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Newer Diagnostic tests for tuberculosis, their utility and their limitations

K.K. Chopra, Director, Shweta Singh, Medical Consultant

PII: S2352-0817(20)30005-2
DOI: https://doi.org/10.1016/j.cmrp.2020.01.004
Reference: CMRP 464

To appear in: Current Medicine Research and Practice

Received Date: 4 January 2020

Accepted Date: 20 January 2020

Please cite this article as: Chopra KK, Singh S, Newer Diagnostic tests for tuberculosis, their utility and
their limitations, Current Medicine Research and Practice, https://doi.org/10.1016/j.cmrp.2020.01.004.

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Title page:

Title: Newer Diagnostic tests for tuberculosis, their utility and their limitations

Type of Article: Review Article

Authors:

Author: K. K. Chopra1 and Shweta Singh2

Affiliations:
1 Director, New Delhi Tuberculosis Center, Jawaharlal Nehru Marg, Delhi Gate, New Delhi-
110002, India, chopra_drkk@yahoo.co.in
2 Medical Consultant, RNTCP Technical Support Network, World Health Organization,
Delhi-110002, India, drshweta11leo@gmail.com

Authors have no conflict of interest

Corresponding author: Dr. K.K Chopra

Review Article

Newer Diagnostic tests for tuberculosis, their utility and their limitations

Abstract:
TB has remained a disease of Public Health Importance since ages affecting more than 10 million
people globally and taking lives of 2 million people worldwide people every year. Despite of the
dramatic improvements made in providing high-quality TB diagnostic services, since the discovery
of the causative bacilli, many people with TB remain undiagnosed or get diagnose 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 diagnosis since 100 years. The challenge becomes onerous when
disease gets associated with drug resistance, HIV, other diseases, etc. in an environment where
transmission is becoming easier by the day. It becomes of paramount importance to address this
biggest public health challenge delivering timely diagnosis using advanced technologies.
Laboratory based diagnostic approach to manage TB relies upon initial microscopic examination
and clinical confirmations, with newer advanced diagnostic tools coming into play such as
genotypic assays (LPA, CBNAAT, LAMP) that are rapid molecular tests, and culture methods
(liquid culture media) with standard drug susceptibility testing assays. Program envisages
correlating the rapid molecular diagnostics, which offers an impressive turnaround time of as low
as around 2 hours, 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 facilitates 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 technique for the analysis of TB with its increased
sensitivity over all others, but requirement of dedicated facilities & infrastructure pushes it back
the line. Finding a reproducible, efficient, cost effective tool with minimal infrastructure
requirements 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.
Keywords: Tuberculosis, diagnostics, genotypic assays, LPA, CBNAAT, Culture, DST
1. Introduction:
Tuberculosis (TB) is a direful onerous disease, causing affliction among human race since
antiquity. According to 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 worldwide causing TB deaths.
Despite various furtherance made for early diagnosis of the disease, under Revised National
Tuberculosis Control Program (RNTCP), an unsettling percentage of estimated 4.3 million remains
under reported or undiagnosed every year.1 Fighting the disease is a hornet’s nest, which foists a
grave socioeconomic burden on the diseased and their family members.
Since the discovery of causative organism for TB i.e. the bacilli Mycobacterium
tuberculosis (MTB) in 19th century, humankind has been working arduously to win over the
perilous disease. The Sustainable Development Goals (SDGs) 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 of pocket expenditure due to TB to be achieved by 2030
as compared to 2015. Following the vision to eliminate the disease, the first challenge begins with
providing early diagnosis for the disease, which has picked up pace expeditiously 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 tuberculosis.4 The only WHO-recommended rapid diagnostic test for detection of TB
and rifampicin 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 tuberculosis because it could detect the
presence of acid-fast bacilli using Ziehl Neelsen (ZN) staining, in a very short time and was highly
cost efficient. However, it needed a high bacterial load of approx. 5 x 103 –104 bacilli per ml of
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 Diode with Fluorescent Microscopes (LED-FM) further removed need for dark
room and worked in lesser power. Since then smear microscopy has been sinew to the TB control
program.5
