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Newer Diagnostic tests for tuberculosis, their utility and their limitations

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 Current Medicine Research and Practice 10 (2020) 8e11

Contents lists available at ScienceDirect

Current Medicine Research and Practice

journal homepage: www.elsevier.com/locate/cmrp

Review Article

Newer diagnostic tests for tuberculosis, their utility, and their

limitations
K.K. Chopra a, *, Shweta Singh b
a
New Delhi Tuberculosis Center, Jawaharlal Nehru Marg, Delhi Gate, New Delhi, 110002, India
b
RNTCP Technical Support Network, World Health Organization, Delhi, 110002, India

a r t i c l e i n f o a b s t r a c t

Article history: Tuberculosis (TB) has remained a disease of public health importance since ages, affecting more than 10
Received 4 January 2020 million people globally and taking lives of 2 million people worldwide every year. Despite the dramatic
Accepted 20 January 2020 improvements made in providing high-quality TB diagnostic services, since the discovery of the causative
Available online 27 January 2020
bacilli, many people with TB remain undiagnosed or get diagnosed only after long delays. Ten countries
account for 77% of this gap and use only smear microscopy for diagnosis, which forms the backbone of TB
Keywords:
diagnosis since 100 years. The challenge becomes onerous when disease gets associated with drug
Tuberculosis
resistance, Human Immuno Virus (HIV) and other diseases in an environment where transmission is
Diagnostics
Genotypic assays
becoming easier by the day. It becomes of paramount importance to address this biggest public health
LPA challenge by delivering timely diagnosis using advanced technologies.
CBNAAT Laboratory-based diagnostic approach to manage TB relies upon initial microscopic examination and
Culture clinical confirmations, with newer advanced diagnostic tools coming into play such as genotypic assays
DST (line probe assay, cartridge-based nucleic acid amplification test, loop-mediated isothermal amplifica-
tion) that are rapid molecular tests and culture methods (liquid culture media) with standard drug
susceptibility testing assays. The program envisages correlating the rapid molecular diagnostics, which
offers an impressive turnaround time of as low as around 2 h, with conventional standard methods to
reinforce the diagnostic capacities. These also provide with august identification of drug resistance
patterns for few most important first-line and second-line drugs that facilitate the early initiation of
correct treatment. The latest developments have brought these tests to near-patient point of care. Culture
tests (liquid culture media) are highest quality level gold standard techniques for the analysis of TB with
its increased sensitivity over all others but requirement of dedicated facilities and infrastructure pushes it
back the line. Finding a reproducible, efficient, cost-effective tool with minimal infrastructure re-
quirements is an on-going search under TB diagnostics. This review addresses significant advances made
in the diagnosis of infection, clinical disease, and drug resistance over the past decades.
© 2020 Sir Ganga Ram Hospital. Published by Elsevier, a division of RELX India, Pvt. Ltd. All rights
reserved.

1. Introduction Control Program (RNTCP), an unsettling percentage of estimated

Tuberculosis (TB) is a direful onerous disease, causing affliction
among human race since antiquity. According to the Global TB
Report 2019, TB affects approximately 10.4 million people every
year, taking a toll on the lives of around 1.8 million people world-
wide causing TB deaths. Despite various advancements made for
early diagnosis of the disease, under Revised National Tuberculosis
* Corresponding author.
E-mail addresses: chopra_drkk@yahoo.co.in (K.K. Chopra), drshweta11leo@
gmail.com (S. Singh).
4.3 million remains under reported or undiagnosed every year.1
Fighting the disease is a hornet's nest, which foists a grave socio-
economic burden on the diseased and their family members.
Since the discovery of causative organism for TB, that is, the
bacilli Mycobacterium tuberculosis (MTB) in 19th century, human-
kind has been working arduously to win over the perilous disease.
The Sustainable Development Goals for 2030 and End TB Strategy
have set up a common goal to end the global TB epidemic with
targets to reduce TB deaths by 90%, reduce its incidence by 80%, and
zero out out-of-pocket expenditure due to TB, which is to be ach-
ieved by 2030 as compared with 2015. Following the vision to

