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Unitaid Diagnostics Technology Landscape 5th Edition, May 2017.

Diagnostics Technology Landscape
5th Edition, May 2017
© 2017 World Health Organization
(Acting as the host organization for the Secretariat of Unitaid)
The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on
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This report was prepared by David Boyle (PATH, Seattle). All reasonable precautions have been taken by the author to verify the information
contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The
responsibility for the interpretation and use of the material lies with the reader. In no event shall Unitaid or the World Health Organization be liable
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Diagnostics Technology Landscape
Abbreviations and acronyms
Executive summary
Tuberculosis (TB) is an urgent public health problem, but many people do not have
access to critical diagnostic tools
Significant events since the last TB diagnostics technology landscape
Acknowledgements and declarations of interest
Overview of the TB diagnostics technology landscape
Recent policy and recommendation updates for TB diagnostics
Screening and triage tools
Digital chest X-rays
Volatile organic compounds (VOCs)
Immune response-based screening tests for MTB exposure
Solutions for diagnostic sample transport
Automated microscopy
Culture-based tools for the diagnosis of TB and DST
Biomarkers to detect MTB exposure and TB disease
Immune response-based tests
Serologic and antigenic biomarkers of TB
Other biomarker development news
NAATs and sequencing methods for TB diagnosis and DST
Update to the NAAT pipeline
Stratification of diagnostic NAATs in the test continuum
Application of NGS to TB diagnosis and control
Automated batched PCR
Autonomous NAAT reagents for use in open systems
Modular, cartridge-based, fully automated NAATs
NAATs for use at peripheral centres
Status update on NAAT-based technologies for reference and intermediate
Update on technology status of NAAT-based technologies intended for use in
microscopy centres
Figure 1. Current FIND TB diagnostics pipeline listing the development phases and
types of technologies in development or evaluation
Figure 2. Prototype Nanosynth breath test and detector
Figure 3. Hand-held Aeonose™ device
Figure 4. QuantiFERON-TB Gold Plus kit from Qiagen: reagents and ELISA plates (le)
and collection materials (right)
Figure 5. TBDx system
Figure 6. Determine™ TB LAM Ag rapid assay, with strip ready for use shown on the right
Figure 7. Current and emerging automated, semi-modular or non-integrated TB NAATs;
their intended laboratory location and release date (actual or anticipated)
Figure 8. Workflow of ReSeqTB bridging targeted NGS platforms to provide rapid
patient management decisions
Figure 9. Abbott Molecular platforms for automated sample preparation (m2000sp,
le) and real-time PCR analysis (m2000rt, right) for MTBC and first-line DST
Figure 10. Akonni TruTip® Automated Sample Prep Workstation
Figure 11. Hain Lifescience FluoroType® MTB [A] and FluoroType® MTBDR [B] processes
Figure 12. Akonni TruDx®2000 platform with TruTip® extraction pipette, TruArray®
processor and TruArray® scanner (le); TruArray® test, microfluidic valve-
less design for simultaneous on-slide PCR and microarray hybridization in a
closed format (right)
Figure 13. Veredus Laboratories VerePLEX™ Biosystem and VereMTB™ Detection Kit
Figure 14. Hydra 1K hand-held platform (le) and chip (centre)
Figure 15. Cepheid Inc. GeneXpert® IV System (GX-4) with four independent modules for
processing test cartridges (le) and the Xpert® MTB/RIF cartridge (right)
Figure 16. Cepheid Inc. Xpert® MTB/RIF Ultra cartridge
Figure 17. HumaLoopT instrument (from HUMAN Diagnostics Worldwide, top le) and
the Pure DNA Extraction kit
Figure 18. Molbio Diagnostics technologies: current and pending products for NAAT-
based detection of MTBC and drug resistance
Figure 19. Ustar Biotechnologies EasyNAT™ TB assay
Figure 20. GeneXpert® Omni
Figure 21. KGI TBDx system
Figure 22. Scanogen reader and test cartridge for use in microscopy centres
Table 1. Summary of NAATs relating their role in TB diagnosis in terms of intended
location of use, throughput and other key factors
Table 2. Current LPA products and associated equipment marketed for MTBC
diagnosis, mycobacterial speciation and genotypic DST
AMG aminoglycoside
AMK amikacin
BCG Bacillus Calmette-Guérin
BD Becton Dickinson
BRICS Brazil, Russian Federation, India, China and South Africa
CAD4TB Computer Aided Detection for Tuberculosis
CAP capreomycin
CE European Conformity (Conformité Européenne) certification
CE-IVD European Conformity (Conformité Européenne)-in vitro diagnostic
CFDA China Food and Drug Administration
CFP10 culture filtrate protein 10
cfu colony forming units
CXR chest X-ray
DCXR digital chest X-ray
DST drug susceptibility testing
ELISA enzyme linked immunosorbent assay
EMB ethambutol
ESAT-6 early secreted antigenic target
FDA Federal Drug Administration
FIND Foundation for Innovative New Diagnostics
FLQ fluoroquinolone
HBC high-burden country
HIV human immunodeficiency virus
IFN-γ interferon-gamma
IGRA interferon-gamma release assay
INH isoniazid
IRISA InterGam Rapid Immuno Suspension Assay
ISO International Organization for Standardization
KAN kanamycin
KGI Keck Graduate Institute
LAM lipoarabinomannan
LAMP loop-mediated amplification
LF lateral flow
LF-LAM lateral flow urine lipoarabinomannan
LMIC low- and middle-income country
LPA line probe assay
LTBI latent TB infection
MDR multidrug resistant
MGIT™ mycobacterial growth indicator tube
miRNA micro ribonucleic acid
mL millilitre
mm millimetre
MODS microscopically observed drug susceptibility
MOX moxifloxacin
MTB Mycobacterium tuberculosis
MTBC Mycobacterium tuberculosis complex
MTB/RIF Mycobacterium tuberculosis/rifampicin resistance
MTBDRsl Mycobacterium tuberculosis drug resistance second line
NAAT nucleic acid amplification test
NGS next-generation sequencing
NTM non-tuberculous mycobacteria
NTP national tuberculosis programme
NWGHF North Western Global Health Foundation
OFX ofloxacin
PCR polymerase chain reaction
PEPFAR President’s Emergency Plan for AIDS Relief
POC point of care
PLHIV people living with HIV
PTB pulmonary TB
PZA pyrazinamide
Q1, 2, 3, 4 Quarter 1, 2, 3, 4
RDT rapid diagnostic test
RIF rifampicin
RR rifampicin resistant
RR TB rifampicin-resistant TB
SL-LPA second-line probe assay
SLID second-line injectable drug
SSM sputum smear microscopy
STR streptomycin
TB tuberculosis
TOP TB totally optimized PCR for TB
TPP target product profile
TRCR transcription reverse-transcription concerted reaction
TST tuberculin skin test
UK United Kingdom
μL microlitre
US United States
USA United States of America
USAID United States Agency for International Development
USB universal serial bus
VOC volatile organic compound
WGS whole genome sequencing
WHO World Health Organization
XDR extensively drug resistant
Tuberculosis (TB) continues to be a major public health threat despite being a curable
disease. Latest figures from 20151 indicate an estimated 10.4 million people had TB, and
1.8 million people died (1.4 million HIV negative and 400 000 HIV positive). Of further
concern is that 480 000 cases of multidrug-resistant (MDR) TBa and a further 100 000 that
were estimated to be rifampicin-resistant (RR) TB have occurred in the same period.1
The rapid and accurate diagnosis of TB infection and disease is critical for timely initiation
of treatment and, ultimately, control of the disease. Of the 10.4 million people who
developed TB in 2015, 4.3 million cases were not diagnosed or notified and only one
quarter of RR/MDR TB cases (132 000) were detected and reported. The underdiagnosis
and underreporting of TB may be due to limited or delayed access to appropriate
diagnosis and care, large private sectors not reporting cases, and the lack of access to
appropriate diagnostic tools due to geographic and/or financial barriers.2-4 Most of the
currently available TB diagnostics are ill-adapted to resource-limited settings or specific
patient needs; or may be priced out of reach.
Many countries still rely on tools such as sputum smear microscopy but new diagnostics
are slowly changing the TB diagnostics landscape. In 2016, the World Health Organization
made policy guidance statements for five new or improved TB diagnostic products.5-9
Further changes are expected, with unmet needs identified and articulated in target
product profiles,10 and a technology pipeline promising new products to address these
needs. Several of these are currently undergoing evaluation in field studies. This updated
report reviews the status of current, emerging and potential technologies.
a TB strains that are resistant to RIF and INH.
Tuberculosis (TB) is an urgent public health problem, but
many people do not have access to critical diagnostic tools.
In 2016, the World Health Organization (WHO) noted that the TB epidemic was larger
than previously estimated, with 10.4 million new cases of TB in 2015, the increase driven
primarily by improved case notification from the private sector in India. However the TB
incidence rate and the number of deaths from TB continue to fall globally.1,11-13 In 2015,
an estimated 1.4 million people died from this largely curable disease with a further
400 000 deaths from TB reported among people living with HIV.1 The Global tuberculosis
report (2015) noted that TB now ranks above HIV as a leading cause of death worldwide.11
Access to accurate TB diagnostics and drug susceptibility tests enable TB programmes
to identify TB cases and select appropriate treatment. WHO estimates that almost 40%
of all TB cases in 2015 were either not diagnosed or cases not reported to national
tuberculosis programmes. The Global tuberculosis report (2016) noted: “If everyone
with TB had a timely diagnosis and high-quality treatment, the case fatality rate would
be low in all countries”.1 Recognizing the critical importance of drug susceptibility
testing, the WHO End TB Strategy includes universal drug susceptibility testing as a key
component of its first pillar: integrated, patient-centred TB care and prevention. Several
of the molecular tests currently in development also oer genotypic drug susceptibility
testing, either integrated into the diagnostic test or as a reflex assay for TB-positive
Improvements in the diagnosis of multidrug-resistant tuberculosis (MDR TB) are
associated with greater implementation of molecular tests, in particular, line probe
assays and the Cepheid Inc. GeneXpert® MTB/RIF assay (hereinaer Xpert® MTB/RIF).
However, an estimated 75% of all MDR TB cases are still not identified and reported.1
Robust and accurate molecular technologies would have the greatest impact on
improving diagnosis if they were made available at microscopy centres, where most
people with signs and symptoms of TB seek a diagnosis.14 Although candidate products
are described in this landscape report, to date, no molecular test has been suiciently
assessed to demonstrate the potential to do this.
The WHO treatment guidelines for drug-resistant TB were updated in 2016.15 They note
that rifampicin-resistant TB cases, with or without resistance to isoniazid should be
treated with MDR TB regimens, with the recommendation that a shorter regimen (9–12
months) be used for cases that are not resistant to second-line drugs. Furthermore, WHO
issued interim guidance on the use of bedaquiline in 2013 and the use of delamanid in
2014;16,17 and policy guidance on the use of delamanid for the treatment of MDR TB in
children (aged 6–17 years).18
Childrenb accounted for 6.3% of the new and relapse TB disease cases notified globally in
2015.1 An estimated 1 million children became ill with TB in 2015, while 210 000 children
died from TB in 2015. Accurately diagnosing paediatric TB continues to be a challenge.
Conventional TB tests rely on identifying bacteria in a sputum sample. However, children
can find it hard to produce sputum, and when they do, it is oen low in bacterial load.
