Author’s Accepted Manuscript

Recent Technological Advancements in

Tuberculosis Diagnostics- A Review

Shagun Gupta, Vipan Kakkar

www.elsevier.com/locate/bios

PII: S0956-5663(18)30354-3
DOI: https://doi.org/10.1016/j.bios.2018.05.017
Reference: BIOS10475
To appear in: Biosensors and Bioelectronic
Received date: 2 January 2018
Revised date: 26 April 2018
Accepted date: 9 May 2018
Cite this article as: Shagun Gupta and Vipan Kakkar, Recent Technological
Advancements in Tuberculosis Diagnostics- A Review, Biosensors and
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 1

Recent Technological Advancements in Tuberculosis

Diagnostics- A Review
Shagun Gupta*, Vipan Kakkar

School of Electronics and Communication Engineering, Shri Mata Vaishno Devi University, Katra, 182320, India
guptashagun15@gmail.com
vipan.kakar@smvdu.ac.in
*
Corresponding Author.

Abstract
Early diagnosis and on-time effective treatment are indispensable for Tuberculosis (TB) control - a life threatening infectious
communicable disease. The conventional techniques for diagnosing TB normally take two to three weeks. This delay in diagnosis and
further increase in detection complexity due to the emerging risks of XDR-TB (Extensively drug Resistant-TB) and MDR-TB
(Multidrug Resistant-TB) are evoking interest of researchers in the field of developing rapid TB detection techniques such as
biosensing and other point-of-care (POC) techniques. Biosensing technologies along with the collaboration with nanotechnology have
enormous potential to boost the MTB detection and for overall management in clinical diagnosis. A diverse range of portable, sensitive
and rapid biosensors based on different signal transducer principles and with different biomarkers detection capabilities have been
developed for TB detection in the early stages. Further, a lot of progress has been achieved over the years in developing various point-
of-care diagnostic tools including non-molecular methods and molecular techniques. The objective of this study is to present a succinct
review of the available TB detection techniques that are either in use or under development. The focus of this review is on the current
developments occurred in nano-biosensing technologies. A synopsis of ameliorations in different non-molecular diagnostic tools and
progress in the field of molecular techniques along with the role of emerging Lab-on-Chip technology for diagnosing and mitigating the
TB consequences have also been presented.

Index Terms—LoC (Lab-on-Chip), MDR-TB (Multidrug Resistant-TB), MTB (Mycobacterium tuberculosis), LTBI (Latent TB
infections), MTBC (M. tuberculosis complex), NAAT, TB, XDR-TB (Extensively drug resistant - TB).

I. INTRODUCTION
Tuberculosis (TB) is a serious infectious disease caused by the bacterium named Mycobacterium tuberculosis (MTB). The
genetically related group of Mycobacterium species that can cause TB is referred to as M. tuberculosis complex (MTBC) and is
comprised of eight species [1]. Besides MTB, the MTBC contains four other TB-causing mycobacteria: M. Africanum, M. Bovis,
M. Microti, and M. Canetti and the remaining familiar pathogenic mycobacteria are: M. Kansassi, M. Leprae, and M. Avium. M.
Kansasii and M. Avium species are classified as "nontuberculous mycobacteria" (NTM) and are responsible for other pulmonary
diseases that resemble TB [2]. TB mainly impairs our lungs resulting in pulmonary disorder, but can sometimes harm other
organs too causing extrapulmonary TB [3] such as osteotuberculosis, neurotuberculosis, skin TB, genito-urinary tract TB etc. [4].
Multiple organs can also be damaged because of MTB attack resulting in disseminated or miliary TB. TB bacterium can remain
dormant or in an inactive state for years without the possibility of tracing prominent symptoms leading to latent TB infections
(LTBI). Almost about 10% of these latent infections build-up active disease which, if not detected and treated on time, increase
the mortality rate. Blood-containing sputum along with chronic cough, night sweats, fever and weight loss are classic symptoms
of active TB and a wide range of symptoms can be observed in extrapulmonary cases [4-5]. Tuberculosis is contagious as it
spreads via air when infected patient coughs, speaks, spits or sneezes [6]. LTBI are generally not communicable, despite the fact
that HIV/AIDS infected patients and chain smokers are more often prone to the active infection [4].
TB is not a modern lifestyle disease, however, current lifestyle adds up to TB risk. TB has been prevailing since primordial
times [7]. Almost one-third of the entire world's population is infected with this mortal disease [4]. With every passing year, new
infections develop in about 1% of the population [8]. Although since 2000, there is decrease in number of new cases each year
[4]. The infographic of current situation of TB and the strategies to eradicate this disease globally from the roots is presented in
Annexure 1. Developing countries have high mortality rate with 95% of TB deaths, as recorded in these countries. In many
African and Asian countries, about 80% of people test positive, while in the United States, 5–10% of population tests positive
[9]. Moreover, emergence of drug-resistance TB (DR-TB) [10] has threatened to jeopardize the worldwide efforts of controlling
 2

this epidemic. Various types of DR-TB include MDR-TB (Multi drug resistant – TB) or uncomplicated MDR-TB [11]: a
complex form which occurs when MTB strain develops resistance for the two most effective first line drugs: Isoniazid (INH) and
Rifampicin (RIF) [10-14]; and XDR-TB (Extensively drug resistant – TB): a more complicated and dangerous form of MDR-TB
in which MTB strain is also resistant to any Fluoroquinolone (FQ) and to one of the three second–line anti-TB injectable drugs
(Kanamycin, Capreomycin or Amikacin), in addition to RIF and INH [15]. Presently a third type of TB specified as TDR-TB or
XXDR-TB (extremely drug resistant TB) has also been observed [16]. It is characterized by the MTB strains which are resistant
to almost all the efficacious drugs (first and second-line both) currently used to cure active TB [17].
Due to the increasing number of these drug-resistant cases and the inability of current diagnostic techniques to detect TB
bacterium rapidly in the early stages has led to the widespread occurrence of TB globally. Many efforts have been made over the
last few decades in developing the efficient, cheap and rapid point-of-care detection techniques for the worldwide management
of the TB epidemic [19-21]. The purpose of this study is to fill the diagnostic gap between MTB detection and various drug-
resistant strains so as to find the opportunities to intervene in this epidemic in the initial stage. Section II of this review describes
the various conventional TB detection techniques and the challenges associated with them. The objective of this study is to
present a succinct review of the available TB detection techniques that are either in use or under development. Here, we have
presented the benefits of using the various biosensing techniques over conventional techniques. Amelioration in different non-
molecular diagnostic tools and progress in the field of molecular technology along with the role of emerging Lab-on-Chip
technology for diagnosing and mitigating the TB consequences have also been discussed.

