoB gene) which in turn inhibits the protein transcription. Despite
absolute concentration technique and it detects difficult rifampi- there being more than 50 mutations characterised by automated
cin-borderline strains better than commercial liquid culture sys- DNA sequencing codons 516, 526, or 531 involve major mutations.
tems. RIF-resistant TB can also be considered as a good surrogate marker
for MDR-TB as more than 90% of RIF resistant TB is also resistant
Despite being quite affordable and an effective alternative for to INH. These advancements have therefore helped make further
testing sputum samples of TB-suspected individuals to existing development of various methods for rapid detection of RIF-resis-
gold standard liquid mycobacterial culture methods this method tance conferring mutations, one of them being line probe assay.
has its own drawbacks. This method has its shortcomings in re-
source-limited settings due to its concern regarding biosafety and The LPA kit consists of 10 oligonucleotide probes i.e. 5 of S
efficiency in handling a large number of samples. Concerns in asso- probes (overlapping wild-type) and 4 R probes (resistant geno-
ciation with biosafety are due to usage of liquid wells in MODs as- type) for detection of specific mutations and a specific probe for
say, there is a risk of aerosolization, spillage, cross-contamination, M. tuberculosis complex immobilised on nitrocellulose paper strips.
and occupational infection as there is manipulation carried out Direct clinical samples or extracted DNA samples are used to per-
of live liquid cultures, though sealed plates which do not require form LPA by PCR amplification of the RIF- resistance-determining
reopening might reduce this risk. This method also requires indi- region of the rpoB gene. The immobilised probes are then hybri-
vidual wells to be read manually posing a requirement of both time dised with biotinylated PCR products and the results determined
and human resources. There might also be difficulty about MOD’s by colorimetric method. If a positive signal is retrieved from the
ability to discern M. tuberculosis and non-tuberculosis mycobacte- wild-type S probe and negative from R probe the M. tuberculosis
rium [23,24]. isolate is considered RIF susceptible. RIF resistance can be deliber-
ated by the absence of one or more wild-type S probe or a positive
Molecular assays for drug resistance detection reaction obtained from one of the four R probes (Morgan, Kalantri,
Line probe assay Flores, and Pai, 2005).

The emergence of multidrug-resistant tuberculosis has ex- The first line-line probe assay GenoType MTBDRplus (referred
pressed a formidable challenge forcing researchers to come up to as GenoType MTBDRplus V1) was endorsed by the World Health
with swifter methods of detection due to the complex diagnostic Organisation in the year 2008 for the rapid detection of multidrug-
and treatment obstacles. LPA is a rapid drug susceptibility detec- resistant tuberculosis. Newer versions have been subsequently de-
tion test approved by WHO for first and second-line agents which veloped since 2011 of the LPA technology, including) the GenoType
can be used for testing of culture isolates (indirect testing) and MTBDRplus version 2 (referred to as GenoType MTBDRplus V2),
also for smear microscopy positive specimens of acid-fast bacilli and (ii) the Nipro NTM+MDRTB detection kit 2 (referred to as “Ni-
(AFB) as well as including both smear-positive and negative spu- pro”, Tokyo, Japan).
tum specimens.

Citation: Vikas Jha., et al. “An Overview of the Drug Susceptibility Testing for Tuberculosis". Acta Scientific Microbiology 3.8 (2020): 47-56.
An Overview of the Drug Susceptibility Testing for Tuberculosis