Current molecular tests can determine whether a patient has active TB disease or has
been exposed to TB. Combinations of tests can identify whether a patient has latent TB
infection but so far cannot determine the risk of progression to active TB disease. Nor
can tests determine if an active TB case has been cured. Though developers have made
progress with tests to detect bacterial DNA or antigens they continue to face significant
challenges. Target product profiles for tests that predict progression from latent TB
infection to active disease, including evaluation protocols to assess these tests, are
expected in Q2 2017.
Next-generation sequencing technologies combine diagnosis of the bacteria, drug-
resistance genotyping and molecular epidemiology from a single test or sample. To
date, next-generation sequencing for clinical purposes has mainly been used in the
diagnosis and treatment of noncommunicable diseases such as cancers. But the value
of this technique has also been shown for challenging infectious diseases such as TB.
While this technology is not available in resource-limited settings and is aimed at tertiary
reference-level facilities, smaller platforms are available. Yet, a number of challenges
remain with the use of next-generation sequencing, including acquiring and extracting
of enough bacterial DNA, especially from compromised samples, or those with low
bacterial load. A number of groups are developing soware to help people without
bioinformatic skills to process and analyse large sets of raw data. The Foundation for
Innovative New Diagnostics (FIND) is reviewing current technologies and methods
to identify where an “end-to-end” next-generation sequencing system (a device that
processes samples from insertion to results) could be implemented in laboratories in
low- and middle-income countries.
Significant events since the last TB diagnostics
technology landscape
Since the publication of the fourth edition of the Unitaid Tuberculosis diagnostics
technology and market landscape report in October 2015, there have been a number of
significant events.19
WHO has released policies on the lateral-flow urine lipoarabinomannan assay (highly
restricted use recommendation),5 as well as an updated policy on use of molecular
line-probe assays for the detection of resistance to isoniazid (INH) and rifampicin
(RIF).7 WHO has also recently released policy statements on the use of molecular
line-probe assays for the detection of resistance to second-line anti-TB drugs,8 and a
robust molecular assay to detect pulmonary TB in sputum (the TB-LAMP assay6 from
b Aged <15 years.
the Eiken Chemical Corp., Japan). Most recently, WHO recommended the Cepheid
GeneXpert® MTB/RIF Ultra assay (hereinaer Ultra MTB/RIF),9 a more sensitive
cartridge-based assay that is intended to ultimately replace the Xpert® MTB/RIF assay
currently used by many national tuberculosis programmes. In addition to these key
events, there have been two large company acquisitions involving key manufacturers
in the TB diagnostic space. Alere Inc. (USA) is currently in the process of being acquired
by Abbott Laboratories (USA). In September 2016, Cepheid Inc. (USA) was purchased
by the Danaher Corporation (USA) for US$ 4 billion.
The molecular test pipeline appears to be contracting. Some products have
underperformed in trials, whilst others do not yet have independent field evaluations
or have reported major delays. As a result, molecular testing at microscopy centres in
the near future may involve the roll out of the Loopamp™ MTBC assay (Eiken Chemical
Corp, Japan) in addition to the GeneXpert® platform. FIND have negotiated pricing for
this new assay and its associated equipment.20 Previous editions of this landscape
report stated that Alere™ q and the Cepheid Inc. Omni systems would be expected in
the near future. However, both of these products have experienced challenges to their
development. Alere Inc. has stopped development work for its q instrument to host
assays for Mycobacterium tuberculosis (MTB) and drug resistance. Meanwhile, Cepheid
Inc. has announced that production of the GeneXpert® Omni (hereinaer Omni) is
delayed due to technical redevelopment challenges associated with manufacturing to
ensure compatibility of test cartridges with the Omni platform. Omni will be assessed
for equivalent performance with the current GeneXpert® platform and WHO will issue
implementation guidance. Field testing in a variety of settings, led by FIND, for the new,
high-sensitivity cartridge, the Ultra MTB/RIF, started in Q1 2016. In March 2017, WHO
released a statement extending the recommendations for the use of Xpert® MTB/RIF to
Ultra MTB/RIF, aer the WHO Technical Expert Group concluded that the Ultra MTB/RIF
is non-inferior to Xpert® MTB/RIF.9 A policy update is expected in 2018 and the timeline
for this product’s release is not known.
Other developers have noted European Conformity-in vitro diagnostic (CE-IVD)
registration of their products in 2016, including Autoimmun Diagnostika (Germany) with
three line-probe assays for drug resistance genotyping and Hain Lifescience (Germany)
with its FluoroType® real-time polymerase chain reaction assays for detection of MTB
and genotyping of drug resistance.
Molbio (India) has increased its portfolio of technologies for genotyping of drug
resistance and made improvements to its sample processing or detection technologies.
It has received or is about to receive CE-IVD markings for them. However, many of the
developers with products in phases 1 and 2 have noted delays in the release dates
scheduled for their products. The reasons are varied but typically reflect a lack of
funding and/or unanticipated technical challenges during development work.
This diagnostics technology landscape outlines the progress with the development of
tools for screening and triage, sample transport, automated microscopy, culture-based
tools for diagnosis of TB and drug susceptibility testing, molecular tests to detect MTB
exposure and TB disease, including sequencing methods.
Recent World Health Organization (WHO) estimates indicate, in 2015 alone, over 10.4
million people fell ill with active tuberculosis (TB), including 580 000 people with a
rifampicin-resistant (RR) form of TB, and over 1.4 million people died.1 Access to rapid,
simple, aordable and reliable TB diagnostics at the point of care (POC) is crucial to
accelerating a reduction in TB incidence and achieving the global goals of ending TB.
As new regimens for drug-resistant TB develop, screening and diagnostic cascades
and technologies need to evolve to keep up with needs of patients to receive a timely
and accurate diagnosis of TB and initiation of treatment. In 2015, only 6.1 million TB
cases were notified to national tuberculosis programmes (NTPs), 57% of which were
bacteriologically confirmed via a WHO-recommended test.1 The remaining 4.3 million
cases were either not diagnosed, or not notified to TB programmes.
Timely access to accurate and reliable diagnostics is necessary for the rational and
responsible use of antimicrobials. Universal access to drug susceptibility testing
(DST), as called for in the WHO End TB Strategy, can include both phenotypic and
genotypic testing methods. The need for access to diagnostics for children with
TB and DST to identify cases of drug-resistant TB is crucial in ensuring patients
receive timely and appropriate treatment. In 2015, only 30% of the 3.4 million of the
biologically confirmed TB cases notified globally were reported to have had DST for at
least rifampicin (RIF).1 Overall, there has been a small increase in DST coverage (22%
in 2014) but this varies substantially between countries. Only 23% of the estimated
580 000 cases with RR TB, were detected and notified, with 36% of these notified
cases also receiving DST for fluoroquinolones (FLQ) and second-line injectable (SLID)
Diagnostics for the detection of latent TB infection (LTBI) are of growing interest.
Studies have shown that treating patients with LTBI reduces the risk of active TB in
people living with HIV (PLHIV) especially when a diagnostic is used to identify those
infected with TB.27 However, those at greatest risk of developing active TB aer an
infection, e.g. PLHIV and those who are immunosuppressed, oen are not detected
by using the current immunobased assays.
Therefore, a dynamic understanding of existing and forthcoming technologies is
key for stakeholders (including ministries of health, Unitaid and other funders) in
facilitating access to appropriate TB diagnostic tools and improving TB care in high-
burden TB and/or HIV settings. This edition of the Unitaid Tuberculosis Diagnostics
Technology Landscape report is intended to complement earlier reports, and presents
a comprehensive overview of TB diagnostic technologies that are commercially
available or close to market. Previous editions of this landscape report are available
The Unitaid Tuberculosis Diagnostics Technology Landscape (2017) was developed by
David Boyle (PATH, Seattle). The material in this landscape report was gathered by the
author from primary sources (e.g. surveys and interviews with technology developers;
targeted analyses where needed) and extensive review of secondary sources (e.g.
published and unpublished reports; WHO policies and systematic reviews; corporate
prospectuses; developer websites).
The technologies described in this landscape report were derived by continued outreach
to known diagnostic manufacturers and leading technology developers working
within the TB diagnostic market, ranging from established multinational diagnostic
companies to startups and academic groups. Information was provided through
questionnaires addressing their technology, target population(s), intended market,
pricing and national or regional regulatory approvals and manufacturing standards.
Only photographs received from manufacturers and developers were included in this
report. Further information on product development and diagnostic validation studies
was acquired via press releases, online technology updates, the peer-reviewed literature
and accessing clinical study websites. The author continually assesses peer-reviewed
literature to identify new technologies, assays or validation studies on existing tools to
update the landscape reports.
With the dissemination of Unitaid landscape reports since 2012, diagnostics developers
also now approach the author with unsolicited product information to be included
in the reports. While information on cost per test or device and intended markets is
provided solely at the discretion of the manufacturer, performance data of any product
described in this landscape report are derived only from independent studies that have
been published in peer-reviewed literature in an attempt to validate the veracity of
claims from developers regarding test accuracy. All images have been reproduced with
permission of the respective companies or agencies.
The author and Unitaid are grateful to all the industry representatives who shared
information (and images) on their products, and also acknowledge technical input
from Madhukar Pai (McGill University, Montreal). Industry and FIND contributions were
technical in nature. Unitaid acknowledge contributions and review of the document by
Laboratories, Diagnostics and Drug-Resistance unit at WHO. The diagnostic technology
pipeline was developed independently by David Boyle with support from Unitaid.
David Boyle serves as a consultant to the Keck Graduate Institute (KGI) TBDx System
project (R01AI111477) and receives funding from Ustar Biotechnologies (China) on a TB-
related project. He has no other commercial/financial interests to declare pertaining to
information described in this landscape report.
This landscape report is an update to the fourth edition,19 which detailed the primary
methods for the diagnosis of pulmonary TB (PTB) and many of the technologies and
products associated with these methods. Since the publication of the fourth edition
in October 2015, there have been a number of significant events. This landscape
report highlights these events, provides the latest updates from developers and
diagnostic manufacturers, with a focus on newer products, regulatory news and
reports on the unbiased validation of technologies that are now on the market or
close to market release. The product categories discussed in this landscape report
include screening and triage tools, solutions for diagnostic transport, automated
microscopy, culture-based tools for the diagnosis of TB and DST, biomarkers to
detect exposure to Mycobacterium tuberculosis (MTB) and TB disease. In particular,
this landscape report features information on the advances in “–omics” research and
next-generation sequencing (NGS) tools for their potential to identify biomarkers
with strong predictive value for TB infection and provide a highly accurate diagnostic
method for TB infection, genotypic DST and molecular epidemiologic information.
As with earlier editions, this landscape report places a strong emphasis on the
development of nucleic acid amplification tests (NAATs) because of their potential to
replace sputum smear microscopy (SSM) and/or oer faster, more eective diagnosis
of PTB and multidrug-resistant (MDR) TB. Other areas of TB diagnostics are noted, but
unless there are significant changes from earlier landscape reports, these areas are
only discussed briefly.
This diagnostics technology landscape outlines the progress with the development
of tools for screening and triage, sample transport, automated microscopy, culture-
based tools for diagnosis of TB and DST, biomarkers to detect MTB exposure and
TB disease; and NAATs and sequencing methods for TB diagnosis and DST. Figure 1
shows the updated overall diagnostic pipeline, including the technologies used in the
patient screening and diagnosis cascade.
