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The following molecular tests are presently being used in the routine diagno-
sis of TB.
S. S. Negi etal.
6.4.1 Loop-Mediated Isothermal Amplication (LAMP)
LAMP is a molecular test based on the isothermal nucleic acid amplication technique developed by Notomi etal. [24]. In LAMP, amplication is characteristically
carried out at one particular temperature to help avoid any requirement of a thermal
cycler, which is mandatory for PCR.It employs a set of four specic primers targeting the six sites of selective DNA fragment and a strand displacing DNA polymerse
to provide a rapid, sensitive, and specic amplication of the target DNA under
isothermal conditions without requiring expensive instruments and reagents. The
isothermal assay is an ultrasensitive nucleic acid amplication method and can produce large amounts of nucleic acid with minute quantities of DNA or RNA templates within a short time. The negative and positive results can easily be detemined
by the color differentiation of the assay.
Several LAMP-based tests have been developed and is being used in the diagnosis of TB.It has been recommended by the World Health Organization (WHO) in
August, 2016, as an alternate approach to smear microscopy for the diagnosis of
pulmonary TB in adults [25]. It is recommended by WHO as a follow-up test in
adult patients with signs and symptoms consistent with pulmonary TB, especially in
smear-negative sputum specimens(Table 6.2). Various targets, like gyrB, rrs, rimM,
IS6110, hspX, mpb64, and sdaA genes, have been used in TB-LAMP assay [26–29].
Various earlier studies have found it around 92% to 100% more sensitive for smearpositive samples than smear-negative samples, where sensitivity is reported between
52.1% and 90.3% [30–33]. The sentence appears ok as its adequetly emphasizing
that LAMP test has also shown promising result in extra pulmonary samples when
compared to conventional microscopy, solid and liquid culture. We thus request to
retain the sentence as such extrapulmonary samples where its sensitivity of 95.6%
was found statistically signicant to smear microscopy, solid and liquid culture
showing sensitivity of 17.4%, 65.2%, and 69.6% [34]. It is, however, found with a
shortcoming of exposure of reaction tubes to aerosol contamination, showing a
false-positive result. WHO accordingly has excluded all data obtained from extrapulmonary samples to keep them still under investigation [25] (Fig.6.1).
6.4.2 COBAS TaqMan TB
This test is developed by Roche Diagnostics, Tokyo, Japan, to replace the COBAS
amplicon assay [35]. This is a real-time PCR assay that amplies specic Taqman
probes and primers to amplify the specic segment of 16S rRNA to detect the presence or absence of MTB complex in the clinical specimen in around 2.5h for a
65°C for 40 min
Visual Detection
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Reaction Mix
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sample tube
reaction mix in
Addition of LAMP
for 10 min
Sample lysis
heating at 100°C
LAMP amplification at
Transfer sample
tube in water bath
Sample for
LAMP Test
Specimen
Clinical Sample
PositiveNegative
Fig. 6.1 Loop-mediated isothermal amplication (LAMP)
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Clinical Sample
TB Patient
Sample forloading in
COBAS
Fig. 6.2 COBAS TaqMan TB
S. S. Negi etal.
maximum of 48 samples. This test is approved by the US Food and Drug
Administration (FDA) for smear-positive and/or smear-negative pulmonary disease.
Although this assay is limited by the manufacturer to respiratory specimens only, it
has also been evaluated in extrapulmonary specimens and found its diagnostic sensitivity (63.6%) and specicity (94.6%) low for the extrapulmonary specimens in
comparison to pulmonary specimens having the sensitivity of 88.4% and specicity
of 98.8% [36, 37]. It was further found that this assay has higher sensitivity in
smear-positive specimens than in smear-negative specimens. A sensitivity parameter was reported between 96.9% and 98% in smear-positive specimens and between
34.9% and 79.5% in smear-negative samples [36, 38, 39] (Fig.6.2).