https://doi.org/10.1016/j.cmrp.2020.01.004
2352-0817/© 2020 Sir Ganga Ram Hospital. Published by Elsevier, a division of RELX India, Pvt. Ltd. All rights reserved.
 K.K. Chopra, S. Singh / Current Medicine Research and Practice 10 (2020) 8e11
eliminate the disease, the first challenge begins with providing
early diagnosis for the disease, which has picked up pace expedi-
tiously with fervent advancements being made under laboratory-
based diagnostics. Early diagnosis improves survival by identifying
infectious cases to prevent further transmission and various public
health actions.2
The high TB burden countries (around 30) accounted for 87% of
the global TB cases,3 where most cases occur in low- and middle-
income countries. In addition, ten of these countries accounted for
77% of the total estimated gap, where sputum microscopy is the
primary method for diagnosing pulmonary TB.4 The only World
Health Organization (WHO)erecommended rapid diagnostic test
for detection of TB and rifampicin (R) resistance currently available
is the Xpert MTB/RIF® assay. Globally, the use of rapid molecular
tests is increasing, and many countries are phasing out use of smear
microscopy for diagnostic purposes (although microscopy and
culture remains the mainstay for treatment monitoring).1
2. Background
Sputum microscopy was developed as a diagnostic test more
than 100 years ago. It became the backbone of TB control program
for diagnosing pulmonary TB because it could detect the presence
of acid-fast bacilli using Ziehl Neelsen staining, in a very short time
and was highly cost efficient. However, it needed a high bacterial
load of approximately 5
103e104 bacilli per ml of the sputum
sample. Overcoming its shortcomings, florescent dyes came into
play, with increased 10% sensitivity, but required a dark room with
conventional microscopes. Then the combination of light emitting
diodes with fluorescent microscopes further removed the need for
darkrooms and worked in less power. Since then, smear microscopy
has been sinew to the TB control program.5
Low sensitivity (25e75% in respect of culture) is the major
limitation of the test making it difficult to diagnose paucibacillary
cases and resulting in false negatives in most cases. Other limita-
tions include inability to differentiate between different species of
mycobacterium, and bacilli viability is the major limitation of the
test. For extrapulmonary (EP) cases, histopathology is used, speci-
ficity of which was dependent on the sampling. Limitations of
processing EP samples through histopathology were lack of experts
and poor resource settings.
The gold standard diagnostic test for TB was culture using solid
media, which had a very high sensitivity (requiring approximately
10 bacilli per ml of the sample). The test had a time consumption of
6e8 weeks to give the result that lead to delayed diagnoses, making
it a major drawback for it. With the increased pace the program has
attained over the past few decades, the requisite for more sensitive
diagnostic tests which produce results in shorter span of time is
imperative. The liquid media supports growth faster than solid
media, and since 1980s, semiautomated and automated liquid
culture systems became available.
3. Newer diagnostic tests
3.1. Genotypic assays
The exigent demands of early diagnosis became satisfied with
august rapid screening/diagnostic methods offered by molecular
technologies based on capturing nucleic acid and detecting myco-
bacterium. It has very high sensitivity and specificity. Using mo-
lecular technologyebased rapid diagnostic tests have reduced the
turnaround time (TAT) significantly and aided prompt initiation of
treatment/preventive services. Program envisages correlating the
rapid molecular diagnostics with conventional standard methods
to reinforce the diagnostic capacities. Two most important
9
polymerase chain reaction (PCR)-based genotypic assays relied
upon by programs are line probe assays (LPAs) and nucleic acid
amplification tests.
3.1.1. Line probe assays
Rapid LPA for first-line drugs tests for the resistance to two first-
line drugs such as R and isoniazid (H) and second-line drugs, such
as fluoroquinolones (FQs), and second line injectable drugs (SLDs),