Since the fourth edition of this landscape report, the molecular diagnostic pipeline
appears to be contracting, with some products underperforming in trials (or with
insuicient evidence) and others reporting major delays in timelines (see Figure 7 in the
section “Update to the NAAT pipeline”). Recently, WHO conditionally recommended
the use of loop-mediated amplification (LAMP) as a low complexity NAAT suitable for
use at microscopy and higher-tier levels.6 The broader use of NAATs has improved
the diagnosis of MDR TB.11 In March 2017, WHO recommended Cepheid GeneXpert®
MTB/RIF Ultra cartridge (hereinaer Ultra MTB/RIF) that can both diagnose MTB and
indicate MDR TB.9 This landscape report highlights the development of the extant
NAATs assays in terms of product release, regulatory approvals and technologies that
are no longer on the market. The route to market of NAATs is generally impeded by
a lack of or delay in independent validation studies to inform on the performance
of the products in their intended use-case settings. This landscape report notes
LAM in sputum (Standard Diagnostics)
IRISA-TB -pleural/pericardial/ascitic fluid
(Antrum Biotec)
ß-lactamase reporter
(Global BioDiagnostics)
New TruArray MDR-TB (Akkoni)
COBAS TaqMan MTB + DST(Roche)
Hydra 1K (Insilixa)
Mycobacterium Real-time MDR
MTB Detect (Great Basin Scientific)
Aries (Luminex)
PNAClamp (Panagene)
AccuPower TB&MDR (Bioneer)
BNP Middlebrook (NanoLogix)
Rapid colorimetric DST
GenoTYPE MTBDRsl (Hain)
REBA MTB-Rifa (YD Diagnostics)
TRC Rapid MTB (Tosoh)
VereMTB (Veredus Laboratories)
LiPA Pyrazinamide (Nipro)
Fluorotype MTBDR (Hain)
TBMDx (Abbott)
Meltpro (Zeesan)
Mycobacteria RT PCR (CapitalBio)
REBA MTB-XDR (YD Diagnostics)
EasyNAT TB (Ustar)
BD Max (BD)
Anyplex series (Seegene, Korea)
(Thermo Fisher)
Sensititre System (Thermo Fisher)
Xtend XDR (Cepheid)
Alere Q (Alere)
Enigma ML (Enigma Diagnostics)
Q-POC (QuantuMDx)
EOSCAPE (Wave80)
TBDx system (KGI)
X1 (Xagenic)
MTB Detection (Tangen Biosciences)
TB POC (Qiagen)
Savanna (NWGHF/Quidel)
T-Track TB (Lophius)
TAM-TB (LMU/Alere)
ESAT-6/CFP-10 skin test (SSI)
BreathLink (Menssana)
Prototype breathanalyzer (Next
Dimensions Tech)
TB Breathalyser (Rapid Biosensor
Aeonose (The eNose Company)
Breath analysis instrument
Breath analysis instrument (Avisa)
Breath analysis instrument (Technion)
TBDx (Applied Visual Sciences)
Fluorescent microscopy (ID-FISH Tech.)
Automatic TB Screener (Fluorobot)
Cellscope (UCSF)
TB LAMP (Eiken)
Genedrive MTB/RIF (Epistem)
Truelab/Truenat MTB (Molbio)
Xpert Ultra/Omni platform (Cepheid)
QuantiFERON-TB PLUS (Qiagen)
Diaskin (Generium)
Microimager (BD)
CAD4TB (Delft Imaging Systems)
Figure 1. Current FIND TB diagnostics pipeline listing the development phases and types of
technologies in development or evaluation
Source: Image reproduced with permission of FIND.
several key policy decisions and peer-reviewed articles describing the performance of
certain NAATs, including two of the next-generation tools (previously called the “fast
followers”). These documents may play an important role in helping the TB community
assess if the performance of these tools matches the needs and expectations of the TB
The application of NGS is becoming more aordable and is increasingly being used
for high-resolution molecular epidemiology of TB and genotyping of drug resistance.
This will further aid the diagnosis of MDR TB and extensively drug-resistant (XDR)
TB and more accurately establish which genotypic markers can be used in DST
assay development. The introduction of new anti-TB drugs or regimens needs to be
paralleled with the ability to genotype for the molecular mechanisms of resistance
that will emerge with scaled use of new drugs and to then use this information to
develop appropriate genotypic assays to identify drug-resistant alleles to the new
Since late 2015, the following policies and associated recommendations have been
WHO policy on LF-LAM assay5
WHO published a policy statement for the use of the Alere Determine™ LAM assay in
November 2015. This is an immunologic lateral flow (LF) strip-based immunodiagnostic
test for the detection of LAM antigen in urine. The key recommendations are:
1. Except as specifically described below for PLHIV infection with low CD4 counts or
who are seriously ill, LF-LAM should not be used for the diagnosis of TB (strong
recommendation; low quality of evidence).
2. LF-LAM may be used to assist in the diagnosis of TB in HIV-positive adult inpatients
with signs and symptoms of TB (pulmonary and/or extrapulmonary) who have
a CD4 cell count less than or equal to 100 cells/μL, or HIV-positive patients who
are seriously ill regardless of CD4 count or with unknown CD4 count (conditional
recommendation; low quality of evidence).
i. These recommendations also apply to HIV-positive adult outpatients with signs
and symptom of TB (pulmonary and/or extrapulmonary) who have a CD4 cell count
less than or equal to 100 cells/μL, or HIV-positive patients who are seriously illd
regardless of CD4 count or with unknown CD4 count, based on the generalization of
data from inpatients.
ii. These recommendations also apply to HIV-positive children with signs and
symptoms of TB (pulmonary and/or extrapulmonary) based on the generalization
of data from adults, while acknowledging very limited data and concern regarding
low specificity of the LF-LAM assay in children.
c More detailed technical and performance descriptions are provided later in this landscape report.
d As defined, “seriously ill” is based on four danger signs: respiratory rate >30/minute; temperature >39 °C; heart rate >120/minute; and unable to walk unaided. Source:
Improving the diagnosis and treatment of smear-negative pulmonary and extrapulmonary tuberculosis among adults and adolescents: recommendations for HIV-prevalent
and resource constrained settings. Geneva: World Health Organization; 2007 (
iii. LF-LAM should not be used as a screening test for TB (strong recommendation; low
quality of evidence).
Based on the above, the existing commercial LAM assay has a very specific indication
for use among severely immunosuppressed, HIV-infected patients only. Hospitalized
patients with AIDS are the most likely setting for the use of this technology. Thus far,
no NTP has adopted this technology. However, South Africa is considering it, given its
TB-HIV co-infection burden, and data from a clinical trial that reported LAM-guided
initiation of anti-TB treatment in HIV-positive hospital inpatients with presumed TB
were associated with reduced 8-week mortality.28 Implementation of LAM testing is
likely to oer the greatest benefit where diagnostic resources are most scarce and where
patients present with severe illness, advanced immunosuppression and an inability to
self-expectorate sputum.
WHO policy on LAMP6
In August 2016, WHO issued a policy recommendation on the TB-LAMP Mycobacterium
tuberculosis complex (MTBC) assay. This low-complexity NAAT technology is designed
for use in microscopy centres and in higher-tiered test facilities. This recommendation
is highly significant as this is the first recommendation for the use of a NAAT-based assay
targeting the microscopy centre level. The two policy recommendations are:
1. TB-LAMP may be used as a replacement test for SSM for the diagnosis of PTB in
adults with signs and symptoms consistent with TB (conditional recommendation;
very low quality of evidence).
2. TB-LAMP may be used as a follow-on test to smear microscopy in adults with signs
and symptoms consistent with PTB, especially when further testing of sputum
smear-negative specimens is necessary (conditional recommendation; very low
quality of evidence).
i. These recommendations apply to settings where conventional SSM is able to be
ii. TB-LAMP should not replace the use of rapid molecular tests that detect TB and
resistance to RIF, especially among populations at risk of MDR TB.
iii. Due to limited evidence, it is unclear whether TB-LAMP has additional diagnostic
value over SSM for the testing of PLHIV with signs and symptoms consistent with TB.
iv. These recommendations apply only to the use of TB-LAMP in testing sputum
specimens from patients with signs and symptoms consistent with PTB.
These recommendations are extrapolated to the use of the TB-LAMP assay in children,
based on the generalization of data in adults, while acknowledging diiculties in the
collection of sputum specimens from children.
WHO updated policy on the use of molecular
LPAs for the detection of resistance to
isoniazid (INH) and RIF7
In November 2015, WHO published an update to the first-line LPA policy, endorsing the
Hain Lifescience (Germany) GenoType® MTBDRplusv2.0 and the NIPRO Corporation
(Japan) NTM+MDRTB Detection Kit 2. The key recommendation is:
1. For persons with a sputum smear-positive specimen or a cultured isolate of MTBC,
commercial molecular LPAs may be used as the initial test instead of phenotypic
culture-based DST to detect resistance to RIF and INH (conditional recommendation;
moderate certainty in the evidence for the test’s accuracy).
i. This recommendation applies to the use of LPAs for testing sputum smear-positive
specimens (direct testing) and cultured isolates of MTBC (indirect testing) from
both pulmonary and extrapulmonary sites.
ii. LPAs are not recommended for the direct testing of sputum smear-negative
iii. This recommendation applies to the detection of MTBC and the diagnosis of MDR
TB but acknowledge that the accuracy of detecting resistance to RIF and INH diers
and, hence, the accuracy of a diagnosis of MDR TB is reduced overall.
iv. This recommendation does not eliminate the need for conventional culture-based
DST, which will be necessary to determine resistance to other anti-TB agents and to
monitor the emergence of additional drug resistance.
v. Conventional culture-based DST for INH may still be used to evaluate patients when
the LPA result does not detect INH resistance. This is particularly important for
populations with a high pre-test probability of resistance to INH.
vi. This recommendation applies to the use of LPA in children based on the
generalization of data from adults.
Based on the above, either tool can be used to detect TB and also to genotype the
alleles that are known to confer resistance to RIF and INH from either a sputum smear-
positive sample or from culture-based isolates. These tests are designed for reference
and intermediate facilities. The test procedures, while requiring general equipment, can
be performed manually or scaled with LPA-specific instrumentation to permit larger
numbers of tests performed.
WHO policy guidelines on the use
of molecular line-probe assays (LPAs)
for the detection of resistance to
second-line anti-TB drugs8
In May 2016, WHO issued a policy recommendation on second-line LPA (SL-LPA)
assays (e.g. the Hain Lifescience GenoType® MTBDRslv1.0 and v2.0). The key
recommendations are:
1. For patients with confirmed RR TB or MDR TB, SL-LPA may be used as the initial test,
instead of phenotypic culture-based DST, to detect resistance to FLQs (conditional
recommendation; moderate certainty in the evidence for test accuracy for direct
testing of sputum specimens; low certainty in the evidence for test accuracy for
indirect testing of MTB cultures).