II. CONVENTIONAL TB DIAGNOSTIC TECHNIQUES

The symptoms of TB appear slowly and many times it is difficult to diagnose whether a patient is suffering from active TB or
not [22], however, lungs disease and constitutional symptoms lasting longer than two weeks indicate the requirement of
diagnosing TB [23]. Initial evaluation of active TB diagnosis is based on microscopic examination (Ziehl–Neelsen (ZN) method
having 22–43% limited sensitivity for a particular smear, AFB (Acid Fast Bacilli) microscopy with maximum detection limit of
of 104 and 105AFB /mL [24]), culture of body fluids (generally multiple sputum cultures) and detection by Chest X-rays where
as latent TB detection relies on blood tests and tuberculin skin test (TST) [25]. Identification of MTB in an analytical sample
(such as sputum, pus or a tissue biopsy) is a definitive means for diagnosing TB. Though, the detection of this slow-growing
bacterium by complicated blood or sputum culture takes two to six weeks [26]. Patients with critical medical condition cannot
wait that long; so their treatment is often started well before the confirmed culture results [27]. Rapid diagnoses of TB is possible
by testing Adenosine Deaminase (ADA) enzyme level and by nucleic acid based amplification tests [22] but these tests are
usually not recommended routinely as they hardly have any impact on the treatment of a patient [27].
TB can be prevented by early detection and on-time and appropriate treatment [28]. Early detection requires regular
screening of the persons who are at high risk [29]. Primary control effort to prevent TB relies on vaccination of infants with the
vaccine named Bacillus Calmette-Guerin (BCG) [30]. Treatment of TB involves multiple antibiotics usage over a prolonged
span of time. MDR-TB and XDR-TB cases are increasing because of the disseminating problem of antibiotic resistance [31] and
traditionally, the identification of such drug resistant mutants is done through Drug Susceptibility Test (DST) which requires the
culturing of bacteria to yield the results in few weeks [32].
To recognize the disease and disease causing agents in the early stages is a crucial task due to the lack of particular
symptoms. The slow onset of disease cause the patients to suffer for weeks and even for months before seeking medical
assistance, and in the meanwhile they transmit the disease to others [33]. Thus, modernized tools for detecting and diagnosing
TB are required to achieve accurate results in a rapid manner keeping in view the cost constraint.
For rapid detection of MTB, many techniques have been developed till date such as radiometric detection [34],
immunoassays like Enzyme Linked Immunospot – ELISPOT [35], Polymerase chain reaction (PCR) [36], TB rapid cultivation
detection systems like MB/BacT system, Septi check, BACTEC MGIT (Mycobacteria growth indicator tube) 960 system [37]
[35] and so on. The sensitivity of few of the techniques [34] with their specific features is summarized in the table I.

TABLE I. SOME NON-BIOSENSING TECHNIQUES WITH THEIR SENSITIVITY [34] FOR TB BACTERIA
Technique
PCR [41]
ELISA (enzyme-linked
immunosorbent assay) [40]
Latex agglutination [42]
DETECTION
Highlights and challenges Sensitivity
 Rapid with high sensitivity.
 Extracting genomic DNA (Deoxyribonucleic acid) for
analysis is complicated [39]. with the true positivity of 95.5%
 Existence of inhibitors of the PCR may promote false-
negative results [38].
 Interferon-assays (IFN-γ) Based test [35].
with the true positivity of 68%
 Sensitivity is poor for extrapulmonary TB cases.
 Affordable, fast and easy to perform.
 Careful interpretation of results is required. and has low with the true positivity of 73.6%
specificity due to the presence of interfering substances.
 3

 Direct detection and amplification of RNA (Ribonucleic

The AMTDT (Genprobe amplified
M. Tuberculosis direct test) [43]
Flow cytometry [44]
with the sensitivity of 94.3% and
acid) of MTBC from blood specimens is possible.
specificity of 100% using simple Lysis
 No inhibitory compounds present as 10% SDS (sodium
method
dodecyl sulphate) is used to remove them.
 Higher sensitivity can be achieved.
3.5× cells/mL
 Rapid and reduces false-positives.

When compared with the traditional microbial culture-based techniques, these methods results in high sensitivity in less time
[34]. However, lack of detection values in real-time and requirements of costly centralized laboratories and skilled technical staff
are the main limitations of these methods. So, it becomes essential to develop such real-time, portable and sensitive techniques
that can detect MTB and drug-resistant mutants in a rapid manner at an affordable cost. Owing to the improvements in the
research area of sensing technology, biosensing technology, discussed in the subsequent section III of this review, is well suited
to achieve the above mentioned objectives.

III. BIOSENSING TECHNIQUES FOR TB DETECTION

Most of the non-biosensing techniques discussed above for MTB detection require highly skilled staff and complex
instrumentation available in large stationary laboratories. So, it becomes essential to design a sensitive, real-time, rapid and
portable system in order to accurately diagnose and prevent TB infection well on time [45-46]. Advancements in biosensing
technology proved to be a fighting chance in diagnosing this fatal disease. Biosensors have been useful in various areas such as
disease diagnostics, clinical assay, environmental monitoring, food and agriculture security etc. Biosensors require little amount
of sample and show high sensitivity and specificity in detecting analytes (biomarkers) in complex sample matrices (urine, serum,
saliva, blood etc.) [47-58]. A biosensor is basically a compact analytical device comprising a bio-recognition component and a
bio-transducer (physico-chemical transducer) component [34]. The bio-recognition element used in designing a biosensor can be
an enzyme, antigen-antibody, nucleic acid, whole cell etc. and by means of different chemical or physical immobilization
processes, this element is bound tightly onto the bio-transducer which can then measure the arising signals precisely [59-60]. The
working principle of a biosensor is shown in Fig. 1.

SIGNAL ANALYZER
BIORECEPTOR (ELECTRIC
TRANSDUCER
SIGNAL)

Fig. 1: Working Principle of Biosensor

It has been seen that the biosensing technology show remarkable features as compared to the conventional techniques
available for MTB diagnosis [34]. For the identification and isolation of MTB, chemical methods used for analysis can be based
on hybridization with specific gene probes (DNA, Ribosomal rRNA based probes), lipid profiles, PCR-RFLP (PCR-Restriction
fragment length polymorphism) technique, gene amplification methods etc. [61-74]. According to the signal transduction method
employed for designing, biosensors can be classified as electrochemical (amperometric, potentiometric, impedometric,
conductometric) [75-78], mass-based (piezoelectric [79-81], magnetoelastic [82], electrostatic), optical [83-85], magnetic [86-
87], etc. [60] [88]. Biosensor based on any transduction principle can analyze any sample for the isolation of required biomarkers
based on targeted application. Fig. 2 shows the general classification of TB diagnostics biosensors on the basis of transducer
employed as well as on the basis of sample analyzed. Various biomarkers of TB can be identified through different samples
(urine, blood or sputum) as mentioned in Fig. 2.
 4

BIOSENSORS
CLASSIFICATION FOR TB
DIAGNOSIS

On the Basis of Analyte Used for

On the Basis of Transducer
Detection
TECHNIQUE/
SAMPLE BIOMARKERS TRANSDUCER
PRINCIPLE
 Volatiles from  Potentiometric
MTB cells  Conductometic
 Electrochemical
Whole MTB cells  Amperometric
 DNA  Impedimetric
Sputum  Secreted and Cell
Internal Antigens
 Wavelength Based
 Other TB
 Evanescence Based
Biomarkers
(Surface Plasmon
Optical Resonance)
 Host antibodies  Optical Fibre
against TB  Chemiluminescence
Blood  Other TB  Fluorimetric
biomarkers
 Piezoelectric
Quartz Crystal
 Volatiles from Mass Based Microbalance
MTB cells
Surface Acoustic
 Whole MTB cells
Wave(SAW)
 DNA
Urine  Magnetoelastic
 Secreted and Cell
Internal Antigens
NMR (Nucleic
 Other TB
Biomarkers
Magnetic Magnetic Resonance)
Based

Fig. 2: Classification of Biosensors for TB Diagnostics [59][88-89]