6.4.3 GeneXpert MTB/RIF Assay
This is an automated cartridge-based nucleic acid amplication test (CBNAAT)
developed by Cepheid Inc., Sunnyvale, California, United States of America. This
test simultaneously provides the result for the detection of MTB and rifampicin
resistance/sensitive pattern within 2h of the test, with the advantage of minimum
human intervention [40]. It has also been approved by WHO and US FDA [41,
42](Table 6.2). For this reason, this assay is also known as Xpert MTB/RIF test.
This assay has been reported as a sensitive test for the rapid diagnosis of TB in
comparison to conventional techniques [43]. Its sensitivity has been reported at
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Fig. 6.3 The rpoB gene 81bp RIF resistance determining region and Xpert MTB/RIF probes
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100% in smear-positive samples [43, 44]. The added advantage of the assay is providing results for the RIF sensitivity/resistance pattern to the tune of 94% sensitivity
and 98% specicity. The biggest advantage of THE GeneXpert has been observed
in smear-negative specimens, wherein it has also been found to be a good tool for
the diagnosis of TB.Earlier studies have shown a sensitivity of 47.8% to 73% in
smear-negative pulmonary specimens while a sensitivity of 28.2% to 73.2% in
smear-negative extrapulmonary specimens [43, 44]. It is, however, reported by
some studies as less sensitive than liquid culture in children and adults for the diagnosis of TB [45, 46]. The mutations yielding the expected probe reactions are in
different colors [47]. Further, in detecting rifampicin drug resistance, it has been
reported that since its probe is specically designed to detect mutation in 81bp
stretch of RRDR of the rpo B gene, it may fail to detect resistance against rifampicin
if the resistance-conferring mutation occurs outside this region, like I491F mutation. It was conrmed in one of the earlier studies that it only detected rifampicin
resistance in 38 (30%) multidrug resistance cases out of 125 cases in comparison to
DNA sequencing [48]. Thus, the inability of the GeneXpert to detect resistanceconferring silent mutations within and outside of the RRDR region may render the
treatment ineffective, causing a serious jolt to optimistic WHO TB elimination
(Fig.6.3).
6.4.4 Gene Xpert MTB/RIF Ultra Assay
To improve on these shortcomings, a new version of the test, known as Xpert Ultra,
has been developed. Xpert Ultra has been provided with two amplication targets,
IS6110 and IS1081, and by doubling the size of the DNA delivered to PCR and
covering the RRDR region from codon positions 507 to 533 by probes for the detection of 27 different mutations, the limit of detection has been reduced from
112.6CFU/mL in sputum for GeneXpert to 15.6CFU/mL in sputum for Ultra [49].
The sensitivity of Xpert Ultra has been reported signicantly higher than that of
Xpert MTB/RIF in both smear-negative, culture-positive TB patients and HIVinfected patients in earlier published studies. Xpert Ultra has been reported for
improved detection of rifampicin drug resistance by detecting mutants at codon
533, silent differentiated mutations at codon positions 513 and 514, and heteroresistant mutants that were earlier missed by susceptibility testing as well as
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MTB/RIFCartridge
startassay
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S. S. Negi etal.
TB Patient
Fig. 6.4 GeneXpert MTB/RIF Ultra assay
Clinical Sample
Mixsample reagentwith sample
andincubate15min at RT
Transfer into theopen
port of the Xpert
Insert the Xpert
MTB/RIFCartridge and
GeneXpert [49]. However, mutation outside the RRDR regions, like Ile491Phe, is
still a concern as its not detectable by Xpert Ultra [50]. Nonetheless, due to its
higher sensitivity in comparison with GeneXpert, Xpert Ultra is recommended by
WHO to replace GeneXpert to improve the diagnosis of TB, especially the paucibacillary forms of TB, like extrapulmonary TB, pediatric TB, or HIV-associated TB
[51](Table 6.2) (Fig.6.4).