2. For patients with confirmed RR TB or MDR TB, SL-LPA may be use as the initial test,
instead of phenotypic culture-based DST, to detect resistance to SLIDs (conditional
recommendation; low certainty in the evidence for test accuracy for direct testing of
sputum specimens; very low certainty in the evidence for test accuracy for indirect
testing of MTB cultures).
i. These recommendations apply to the use of SL-LPA for testing sputum specimens
(direct testing) and cultured isolates of MTBC (indirect testing) from both pulmonary
and extrapulmonary sites. Direct testing on sputum specimens allows for the earlier
initiation of appropriate treatment.
ii. These recommendations apply to the direct testing of sputum specimens from
RR TB or MDR TB, irrespective of the smear status, while acknowledging that the
indeterminate rate is higher when testing sputum smear-negative specimens
compared with sputum smear-positive specimens.
iii. These recommendations apply to the diagnosis of XDR TB, while acknowledging
that the accuracy for detecting resistance to FLQs and to SLIDs diers and hence the
accuracy of a diagnosis of XDR TB overall is reduced.
iv. These recommendations do not eliminate the need for conventional phenotypic
DST capacity, which will be necessary to confirm resistance to other drugs and to
monitor the emergence of additional drug resistance.
v. Conventional phenotypic DST can still be used in the evaluation of patients with a
negative SL-LPA result, particularly in populations with a high pre-test probability
for resistance to FLQs and/or SLIDs.
vi. These recommendations apply to the use of SL-LPA in children with confirmed RR
TB or MDR TB based on the generalization of data from adults.
vii. Resistance-conferring mutations detected by SL-LPA are highly correlated with
phenotypic resistance to ofloxacin (OFX) and levofloxacin; however, the correlation
of these mutations with phenotypic resistance to moxifloxacin (MOX) and
gatifloxacin is unclear and the inclusion of MOX or gatifloxacin in a MDR TB regimen
is best guided by phenotypic DST results.
viii. Resistance-conferring mutations detected by SL-LPA are highly correlated with
phenotypic resistance to SLID and are an indication to use an MDR TB regimen that
is appropriately strengthened.
ix. Given high specificity for detecting resistance to FLQs and SLID, the positive results
of SL-LPA could be used to guide the implementation of appropriate infection
control precautions.
This test is designed to be a reflex test for samples testing positive for MDR TB, e.g. aer
using molecular LPA for the detection of INH and RIF. Similarly, this test is intended for
use in reference and intermediate facilities. The test procedure, while requiring general
laboratory equipment, can be performed either manually or scaled with LPA-specific
instrumentation to permit high volume throughput.
WHO recommendation
of GeneXpert® MTB/RIF Ultra9
In March 2017, WHO issued a report from the technical expert review of Ultra MTB/RIF.
The dossier of evidence was prepared by FIND in collaboration with the Tuberculosis
Clinical Diagnostics Research Consortium. The data was derived from a multi-country
non-inferiority study. A total of 1520 patients with signs and symptoms of TB were
enrolled in these countries for a direct comparison of the performance of Ultra MTB/RIF
assay against the current GeneXpert® MTB/RIF assay (hereinaer Xpert® MTB/RIF) on the
same specimen. The accuracy of these assays was assessed via with four cultures as the
reference standard for TB detection (two liquid culture tubes + two Löwenstein–Jensen
slants, performed on two specimens obtained on separate days). Phenotypic drug-
susceptibility testing as well as sequencing was performed for rifampicin resistance
detection. The redevelopment of the cartridge and assay designs has resulted in
Ultra having a limit of detection (LOD) of 16 bacterial colony forming units (cfu) per ml
compared to 114 cfu/ml for Xpert® MTB/RIF.
The technical expert group agreed Ultra MTB/RIF was not inferior to the current Xpert®
MTB/RIF for the detection of MTB and the detection of RR TB. The greatest benefit of
the Ultra MTB/RIF was in the increased yield for the detection of MTB in smear-negative
culture positive specimens, paediatric specimens, extra-pulmonary specimens (notably
cerebrospinal fluid) and especially for HIV positive individuals whose specimens are
frequently paucibacillary.
In the report, WHO listed a series of implementation considerations for the Ultra MTB/
1. The interpretation of Ultra MTB/RIF results for MTB detection are the same as for
Xpert® MTB/RIF with the exception of “trace calls”.
2. Ultra MTB/RIF has high sensitivity for MTB detection and incorporates a new
semiquantitative category “trace call” that corresponds to the lowest bacillary
burden for MTB detection.
3. Ultra MTB/RIF has both high sensitivity and specificity for rifampicin resistance
4. All persons with RR TB, identified by Ultra MTB/RIF should undergo further testing
as per current WHO policy guidance to determine if there is additional resistance to
the class of fluoroquinolones and/or the group of second-line injectable drugs.
5. Ultra MTB/RIF can be used on all GeneXpert® instrument platforms and is suitable
for use at central or national reference laboratory level, regional and district
levels. GeneXpert® has the potential to be used at the peripheral level, provided
uninterrupted electricity supply and temperature conditions can be ensured.
The Ultra MTB/RIF cartridge is envisaged to eventually replace the current Xpert® MTB/
RIF assay due to its greater sensitivity in detecting MTB. The new cartridge also has the
potential to provide more accurate test data with its capability to discriminate silent
mutations in rpoB that do not confer RIF drug resistance (see Modular, cartridge-based,
fully automated NAATs).
There are a number of TB disease screening triage tools available ranging from symptom-
based screening checklists to automated digital chest X-rays (DCXR). The purpose of such
tools is to triage patients most likely with active TB disease or TB infection, thereby reducing
the need for laboratory testing and saving money with the intent of improving TB diagnosis.
The next sections provide an update on such screening technologies in development.
In 2016, WHO released a factsheet and a summary of current recommendations and
guidance on programmatic approaches on chest radiography for TB detection. The
factsheet highlighted the essential role of a chest X-ray (CXR) as a sensitive tool for
screening active TB disease, diagnosing childhood TB (pulmonary and extrapulmonary)
and excluding active TB before initiating treatment of latent TB.29,30 A recent survey in
14 TB high-burden countries (HBCs) reported that chest radiology was available at
over 90% of district hospitals or community health centres and in 100% of reference
or tertiary hospitals. At lower levels, only two countries noted that they oered CXR at
local health posts or in the community and only three countries noted that CXR was
broadly available at microscopy centres or primary health centres.31
As digital technology becomes more aordable and accessible, the use of DCXR is an
area of increasing interest for clinicians and TB programmes. The potential of portable
DCXR as a tool for TB screening in low-resource settings was first introduced in the 2014
landscape report.32 DCXR oers rapid and low-cost screening when compared with
the older film-based method. In addition, the digital images can now be analysed via
computer-based algorithms, which precludes the need for a radiographer to look for
lesions and other indicators of TB disease thus reducing cost, oering greater flexibility
and making more rapid diagnosis. As detailed in the fourth edition of this landscape
report, the Computer Aided Detection for Tuberculosis (CAD4TB) automated analysis,
commercialized by Del Imaging Systems (Netherlands) with European Community
(CE)-certification, can be used with digital images provided by DCXR equipment.
The use of DCXR may be used as a triage tool and for patients with abnormalities
consistent with TB followed up with bacteriological tests. A recent study in South Africa
demonstrated that prescreening by DCXR increased throughput of patient testing and
decreased the amount of Xpert® MTB/RIF tests required with only a slight decrease in
sensitivity.33 The study noted the performance of the diagnostic algorithm among HIV-
uninfected patients was considerably better than in HIV-infected patients, therefore
consideration should be applied for dierent thresholds for both groups. In May 2016,
the Ministry of Health of Ghana announced that it would roll out 52 digital X-ray systems
with CAD4TB soware and a platform for teleradiology. The Accelerating Tuberculosis
Case Detection in Ghana project is aimed at improving TB diagnosis in addition to
providing improved health-care services for other general needs for X-rays, including
traumatic injuries.
Meanwhile, Advenio (Chandigarh, India) is currently developing Ri-View TB, another
automated image analysis soware package for use with DCXR. The company is working
with the National Institute for Research in Tuberculosis (Chennai, India) with support from
the Bill & Melinda Gates Foundation but the current status of development is unknown.
The rapid and non-invasive screening of PTB disease via metabolic compounds in
breath and urine has been an area of research. Giant African pouched rats have
accurately predicted active PTB disease by smelling sputum samples.34-36 The nematode
Caenorhabditis elegans has a measurable chemotactic response upon exposure
to MTB-specific VOCs.37 A number of companies are developing instrumented VOC
products via a range of methods, including gas chromatography (Menssana Research
Inc., USA), metal-oxide sensors (Nanosynth Materials and Sensors, USA; and The eNose
Company, Netherlands) and metabolite detection by chemical reaction (Metabolomx,
USA). The Rapid Biosensor Systems Ltd (UK) has also developed a breath test but as it is
antigen-based for the detection of MTB, it is represented in the “Serologic and antigenic
biomarkers of TB” section below.
Figure 2. Prototype Nanosynth breath test and detector
Notes: The exhaled air is passed over the detection chip (red square) that is inserted in the test instrument. Bluetooth capability on the instrument then sends the
test data to a smartphone that hosts the data analysis application and subsequently processes the test result.
Source: Image reproduced with permission of Nanosynth and the University of Utah.
Products are at varying stages in the development pipeline. While Menssana Research
Inc. was one of the first companies in this space, the current status of its Breathscanner™
tool is unknown. Nanosynth has developed a prototype device that is currently
undergoing pilot studies in Uganda and also in four districts of Mumbai (Figure 2).
The Mumbai study intends to screen up to 1000 TB cases. In terms of time to market,
Nanosynth expects to be ready in Q1 2019, with an initial target of India, South Africa
and Uganda as its entry markets. This portable device involves a patient to breathe
into a tube, and if the patient has TB then the VOCs, produced by MTB in the lungs,
bind to the titanium oxide nanotubes present in the device. This binding results in an
electrical current being produced that is detected and read by a hand-held electronic
device such as a smartphone. Nanosynth claims this screening device takes 10 minutes
to run a single test. Patients with a positive result are then recommended to undergo
confirmatory testing to establish diagnosis and DST.
The eNose Company recently received ISO 13485 certification and its Aeonose™ device
obtained CE registration on 1 December 2015 (Figure 3). Pilot and validation studies
in several countries, including Bangladesh, Indonesia, Kenya, Paraguay, South Africa
and Venezuela are ongoing. Data from Bangladesh have demonstrated a sensitivity
and specificity (compared to SSM and solid culture) of 76.5% and 87.2%, respectively.38
The intended population for Aeonose™ is adults and children older than 4 years of
age. The environmental conditions for use include operational temperatures from 10–
40 °C, storage temperature range of minus 40 °C to 70 °C and 10– 85% non-condensing
humidity. The device contains a rechargeable battery and is independent of mains
electricity. Users of Aeonose™ should be qualified operators, and a nurse practitioner
can easily be trained to operate the device.
Figure 3. Hand-held Aeonose™ device
Notes: The device contains three metal oxide sensors (le). Carbon fibre filters are placed onto the mouthpiece and in the device entry point (top centre). Test
data are analysed by transferring data via an iOS system to a secure server in Amsterdam through a 3/4G connection via an application tool (lower centre) and
returned data are presented as an interpretable test result (right).
Source: Images reproduced with permission of The eNose Company.