A. Advancements in Transduction Mechanisms:

The field of biosensors has been revolutionized during the last decade owing to the significant growth in micro-system
technologies for designing reliable, integrated and miniaturized micro-transducers together with the biological sensing elements.
Such biosensor devices have raised the possibility of getting a complete understanding of dynamic cellular metabolic events and
to achieve a comprehensive insight into metabolism of human biology [90]. A remarkable progress has also been achieved in the
different biosensing techniques as mentioned below for TB detection in the time span ranging from 2000-2010.
1) Mass based detection techniques:

a) Piezoelectric Biosensors:
The transduction principle of Mass/piezoelectric sensors is to detect the changes in mass and surface characteristics with high
sensitivity by utilizing quartz crystals. Such sensors have the capability of analyzing the molecular interactions occurring
between ligand and target, and can also monitor the sensing platform surface for different biochemical reactions. In order to
detect MTB, two main biosensing platforms based on quartz crystal have been exploited: (i) quartz crystal microbalance (QCM)
based sensors, and (ii) series piezoelectric crystal technology [91].
QCM technology works on the principle that a quartz crystal resonator experiences a frequency shift when the gravitational
load changes on the sensor surface and also due to the variations in the viscoelastic properties of the sample cause [92-93]. F. He,
L. Zhang et al.; developed a Piezo-immunological sensor which can process the sputum samples for MTB detection with the
sensitivity of cells/mL [79]. In order to immobilize the antibodies against MTB on its surface, this sensor employed the
coating of a QCM sensing surface by a copolymer named styrene-butadiene-styrene as a membrane-mimicking layer and while
incubating with MTB on the platform, the real-time monitoring of captured MTB cells is performed by observing the shift in
 5

frequency due to the change in mass loading. Despite the merits of this QCM technology such as label-free, simple and rapid, the
main limitation is that the accuracy achieved by this technology can get affected by numerous factors such as electrical
conductivity, viscosity, dielectric constant and density of the sample [91].
The series piezoelectric quartz crystal biosensor and an acoustic wave impedance based biosensor with the detection limit of
2× cells/mL for the detection of MTB H37Ra biomarker and MTB cells respectively have been presented by F. He, J.
Zhao,et al [80]. Another piezoelectric biosensor employing multichannel series quartz crystal (MSPQC) and having the
capability of extracting volatiles produced by MTB cells growth (NH3, CO2) from sputum samples has been illustrated by J. Ren,
F. He, et al. This biosensor has the detection limit of about 10 CFU / ml (colony-forming units per millilitre) and has wide linear
range from 102 to 107 CFU/mL [81]. This MSPQC assay is less expensive and more sensitive as compared to the conventional
Lowenstein–Jensen (L–J) slants and BACTEC™ MGIT™ 960 system. The major drawback of MSPQC is that it is not suitable
for point-of-care testing as the culturing of MTB requires 2-3 days and also sample pre-treatment is required in order to avoid the
contamination with other potential bacteria [91].

b) Magnetoelastic Biosensors:
A magnetoelastic biosensor is a free-standing structure comprising a ribbon-like magnetoelastic film which is coupled with a
biochemical or chemical sensing layer such as enzyme. Its working principle is that when an external magnetic field is applied,
the sensor experiences mechanical vibrations at a particular resonant frequency and a return magnetic field is launched which can
be monitored by a pickup coil remotely. As there is lack of physical association between the detection system and sensor, such
biosensors can be used for detecting various chemical (e.g., microorganisms) and physical parameters (flow velocity,
temperature, pressure, elasticity, mass loading, density, and liquid viscosity [34]. Pang, Q. Cai, et al.; have described a
magnetoelastic biosensor having the detection limit of cells/mL for the direct and real-time identification of the MTB cells
from sputum [80].

2) Electrochemical Detection Techniques:

The working of Electrochemical Biosensors is based on the measurement of electronic current or on the detection and
analysis of conductance or ionic variations carried by bio-electrodes [94]. Sensitive detection of nucleic acid, enzymes,
microorganisms and assays are the potential application of these sensors [34]. A.K. Pavlou et al.; described an electronic nose
detection system, as shown in Fig. 3 (A), for MTB and it has been shown that electro-conductive polymers based gas-sensing
system can be used to differentiate between non-infected liquid cultures from those that are infected with TB. With the use of
this system, different mycobacterial species can also be identified in-vitro [75]. An electrochemical immunosensor which can
analyze the proteic matrix for extracting MTB antigens with 1.0 ng/mL sensitivity has been developed by M. Dıaz-Gonzalez et al
[76].

3) Optical Detection Techniques:

The transduction principle utilized by an optical biosensor depends on the optical measurements like absorbance, light
scattering, fluorescence, and etc. [94]. A highly sensitive optical biosensor based on fibre-optic sensing technique has been
presented by A.M. Pariwono et al. and a different sample collection strategy has been explained which allows the onsite
screening of TB patients in a rapid manner. This strategy leads to the development of an easy to use, safe, and a portable
diagnostic technique for MTB [83]. Duman, et al.; and McNerney, Ruth, et al.; have illustrated optical sensors with surface
plasmon resonance (SPR) sensing technique having sensitivity of 30 ng/μL [84] and breathalyzer as sensing technique with
sensitivity of 50–75 CFU /mL [85] for detecting MTBC and sputum processed MTB cells respectively.

4) Magnetic Detection Techniques:

Magnetic particles are utilized in designing these biosensors. The surfaces of these particles are accordingly functionalized
and modified so as to identify and recognize specific bio-molecules [95]. In the year 2008-2009, a DMR (diagnostic magnetic
resonance) based magnetic sensor platform has been described by Pand Lee, Hakho, et al. for identifying and detecting the MTB
cells and by-products in just 30 minutes from unprocessed sputum sample with the detection limit of 20 CFU/ mL [86-87]. The
working principle of DMR technique is based on the monitoring of surrounding water molecules so as to detect variations in the
spin relaxation time. This DMR system is a high- throughput, miniaturized and automated lab-on-chip scale adaptation of the
conventional NMR (nuclear magnetic resonance) technique which can analyze the chemical characteristics of bio-molecules and
can also determine the magnetic properties of atomic nuclei [88][96].

B. Role of Nanotechnology in Biosensors:

Efficient transduction specifically the precise signal capture of biological recognition phenomenon is the major challenge in
developing a biosensor [88]. Nano-material based biosensing technology has proven to be a promising technique to combat this
challenge. Advancements in nanotechnology have helped in developing the fast, accurate, versatile and highly sensitive nano-
biosensors. Many novel nanomaterials have been fabricated in the recent years and their applications in biosensors have also
been explored. The detection of any biophysical or biochemical signal associated with a particular disease at a single molecule or
 6

cell level is the ultimate goal of these nano-biosensors. In order to facilitate molecular diagnostics, such nano-biosensors can be
integrated into other technologies like lab-on-a-chip [90]. The biosensor systems based on the amalgamation of biosensing
technology and nanotechnology are proving their importance in various fields like clinical medicine, drug delivery, medical
diagnostics, environmental monitoring etc.The sensitivity of these biosensors can be improved and detection limits can be
reduced by using various nanoparticles (gold, silver, graphene, nano-beads etc.) due to the ease of immobilization of large
amounts of bioreceptor units owing to their high specific surface area [97]. Now- a- days, nanotechnological cantilever sensors
are also gaining popularity due to their capability of detecting the multi drug resistant mutants with an incredible sensitivity [60].
Schematic representation of one of such sensor array is shown in Fig. 3 (B).
Phunpae, P. et al.; described a Sandwich ELISA platform based on colorimetric technique which analyse Ag85B antigen for
MTB detection with the detection limit of 8 ng/mL in the linear range of 8-400 ng/mL [98]. A new Fluorimetry based Sandwich
ELISA technique for analysing Ag85B antigen using gold nanorods and quantum dots has been studied by Kim, E. J et al. This
latest technique can detect 0.013 ng/mL of Ag85B in the linear range of 0.013-1 ng/mL [99]. A carbon nanotube (CNT) based
biosensor for MTB detection has been presented by Maumita Das et al. This study reported a controllable electrophoretic
deposition (EPD) of CNT and (Zirconia) Zr (Nano Zr -CNT) nanocomposite films onto an ITO (Indium-tin-oxide) coated
glass substrate. Aforesaid Nano Zr -CNT/ITO electrode has been used for analysing single stranded DNA probe (ssDNA)
specific to MTB [100].
Anna Miodek et al.; developed an electrochemical sensor based on the assembly of nanomaterials
(MWCNTs/PPy/PAMAM) i.e. array of composite nanomaterials built with multiwalled carbon nanotubes (MWCNTs) enamelled
with polypyrrole (PPy) and redox PAMAM (Polyamidoamine) dendrimers. It is specified in this article that the nanomaterials
show a tremendous performance with a very low detection capability of 0.3 fM when electrochemical phenomenon is used for
the detection of DNA hybridization. This E-DNA sensor has been demonstrated to detect real samples of DNA from MTB and it
results in appreciable selectivity and sensitivity. Thus, this biosensor possesses a potential for specific detection of single
nucleotide polymorphism (SNP) [77].
Chang Liu et al.; presented an electrochemical DNA detection biosensor with exceptional sensitivity. An electrode modified
with reduced graphene oxide (rGO) and gold nanoparticles (AuNPs) is utilized as a sensing platform. This biosensor uses a
signal amplification strategy and a probe-labelled Au–PANI (Gold-Polyaniline) nano-composite for amplification. The
sensitivity has shown to be in the range of fm (between 1.0 x and 1.0 x m) when it is demonstrated for the detection
of MTB DNA [78]. A paper-based analytical device based on colorimetric sensing strategy using gold nanoparticles has been
developed by Tsung-Ting Tsa et al. In this study, it is demonstrated that the hybridization of single-stranded DNA probe
molecules with targeted double-stranded TB DNA results in the variation of colour of gold nanoparticle colloid. This colour
variation can be monitored by utilizing surface plasmon resonance effect. For TB DNA sequences, the limit of detection
achieved by this device is around 1.95× ng/mL [101].
A rapid and flexible Ag–C60 (FLAG-C60) nano-biosensor for detecting MTB in the initial stage of disease has been developed
by Mulpur, Pradyumna, et al. This biosensor employs the surface plasmon coupled emission (SPCE) phenomenon resulting in
highly sensitive detection of specific targets when compared to the traditional fluorescence sensors. The limit of detection of this
FLAG-C60 comes out to be around 20 MTB/mm2. The proof-of-concept of using this flexible biosensing platform with the smart
phones for point-of-care detection of MTB has also been demonstrated [102]. Fig. 3 (C) shows the photograph of this flexible
AG-C60.
 7