6.4.5 Line Probe Assay (LPA)
Line probe assay (LPA) is a qualitative invitro diagnostic test based on DNA-STRIP
technology for the identication of the MTB complex and its resistance to RIF
(mutations within the rpoB gene) and INH (mutations within the katG gene and the
inhA promoter) [52]. The LPA test includes DNA extraction, master mix prepara-
tion and addition, multiplex amplication with biotinylated primers, and detection
with reverse hybridization. The test is an aid in the rapid diagnosis of MDR-TB,
which is a prerequisite for the appropriate treatment initiation [53, 54]. Resistance
to rifampicin and isoniazid diagnosis is based on detected probe reactions for each
individual mutation. Hence, mutated strains with low-level resistance and double or
triple mutations, including noted delayed reactions, problems of hybridization, partial inhibition of color development, and mutations outside the region from RRDR,
are not diagnosed [11, 55].
To characterize rst-line drug-resistant MTB bacilli, WHO has endorsed the
existing reverse hybridization-based line probe assay, namely the GenoType
MTBDRplus assay for drug resistance [11](Table 6.2). The drug resistance diagno-
sis by this assay depends on the amplied gene regions containing resistanceassociated mutations determined by reverse hybridization to the immobilized
mutated and wild-type sequences on the strip. In the presence of uncommon or rare
mutations, the MTBDRplus assay shows its limitation during diagnosis. The
GenoType MTBDRplus works on a very short piece of a targeted gene, which leads
to failure in the proper diagnosis of resistant or susceptible MTB, mainly when
novel or rare nonsynonymous mutations are outside the targeted drug resistance
n
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Chromogen
Colour Reactio
Nitrocellulose
Paper
Fig. 6.5 Line probe assay (LPA)
Alkaline
Phosphate
Streptavidin
Biotin
Biotin-labelled
single stranded
amplified target
DNA Probe
Immobilized DNA
Probe on paper strip
regions. Nucleic-acid-based assays, such as the Genotype MTBDRPlus assay, have
failed to differentiate synonymous and nonsynonymous mutations that may or may
not lead to phenotypic drug resistance [56].
GenoType MTBDRsl provides additional information on MDR strains to detect
and characterize the resistant strains against second-line drugs such as uoroquinolones (FLQ) and second-line injectable drugs (SLID) (capreomycin, kanamycin, or
amikacin). This assay detects and differentiates the preextensively drug-resistant
(pre-XDR) and extensively drug-resistant TB (XDR-TB). XDR-TB occurs when
MDR-TB inherits additional resistance to any FLQ (ooxacin, levooxacin, gatioxacin, and moxioxacin) among any of the SLID aminoglycosides (capreomycin, amikacin, and kanamycin) [57–59]. GenoType MTBDRsl VER 2.0 is a
redesigned and improved version of the previous one and bears the ability to detect
the most common mutations at gyr A and gyr B for FLQ resistance and rrs and eis
genes for SLID resistance. The gyr A probes target codons 85 to 97, rrs probes target
the positions 1401 to 1484, codons 536 to 541in the gyrB gene and −10 to −14
position in the eis promoter (Fig.6.5).
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S. S. Negi etal.
6.4.6 Truenat MTB-RIF Dx
Novel fast molecular diagnostics will possibly dramatically increase MTB detection
and point of care, which are key mechanisms of the WHO End TB Strategy. Truenat
MTB, Truenat MTB Plus, and Truenat MTB-RIF Dx assays have been manufactured by Molbio Diagnostics/Bigtec Labs, Goa/Bengaluru, India. Truenat assays are
an innovative micro real-time PCR assay based on a chip that detects MTB in clinical samples within just about an hour [60](Table 6.2). After getting positive assay
results, one more additional chip should be required to identify RIF drug resistance,
with an additional time period. The assay is ready to run in a Truenat lab system,
which is a battery-powered preparation system (device for DNA extraction) and an
automatic PCR machine, accessible in one, two, or four module congurations. The
Truenat system sensitivity for MTB diagnosis is 68–100%, and linkage-to-care is
84–100%. The Truenat system is a very popular and widely used point-of-care
(POC) machine that is cost-effective with >88% sensitivity and is decremental costeffective when sensitivity is 74–88% and specicity is 80–100% (Fig.6.6).