Low sensitivity (25–75% in respect of culture) is the major limitation of the test making it difficult
to diagnose pauci-bacillary cases and resulting in false negatives in most cases. Other limitations
include inability to differentiate between different species of mycobacterium and bacilli viability
are the major limitations of the test. For Extra-pulmonary (EP) cases, histopathology is used,
specificity 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 sample). The test had a time consumption
of 6-8 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, semi-automated 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
mycobacterium. It has a very high sensitivity and specificity. Using molecular technology based
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 PCR based Genotypic assays relied upon by program are Line Probe Assays and Nucleic
Acid Amplification Tests.
3.1.1. Line Probe Assays (LPA): Rapid LPA for first line drugs (FL-LPA) tests for the resistance
to two first line drugs viz. rifampicin and isoniazid, and that test for resistance to second line drugs
viz. fluoroquinolones and second line injectable, referred to as a second-line LPA (SL-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-LiPA (RIF & MYCOBATCERIA v2) based on their nucleotide differences
and DNA strip technology (Hain CM test, MTBDRplus v2 and MTBDRsl v2).
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 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 DST.1
3.1.2. Cartridge Based Nucleic Acid Amplification Test (CBNAAT):
Recent rapid diagnostic tests like Nucleic Acid Amplification Tests (NAATs) have turned up as
significant diagnostic tests in TB because of their shortest TAT with increased sensitivity and
specificity as compared to age old gold standard methods. This technology has thrusted the
program to take a colossal leap in diagnostics and the uniqueness of this test has facilitated its easy
implementation. This simultaneously tests for TB primarily and resistance to rifampicin as a
byproduct. The test is commercially available and now used globally for diagnosis of pulmonary
TB, pediatric TB, specific forms of Extra-pulmonary TB. The test has much better accuracy than
microscopy.1
3.1.2.1. Xpert® MTB/RIF assay from Cepheid, Sunnyvale USA, is the only rapid test for
diagnosis of TB currently recommended by WHO presently and recommended by program as
well. It is fully automated and submits the result in approximately two hours of sample subjected
to the machine. The program, under Universal Drug Susceptibility Testing (UDST) scheme, offers
the test as diagnostic screening test for individuals at risk of Multi Drug Resistant TB (MDR-TB).
It has limit of detection (LOD) of 114 colony-forming units (CFU) per ml of sample. It demands
minimal infrastructure requisites than 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 reported for Xpert when compared with reference standard of
combination of culture and clinical diagnosis of TB8. For biopsies, urines, pus and cerebrospinal
fluids the sensitivity exceeded 85%, while it was slightly under 80% for gastric aspirates. 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 diagnostic tests
viz. smear microscopy and culture are still used for monitoring the treatment outcomes and
resistance patterns for other drugs.9
Limitations: The test is expensive which raises concerns 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 substantially in high burden countries as
compare to smear microscopy and clinical diagnoses.
3.1.2.2. TruNAAT is semi-automated in-house test from India that has shown exemplary results in
diagnosing the mycobacterium and resistance pattern for Rifampicin. The can be made available at
point of care as nearer to the patients into the peripheral areas, being a hand held 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 rifampicin resistance.
Limitations: GeneXpert does not obliterate the need of conventional microscopy, culture and anti-
tubercular drug sensitivity that are required to monitor the progression of treatment and for drug
resistance determination to drugs other than Rifampicin.9
3.1.3. Loop-mediated isothermal amplification (LAMP) performs DNA amplification and gene
detection in single step and can be observed directly through naked eye under UV light, with TAT
of 40 minutes. Without any high-end infrastructure requirements, it can be used as near patient
assay. It has higher sensitivity as compared to smear microscopy and increases case detection by
40% from smear negative samples. Hence, WHO recommends it as an add-on test after smear.
Limitation: Inability to identify drug resistance along with reduced specificity makes it a less
preferred test for initial diagnosis.
3.1.4. Genedrive (Epistem) with is new development on the NAAT platform for detection of TB
and Rif-Resistance, but has lower accuracy and does not support WHO requirements.