referred to as a second-line LPA. The sensitivity and specificity of
PCR were 93% and 84%, respectively.6 They can distinguish between
different species of the mycobacterium along with the resistance
patterns. Technologies used for the same are Inno Genetics Line
Probe Assay (INNO-LiPA) (RIF & MYCOBATCERIA v2) based on their
nucleotide differences and DNA strip technology (Hain life sciences
Common Mycobacteria (Hain CM) test, Mycobaterium Tuberculosis
Drug Resistant, MTBDRplus v2, and Mycobaterium Tuberculosis
Drug Resistant Second Line, MTBDRsl v2).
3.1.1.1. Limitations. DNA probe technology cannot discern mixed
cultures and additional probes capable of the same are to be used.
In addition, LPA tests cannot be performed onto smear-negative
specimens, and hence, such specimens need to be put on cultures
and then growths could be subjected for LPAs. This begets culture-
based methods to remain the reference standards for Drug Sus-
cetibility Testing (DST).1
3.1.2. Cartridge-based nucleic acid amplification test
Recent rapid diagnostic tests such as nucleic acid amplification
tests (NAATs) have turned up as significant diagnostic tests in TB
because of their shortest TAT with increased sensitivity and spec-
ificity as compared with age-old gold standard methods. This
technology has thrusted the program to take a colossal leap in di-
agnostics, and the uniqueness of this test has facilitated its easy
implementation. This simultaneously tests for TB primarily and
resistance to R as a by-product. The test is commercially available
and now used globally for diagnosis of pulmonary TB, pediatric TB,
and specific forms of EP TB. The test has much better accuracy than
microscopy.1
3.1.2.1. Xpert® MTB/RIF assay. Xpert® MTB/RIF assay from Cepheid,
Sunnyvale USA, is the only rapid test for diagnosis of TB currently
recommended by WHO and by program as well. It is fully auto-
mated and submits the result in approximately 2 h of the sample
subjected to the machine. The program, under universal drug
susceptibility testing scheme, offers the test as a diagnostic
screening test for individuals at risk of multidrug resistant TB. It has
limit of detection (LOD) of 114 colony-forming units (CFUs) per ml
of the sample. It demands minimal infrastructure requisites that
can even be a mobile unit, taking the screening/diagnostic and
resistance tests nearer to the patient7 in remote locations as well.
An overall sensitivity and specificity of 81.3% and 99.8% was re-
ported, respectively, for Xpert when compared with reference
standard of combination of culture and clinical diagnosis of TB.8 For
biopsies, urines, pus, and cerebrospinal fluids, the sensitivity
exceeded 85%, while it was slightly lower than 80% for gastric as-
pirates. It was, in contrast, lower than 50% for cavitary fluids. High
sensitivity and specificity (86.9% and 99.7%, respectively,) were also
obtained for pediatric specimens. However, conventional diag-
nostic tests, such as smear microscopy and culture, are still used for
monitoring the treatment outcomes and resistance patterns for
other drugs.9
3.1.2.1.1. Limitations. The test is expensive which raises con-
cerns about its affordability in low- and middle-income settings.
However, according to a study, it has been found as a cost-effective
method10 for TB diagnosis by increasing the case finding
10 K.K. Chopra, S. Singh / Current Medicine Research and Practice 10 (2020) 8e11
substantially in high burden countries as compared with smear
microscopy and clinical diagnoses.
3.1.2.2. TrueNAT. TrueNAT is semiautomated in-house test from
India that has shown exemplary results in diagnosing the myco-
bacterium and resistance pattern for R. This can be made available
at the point of care as nearer to the patients in the peripheral areas,
being a handheld device that works without electricity. The test
needs to be performed in two rounds, where first, it identifies the
presence of mycobacterium using DNA extraction in the process
and then positive sample is subjected for establishing R resistance.
3.1.2.2.1. Limitations. GeneXpert assay does not obliterate the
need of conventional microscopy and culture and antitubercular
drug sensitivity that are required to monitor the progression of