A)

B) Electric Signal
Microelectronic
Process C)

Analytes (Targets) Sensors Sensitive Layer

Fig. 3: Schematic Representation of different developed biosensors. A) Biosensor based on electronic nose detection system comprising three major
components: a) delivery chamber for sample, b) a sensor array for detection along with pattern recognition system and c) a computing system for interpretation of
result and for analysis of data analysis [88]. B) Cantilever sensor array for developing biosensor [59]. C) Flexible Ag- C60 [103].

Further, the growing number of drug-resistant TB cases (MDR and XDR-TB) is threatening the public health and prompt
diagnosis of such drug resistant (DR) mutants is of utmost importance and is required to mitigate their consequences in order to
reduce the mortality rate. The progress in nano-biosensing technology has made it possible to detect the emergence of these DR
strains at point-of-care. Based on biosensors, many new diagnostic tools particularly specific to MDR and XDR-TB targets have
been developed. A novel diagnostic platform, known as TB-DzT, combining binary deoxyribozyme (BiDz) sensors and
multiplex PCR technique for MTB detection and for the identification of MDR and XDR mutants has been illustrated by
Bengtson, Hillary N., et al. This rapid diagnostic tool with BiDz sensors can indicate and identify the mutations associated with
RIF, INH and FQs in just 4 hours with the limit of detection of 10-15 genomes in 60 minutes [104]. Engström, Anna, et al. have
developed a flexible and multiplexed molecular diagnostic tool for the detection of rifampicin resistance (RR-TB) in MTB by
using padlock probes and magnetic nano-beads based readout. These padlock probes can specifically target rpoB gene mutations
associated with rifampicin resistance [105].
Owing to the vast improvements in microfluidics and nanotechnology, a variety of biosensors have been developed for
detection of MTB (Table II), thus foreshadowing the inevitable era of microfluidics and nanotechnology in TB diagnostics [91].
Table II specifies the detection limits of various recently developed nano-biosensors along with the different biomarkers of TB.
TABLE II. VARIOUS TYPES OF BIOSENSORS FOR THE DETECTION AND IDENTIFICATION OF MTB AND DIFFERENT DRUG RESITANT
MUTANTS
Sample Analyzed
Biosensor devices Limit of Detection References
Sample Biomarker

Amperometric biosensor Sputum MTB 100 CFU/mL [106]

DNA detection biosensor employing

- MTB DNA 0.065 ng/μL [107]
electrochemical phenomenon
Optical sensor based on SPR sensing
Tissue fluids (Blood) CFP-10 MTB antigen 0.1 ng /mL [108]
technique
Lipoarabinomannan (LAM), 78 nM (1.48 mg/L) for
Antigen 85 complex (Ag85) urinary LAM, 183 nM for
Waveguide-based optical biosensor Urine, Serum [109]
and Early Secretory Serum Ag85, 125 nM for
Antigenic Target 6 (ESAT6) Serum ESAT6
100 CFU /mL
Magnetic barcode platform based biosensor Sputum RNA from MTB cells [110]
 8

Padlock Probes based Lateral flow nucleic 3 ng for katG 315 WT probe,
MTB DNA/ rpoB and katG
acid biosensors (LFNAB) for MDR-TB - and at least 30 ng d for the [111]
mutations
detection rpoB 531 WT probe
Electrochemical biosensor employing gold
- MTB DNA 0.05 ng/mL [112]
nanotubes array electrode
28 fM (femtometre) of
Graphene-based portable SPR sensor - MTB DNA cssDNA (circular-ssDNA) [113]
target in the salt buffer solution
Gold nanoparticles/toluidine blue–graphene
oxide (Au NPs/TB–GO) nanocomposites
- DNA/ MDR1 gene 2.95 × 10−12 M [114]
based label-free electrochemical DNA
biosensor for MDR detetction
Lable-free DNA based microcantolever
Biosensor for MTB detection with - MTB DNA/rpoB 2 pg/mL [115]
identification of Rifampicin resistance
Impedimetric DNA biosensor for detecting
- MTB DNA/rpoB - [116]
(rifampicin resistance) RR-TB
Electrochemical PNA (Peptide nucleic acid)
biosensor based on NH2-GO/QDs (amine
- MTB DNA 8.948 × 10−13 M [117]
functionalized graphene oxide composited
with CdS quantum dots) modified electrode
Aptamer based voltametric biosensor - MPT64 Antigen 0.9 fg/mL [118]
LTBI biomarkers (tumor
Multiplexed Electrochemiluminescence necrosis factor-alpha (TNF-
Serum 1.6− 200 pg/mL [119]
(ECL) immunosensor α), interleukin (IL)-2 and
interferon-gamma (IFN-γ))

Thus, from the analysis of above study, it can be concluded that the biosensing technology has helped in unfolding enormous
opportunities for fast and accurate TB detection as compared to the conventional techniques used in the laboratory settings.
Further, successful integration of nanotechnology with biosensing technology yields even better results in controlling TB
epidemic. Moreover, the portability of these biosensors also makes them ideal for pathogenesis of various diseases like cancer,
influenza, TB etc.
Current research scenario has entered into an era where collaboration of diversified fields (including molecular biology,
microarray chemistry, biosensing, nanotechnology, electronics etc.) has been made feasible and has led to the development of
various point-of-care techniques. The subsequent sections of this review present the numerous developments in the point-of-care
TB diagnostic technology and explore the precision and potential impact of entrenched and state-of-art POC TB diagnostic tools.