52

Regardless of being approved by WHO, line probe assay has LATE-PCR with lights-on/lights-off probes
its own limitations. Resistance cannot be ruled out entirely even
in the existence of all WT probes as mutations which are respon- Though the above mentioned diagnostic techniques are well
sible for conferring resistance are outside the region covered by established and approved by WHO, newer technologies are al-
the test thus necessitating auxiliary phenotypic DST to provide ways being invented and tested to overcome existing drawbacks
a full assessment. Identification of mutations can be inferred by and to increase efficiency. One such new technique is LATE-PCR
the specific MUT probes or by the absence of amplicons binding to with Lights-On/Lights-Off Probes. This PCR technique has been
wild type probes. However, the presence of synonymous and non- described as an enhanced non-symmetric PCR technique (uses
synonymous mutations (e.g. phylogenetic mutations) could cause limiting and excess primers to generate larger amount of single-
systematic errors. Another one of the shortcomings is the inability stranded amplicons). The technique uses two pairs of Lights-On/
of the test to detect resistant bacteria if the resistant population is Lights-Off probes of the same fluorescent colour to detect mutations
less than 95% of the total bacterial population. The cost of the LPA in specific regions of the generated single-stranded amplicons. The
kit also renders the test impractical for widespread use in those Lights-On probe is labelled with a fluorophore and quencher while
regions of the world most affected by MDR-TB and most in need of the Lights-Off probe is labelled only with a quencher. The Lights-
a method for its rapid diagnosis [25]. Off probe binds to a sequence adjacent to the Lights-On probe and
absorbs the energy from the fluorophore. Since the probes have
Real time PCR assays a short length, variance of even one base pair results in different
Xpert MTB/RIF fluorescent signatures. This is useful to assess any mutations in
the DNA. As the signal from each Lights-On probe is extinguished,
Another DST approved by WHO in 2011 for the initial diagnosis multiple probe pairs of the same colour or different colours can be
of MDR-TB suspected individuals is the Xpert MTB/RIF [26]. The utilized to analyse sequences several hundred nucleotides in length
Xpert MTB/RIF is a fully automated, cartridge based, heminested [32].
real-time polymerase chain reaction (PCR) analysis. The cartridge
is manufactured from plastic, is multichambered and contains all This new technique has been used to develop a new diagnostic
the reagents for sample processing and the subsequent real-time tool called FluoroType MTBDR VER1.0 by Hain lifesciences. This
PCR run [27,28]. The analysis is carried out on the GeneXpert, Ce- technique can detect mutations in rpoB, katG, and inhA regions
pheid platform which is a software-driven cartridge processor and in a single tube. It has the advantage of hands-on time, faster re-
integrated fluorescence-based quantitative thermal cycler [28,29]. sults (within 3h), no DNA contamination, and automatic result
interpretation when compared to GenoType MTBDRplus and has
In this assay, amplification of aforementioned rpoB gene is comparable sensitivity and specificity of RIF resistance detection
carried out to analyze the region for mutations in the rifampin to Genotype MTBDRplus and Xpert MTB/RIF. However, sensitivity
resistance-determining region (RRDR). The cartridge contains five and specificity of INH resistance detection is lower than GenoType
molecular beacons for detecting these mutations. If at least two MTBDRplus [33]. The tool has been further developed to produce a
of the five probes are positive, then the samples are positive for newer model called FluoroType MTBDR VER2.0 [34].
the presence of M. tuberculosis and failure of one or more beacons
to hybridize with the rpoB amplicon can be inferred as rifampicin Sequencing
resistance [27]. Susceptibility testing has undergone a prodigious innovation
with its leap to molecular methods of resistance detection espe-
While there are several advantages of the Xpert MTB/RIF test cially in relation to the development of various molecular tests en-
such as short time required for the results and minimal techni- dorsed by WHO such as Xpert MTB/RIF and the MTBDRplus, MTB-
cal training required for handling the system [30], there are few DRsl, and Nipro line probe assays [35]. One of the best technologies
drawbacks too. One of the drawbacks is that the assay could over- for the rapid analysis of the genotype of an organism is sequencing.
estimate MDR-TB in regions of RIF mono-resistance since it only Targeted whole genome sequencing or Xpert Ultra (Cepheid) and
detects RIF resistance [31].

Citation: Vikas Jha., et al. “An Overview of the Drug Susceptibility Testing for Tuberculosis". Acta Scientific Microbiology 3.8 (2020): 47-56.
An Overview of the Drug Susceptibility Testing for Tuberculosis