Carbon fibre filter discs are attached to both the Aeonose™ instrument and the
mouthpiece to prevent risk of aerosolization of TB cells. A bacterial filter located inside the
mouthpiece and a one-way valve system prevent contamination of the instrument by TB or
by breath-associated microbes (Figure 3). A single test takes a minimum of 15 minutes to
complete, including yielding results. The mouthpiece is gently inserted into the sample port
on the top of the Aeonose™. The patient wears a disposable nose clip and slowly breathes into
the device via a disposable mouthpiece for 5 minutes, then the device performs additional
actions for another 10 minutes to clean the sensors. An audio signal indicates completed
collection. The collected data are sent via Bluetooth to an iOS application hosted by an
iPhone or iPad and then transmitted by the iOS device to a secure server to be analysed.
The results are sent back immediately and displayed on the iOS application. The disposable
mouthpiece and filters are removed and new ones inserted prior to further use. The device
should be cleaned at the end of each day with 70% alcohol. The eNose Company currently
recommends each device is returned yearly for a service and calibration check. Dedicated
soware to perform the service at the test location is close to finalization. While current
costs are unknown, The eNose Company is reported to be working with larger organizations
in order to reduce the currently high cost of production due to instrument complexity.
Finally, Metabolomx recently reported results from a small pilot study of its prototype array
under a variety of urine test conditions, with 85.5% sensitivity and 79.5% specificity.39 The
prototype uses a colorimetric sensor array to indicate TB infection from VOCs in urine.
Inhalation of MTB can lead a person’s immune system to respond by eliminating the inhaled
bacilli and controlling the multiplying inhaled bacilli. If the immune system successfully
contains the bacilli, halts their multiplication and prevents further progression in the body,
then LTBI is established. In this state, the person is infected with TB but is neither infectious
nor symptomatic. However, if MTB overwhelms the immune system and continues to
multiply, then the person progresses to TB disease. People who are infected with latent TB
and who are not immunocompromised typically have a 10% lifetime risk of developing TB
disease. If the person becomes immunocompromised, or is a child under the age of 5 years,
then this risk can increase significantly, e.g. 20–37 times greater risk for PLHIV.40 Therefore,
as indicated in the WHO End TB Strategy, the diagnosis and treatment of LTBI are important
elements in the control and elimination of TB disease worldwide.41
Detecting exposure to MTB can be currently performed using two immune response-based
screening methods: the tuberculin skin test (TST); and interferon gamma release assays
(IGRAs). However, both methods have shortcomings and are of insuicient performance to
discriminate between active TB disease and LTBI.
Currently, the Mantoux TST is commonly used and is performed by an intradermal infection
of a purified protein derivative from MTB into the skin of the forearm. The challenges that
limit the performance of TST include patient age, immune status, recent Bacillus Calmette-
Guérin (BCG) vaccination history (BCG limits the specificity of TST), exposure to non-
tuberculous mycobacteria (NTM) and the cut o for reading the test.42 In addition, shortages
of supply of the TST antigens have been reported in Europe and the United States.43,44
IGRAs are another immune response-based laboratory test used to indicate MTB exposure.
The assays have higher specificity than the TST, have less cross-reactivity with BCG than
the TST and correlate well with MTB exposure. Additionally, they require more complex
infrastructure and highly skilled sta. This method requires CD(4)+ T-lymphocytes (T-cells)
harvested from a patient’s whole-blood sample to be incubated with MTB-specific antigens;
the early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP 10). These
antigens stimulate the release of interferon-gamma (IFN-γ) from the CD(4)+ T-cells, which is
then quantified by the assay.
The QuantiFERON-TB (QFT) Gold In-Tube assay (Qiagen, USA) and the T-SPOT.TB® assay
(Oxford Immunotec, UK) are two widely available IGRAs that have US Food and Drug
Administration (FDA) and Health Canada clearance and are CE marked for use in Europe. The
QFT assay also includes a third MTB antigen, the TB7.7 antigen. In addition to these, several
other IGRA products are available from manufacturers from China (TB-IGRA, Beijing Wantai
Biological Pharmacy Enterprise Co. Ltd; ASACIR TB, Haikou VTI Biological Institute), Republic
of Korea (SD Biosensor Inc.) or India (TB Platinum, Immunoshop India Pvt Ltd). Qiagen has
released the QFT gold plus, which also stimulates IFN-γ production from CD(8)+ cells,
improving the identification of TB infection in immunodeficient individuals, including
HIV-positive patients and small children (Figure 4). This assay involves larger MTB
peptide antigens to stimulate CD(4)+ cells and shorter peptide antigens to stimulate the
Figure 4. QuantiFERON-TB Gold Plus kit from Qiagen: reagents and ELISA plates (le) and collection
materials (right)
Source: Images reproduced with permission of Qiagen.
CD(8)+ cells. Qiagen claims greater sensitivity by its assay oering greater stimulation
of IFN-γ release from two subsets of T-lymphocytes rather than just CD(4)+. Since the
previous landscape report, there have been five studies to evaluate the performance of
the newer test that note similar performance to the QFT gold.45-49
Meanwhile, the Statens Serum Institut in Denmark has developed a novel skin test
named C-Tb for detecting LTBI. The basic concept behind the test is to combine the
ease-of-use of the TST with the high specificity of the IGRAs. The test measures the
body’s immune response to two specific MTB antigens that are not contained in the BCG
vaccine: ESAT-6; and CFP10. The C-Tb test is designed to be used in the exact same
way as the TST for the end user. Contrary to the TST where the cut o of induration
varies depending on local guidelines, age and immune status (from 5–15 mm), the
C-Tb test has a fixed cut-o value of 5 mm of induration.50A double-blinded phase 3
study of C-Tb in 979 adults, ranging in TB status from presumed uninfected through
intermediate and high risk of latent TB to active disease, was carried out by the
Statens Serum Institut in Spain. Both C-Tb and IGRA had a specificity of 97% (Statens
Serum Institut, unpublished information, 2016). C-Tb was strongly associated with
MTB exposure and the C-Tb test was concordant to IGRA in 95% of study participants.
A recent study in South Africa reported sensitivity of C-Tb to be similar to IGRA (73.9%
and 75.1%, respectively) in patients with confirmed TB disease, with the sensitivity
unaected by HIV status. However, C-Tb sensitivity was significantly reduced for PLHIV
with CD4 counts <100 cells/mm3.51
A recent independent review of TST, IGRAs and the C-Tb assay noted that while both IGRAs
and C-Tb assays have improved specificity compared to the purified protein derivative
TST, this came with a decrease in sensitivity.52 A similar review also noted that based on
current technologies, the TST is suitable for use in resource-constrained settings as the
test is low cost, relatively easy to perform and does not require facilities of highly skilled
sta.42 The price of the new C-Tb test has yet to be determined but it is anticipated to be
significantly lower than the IGRAs, making it a more cost-eective and simple-to-use
test for LTBI. The C-Tb test will be manufactured under good manufacturing practice
standards in compliance with both European and US guidelines. Thus, a high, consistent
quality of the C-Tb product is ensured and, due to the simpler production process for
recombinant production, supply will be stable avoiding the periodic shortages of TST
that have been frequent in the past.43,44 The Statens Serum Institut is now in the process
of submitting an application for marketing authorization to the European Medicines
Agency. Other groups manufacturing or developing improved TSTs include Generium
Pharmaceutical (the Diaskintest, Russian Federation), which is available commercially
in Kazakhstan, the Russian Federation and Ukraine, and an ESAT-6-based skin test that
has undergone phase II trials in China.53
While these immune-response based screening tools can indicate if a person has been
exposed to TB, challenges still remain to dierentiate people with TB infection and TB
disease,54 predict and identify people beginning to progress from infection to active TB
disease,55,56 and assess if a patient has fully recovered from TB disease (and no longer
requires treatment).57 (See also the “Biomarkers to detect MTB exposure and TB disease
section below).
Aer the screening and triaging of patients at risk of TB, a diagnosis is necessary.
Currently, PTB diagnostic tools require adequate amounts of quality sputum specimens
collected from patients presumed to have TB. If a sputum sample is unable to be tested
in the facility where the collection took place, it may be shipped to a dierent laboratory
for diagnostic testing via culture, LPA or Xpert®. Certain tests are only available in larger
facilities (e.g. Xpert®, culture or DST LPAs), therefore transportation of the specimen
while maintaining its integrity is critical. As sputum is not sterile, prolonged storage at
ambient temperatures encourages growth of commensal microflora, which can aect
subsequent culture and also aect the quality of nucleic acids. Typically, samples are
shipped via a courier under cold chain but this becomes more challenging in peri-
urban or rural areas, thereby compromising quality of diagnosis and limiting access
to care. FIND has draed a preliminary target product profile (TPP) describing the key
components and attributes required.58 A systematic review of available solutions is
under way and a technical consultation to review additional data is planned for June
2017. There are two companies that are oering products in this space.
DNA Genotek Inc.
(Ottawa, Canada; a wholly owned subsidiary of OraSure Technologies
Inc., USA) has developed the OMNIgene® SPUTUM (OM-S), to liquefy and decontaminate
the sample and permit its transport without cold chain. The company claims viability
for at least 8 days in temperatures as high as 40 °C. The OM-S treated sample may be
used for microscopy, culture or nucleic acid testing; the primary diagnostic methods
for TB. A pilot study in Nepal compared cold chain shipping with OM-S treated sputum
and noted that the OM-S transport medium greatly reduce contamination of samples
from 12% to 2% and increased TB diagnosis by 9% when using culture.59 The smear
microscopy results with both methods were identical. In another study, also in Nepal,
OM-S treated sputum samples shipped at ambient temperatures gave identical results
to samples shipped via cold chain when using Xpert®.60 The study further demonstrated
the OM-S transport medium may be suitable for use with Xpert® when using either
the sediment or sputum protocols provided with the Mycobacterium tuberculosis/
rifampicin resistance (MTB/RIF) assay. FIND is working with DNA Genotek Inc. to validate
the performance of OM-S in a larger multisite study, aer a small study demonstrated
100% concordance with high- and medium-characterized MTB loaded sputum panels
stored in OM-S sputum.61
Longhorn Vaccines and Diagnostics LLC (USA), are oering the second product, the
PrimeStore® Molecular Transport Medium (PS-MTM). Unlike OM-S, which liquefies and
keeps MTB cells viable outside of the cold chain, PS-MTM liquefies the sample but also kills
all viable microorganisms and stabilizes the nucleic acids in the sample.62 The nucleic acids
remain stable over 28 days at ambient temperature in the medium and is compatible with
a range of nucleic acid extraction systems. PS-MTM is intended to aid microscopy centres
to send specimens to higher-tier laboratories for further NAAT analysis. An independent
study using PS-MTM with real-time PCR targeting IS6110 on clinical sputum samples were
compared with Xpert® and liquid culture data. PS-MTM with real-time PCR was concordant
with culture at 82% (McNemar, P = 0.55) and 84% (McNemar, P = 0.05) for Xpert®.63 A pilot
study has demonstrated PS-MTM may also be used with the Xpert® method but further
studies are necessary to build a stronger body of evidence to confirm this.64 The company
is currently working with manufacturers in the USA and Europe to produce its product at
scale intend to seek European Conformity-in vitro diagnosis (CE-IVD) certification once
these are established. While cost of the PS-MTM is unknown, the developer claims to have
a pricing scheme for low- and middle-income countries (LMICs).