IV. DEVELOPMENTS IN DEVICE PLATFORMS FOR POINT-OF-CARE (POC) TB DETECTION

To ensure the fulfillment of unmet requirements in TB diagnostics such as rapidity, affordability, simplicity, precision and
high sensitivity, at point-of-care, necessitates the development of novel techniques that can generate the same-day test results
with the ability to detect various drug resistant mutants [120]. Rapid progression in the various fields like nanotechnology,
molecular biology, biosensors, VLSI etc. has empowered the integration of all such processes on a single platform for POC TB
detection so as to pacify the TB consequences and to minimize its transmission chances. In this section, various commercialized
and emerging non-molecular and molecular techniques [18] based POC device platforms have been evaluated [18] [91] [120]
and Fig. 4 represents some of these POC-TB diagnostic kits.
A. Non-molecular Diagnostic techniques:
To a great extent, the non-molecular methods are the modifications or refinements of conventional techniques, with the main
objective of ensuring more-rapid test results. Many such methods have been developed till date. These include microscopy
(fluorescent Light Emitting Diode (LED) Microscopy having sensitivity of 83.1% [24]) and imaging tools (magnetic resonance
imaging, positron emission tomography–computed tomography (PET-CT) for diagnosing extrapulmonary cases [121]), digitized
radiography, MDR-XDR TB colour tests, Microscopic Observation Drug Susceptibility (MODS) assay, colorimetric assay,
antigen/antibody-based detection tests etc. [122-123]. Among the above mentioned tests, the first non-molecular POC test
developed was Smear microscopy [124] and over the period of time, many prototype devices have been developed due to the
advancements in automated microscopy technique, but still successful devices have not yet been emerged [125]. As per WHO’s
2017 Global Tuberculosis Report [18], following two tests have recently been endorsed by WHO for point-of-care testing of TB
[126-127].

1) Urine based antigen detection assay:

Detection of MTB antigen is one of the main options for diagnosing TB as this method can reflect the global burden of TB
mycobacteria without depending on the individual’s immune status [125]. A point-of-care TB antigen-detection based diagnostic
platform named as Alere Determine TB-LAM Ag Test (Alere Inc., Waltham, USA) has been endorsed by WHO recently [126].
This test is based on the detection of active TB specific mycobacterial lipoarabinomannan (LAM) antigen present in urine
sample. Urine-based testing is preferable over sputum-based testing since it is easy to accumulate and store urine and also the
 9

chances of infection associated with sputum collection are less in urine-based tests. This point-of-care test can be used for the
screening of active TB in HIV-positive people [123,128]. The sensitivity of this test in HIV-infected patients with CD4 cell count
<100 cells/µL is around 56 % and with CD4 cell count > 100 cells/µL, the sensitivity reduces to 26% [126]. The photograph of
Alere Determine TB-LAM Ag rapid assay with a ready-to-use test strip is shown in Fig. 4 (G).

2) Interferon-Gamma (IFN-γ) Release Assays (IGRA):

In order to find out the presence of TB germs in an individual’s body, WHO has endorsed a TB blood test known as Interferon-
Gamma Release Assays (Oxford Immunotec, UK, Qiagen, USA) [127]. IGRAs are basically ex vivo assays having the potential
of measuring T-cell response following nightlong stimulation with the MTB specific antigens. This blood-based test has the
capability of differentiating active TB from latent TB infections [123,127]. IGRAs are available in two formats:
QuantiFERON®-TB (an ELISA based platform), which is used to quantify the IFN-γ concentration; and TSPOT® TB test (an
ELISPOT assay), which is used for counting the number of antigen-specific T lymphocytes produced by IFN-γ [91] [120].
Initially, the IGRAs used to take 24 hrs to provide results and required skilled personnel and complex instrumentation [91] but in
the recent years, new POC approaches such as assay system using Lab-on-a-Printed Circuit Board (LoPCB) [130] have been
emerged in order to overcome aforesaid limitations [131].
B. Molecular techniques:
Progression in cell biology has led to the development of a series of novel infectious disease diagnostic techniques. The aim
of such diagnostic tools is to identify the specific molecular fingerprint which is unique to individual pathogenic microorganism.
The significant benefits of using these molecular methods include the potential of designing the more specific and highly
sensitive assays, the possibility of yielding the fast results and the requirement of less technical staff when compared to the
conventional techniques. Earlier in TB endemic countries, low-cost smear microscopy technology was used to detect TB but this
technique has limited diagnostic utility because of insufficient amounts of bacteria in clinical specimens [132]. This leads to the
emergence of new diagnostic tools known as whole genome sequencing (WGS) and nucleic acid amplification tests (NAATs)
[133].

1) Sequencing:
Sanger dideoxy DNA sequencing method is the current gold-standard and most widely used sequencing technique for the
objective identification and detection of different species of mycobacteria. Till today, complete genome sequences have been
used for over 40 species including MTB. Sequence specific targets such as rpoB gene, the heat shock protein hsp65 gene etc. are
being identified [134] and then PCR primers are used to amplify DNA broadly from different mycobacterial species and finally,
this amplified DNA is sequenced and compared to the already established sequences from sequence database. After an organism
growth using culture, Sequencing takes 8-24 hours to report the results. The way in which the samples are batched influences the
results of sequencing. It is a labour-intensive procedure and requires trained technical staff. It is generally performed when less
expensive techniques such as MALDI-TOF MS and hybridization probes fail to produce desired results [1]. Recently, the next
generation sequencing (NGS) methods [135] such as whole genome sequencing (WGS) technique [133] has supplanted the
traditional Sanger method [1].
Infection prognosis and treatment may be influenced by genetic markers of organisms and their investigation has been made
possible by microbial genomics. WGS allows the identification of microevolution within MTB lineages by avoiding false
positives and can also detect various drug resistant mutations in a much less duration than the conventional drug susceptibility
test (DST) [136] and better than Xpert MTB NAAT assay [133]. However, to extract an adequate amount of DNA in WGS, prior
culturing of this slow-growing MTB is required which can take few weeks. Thus, extraction of DNA without re-culturing using
frozen isolates is very important. A low-cost and a reliable technique of DNA extraction sufficient to WGS for predicting
antibiotic resistance and for identifying mycobacterial species in clinical samples is performed using 1 mL of early positive
MGIT (mycobacterial growth indicator) tube cultures [137]. An important technique has been discovered recently with which
WGS can be used without performing the prior specimen culture. In order to capture the whole MTB genomes directly and
completely from uncultured samples of sputum, this method utilizes biotinylated RNA baits specific to the MTB DNA [138].
The major limitation of WGS technique is its prize which has limited its routine use only to high-income countries [133].

2) Nucleic Acid Amplification Techniques (NAAT):

Although, WHO has approved various phenotypic techniques for TB diagnostics such as testing of drug susceptibility by
commercial liquid culture technique, use of nitrate reductase assay, colorimetric redox indicator technique, microscopic
observation method of drug resistance etc., but these tests are time-consuming as single visit to clinic cannot provide appropriate
results and also require highly skilled operators and complex infrastructure. In contrast to these phenotypic methods, NAATs
have the capability of providing the results in a rapid manner and can detect even small amounts of DNA by utilizing different
amplification strategies like PCR, loop-mediated isothermal amplification etc. [120]. It is found that for diagnosing TB, NAATs
are less sensitive than culture, as extensive physical and chemical treatment is required for releasing the DNA, then for extracting
the bacteria, removing the inhibitors, and to concentrate the samples. But the advantage of NAATs is that they are highly specific
 10

and can identify new TB cases in few hours [139]. For the identification of drug resistant mutants and for detection MTB rapidly,
the two most commonly used NAAT techniques are Line probe assays (LPAs) and reverse-transcription polymerase chain
reaction (RT-PCR) [140-141]. NAAT diagnostic methods involve high costs and require a lot of technical guidance but universal
acceptance of this technology for diagnosing TB especially in low-income and epidemic countries encouraged the investments
from philanthropic and public resources. Efforts are being made and new NAAT technologies are being developed in order to
reduce the cost, to decrease the time to result and to improve the portability and robustness. Some of these NAAT techniques
including LPAs, loop-mediated isothermal amplification (LAMP), Cartridge based NAAT (CB-NAAT), RT-PCR and other
automated NAAT platforms [91][120][140-141]are explained below:

(a) Line Probe Assays:

Line probe assays (LPAs) utilize PCR amplification strategy for amplifying MTB-specific DNA sequences. It can also
amplify drug-resistant genetic mutation sites so as to detect TB and different drug-resistant cases either directly from clinical
samples or through the testing of culture isolates. WHO has endorsed the use of LPAs in 2008 in order to enhance the rapid
molecular detection of MTB and for screening the various genotypic drug resistant mutations [142]. The LPAs provide the best
results in smear-positive samples having high bacillary load but fail in the cases of smear-negative samples owing to the high
risk of getting contaminated by bacteria and so are not recommended in such cases because of low sensitivity. Cost constraint,
skilled staff and complicated infrastructure due to the requirement of controlled temperature are also the main limitations of these
LPAs. Inno-LiPA Mycobacteria assay (Innogenetics NV, Ghent, Belgium) is one of the commercially available LPA for
detecting the MTB and can also identify both rifampicin and isoniazid drug resistant mutations [125]. Advancements in the LPA
technology have now permitted to detect the resistance to various second-line TB drugs by genotypic testing methods with high
specificity and with moderate sensitivity (GenoType MTBDRsl assay, Hain Lifescience, Germany). Further the newer assays
have been developed which can directly analyze smear-negative sputum samples with lower detection limits and can be used to
screen the resistance to important second-line drugs at reference laboratory level (MTBDRplus version 2.0, Hain Lifescience,
Germany) [142].

(b) Loop-Mediated Isothermal Amplification (LAMP):

Further advancements in the POC NAAT technology has led to the development of a loop-mediated isothermal amplification
test (TB-LAMP, Eiken Chemical Co, Japan) for use in resource- limited settings and this test has been endorsed by WHO in
2016 [143-144]. This test is a temperature-independent and a unique way of amplifying DNA from TB microorganism [143].
This LAMP assay can specifically synthesize large quantities of DNA without the prior requirement of thermal cycling by
utilizing only 4 primers, thus aids the performance of NAAT at POC by eliminating the need for an expensive thermal cycler
[91]. Batch processing of upto 14 specimens can be done within 2 hours, thus helping in the rapid MTB detection by amplifying
sufficient amounts of DNA and finally the results can be interpreted by simply visualizing the fluorescence patterns (shown by
negative and positive controls). Although this test provides better results in comparison with the sputum smear microscopy
technique, but its main limitation is that it is not capable of identifying the drug resistant mutants thus limiting its use to only
those cases having less chances of developing MDR- TB [142-143].

(c) Miniaturized cartridge-based NAAT (CB-NAAT):

A NAAT platform known as the GeneXpert analyser (Cepheid, USA) eliminates the need for specialist technical skills or for
laboratory facilities by integrating the different procedures of sample processing, amplification, and DNA detection in one
instrument [91][145]. Another NAAT test named as the Xpert MTB/RIF assay (shown in Fig. 4 (H)) has the capability of
detecting the MTB DNA in less than 2 hrs and can also identify the mutations accountable for the resistance to the key drug
rifampicin. The sensitivity of this test is better than that of microscopy but less than that of culture [146-148].
 11

A) B) FluoroType MTBDR (Hain Lifescience, Germany) BD Max MDR-TB, (Becton Dickinson, USA)

E) F) G)
C) D) H) I)

VereMTB multiplexed lab-on-a-chip

Xpert MTB/RIF (Cepheid, USA)

Fig. 4: Various Point-of-Care TB Diagnostic Tools. A) Visual Interpretation of TB Test Results by TB LAMP technique (Eiken, Japan) [165], B) Truenat
MTB diagnostic kit (Molbio/bigtec Diagnostics, India) [146], C) Genedrive MTB/RIF ID platform, (Epistem, UK)[165] , D) EasyNAT MTBC Diagnostic
Kit(Ustar Biotechnologies, China) [165], E) FluoroType® MTB platform- a real time PCR assay for MTB detection (Hain Lifescience, Germany) [161], F) BD
Max MDR-TB detection kit, (Becton Dickinson, USA) [166], G) POC non-molecular Determine TB LAM Ag rapid assay with ready to use test strip shown on
the right (Alere, USA) [161] H) A disposable Xpert MTB/RIF assay cartridge and a Gene Xpert IV machine having four independent modules for processing test
cartridges [120,161], I) VerePLEX Biosystem Lab On-chip Platform [159].

(d) Other Automated and RT- PCR NAAT Techniques:

Extensive research is in progress for exploring the potential of NAAT technology in case of extrapulmonary and paediatric
infections. In order to enhance the speed of the amplification reaction and for minimizing reagent costs, miniaturization is being
exploited. Isothermal amplification methods now utilize a single constant temperature step for PCR instead of different
thermocycling steps resulting in reduction of complexity of device and shortening of assay time [149]. An enzyme-dependant
reaction known as Recombinase polymerase amplification (RPA) is an alternative development. With these methods, MTB
detection in a processed sputum sample has become possible in less than 20 minutes with high specificity at 39 ºC. Depending on
the targeted DNA insertion element, the range of sensitivities reported are from 91.4% (95% confidence interval (CI) 85–97.9%)
to 87.5% (95% CI 81.7–93.2%) [150].
Inspite of the advances in nucleic acid amplification technology, DNA extraction and sample handling remain stumbling
blocks [151]. Many attempts have been made in order to provide the cheaper substitutes of the expensive and sophisticated
technique utilized by the Xpert MTB/RIF. Three new fast diagnostic tools have been released to the market till date. These are
Truenat MTB (Molbio Diagnostics, India), The EasyNAT Diagnostic Kit and The VereMTB assay test.
The EasyNAT Diagnostic Kit as shown in Fig. 4 (D) has been developed by Ustar Biotechnologies, China for detecting the
Mycobacterium tuberculosis Complex. This kit utilizes an isothermal amplification process in which amplification reaction step
takes 60 minutes at 63 ºC and then a lateral flow device taking almost 30 minutes is used for visual detection purpose [152]. A
separate kit is used for sample extraction by this technique. Compared to the culture technique, the sensitivity of this method is
97.8% when tested for concentrated sputum and 84.1% when thinned sputum is examined and in smear-negative cases the
sensitivity comes out to be 59.8% [153].
Truenat MTB is a real-time PCR technique performed on a semi-automated miniaturized chip. A handheld sample
preparation device, shown in Fig. 4 (B)), operating on a battery and employing a nano particle-based protocol to extract nucleic
acids, is used to process sputum. Thus, there is no need for any additional equipment. The results with this diagnostic tool for
MTB detection can be reported within an hour [154]. The sensitivity and specificity of Truenat MTB and Xpert MTB/RIF are
comparable [155] and these values reach upto 91.1% (CI: 86.1-94.7) and 100% (CI: 90.0-100) respectively, in comparison to
90.58% (CI: 85.5-94.3) and 91.43% (CI: 76.9-98.2) respectively values for the in-house nested PCR protocol [156].
In order to overcome the time constraint and for the early stage TB diagnosis, a new fully automated technology named Lab-
on-Chip (LoC) technology can be used which will result in the overall reduction of mortality and morbidity rate because of TB.
With this technology, several laboratory operations can be performed on a single chip resulting in a handheld and portable
device. This becomes feasible due to the possibility of integration of large number of interdisciplinary modules on a single chip
can now be achieved by using available NEMS (Nanoelectromechanical systems), MEMS (Microelectromechanical systems)
techniques. LoC is basically the combination of fluidics, biosensors, electronics and VLSI as shown in Fig. 5 [157]. LoCs
devices are suitable for point-of-care diagnosis as they provide the fast and on-time diagnosis results and have the advantage of
portability, modularity, low power consumption, reconfigurability etc.
 12

BIOSENSORS/
ELECTRONICS
BIOTECHNOLOGY

LAB-ON-CHIP
(LoC)
MICROFLUIDICS
(SPUTUM, VLSI
BLOOD, URINE
etc.)