53

Next Generation Sequencing (NGS) has the ability to provide both Phage amplified biologically assay (PhaB assay)
diagnosis for an individual patient and dispense mutation-specific A range of rapid molecular assays is available commercially for
information and phylogenetic data hence becoming a major break- the detection of mutation associated with multi-drug resistance
through in molecular biology [33,35]. M. tuberculosis. However, these assays are impractically expensive,
complex and have 90 - 95% predictive. Such assays also harbour
This allows an opening of attractive options of monitoring the
the drawback of being less predictive of resistance for drugs other
surveillance of drug resistance by examination of all loci, deliv-
than rifampicin and isoniazid. In the study conducted by IJ Eltring-
ering information in regard of changes both small or large in the
ham., et al. PhaB assay was extended to the drugs ethambutol, pyra-
genome, predict evolution of organism, detect epidemics thus
zinamide, streptomycin, and ciprofloxacin for 157 isolates in com-
helping in the management of patients with MDR-TB and provides
parison to resistance ratio method retrieving significantly better
guidance for suitable drug regimen selection [35,36].
correlations for ciprofloxacin, ethambutol, and pyrazinamide. The
Despite the availability of several nucleic acid based assays turn-around time for this assay is 2 - 3 days in comparison to the 10
each have their own sets of drawbacks such as the Genotype MT- days required for resistance ratio method [41].
BDR plus method has the ability to only detect MDR strains via
characterising mutations in katG gene (S315T), rpoB gene (D516V, This assay is based on the principle of the ability of M. tubercu-
H526Y/D, and S531L) and inhA promoters failing to detect XDR M. losis to prevent the inactivation of mycobacteriophage from phagi-
tuberculosis strains. The LPA method despite being both specific cidal chemicals thus protecting it. Therefore, when incubated with
and sensitive can never reach 100% sensitivity as many mutations drugs the mycobacteriophage can only be protected by viable M. tu-
associated with resistance are yet to be discovered [37]. LPA also berculosis that is, resistant bacilli. The viable bacilli protecting the
fails to detect some drug-resistant isolates mutations present in phage within are then lysed after the rapid cycle of infection and
the genome of a minor population in case of co-infection [38]. A replication of the phage. Lysis is observed as clear areas or plaques
study conducted has also found that WGS has better performance on a rapidly growing lawn of M. smegmatis [41,42].
than LPA in prediction of phenotypic DST in terms of sensitivity
The phage amplified biologically (PhaB) assay was developed
(94.2% vs. 84.0%) [39]. Therefore, various NGS-based kits are now
by Wilson., et al. using D29 to detect viable M. tuberculosis, demon-
available in the market one of them being the Ion Torrent Personal
strating the blocking ability of rifampicin to halt productive infec-
Genome Machine (PGM) a rapid (2 days) full length Mycobacterium
tion in sensitive strain but not in resistant [43].
tuberculosis gene analysis equipment following a novel protocol
developed by Life technology [37]. In comparison to the BACTEC system, the PhaB assay has about
one-sixth of the reagent cost. It also cuts down the requirement of
In middle- and low- income countries there are various chal- purchasing and maintenance of high-cost monitoring equipment.
lenges of large-scale sequencing. Sequencing as in such requires Further no hazard or costs are incurred in regard to handling of
robust software and database tools for the complete exploitation radioactive material [44-47].
of this technology, the experiments conducted, data acquiesced
and analysed needs specialised personnel and bioinformatics fa- Conclusion
cilities for proper functioning, adding to this is the high GC con- Drug-susceptibility testing is a widely practiced ritual followed
tent and repetitive nature of the genome of M. tuberculosis mak- to successfully treat tuberculosis patients and for the progression
ing sequencing quite challenging. This technology requires high of developing new and effective strategies to overcome the hurdles
amounts and high-quality DNA and determination of whether new faced due to the emergence of drug-resistant tuberculosis. Most
mutations can confer anti-TB drug resistance. The major drawback people who develop TB can be cured with a timely diagnosis and
for NGS in middle- and low- income countries is the heavy expens- treatment with antibiotics curtailing the onward transmission. As
es presented by NGS platforms [35,36,40]. a result, various methods have been developed over the years for

Citation: Vikas Jha., et al. “An Overview of the Drug Susceptibility Testing for Tuberculosis". Acta Scientific Microbiology 3.8 (2020): 47-56.
An Overview of the Drug Susceptibility Testing for Tuberculosis

54

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method developing to the contemporary molecular methods. The susceptibilities of Mycobacterium tuberculosis strains by us-
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9. Martin A., et al. “Colorimetric redox-indicator methods for the
deaths per year. This demands the instigation of newer methods
rapid detection of multidrug resistance in Mycobacterium tu-
with advantages allowing accurate, easy, swift and an economic
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upper-hand to the global population.
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