3.2. Culture methods and Drug Susceptibility Testing:

The World Health Organization (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 requires as few as 10-100
cells/ ml of processed concentrated material
Culture can be done over solid or liquid culture media, but solid culture has lost its preferability
due to high turn-around time of around 3 weeks days for giving a positive result11 and around 12
weeks for DST when 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 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 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 WHO
are BACTEC 460 system, MGIT (Mycobacteria Growth Indicator Tube) and VersaTREK system.
BACTEC is a semiautomatic 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 (7-12 days) of detection and drug screening, which
makes it highly useful for rapid phenotypic drug susceptibility.16
Mycobacterium tuberculosis complex (MTBC) are differentiated from positive cultures using
qualitative identification method of chromatographic immunoassay, using MPB64 protein
predominantly secreted by MTBC.17 Phenotypic DST methods also are performed as direct or
indirect tests, of which indirect assays are extensively validated and are honored as the gold
standard. First Line DST is done for first line anti TB drugs i.e. isoniazid (H), rifampicin (R),
ethambutol (E), streptomycin (S). Second Line DST uses automated liquid systems as gold
standard for second line drugs e.g. the injectables (Kanamycin, Capreomycin and Amikacin), the
fluoroquinolones (Levofloxacin and Moxifloxacin), Ethionamide Clofazimine, Cycloserine and
Linezolid. DST for newly introduced SLD viz. Bedaquiline and Delamanid using MGIT method is
still under trial phase.
Limitations: Apart from its high turn-around time, limitations of culture methods include low
sensitivity in EP-TB and pediatric TB disease due to the pauci-bacillary nature of test. Culture are
highly prone to growth of other microorganisms and needs decontamination, which effects
mycobacterium as well. It requires dedicated facilities & infrastructure along with skilled
laboratory personnel and specialized biosafety conditions.
4. Future prospects in TB diagnostics:
Finding a reproducible, efficient, cost effective tool with minimal 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 pipe-line are LAM assay,
GeneXpert Ultra, TRCReady 80, VOCs, Assays for diagnosis of LTBI, Automated Microscopy
Platforms, SPR sensing, Nano-particles, Lab on a chip and Future NGS Platforms.
4.1. LAM assay (Alere DetermineTM): It’s a lateral flow based immunodiagnostic strip test which
detects lipoarabinomannan (LAM) antigen present in urine. It would be helpful in diagnosing TB
in patients who are unable to expectorate sputum, PLHIV, immunocompromised and bed ridden
patients.
4.2. GeneXpert Ultra (Cephied): It is an updated version of CBNAAT with improved assay
performance, providing LOD of 16 bacterial cfu/ml. Based on the same principle of CBNAAT, it
has advanced sensitivity over smear negative, pediatric samples and other paucibacillary
specimens including HIV positive cases. Another advantage it offers is detecting mutations
causing resistance for SLDs viz. INH, FQ and AMG with its XDR TB assay. The same is under
pilot run and actual release date is not known yet.
4.3. TRCReady® 80: This fully automated molecular diagnostic tool is based on transcription
reverse-transverse concerted reaction (TRCR) amplification technology. It has a capacity to detect
8 samples at once in 30 minutes by measuring the total fluorescence in real time.
4.4. Volatile Organic Compounds (VOCs): It works using mycobacterium metabolites derived
from 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 presumptive TB patient breathes into the device’s tube, the MTB
VOCs binds with titanium oxide compounds in the gadget, generating electrical impulse that is
read using smartphone applications.
4.5. Assay for diagnosing LTBI: A skin test named C-Tb (Statens Serum Institute in Denmark)
measures body’s reaction to two explicit MTB antigens i.e. ESAT-6 and CPF10. Improved TST
assays are also in the pipeline. At present, Tuberculin skin tests (TSTs) and new interferon-γ–
release assays (IGRAs) are alternative tests for detecting latent tuberculosis.18
4.6. Automated Microscopy Platforms: Conventional microscopy also gets an update with
advanced automated imaging techniques. One such example is TBDx system, which is a
computerized microscopy-imaging framework with high-resolution power magnifying lens to look
up to 200 arranged smears utilizing fluorescent microscopy in a single run.19, 9 Another gadget
named QuantuMDx (UK) based on MTB cell concentration as a standalone assay (Capture-XTTM)
is in development stage.
4.7. Surface plasmon resonance (SPR) sensing: It is a fully integrated point of care SPR
imaging-based diagnostic system.20
4.8. Use of Nano-particles: Nanotechnology based technologies for multiplex identification is
being developed. It works using antibody coated with capture probe or magnetic and bar-code
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-chip frameworks e.g. Cantilever beams and nanotube paddle resonators.23
4.10. Future NGS Platforms: Smaller, robust and user friendly stages for use in comparatively
smaller research facilities getting developed based on light cartridge based frameworks which
would be cost effective as well.24