SIMULATION
TOOLS (MEMS,
NEMS etc.)

Fig. 5: Interdisciplinary Fields of LoC [157]

A nucleic-acid based Lab-on-Chip (LoC) device known as VereMTB (shown in Fig. 4(I)) has been developed by Veredus
Laboratories, Singapore but it is not commercialized yet and its performance is still under supervision and at this stage, it has
been released for only research use. VereMTB is basically a fast, multiplexed PCR-microarray based test and use the VerePLEX
Biosystem Lab-on-Chip platform. The VerePLEX platform comprises an exclusive disposable device constituting microfluidic
and microarray PCR modules. This method has the capability of automatically analyzing the microarray by using an optical
reader (OR) and a temperature control system (TCS). Thus, a user- friendly and easily understandable diagnostic report is
possible by this system. The VereMTB operates by combining microarray hybridization assay with multiplex PCR to detect,
identify and differentiate 10 different mycobacterium strains with special emphasis on MTBC [158]. It can also detect the
common non-tuberculous mycobacteria (NTM). In less than 3 hours, the result (without including sample extraction) can be
reported. The diagnostic accuracy comes out to be over 97.8% compared to sequencing. It also has the capability of diagnosing
MDR-TB by detecting MTBC’s resistance to Isoniazid and/or Rifampicin from smear-positive pulmonary clinical specimens or
from cultivated samples (liquid or solid cultures). Its use is even reasonable for screening purposes to develop country-specific
TB action plans and also for diagnosing patients after treatment failure and relapse.
The results from this semi-automated VerePLEX platform specific to MDR-TB targets exhibit high sensitivity and specificity
leading to a possibility of simple and rapid analysis of MDR patients in centralized laboratories. Specificity and sensitivity of
NTM probes can also be evaluated on the same platform. Spoligotyping, a state-of-the-art technique for the detection and typing
of MTB or MTBC bacteria simultaneously, is based on PCR amplification method. This strategy can provide the epidemiologic
information and can detect the causative bacteria in a rapid manner as compared to culture. Implementation of such a method in
clinical settings is possible by this LoC technology. An independent layout for this spoligotyping strategy of MTBC has been
developed within the TM-REST Project [160] but still a common platform is needed to be designed for overall detection process.
The various POC NAAT technologies and products in development are shown in the table III. In the table, CE mark means
conformity mark for products sold within the European Economic Area; and US FDA stands for United States Food and Drug
Administration [151].

TABLE III. POINT- OF- CARE TESTING TECHNOLOGIES FOR TB DIAGNOSTICS

Type of Reaction used Time to Result (in Product Development

Technology Characteristic features References
for amplification minutes) Stage

 Automated extraction of sample.

 Rifampicin Resistance detection  Endorsed by WHO.
Xpert TB/RIF
(Cepheid, USA) in pulmonary, extrapulmonary  Approved for CE
mark and also by US [145] [151]
and paediatric samples is possible.
FDA
<90 (including
Amplification using PCR
sample extraction)

 Automated post-PCR melt curve

analysis.  WHO guidance
Xpert MTB/RIF Ultra [161] [163]
 Ultra sensitivity is up to 17% issued in March 2017
higher than Xpert MTB/RIF
 13

FL-LPA  Line probe assays.

( Hain Lifescience,  Detection of Mycobacterium 24-48 hours.
Germany and Nipro, tuberculosis (MTB), isoniazid and
Japan)
Amplification using PCR rifampicin resistance in acid-fast Batching of samples Endorsed by WHO [125] [161]
Test Name- GenoType®
bacilli smear positive sputum or may increase
MTBDRplus and
MTB cultures is possible. turnaround time.
NTM+MDRTB
Detection Kit
 Line probe assays.
SL-LPA  A rapid DNA-based
(Hain Lifescience,  Used for the detection of
test.
Germany) resistance to fluoroquinolones and 24-48 hours Endorsed by WHO [125] [161]
 Amplification Using
Test name- Genotype® second-line injectable agents.
MTBDRsl PCR.

 Detects MTB in sputum

TB LAMP Loop-mediated
specimens. 90 (1.5 hours) Endorsed by WHO [125] [161]
(Eiken, Japan) isothermal amplification
 Does not assess drug resistance
 Multiplexed assay that detects
On the market but Not
TB and nontuberculous
Multiple Targets can be Endorsed By WHO as
iCubate System (iCubate, mycobacteria, and rifampicin-,
amplified using array - evidence to support its [18, 162]
USA) isoniazid-, ethambutol-, and
detection technology use has not been
streptomycin resistance in a single
submitted to WHO.
cartridge.
 Detects 16 mutations in 3 genes On the market but not
Genechip, TB drug Amplification using real- associated to drug resistance to Endorsed By WHO as
resistance array, Capital time fluorescence PCR the two first-line anti-TB drugs, 6 hours evidence to support its [18, 125]
Bio, China technology. rifampicin and isoniazid with use has not been
only one test submitted to WHO
 Isothermal Process at 65ºC.
EasyNAT Placed on the market.
CPA (Cross-Priming  Instrument-free approach for
(Ustar Biotechnologies, <90 Not Endorsed By [152-153]
Amplification) Reaction DNA extraction and visual
China) WHO
detection.
 Released to market.
Truenat MTB DNA extraction using  Not Endorsed By
Amplification using
(Molbio/bigtec miniaturized semi-automated <60 WHO [154-156]
PCR
Diagnostics, India) chip.  Approved for CE
mark.
 CE marked
Simultaneous detection of the M.  Released to the
FluoroType MTBDR tuberculosis complex as well as market in April 2017 [125] [161]
(Hain Lifescience, Multiplex PCR of the resistance-mediatins 3 hours
Germany) mutations in the and scheduled for
genes rpoB, inhA and katG. WHO evaluation in
2018/2019.
 High sensitivity and specificity
even in smear negative, culture  CE marked
M2000 RealTime MTB positive samples, for reliable [18] [161]
Automated real time –  Scheduled for WHO
System, detection of eight members of the - [163]
PCR evaluation in
(Abbott, USA) MTB complex.
2018/2019
 Can be used alongside HIV
RNA platform.
 CE marked, and
 An automated qualitative in
launched for marketing
vitro diagnostic test for the direct
BD Max MDR-TB, in Europe in April
Real-time polymerase detection of MTBC DNA [18] [161]
(Becton Dickinson, <4 hrs 2017.
chain reaction (RT- PCR)  Can also detect Rifampicin and
USA)  Scheduled for WHO
Isoniazid Resistance.
evaluation in
2018/2019
 A battery-operated, wireless,
and web-enabled, allowing
 Expected to launch
instrument and test information to
in Q2 2018.
GeneXpert Omni, be transmitted in real time. [161]
Quantitative PCR -  Scheduled for WHO
(Cepheid, USA)  Utilizes the same cartridge-
evaluation in
based system as the original
2018/2019
GeneXpert® model.

DNA extraction is done using a

Genedrive MTB/RIF ID, Paper-based digestion technique.
Amplification using PCR 60 Field trials [151]
(Epistem, UK) Possible to detect resistance to
Rifampicin.
 14

NAAT cartridge for GeneXpert

platforms that can detect
Xpert XDR-TB cartridge,
RT- PCR resistance to 14soniazid, - Under Development [162]
Cepheid, USA
fluoroquinolones, and the second-
line injectable agents
On-slide PCR and gel-drop
microarray technology.
TruArray MDR-TB, A portable, bench-top assay that
PCR detects mycobacterial Few hours In development [18] [161]
(Akkoni, USA)
susceptibility to rifampin and
isoniazid, using a single,
integrated-workflow approach
Scanning time of
Can detect MTBC and genotypes
less than 15
INFINITIMTB Assay, resistance to Rifampin, Isoniazid, In development.
PCR minutes for 96 [18] [162]
(AutoGenomics, USA) and Pyrazinamide in a single For research use.
multiplexed
assay.
microarrays
FluoroType XDR-TB
assay
PCR - - Under development [18]
(Hain Lifescience,
Germany)
Based on melting curve analysis.
Can detect XDR and MDR-TB in Approved by the China
MeltPro TB assay sputum samples. Food and Drug
PCR amplification 3.5 hours [18] [164]
(Zeesan Biotech, China) Can also perform spoligotyping to Administration
provide epidemiologic (CFDA)
information on strain identities.
A portable molecular diagnostic
device with inbuilt DNA Due for
QuantuMDx Amplification using sequencing for highly multiplexed < 20 minutes commercialisation in
[161]
(POC, UK) qPCR diagnostic and drug susceptibility 2018
testing.