The simplicity and low costs associated with direct visual examination for acid-fast bacilli
(AFB) SSM make it the most common diagnostics test used globally, especially in resource-
constrained settings. Despite its widespread use, SSM is not ideal for the diagnosis of MTB
as it has poor sensitivity and variability of results. The poor sensitivity of SSM is a challenge
in diagnosing paediatric and PLHIV TB cases as the specimens are oen paucibacillary, and
thus not detected by this method. SSM is also time consuming, has low throughput and
requires well-organized quality assurance programmes to maintain user performance. It
also does not detect drug-resistant (DR) forms of TB. A market estimate of the number
and cost of SSM in HBCs noted 77.6 million smears at a median cost of US$ 109 million.65
Previous landscape reports have documented the development of the Microimager, an
automated reader that was in development by Becton Dickinson (USA; hereinaer BD)
as a replacement tool for manual SSM. BD is no longer developing this tool and so it
will not come onto the market. The TBDx system is an automated digital microscopy
platform that is available from Signature Mapping Medical Sciences Inc. (a wholly owned
subsidiary of Applied Visual Sciences Inc., USA). This platform consists of a high-quality
microscope and imaging system that in conjunction with a slide holding carousel can
Figure 5. TBDx system
Note: Automated slide loader (le), fluorescence microscopy with digital camera and automated stage (centre) and laptop to operate the reader and employ the
scoring algorithm (right).
Source: Image reproduced with permission of Applied Visual Sciences Inc.
read up to 200 prepared smears using fluorescent microscopy in a single run (Figure 5).
The proprietary soware reads the images to detect stained cells. SSM slides can
be prepared via commercial automated or high-throughput staining platforms to
permit scaled preparation of slides with more consistent staining and washing prior
to reading – e.g. the RAL stainer (RAL Diagnostics, France) or Aerospray® TB Series 2
(ELITechGroup, France). Without the carousel, the system can automatically process
1–4 slides. The robotic carousel can host 50–200 slides with each slide being read in 5
minutes, meaning 200 slides would take about 16 hours to read. The developer notes
the scanning algorithm can be changed to permit faster reading of strongly SSM positive
samples to shorten the overall time of a run.
A pilot study in South Africa concluded that “as a standalone diagnostic system, it proved
to be comparable to highly experienced microscopists and oered a diagnostic solution
that could provide quality-assured microscopy in settings where trained microscopists
are diicult to find”.66 One area of interest for the application of this product is where
molecular testing is too expensive to routinely perform, for example, prescreening
sputum samples prior to Xpert® analysis in order to reduce the number of Xpert®
tests to screen negative or indeterminate samples only. This has been investigated in
a study where over 1000 samples were screened using SSM, automated SSM, Xpert®
and mycobacterial growth indicator tubes (MGIT™, liquid culture).66 A primary finding
was that using the TBDx to screen specimens prior to using Xpert® could detect 90% of
patients with subsequent Xpert®-positive results. The cost-eectiveness of automated
digital microscopy has been modelled to investigate the possible savings and impact
on TB diagnosis.67 The study investigated costs and eectiveness of dierent algorithms
for automated digital microscopy, including as a standalone test and with confirmation
of positive results with Xpert®. If screening more than 30 slides per day, the primary
conclusion was that while universal Xpert® is the preferred diagnosis, when resources
are scarce and MDR TB is not common, the application of automated digital microscopy
can identify the majority of cases and halve the cost of diagnosis and treatment. There
are no further cost updates since the previous landscape report, when the microscope
and computer oered was available at US$ 23 000 and the (optional) 200-slide robotic
loader at US$ 21 000.
(UK) is developing a MTB cell enrichment device to improve the sensitivity
of SSM beyond current basic concentration methods such as sputum sedimentation.
The company has started to investigate the use of an MTB cell concentration technology
as a standalone diagnostic called Capture-XT™. This technology is able to concentrate
low numbers of cells into a 10 μL fluidic capture chamber from an initial much larger
sample volume, therefore allowing the captured cells to then be visualized by either a
microscopy technician or a device with a camera. The presence of cells in the viewing
window will be positive for MTB as the proprietary capture mechanism is claimed to be
specific to MTB cells. QuantuMDx also notes that they are developing a process where
these concentrated cells could then be reflexed to the Q-POC™ molecular confirmatory
and DST assay that they are also developing (see below the section on NAATs for use in
microscopy centres). The potentially low-cost format of the Capture-XT™, which claims
to have sensitivities close to that of culture, is seen as a replacement technology for SSM.
Including the reflex molecular test as a replacement for culture, this technology might
potentially be implemented at the lower levels of the health-care system, including
microscopy centres. The Capture-XT™ assay is projected to take 45 minutes from raw
sputum. The current prototype processes samples individually but a higher-throughput
device is under investigation. The device will be battery operated and will be able to
operate for 8 hours on a single charge. Pilot testing of the technology has started. The
Capture-XT™ diagnostic disposable assay has a target price of between US$ 1–2 and
QuantuMDx estimates the initial product release for Q4 2017–Q1 2018.
There are no new updates regarding the culture-based diagnosis of MTB. Due to the risks
associated with laboratory-acquired infection, culture of MTB requires higher biosafety
requirements as opposed to methods that simply manipulate infected material (e.g.
SSM). Therefore, most culture is performed in reference facilities or some intermediate
laboratories dependent on appropriate biosafety facilities and trained sta. While
culture can take a long time to generate a test result (typically up to 4 weeks), it is a
highly sensitive assay and can also be used for the further phenotypic determination of
drug resistance with culture-positive cases. Three manufacturers oer automated liquid
culture-based systems: the BacT/ALERT® 3D instrument from bioMérieux (France); the
BACTEC™ mycobacterial growth indicator tube (MGIT™) platform from BD (USA); and
the Mycolor TK platform from Salubris (Turkey). While the Salubris product is new to
the market, there has been a small comparative study of the performance of TK-SLC-L
media and the MGIT™ platform.68 The data showed very similar performance with a
longer time for detection with TK-SLC-L (3 to 5 days); however, the group noted that
contamination events were significantly reduced with the TK-SLC-L media (1.3% versus
13.7%). Pricing is not available for the bioMérieux and Salubris products, although FIND
has negotiated preferential pricing with BD for HBCs for the procurement of the MGIT™
instruments, culture reagents and servicing.20
In addition to commercial liquid culture systems, WHO has recommended an interim
measure regarding the use of lower-cost and non-commercial methods such as
microscopically observed drug susceptibility (MODS) assays, which can be used
to simultaneously screen for MTB in addition to drug resistance in parallel culture
assays.69 Laboratories can procure individual reagents from local vendors to make their
own test media or procure a MODS kit from Hardy Diagnostics (USA), which includes
all key materials such as plates, sealed lids, media and drugs. This has been shown
to have similar performance to MODS assays in Peru.70 An advantage of MODS over
conventional culture is that the algorithm typically screens for INH and RIF resistance
as well as for growth of MTB; essentially speeding the time to a result for MDR TB from
culturing sputum samples. One multicentre study assessed the performance of several
methods to detect MDR TB or XDR TB from clinical samples specifically investigating
resistance to INH, RIF, MOX, OFX, amikacin (AMK), capreomycin (CAP) and kanamycin
(KAN).71 At 14.3 days, the mean time to result of the MODS assays was slower than LPAs
or pyrosequencing (both 1.1 days) but faster than MGIT™ (24.7 days) and having the
best performance in detecting RIF and KAN resistance.
The search for new biomarkers to indicate TB is ongoing.72-75 WHO convened a meeting in
April 2014 to determine the priorities for research in the TB diagnostics field.10 Four TPPs
for diagnostic tests were prioritized, including a rapid biomarker-based instrument-free
POC test for non-sputum samples (that can also detect childhood and extrapulmonary
TB). The identification of an appropriate biomarker is key to this need being realized
with a good commercial product based on the TPP metrics. The following section notes
the current commercial development work using serologic, immunologic, chemical and
other methods to identify biosignatures of TB infection.
Saliva, urine, serum and whole blood biomarkers provide the advantage of easy
sample collection, and have the potential to detect MTB exposure and dierentiate
between active TB disease and TB infection. There have been a number of new studies
investigating the combination of genes or transcriptomes to uniquely identify and
dierentiate between TB infection, TB disease and other diseases. A recent study found
a set of seven plasma inflammatory biomarkers that indicate TB infection.76 Dierences
in biosignatures in saliva have also been found to potentially discriminate TB from other
respiratory diseases and change with response to treatment.77 Another study using serum
has reported a set of six biomarkers strongly associated with active TB infection that also
change aer treatment, indicating a potential method to monitor treatment therapy.78
Studies investigating miRNA signatures from serum and peripheral blood mononuclear
cells may have diagnostic value.79,80 A recent meta-analysis using gene expression data
from TB study cohorts indicated a three-gene signature may be suicient to diagnose
PTB.81 In contrast, one small study demonstrated these same three genes may have
poor sensitivity to diagnose TB but could be used to rule out TB infection with very high
There have also been some developments in the search for TB pathogen-specific
biomarker molecules.83 A recent review article provides an excellent overview of the
current methods and challenges faced in the use of “–omicse” techniques to diagnose
active TB.84 One case control study identified a 27-transcript signature that distinguished
active TB disease from LTBI and a 44-transcript signature distinguished active TB from
other diseases from the whole blood of both HIV-infected and -uninfected adults.85 The
data were used to create disease risk scores to classify PTB from LTBI and other diseases
with a sensitivity and specificity of 90% or more for each.
e “-omics” is a term used to describe of a range of molecular biology techniques including genomics, transcriptomics, proteomics and metabolomics.
A prospective cohort study of adolescents with LTBI were followed with regular
transcriptomic profiling of target genes.86 A 16-gene signature was found that predicted
TB progression with a sensitivity of 66.1% and a specificity of 80.6% in the 12 months
preceding TB disease diagnosis. When used with independent cohorts of unscreened
adolescent and two cohorts of adults, the 16-gene signature had a sensitivity of 53.7% and
a specificity of 82.8% in the 12 months preceding TB disease diagnosis. This signature may
be of use to measure progression towards active TB while the patient is asymptomatic.
There is a potential for biomarkers to be employed in a direct and rapid diagnostic method
using non-invasive or minimally invasive specimens (e.g. breath, urine, finger stick whole
blood). This would be of particular benefit in a rapid triage test to rule out MTB infection.
A three-gene signature could easily be translated onto the Xpert® platform and miRNA
are oen qualified by quantitative reverse transcription PCR, a method commonly used
for viral HIV RNA measurement.81 The challenge to the measurement of these transcript
signatures is that the cost of a rule-out assay would unlikely be less expensive on the
Xpert® platform. Similarly, while the binary detection of analytes can use lateral flow
strips, there is significant further complexity in pooling multiple protein biomarkers onto
a single assay with qualitative readout via a device. These add great cost and complexity
to assay development and it would be unrealistic for such technology to remain within
cost for a triage test recommended by the recent WHO TPP.10
Further information on diagnostic methods for LTBI and developments in non-invasive
specimens can be found above in section Immune response-based screening tests for
MTB exposure.