Conflict of Interest: None

References:
1. WHO Global TB Report 2019. Available from
https://apps.who.int/iris/bitstream/handle/10665/329368/9789241565714-eng.pdf?ua=1
2. Drobniewski FA, Caws M, Gibson A, Young D. Modern laboratory diagnosis of
tuberculosis. Infectious Diseases. The Lancet. 2003;3:141-147.
3. WHO Global TB Report, 2018. Available from
https://www.who.int/tb/publications/global_report/gtbr2018_main_text_28Feb2019.pdf
4. Steingart KR, Henry M, Vivienne Ng, et al. Fluorescence versus conventional sputum
smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis. 2006;6:570-81.
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 Microbiol. 2004;42:1012-5.
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;102:805-7.
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;40:442-447
9. Vashistha H, Chopra KK. TB Diagnostics: Journey from Smear Microscopy to Whole
Genome Sequencing. In: Hasnain S., Ehtesham N., Grover S. (eds) Mycobacterium
Tuberculosis: Molecular Infection Biology, Pathogenesis, Diagnostics and New
Interventions. Springer, Singapore. 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 Observation 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;30:64-8.
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;42:2321–2325.
15. Siddiqui MAM, Anuradha PR, Nagamani K, Vishnu PH. Comparison of conventional
diagnostic modalities, BACTEC culture with polymerase chain reaction for diagnosis of
extra-pulmonary tuberculosis. J Med Allied Sci. 2013;3:53-58.
16. Morcillo N, Imperiale B, Di Giulio B. Evaluation of MGIT 960 and the colorimetric-based
method for tuberculosis drug susceptibility testing. Int J Tuberc Lung Dis. 2010;14:1169-
75.
17. Hillemann D, Rüsch-Gerdes S, Richter E. Application of the Capilia TB assay for culture
confirmation of Mycobacterium tuberculosis complex isolates. Int J Tuberc Lung Dis.
2005;9:1409-1411.
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;149:177-84.
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;191:1443–1449.
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;5:763–775.
22. Cheng MM, Cuda G, Bunimovich YL, et al. Nanotechnologies for biomolecular detection
and medical diagnostics. Curr Opin Chem Biol. 2006;10:11-9.
23. Witkamp B, Poot M, Pathangi H, et al. Self-detecting gate tunable nanotube paddle
resonators. Applied Physical Letters. 2008;93:111909–1-3. DOI: 10.1063/1.2985859.
24. Pankhurst LJ, Del Ojo EC, Votintseva AA, et al. Rapid, comprehensive, and affordable
mycobacterial diagnosis with whole-genome sequencing: a prospective study. Lancet
Respir Med. 2016;4:49–58.