Enzyme-based Study showing proof

RPA Recombinase Polymerase Isothermal Process at 39 ºC. <20 minutes of concept is [150]
Amplification Reaction published.
 Isothermal Process.
NEAT (Nicking Amplification  Nicking Enzyme is used to Product under
Enzyme Amplification reaction based on Nicking amplify the DNA exponentially - [151]
development.
Test) enzyme (NEAR) within temperature range from 55
ºC to 59 ºC.
 Microarray technology.
 Detection of Resistance to
VerePLEX Biosystem Amplification using Rifampicin and Isoniazid is Available for research
<180 [151] [158]
Lab On-chip Platform PCR possible. use
 Can also detect other nine non-
TB mycobacteria.

V. CONCLUSION
TB is a dominant global health challenge and is one among the top ten leading infectious causes of death, thus considered as
a major burden-inflicting disease in the world. For achieving the technological breakthroughs as per the End TB strategy by
2025, the slow and fragile progress in TB prevention and control need to be accelerated and this requires the major
improvements and investments in the pipelines for novel drugs and vaccines, advanced treatment regimens and for state-of-art
diagnostic tools. Conventional TB diagnostic techniques like culture and microscopy are either too labor-intensive or insensitive
respectively. The successful collaboration of nanotechnology with biosensing technology have potential to boost the MTB
detection and for overall management in clinical diagnosis. Further, the ongoing research in integrating distinct interdisciplinary
fields has led to the development of various portable, hand-held, and point-of-care diagnostic tools. The improvements in non-
molecular methods, whole genome sequencing and NAAT based molecular techniques have shown promising results in TB
diagnostics. NAATs do not rely on culture growth and can be used for the direct identification of MTBC within respiratory
specimens resulting in a more rapid diagnosis and appropriate patient care. Cost constraint and incapability of differentiating
between live and non-viable MTBC are the major challenges limiting the usefulness of these tests for monitoring response to the
given treatment leading to the requirement of other potential detection techniques. The emerging Lab-on- Chip technology with
biosensors as one of the component is one such potent diagnostic technique having capability of integrating several laboratory
components and can perform different laboratory functions on a millimetre-size single chip, thereby minimizing the technical
 15

requirements and infrastructure with the benefit of preserving analytical capabilities. The customization of the array design has
become easier and this impels the LoC layout to be a resourceful tool. The specific targets for local genetic variants, new
therapeutic regimens including neoteric key-drugs, and new genes and/or mutations can be easily integrated by using this
technology. But successful validation of this technique into a market product is still pending and hence cannot be benchmarked
for TB detection. Thus, the ongoing research involves exploration of several technologies and new diagnostic tools have been put
forth for utility in clinics and laboratories, but as yet meagre is accessible in real practice to aid the initial stage detections in the
society.

VI. FUTURE PERSPECTIVES

Implementation of new techniques in an economical way and magnifying their impact for TB diagnosis is the need of the
hour, since scant is actually available for assisting the early stage TB infections and for identifying drug-resistant cases
especially in developing countries. Nano-biosensors are the potential diagnostic tools for early stage diagnostics of TB with high
sensitivity but detection of different drug-resistant strains and other NTM strains in one medium is still a challenging task. There
is also the urgency for some novel innovative techniques that can diagnose the extrapulmonary and pulmonary cases on the same
platform. A breakthrough achievement can be made if it becomes feasible to integrate spoligotyping probes and to detect XDR-
TB and MDR-TB and other NTM probes rapidly in one medium-density microarray design by employing independent
multiplex-PCR. Owing to the advancements in nanotechnology, LoC technology employing nano-biosensors can serve as a
promising tool to accomplish all the above mentioned objectives and can act as filler for the aforesaid diagnostic voids,
particularly in low-income countries. Hence, this technological growth has shown a glimmer of hope for controlling and
eventually for complete eradication of TB. Further, the same LoC system can be acclimatized according to different research
requirements, thus suggesting a multi-purpose platform relevant for diagnosing various crucial diseases such as viral diseases,
malaria, influenza (like seasonal flu, swine flu, etc.), other bacterial diseases and so on.
ANNEXURE 1: Infographic: Global TB status (Data taken from WHO Global TB Report 2017 [18])
 16

GLOBAL TB EPIDEMIC STATUS AND

ELIMINATION STRATEGIES
(United Nations’ (UN) Sustainable Development Goals (SDGs) and WHO’s End TB Strategy )

By 2030, WHO’s End TB Strategy promise to reduce TB deaths by 90% and an overall reduction of 80 % in TB
occurrence. But TB is still a high burden disease. It is the ninth leading killer disease worldwide and the foremost
cause of death from a single infectious agent, even ranking above HIV/AIDS.

6.3 million new incidents of TB were

reported in 2016, (almost comparable to 61%
of the estimated figure of 10.4 million
people). Out of 10.4 million, it was
estimated that 10% were HIV-positive (with
56% in the following five countries: China,
India, Indonesia, Pakistan And Philippines
and 74% in Africa) The reported cases of
HIV-positive TB in 2016 were 476,774
(equivalent to 46% of the estimated figure),
of these 85% were on antiretroviral therapy
(ART).
1.3 million HIV-negative people
died from TB in 2016 (Less than
1.7 million in 2000) and further
374,000 deaths were reported
among people with HIV-positive.
600,000 new TB incidents were reported
in 2016 with rifampicin (an immensely
potent first-line drug) resistance (RR-TB),
Out of these, 490,000 suffered from
MDR-TB. The situation was worse in
China, India And the Russian Federation
with almost half (47%) of aforesaid TB
incidents. Worldwide in 2016, 8000
patients had eventually developed XDR-
TB.

GLOBAL FEEDBACK AND IMPACT

201 countries and territories (representing almost 99% of TB cases and overall
world’s population) contributed in WHO’s 2017 round of global TB data collection.

For TB care and prevention, financing and investments

Between 2000 and 2016, the TB mortality rate (per
have been increasing for more than 10 years, but still
100 000 persons) drop by 37% globally and 53
funding gaps exist and these gaps amount to 2.3 Billion
million lives were saved. At present, this rate is
US$ in 2017. For TB research, funding gap is over 1.2
falling at about 3% per year.
Billion US$ per year.
PRIORITY ACTIONS FOR PREVENTION AND CONTROL OF MDR AND
XDR-TB

RAPID DIAGNOSIS:  Ensuring Funding and

 Screening of active TB and stewardship for planning and
LTBI in high risk groups. Supporting Global TB
services of high quality.
 Control
Fast detection of Drug-  Making investments for research
Resistant Cases. and new TB theranostic tools.

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Highlights

 Tuberculosis (TB) is a global burden-inflicting infectious disease and poses a far greater and
difficult diagnostic challenge.
 Recent developments in biosensing technique with the use of nanomaterials have been studied.
 Significant advancements in various point-of-care techniques for early diagnostics of MTB and
drug resistant mutants have been reviewed.
 The emergence of state-of-art Lab-on-Chip (LoC) TB diagnostic technique has been presented.