Antrum Biotech
(South Africa) has developed the InterGam Rapid Immuno Suspension
Assay (IRISA™-TB), an enzyme linked immunosorbent assay (ELISA) designed for use
in a laboratory to specifically diagnose extrapulmonary TB. The tool can detect the
presence of extrapulmonary TB from samples, including pleural, pericardial, ascetic
and cerebrospinal fluid. The 96-well plate format can host low or higher numbers of
samples depending on test workloads. This level of test complexity makes it suitable
for intermediate-level facilities (e.g. hospital laboratories) and requires instrumentation
associated with ELISA assays (e.g. plate shaker, washer, reader). The IRISA™-TB is CE
marked but pricing is currently not known.
(UK) has developed ImmiPrint®, a complex immuno-array technology to
detect soluble cellular dierentiators (sCDs), produced by macrophages in response to
an infection. Dierent infectious diseases can elicit unique sCD fingerprints, therefore
the ImmiPrint® can be used to screen samples to identify sCD biomarker profiles to
discriminate active TB infection from other infectious diseases. The company is now
working to develop a low-cost platform to host the biomarker assays identified by
the ImmiPrint® technology. In 2015, ProteinLogic and partners were awarded a
European Union Horizon 2020 Small- and Medium-sized Enterprises programme grant
to develop a rapid POC test for the diagnosis of active TB disease. Its development
partner, Biosensia (Ireland), is developing the RapiPlex platform, a multichannel
fluidic chip, fluorescence immunoassay system, and an integrated optical reader that
uses fluorescence detection to simultaneously measure up to 24 analytes using either
quantitative and/or qualitative analysis. The test is claimed to require a single sample
addition and testing is completed in 5–15 minutes. No further information is currently
Significant progress in disease management has been made with rapid diagnostic
tests (RDTs) for diseases such as malaria and HIV where disease-specific antigens or
antibodies can be detected with a high degree of confidence via a simple and rapid test.
In 2011, WHO released policy guidance on commercial serologic tests stating that they
should not be used in the diagnosis of TB, despite being available in some markets.87
However, the need for research on new/alternative POC tests for TB diagnosis, including
serological tests with improved accuracy, was also highlighted. Research has focused
on identifying new TB antigens and understanding the immuno-dominant domains of
target antigens in order to increase specificity.88,89
As such, several groups are developing serologic tests for use with blood specimens
TB Biosciences
(USA) was developing a rapid LF immunoassay.88 The product
was initially scheduled for release in Q2 2015, however, development work has been
stopped due to poor performance of the prototype assays. Instead, TB Biosciences
is collaborating with Quidel (USA) to develop an LF assay to detect multiple TB
biomarkers via a fluorescence reader with greater precision and accuracy. FIND and
mBio (USA) have been engaged in a long-term project to develop a serologic assay
for TB infection using over 50 dierent TB antigens for the interrogation of patient
antibodies to TB. An update describing the development and performance of the
prototype tool is expected to be published soon.
Rapid Biosensor Systems Ltd
(UK) has developed the TB Breathalyzer, a tool to detect
TB antigens from a breath specimen. Unlike VOCs, the company claims that antigen
detection from breath sampling has greater specificity. The test detects the Ag85B
antigen, which is MTBC specific and found in sputum or other fluids. The native antigen
(Ag85B) can displace an antibody bound to a fluorescently labelled peptide on the
sensor array. The decrease in fluorescence is measured by evanescent wave fluorimetry
and the device then scores the result. The TB Breathalyzer uses a disposable collector
and reaction tube that is read via a battery-powered reader aer breath collection.
The patient coughs into a collection tube and then depresses an internal concentric
plunger to collect the sample on the inside surfaces of the tube. The plunger is then
twisted to smear the concentrated sample across the peptide-coated prism at the base
of the tube. This coating is dry and no mixing of additional fluids is required. Each test
takes 10 minutes to screen a patient. The limit of sensitivity of the device has not been
determined but is anticipated to be in the range of 50–75 cfu.90 A prototype device was
piloted in a field trial in Ethiopia where it had a sensitivity of 79%.90 Rapid Biosensor
Systems Ltd has designed a unit with a production capacity of >500 000 per batch run,
where the price per test would be close to US$ 5.
Alere Inc.
(USA) introduced the DetermineTM TB Lipoarabinomannan (LAM) Ag rapid
assay (Figure 6), in 2012. This is a LF immunochromatographic strip or RDT that targets
the LAM antigen in urine via a polyclonal antibody capture and detection method on a
nitrocellulose strip. LAM, a lipopolysaccharide, is a metabolite of mycobacterial species
representing a key component of the cell wall and is produced by both growing cells
and the degradation of the cell wall.91 As such, it should be noted that this assay is
not only specific for MTB, but will also detect the presence of other NTMs. LAM from
mycobacterial infection anywhere in the body is ultimately expelled from the body
in urine and can be detected in it. The test requires a 60 μL aliquot of urine, with no
other tools necessary. The test result is visually noted on the test stripe aer 25 minutes
incubation. The simple format of the assay does not require significant training for the
user and the product is stable for 15 months at 30 °C.
Figure 6. Determine™ TB LAM Ag rapid assay, with strip ready for use shown on the right
Source: Image reproduced with permission of Alere Inc.
The Determine™ TB LAM Ag rapid assay has undergone significant independent
evaluation of its potential use as a rapid screening tool for TB infection in high-income
countries and HBCs in Africa, Asia and South America. These data were reviewed by
WHO, resulting in the release of the WHO policy guidance described earlier.
The restricted use of the tool in only critically ill patients with HIV co-infection is necessary
because earlier studies have shown that diagnostic performance of the assay improves as
CD4 counts decrease.91,92 The reasons for this are not fully understood but it is hypothesized
that critically ill HIV patients may have a disseminated TB infection that is very diicult to
rapidly diagnose with current tools. The greater bacterial load associated with widespread
infection and, therefore, antigen load, the greater likelihood of genitourinary tract TB and
greater glomerular permeability to allow increased antigen levels in urine. Alere Inc. has
identified Africa and Asia Pacific as the primary markets for sale of the Determine™ TB LAM
Ag rapid assay. Other groups are currently investigating ways to improve the performance
of the LAM in RDTs and other formats by using more specific antibodies, and improving to
sample preparation and readers to improve test sensitivity.
Earlier landscape reports highlighted the great interest around an assay that could
specifically detect TB via the use of fluorescently labelled beta lactamase analogues that
were enzymatically digested using a TB-specific β-lactamase.94 GlobalBio Diagnostics
Corp. (USA) began developing a POC diagnostic tool but due to a variety of development
and funding challenges, the group is no longer developing the technology at this time.
The application of NAATs has revolutionized rapid and accurate diagnostic testing for most
pathogens. The technology usually involves PCR to specifically amplify a targeted section of
genomic DNA or cellular RNA (e.g. 16s RNA) of a pathogen. Detection methods, to reveal the
presence of pathogenic genetic material, vary across products. Many use fluorescence, while
simpler tests use luminescence or stripes on amplicon capture strips to visual detection.
NAATs are frequently used to identify known genetic alleles that confer drug resistance.
NAAT-based diagnostics in TB can face a number of challenges, including the specimen
type, the relatively low numbers of pathogens present in the specimen and the diiculty in
lysing the TB cell wall to release the nucleic acids. Heterogeneous mixtures of drug-sensitive
and drug-resistant bacteria in a sample can also confound genotyping of drug resistance.
While NAAT methods are more sensitive than most other tests for TB, culture remains the
gold standard for TB diagnosis as it oers the greatest sensitivity, with a limit of detection
of ~10 cells/mL of sputum. However, it can take months to diagnose TB and thereaer MDR
TB by culture-based means. The general accuracy and rapid time to detection make NAATs
an essential tool for the TB community to rapidly and accurately diagnose TB and MDR TB.
Its decentralization to lower levels of the health system, e.g. microscopy centres, will be
necessary to replace or augment SSM methods and for the rapid detection MDR TB.14
Products to identify XDR TB are now in late development phases. Such tools need to be
accurate, low cost, easy to perform and robust.
The introduction of new tools such as NGS
will have a highly significant impact on improving the diagnosis of MDR TB and XDR TB,
increase the understanding of emerging drug resistance mechanisms and permit molecular
epidemiology to be performed at an unprecedented level as compared with current methods.
In the fourth edition of this landscape report,19 the pipeline of NAATs in development
was presented as being highly promising, especially with molecular tests targeting
intermediate laboratories and microscopy-level facilities. Unfortunately, the pipeline
has somewhat contracted, with some product development eort being stopped entirely
or postponed, or projects not demonstrating the performance initially anticipated. A
summary of the status of current technologies on the market or close to market with
revised release dates is shown in Appendix 1. The pipeline for TB-related NAATs is shown
in Figure 7. Other technologies and assays are described below but these are typically
for use with open platforms or more generalized methods and equipment (e.g. NGS).
The Alere™ q assay and platform was originally planned to be released with CE-IVD
marking in 2016, but all TB-related development of this tool has stopped. Enigma
Diagnostics Ltd (UK) were developing the Minilab platform, a standalone system that
hosts a cartridge in which sample preparation and amplification were automatically
performed in a process similar to the Xpert® MTB/RIF assay. It was recently announced
that this group has entered liquidation and so development eort has now ceased.
Several of the NAATs under development for use in microscopy facilities are delayed
due to technical and funding challenges. Tangen Biosciences (USA) is delaying the
development of TB assays to focus on other disease targets, while Qiagen (Germany)
has noted that it is looking for external funding to drive the development of its POC tool
for TB diagnosis if it passes early development milestones.
A recent independent evaluation of the Genedrive® MTBC assay (Epistem Ltd, UK)
highlighted the need for independent evaluations of new assays soon aer they become
marketable.96 The similar products in this class from Ustar Biotechnologies and Molbio
Diagnostics have also undergone limited evaluation since their release.
Figure 7. Current and emerging automated, semi-modular or non-integrated TB NAATs; their intended
laboratory location and release date (actual or anticipated)
Source: Author analysis
Green filled product labels indicate a WHO recommendation is available for the product.
*Eiken Loopamp™ was released in 2012 but endorsed in 2016
Stratification of diagnostic NAATs
in the test continuum
While there are a number of NAAT-based assays and platforms available or in
development (Figure 7), their most eective positioning in country TB programmes is
dependent on the setting, test complexity and user requirements (Table 1). The number
of facilities within an NTP is largely correlated to country income status. High-income
and upper-middle-income countries generally have devoted more financial resources
to TB at upper- and middle-tiered facilities, potentially oering more comprehensive TB
diagnostic testing via NAATs. A survey of basic infrastructural and logistical capacities
of microscopy centres in Brazil, Russian Federation, India, China and South Africa
(BRICS) and LMICs highlighted the inequalities between these countries, observing that
most LMICs have limited temperature control, limited access to mains electricity and
a lack of general laboratory equipment.97 In terms of implementation of NAATs for TB
diagnosis, all countries noted a lack of skilled sta in level 1 facilities. Limited financial
resources in LMICs have generally meant fewer resources invested in TB control with less
dedicated laboratories and instead relying heavily on SSM in microscopy. A more recent
study presented the data from surveys of 14 high-burden TB countries with regard to
diagnostic testing and treatment options available across the four levels of health-
care settings.14 No countries reported any NAAT testing being performed by community
or village health workers or at local health posts (level 0). Only four countries oered
Table 1. Summary of NAATs relating their role in TB diagnosis in terms of intended location of use,
throughput and other key factors
Test Level Throughput Function Complexity Cost Cost/test
POC assays 3, 2, 1 Moderate/low TBDx, DSTaLow Moderate/
3, 2, 1bModerate TBDx, DST High High Moderate
Microarrays 3, 2 Moderate MTB/NTMc Dx, DSTaHigh High High/
LPAs 3, 2 Moderate MTB/NTMc Dx, DSTaMedium Medium Medium
batched PCR
3 High/
MTB/NTMc Dx, DSTaHigh High Low
Open PCR
3 Moderate MTB Dx, DSTaHigh High Low
NGS 3 High MTB/NTMc Dx, DST,
mol epi
High High Moderate
a DST is genotyping of drug resistance that may be multiplexed with TB diagnosis or applied as a reflexive test aer MTB infection is confirmed. Level 1
microscopy centre or community care centre, Level 2 hospital or regional clinical facility, Level 3 reference or tertiary hospital.14
b Testing at this level is performed in few LMICs.
c Some tests are available to rule in other common types of NTM.
routine NAAT or culture testing for DST at microscopy centres or primary health centres
(level 1), while 12 countries oered this service at district hospitals or community health
centres (level 3) and all countries did so at reference or tertiary hospitals (level 3).