treatment and for drug resistance determination to drugs other
than R.9
3.1.3. Loop-mediated isothermal amplification
Loop-mediated isothermal amplification performs DNA ampli-
fication and gene detection in a single step and can be observed
directly through naked eye under UV light, with TAT of 40 min.
Without any high-end infrastructure requirements, it can be used
as near patient assay. It has higher sensitivity as compared with
smear microscopy and increases case detection by 40% from smear-
negative samples. Hence, WHO recommends it as an add-on test
after smear.
3.1.3.1. Limitation. Inability to identify drug resistance along with
reduced specificity makes it a less preferred test for initial
diagnosis.
3.1.4. Genedrive
Genedrive with (Epistem) is the new development on the NAAT
platform for detection of TB and Rif-Resistance but has lower ac-
curacy and does not support WHO requirements.
3.2. Culture methods and drug susceptibility testing
The WHO suggested culture as a highest quality level gold
standard technique for the analysis of TB. It has high sensitivity over
other diagnostic tests, and it can be used for phenotypic DST and to
give adequate DNA rapid molecular tests for epidemiologic studies.
It has a preferred affectability over microscopy because it can
require as few as 10e100 cells/ml of processed concentrated
material.
Culture can be done over solid or liquid culture media, but solid
culture has lost its preferability because of high TAT of around 3
week days for giving a positive result11 and around 12 weeks for
DST when the using L-J medium.12 A WHO Expert Group endorsed
the use of liquid culture media for the identification of
M. tuberculosis and for DST in 2007.13
3.2.1. Liquid culture media
Liquid culture media have demonstrated increased sensitivity
and an expanded recovery yield of 10% in contrast to solid media.
Liquid cultures are performed using automated liquid culture
frameworks, for example, BacT/ALERT MP (bioMerieuxInc, Durham,
NC, USA) and BD BACTEC Mycobacterium Growth Inhibitor Tube
(MGIT) (Becton Dickinson, Sparks, MD, USA).14,15 The TAT for liquid
culture to obtain results is also around 10 days. With its high
sensitivity, it can diagnose TB among smear-negative cases as well.
The framework methods adopted by RNTCP in India and endorsed
through the WHO are BACTEC 460 system, Mycobacteria Growth
Indicator Tube (MGIT), and VersaTREK system. BACTEC is a semi-
automatic framework, which depends on the production of
radioactive carbon dioxide from substrate palmitic acid. This
method produces a nifty quality of distinguishing mycobacterium
from others, hence used as standard now for first-line and second-
line drug DST. MGIT uses fluorochromes giving early growth (7e12
days) of detection and drug screening, which makes it highly useful
for rapid phenotypic drug susceptibility.16
Mycobacterium tuberculosis complex (MTBC) is differentiated
from positive cultures using the qualitative identification method
of chromatographic immunoassay, using MPB64 protein predomi-
nantly secreted by MTBC.17 Phenotypic DST methods also are per-
formed as direct or indirect tests, of which indirect assays are
extensively validated and are honored as the gold standard. First-
line DST is performed for first-line anti-TB drugs, that is, H, R,
ethambutol, and streptomycin. Second-line DST uses automated
liquid systems as gold standard for second-line drugs, for example,
the injectables (kanamycin, capreomycin, and amikacin), the fluo-
roquinolones (levofloxacin and moxifloxacin), ethionamide, clofa-
zimine, cycloserine, and linezolid. DST for newly introduced second
liine drugs (SLD) such as bedaquiline and delamanid using the
MGIT method is still under trial phase.
3.2.1.1. Limitations. Apart from its high TAT, limitations of culture
methods include low sensitivity in EP TB and pediatric TB disease
because of the paucibacillary nature of the test. Cultures are highly
prone to growth of other microorganisms and
need decontamination, which effects mycobacterium as well. It
requires dedicated facilities and infrastructure along with skilled
laboratory personnel and specialized biosafety conditions.
4. Future prospects in TB diagnostics
Finding a reproducible, efficient, cost-effective tool with mini-