6.4.7 Sequencing Platforms fortheIdentication ofNovel
Mutations ofDrug Resistance
Timely and accurate identication of TB diagnosis and drug-resistance mutational
changes in sequences plays an important role in the effective management and elimination of TB.Many techniques for targeted or whole-genome sequencing, including the Sanger (capillary-based) and next-generation sequencer (NGS) and
third-generation sequencing, are employed for the detection of drug resistance in
MTB [61]. Notably, PCR-based targeted and whole-genome sequencing are two
different approaches for the analysis of the targeted gene or genome sequence of
MTB to reveal a clear picture of all mutations in a single assay [62]. Sequencing is
a highly reliable and accurate method used as the gold standard assay for the detection of any mutational changes for the detection of resistance in both reported and
novel mutations. Precise whole-genome sequence or targeted gene sequence data
can be achieved within 48h from an automated sequencer manufactured by Thermo
Fisher (SeqStudio Flex Genetic Analyzers and Applied Biosystems 3500 Series
Genetic Analyzer), Illumina Singapore (MiniSeq, MiSeq, NovaSeq), that use uorescent chemistry methods. Except sequencing for detection of rifampicin drug
resistance which cost reasonably low as it requires mainly identication of nucleotide changes in the RRDR region of 81 nucleotide sequences in the rpoB gene, the
high cost involved in determining drug resistance for other antimycobaterial drugs
wherein multiple genes/targets are required to unravel the mutational changes
restrict their testing in daily routine [63]. The MDR-TB results through sequencing
involve mainly an analysis of mutational sequential changes in specic drug target
genes (Fig.6.7 and Table6.2).
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to Trunat chip
Transfer the nucleic acid
extractor
Insert the extraction cartridge in
BUFFER
into extraction cartridge
Transfer to entire volume
Load the Trunat chip in TrunatReader
with 0.5ml sample
Mix 2.5 lysisbuffer
Clinical Sample
Fig. 6.6 Truenat MTB-RIF Dx
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Load the cartridge into sequencer
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S. S. Negi etal.
QC & Pooling
PCR Clean-up
Library Preparation
Tagmentation Adapter Ligation
Quantification
extraction
Nucleic acid
Clinical
Sample Culture
into the cartridge
Transfer pooled library
Data analysis
TB Patient
Mutation analysis
Fig. 6.7 Sequencing platforms for the identication of novel mutations of drug resistance
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94.6–
98.8%
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(continued)
Efciency of technique
TAT
(turn
around
Gene
Sensitivity Specicity
time) Advantages Limitation
target
90–95% 95–98%
• Aerosol
contamination is
high
• Requires
several manual
• Minimal expertise
required
• Rapid and
cost-efcient
1h
rpoB,
IS6110
MTB
diagnosis
63.6–
88.4%
steps
• This assay is
limited to
• Minimal expertise
required
2.5h
16S
rRNA
MTB
diagnosis
85–96% 96–98%
respiratory
specimen only
• High costs and
• Expensive
equipment is not
required
• Rapid diagnosis of
rpo B 2h
MTB
sophisticated
equipment is
required
• Does not
TB with rifampicin
sensitivity/resistance
pattern
diagnosis
and RIF
resistance
detect all
mutations
conferring
resistance to
anti-TB agents
Loop-mediated
isothermal
amplication
Chemical
Co
Molecular
assay Make Principle Purpose
1 LAMP Eiken
S.
Table 6.2 WHO recommended various molecular diagnostic assays for the rapid detection of MTB and drug-resistance
no.
(LAMP)
Realtime PCR
assay
Roche
Diagnostics
TaqMan TB
2 COBAS
cartridge-based
nucleic acid
amplication test
Cepheid Inc Automated
MTB/RIF
assay
3 Gene Xpert
(CBNAAT)
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