mal infrastructure requirements is an on-going search under TB
diagnostics. Incessant efforts are being made to thwart the disease
from making humankind suffer any further. Few of the future
projects in the pipeline are lipoarabinomannan (LAM) assay, Gen-
eXpert Ultra, TRCReady 80, volatile organic compounds (VOCs),
assays for diagnosis of Latent Tuberculosis Infection (LTBI), auto-
mated microscopy platforms, surface plasmon resonance (SPR)
sensing, nanoparticles, lab-on-a-chip and future NGS platforms.
4.1. LAM assay (Alere Determine™)
It is a lateral flowebased immunodiagnostic strip test which
detects LAM antigen present in urine. It would be helpful in diag-
nosing TB in patients who are unable to expectorate sputum, pa-
tients with Patients Living with Human Immuno Virus (PLHIV), and
patients who are immunocompromised and bed ridden.
4.2. GeneXpert Ultra (Cephied)
It is an updated version of the cartridge-based nucleic acid
amplification test (CBNAAT) with improved assay performance,
providing LOD of 16 bacterial CFU/ml. Based on the same principle
of the CBNAAT, it has advanced sensitivity over smear-negative,
pediatric samples and other paucibacillary specimens including
Human Immuno Virus (HIV)-positive cases. Another advantage it
offers is detecting mutations causing resistance for SLDs such as,
Isoniazid (INH), Fluoroquinolones (FQ), and Aminoglycosides
(AMG) with its Extensive Drug Resistance Tuberculosis (XDR TB)
assay. The same is under pilot run and actual release date is not
known yet.
 K.K. Chopra, S. Singh / Current Medicine Research and Practice 10 (2020) 8e11
4.3. TRCReady® 80
This fully automated molecular diagnostic tool is based on
transcription-reverse transverse concerted reaction amplification
technology. It has a capacity to detect 8 samples at once in 30 min
by measuring the total fluorescence in real time.
4.4. Volatile organic compounds
It works using mycobacterium metabolites derived from the
host's digestion or immune reaction. Various VOC devices are being
developed such as gas chromatography, metal oxide sensors, and
metabolite detection by chemical reaction, which are handheld
portable devices. When a presumptive patient with TB breathes
into the device's tube, MTB VOCs bind with titanium oxide com-
pounds in the gadget, generating electrical impulse that is read
using smartphone applications.
4.5. Assay for diagnosing LTBI
A skin test named CeTb (Statens Serum Institute in Denmark)
measures body's reaction to two explicit MTB antigens, that is,
ESAT-6 and CPF10. Improved tuberculin skin test (TST) assays are
also in the pipeline. At present, TSTs and new interferon-gerelease
assays are alternative tests for detecting latent TB.18
4.6. Automated microscopy platforms
Conventional microscopy also gets an update with advanced
automated imaging techniques. One such example is the TB diag-
nosis (TBDx) system, which is a computerized microscopy imaging
framework with high-resolution power magnifying lens to look up to
200 arranged smears using fluorescent microscopy in a single run.9,19
Another gadget named QuantuMDx (UK) based on MTB cell con-
centration as a standalone assay (Capture-XT™) is in the develop-
ment stage.
4.7. SPR sensing
It is a fully integrated point-of-care SPR imaging-based diag-
nostic system.20
4.8. Use of nanoparticles
Nanotechnology-based technologies for multiplex identification
are being developed. It works using antibody coated with capture
probe or magnetic and barcode probe tag tests.21,22
4.9. Lab-on-a-chip
Assays capable of detecting even single molecules are now being
developed as lab-on-a-chip frameworks, for example, cantilever
beams and nanotube paddle resonators.23
4.10. Future NGS platforms
Future NGS platforms are smaller, robust, and user friendly
stages for use in comparatively smaller research facilities which are
getting developed based on light cartridge-based frameworks
which would be cost-effective as well.24
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11
Declaration of competing interest
None.
References
1. WHO global TB Report 2019. Available from: https://apps.who.int/iris/
bitstream/handle/10665/329368/9789241565714-eng.pdf?ua¼1.
2. Drobniewski FA, Caws M, Gibson A, Young D. Modern laboratory diagnosis of
tuberculosis. Infectious Diseases. Lancet. 2003;3:141e147.
3. WHO global TB Report. Available from: https://www.who.int/tb/publications/
global_report/gtbr2018_main_text_28Feb2019.pdf; 2018.
4. Steingart KR, Henry M, Ng Vivienne, et al. Fluorescence versus conventional
sputum smear microscopy for tuberculosis: a systematic review. Lancet Infect
Dis. 2006;6:570e581.
5. Minion J, Pai M, Ramsay A, Menzies D, Greenaway C. Comparison of LED and
conventional fluorescence microscopy for detection of acid-fast bacilli in a low-
incidence setting. PloS One. 2011;6, e22495.
6. Kivihya-Ndugga L, van Cleeff M, Juma E, et al. Comparison of PCR with the
routine procedure for diagnosis of tuberculosis in a population with high
prevalences of tuberculosis and human immunodeficiency virus. J Clin Micro-
biol. 2004;42:1012e1015.
7. Clouse K, Page-Shipp L, Dansey H, et al. Implementation of Xpert MTB/RIF for
routine point-of-care diagnosis of tuberculosis at the primary care level. S Afr
Med J. 2012;102:805e807.
8. Tortoli E, Russo C, Piersimoni C, et al. Clinical validation of Xpert MTB/RIF for
the diagnosis of extrapulmonary tuberculosis. Eur Respir J. 2012;40:
442e447.
9. Vashistha H, Chopra KK. TB diagnostics: journey from smear microscopy to
whole genome sequencing. In: Hasnain S, Ehtesham N, Grover S, eds. Myco-
bacterium Tuberculosis: Molecular Infection Biology, Pathogenesis, Diagnostics and
New Interventions. Singapore: Springer; 2019. https://doi.org/10.1007/978-
981-32-9413-4_23.
10. Vassall A, van Kampen S, Sohn H, et al. Rapid diagnosis of tuberculosis with the
Xpert MTB/RIF assay in high burden countries: a cost-effectiveness analysis.
PLoS Med. 2011;8, e1001120.
11. Kidenya BR, Kabangila R, Peck RN, Mshana SE, Webster LE. Early and efficient
detection of Mycobacterium tuberculosis in sputum by microscopic observa-
tion of broth cultures. PloS One. 2013;8, e57527.
12. Lazarus RP, Kalaiselvan S, John KR, Michael JS. Evaluation of the microscopic
observational drug susceptibility assay for rapid and efficient diagnosis of
multi-drug resistant tuberculosis. Indian J Med Microbiol. 2012;30:64e68.
13. WHO Expert Group Meeting; 2007. Available from: https://www.who.int/tdr/
publications/tdr-research-publications/immunotherapeutic-interventions-tb/
en/.
14. Cruciani M, Scarparo C, Malena M, Bosco O, Serpelloni G, Mengoli C. Meta-
analysis of BACTEC MGIT 960 and BACTEC 460 TB, with or without solid media,
for detection of mycobacteria. J Clin Microbiol. 2013;42:2321e2325.
15. Siddiqui MAM, Anuradha PR, Nagamani K, Vishnu PH. Comparison of con-
ventional diagnostic modalities, BACTEC culture with polymerase chain reac-
tion for diagnosis of extra-pulmonary tuberculosis. J Med Allied Sci. 2013;3:
53e58.
16. Morcillo N, Imperiale B, Di Giulio B. Evaluation of MGIT 960 and the
colorimetric-based method for tuberculosis drug susceptibility testing. Int J
Tubercul Lung Dis. 2010;14:1169e1175.
17. Hillemann D, Rüsch-Gerdes S, Richter E. Application of the Capilia TB assay for
culture confirmation of Mycobacterium tuberculosis complex isolates. Int J
Tubercul Lung Dis. 2005;9:1409e1411.
18. Pai M, Zwerling A, Menzies D. Systematic review: T-Cell-based assays for the
diagnosis of latent tuberculosis infection: an update. Ann Intern Med. 2008;149:
177e184.
19. Ismail NA, Omar SV, Lewis JJ, et al. Performance of a novel algorithm using
automated digital microscopy for diagnosing tuberculosis. Am J Respir Crit Care
Med. 2015;191:1443e1449.
20. Fu E. Chapter 10. In: Schasfoort BM, Tudos AJ, eds. Handbook of Surface Plasmon
Resonance. Cambridge: Royal Society of Chemistry; 2010.
21. Resch-Genger U, Grabolle M, Cavaliere-Jaricot S, et al. Quantum dots versus
dyes as fluorescent labels. Nat Methods. 2008;5:763e775.
22. Cheng MM, Cuda G, Bunimovich YL, et al. Nanotechnologies for biomolecular
detection and medical diagnostics. Curr Opin Chem Biol. 2006;10:11e19.
23. Witkamp B, Poot M, Pathangi H, et al. Self-detecting gate tunable nanotube
paddle resonators. Appl Phys Lett. 2008;93. https://doi.org/10.1063/1.2985859,
111909e1-3.
24. Pankhurst LJ, Del Ojo EC, Votintseva AA, et al. Rapid, comprehensive, and
affordable mycobacterial diagnosis with whole-genome sequencing: a pro-
spective study. Lancet Respir Med. 2016;4:49e58.