While SSM is low cost and comparatively easy to implement, it is not as sensitive a test as
NAATs, especially with PLHIV, nor for the diagnosis of paediatric TB. In addition, SSM does
not detect drug resistance. It is, however, routinely used in most LMICs as the primary
test for MTB at level 1 facilities.14 Therefore, to improve decentralized TB diagnosis and
oer MDR TB testing, there is a pressing need for aordable high-performance NAATs
that can be implemented at the microscopy centre level.
The following sections highlight the NAATs currently on the market or in development,
and notes aspects of their intended use, performance (where data are available), cost
and current placement in the development pipeline.
Application of NGS to TB diagnosis and control
The application of NGS or massively parallel sequencing is a new area of methodology
that combines MTB diagnosis, drug resistance genotyping and molecular epidemiology
from a “single” test or sample. The technique allows multiple individual DNA strands
from MTB cells to be independently sequenced, thereby allowing for accurate
discrimination of heterogenetic variation (e.g. heteroresistance) and quantitative
analysis. NGS can be applied to the whole genome or to target regions within a genome
aer amplification of the specific target regions of genomic DNA.98 A significant focus of
NGS for clinical purposes has been on its application for the diagnosis and treatment
of noncommunicable diseases such as cancers and screening for genetic inheritable
diseases but the potential value of NGS has now been demonstrated with challenging
infectious disease syndromes such as bacterial sepsis and MTB.
Although sequencing technology is currently not available in most resource-
limited settings, reagent and equipment costs continue to fall and the potential to
run sequencing assays directly from sputum (negating the need for culture) could
dramatically reduce turnaround times. The methods to prepare and sequence samples
can vary significantly and, in addition, the underlying mechanisms by which NGS
systems operate are technically complex.99,100 There are a variety of NGS platforms on
the market and in development, including tools for high and moderate throughputs,
for example: Bio-Rad Laboratories (GnuBio); Illumina (MiSeq, HiSeq and NextSeq);
Oxford Nanopore (minION, PromethION, GridION); PacBio (Sequel and RS2); Thermo
Fisher (SOLiD, S5, personal genome machine [PGM], Proton); Qiagen (GeneReader);
and the Vela Diagnostics NGS platform (Singapore). While most of these products are
designed for use in a reference-level facility, oen at great cost, smaller platforms with
potential application in intermediate-level facilities are available (e.g. MiSeq, PGM
and the minION). Currently, the Illumina platforms appear to be the most popular
among research and clinical groups due to the low error rate, simplicity of sample
preparation and ease of method development. Smaller platforms that are robust and
capable of use in smaller laboratories are currently in development, these include:
Genalysis® from DNAe; Gene Electronic Nano-Integrated Ultra-Sensitive (GENIUS)
from GenapSys; and the Genia sequencer from the Genia Corporation (all USA). These
developing tools are intended to host small cartridge-based systems with integrated
sample amplification and sequencing at a cost target of US$ 100 or less, however,
further details are limited. Currently, the cost of platforms and sequencing available on
the market varies considerably (US$ 80 000–>750 000) and the cost per giga base (US$
A recent prospective, multicentre and international pilot study assessed the real-time
performance and cost of whole genome sequencing (WGS) for the laboratory diagnosis
of mycobacterial infection, including drug-sensitive and -resistant forms of MTB.102 Key
findings included that NGS showed high concordance with the current methods for both
identification of mycobacteria and DST (93% for each), identification of an outbreak,
the identification of an inter-regional cluster of INH-resistant TB and an undiagnosed
case of MDR TB allowing an immediately change in treatment. Cost analysis of both the
traditional (non-NGS) and the NGS algorithms showed a savings of 7% overall if NGS
alone was to be used. The median time to perform NGS and receiving a full report was
only 5 days, which is faster than current diagnostic algorithms.
Challenges of using NGS for TB diagnosis and control
While a highly promising new tool in the fight against MTB, NGS faces a variety of
challenges to its broader implementation for clinical purposes.103,104 The first relates to
acquiring enough MTB DNA to allow suicient coverage or reads of the MTB genome
targets for WGS. Peer-reviewed studies have used cultured MTB as the primary DNA
source in clinical samples for WGS.102,105 Extracting suicient MTB DNA can be challenging
in terms of paucibacillary samples where there are only a few cells to extract DNA from.
Results can be confounded by sequencing the genomic DNA from other commensal
microflora, especially from samples such as stool. The MTB itself can be challenging in
terms of its greater guanine cytosine (GC) nucleotide base pair content and repetitive
regions that may aect some NGS methods.105 Currently, the preparation of samples,
processing and sequencing are a complex process requiring highly trained sta in a fully
functional laboratory. Qiagen and Vela Diagnostics oer fully automated processes from
DNA extraction to NGS via a suite of dedicated machines. Other challenges to NGS include
identifying the best platform to a specific approach as dierent platforms oer dierent
benefits for NGS in terms of read size, cost per read and error rates.101
While WGS cannot be reliably performed without culture of the targeted microbe
to provide suicient DNA, alternative methods that allow NGS to move beyond
the reference laboratory have been described. Shotgun sequencing (random and
incomplete sequencing of the MTB genome) from SSM positive sputum samples could
enable molecular genotyping.106 In addition, the targeted amplification of key drug
resistance alleles by PCR prior to NGS is another method that does not first require
culture. The PCR amplification stage compensates for the initial lack of target DNA
typically extracted from a sputum sample but sequencing is only performed on the PCR
amplified DNAs. Pilot studies have demonstrated the feasibility to use sputum samples
in dierent NGS platforms, and the use of PCR assays to target resistant genotypes to
both first- and second-line drugs within a single test.98,107
The massive amount of raw data generated by NGS and WGS presents a significant
challenge to the user. NGS data management includes mapping or assembly of the
genome, base variant calling and comparative phylogenetic analyses. Soware and
algorithms for each of these steps are typically highly complex and oen unlinked to
each other and thus it is unclear how data could be rapidly processed in laboratories
with users unfamiliar with bioinformatics skills.108 In one high-income country pilot study
assessing the validity of NGS as a diagnostic tool, the group used a single centralized
bioinformatics unit to review, parse, archive and process data into test results for return
to the test laboratories in order to reduce costs.102 Several groups are developing soware
so that users without bioinformatics experience can use NGS data. Data storage and
access are also major challenges to this as local computing infrastructures to support
massive data files and the necessary soware to support analysis are not cost eective
and most likely unable to be supported by HBCs. An alternative is to leverage o existing
cloud-based systems provided by large technology groups such as Amazon, Google and
Microso (all USA), which oer flexibility, scalability, security and lower costs more than
localized systems. Many US-based bioinformatics groups already take this approach to
NGS data and its analysis.109 FIND is reviewing current technologies and methods in
order to identify where an end-to-end (from sample insertion to results) NGS system
could be implemented into HBC laboratory settings.110
Other advantages of using NGS for TB diagnosis and control
NGS facilitates the creation of an accessible repository of assessed and fully annotated
drug resistance alleles, therefore enabling the better understanding of drug resistance of
MTB for current and emerging therapies. The breadth of the current pipeline of potential
new TB drugs and regimens presents a challenge for diagnostic developers trying to
proactively develop tools that can be deployed at the time of launch. The Critical Path
to TB Drug Regimens (CPTR) programme led by the Critical Path Institute (USA) has
recognized the need for an international data-sharing TB drug resistance platform to
enable the accelerated development of safer, faster-acting and more eicacious drug
regimens for TB treatment. The CPTR programme also supports the continuing and
evolving need for public and industry access to high-quality, globally harmonized data
regarding clinically relevant MTB drug resistance-conferring mutations to support next-
generation rapid molecular assays.
The Relational Sequencing TB (ReSeqTB) platform, which was launched globally in April
2016, is a freely available resource to the TB community.111,112 Key elements of ReSeq
include a validated regulatory-grade TB-specific WGS pipeline, implementation of
standards, a robust and thorough curation process to accurately capture contributor
data, a secure platform that has the flexibility to host and access data and meta-data,
including clinical outcomes. ReSeqTB is available through a cloud-based environment
allowing individual laboratories to securely upload and analyse its own data in-house
without the need for bioinformatic expertise (Figure 8). A list of mutations that define
drug-resistant loci and polymorphisms derived through statistical analysis with input
from TB sequencing and bioinformatic experts are freely available to download. These
lists will be updated periodically and published as more data are contributed to the
Figure 8. Workflow of ReSeqTB bridging targeted NGS platforms to provide rapid patient management
Source: Image reproduced with permission of the Critical Path Institute.
In addition to a data browser with the ability to filter data, novel visualization tools
to graphically display genome data with the locations of drug-resistant alleles have
recently been released. Improvements and additional functionality are planned in
the future to meet increasing demands for analysing WGS data. Additional regulatory
compliant TB-specific targeted NGS pipelines and dedicated analytic tools, including
a data mining tool to discover new single nucleotide polymorphisms associated with
drug resistance, are expected to be available in 2017. Reporting language templates
are being developed to standardize sequencing results that are generated and
provided to clinicians. Clinical demonstration studies are needed to ensure that rapid
patient-centred drug-resistant sequencing results are appropriately implemented
and translated to improve patient outcomes.
Summary: The massive volume of actionable data produced by NGS methods will
have a significant impact on TB diagnostics, patient treatment and control. Currently,
many high-income countries are already devoting resources to NGS centres that can
accurate sequence analysis of many pathogens, including MTB. As was seen with
other NAATs in their infancy (e.g. PCR), the complexity and costs of the test processes
initially limited their roll out to the smaller laboratory but technology developers
are working on platforms and methods to make NGS a routine method in clinical
laboratories. Part of this expansion has led to challenges in validation, quality
control and data interpretation beyond what clinical laboratories have previously
encountered.104 However, as these technologies mature, improved quality assurance
methods will also be developed to reduce barriers to implementation. NGS data provide
information at a much higher resolution than that of traditional techniques, allowing
fine-scale epidemiological investigations, while also oering improved diagnosis and
surveillance of drug resistance to both first- and second-line drugs. One pilot study113
showed that DST via WGS genotyping is faster than conventional phenotypic methods
and it is expected to become faster and cheaper in the future. Essentially, diagnosis,
information on the optimal therapy and lineage of infection may all be available in one
test method. The creation of the ReSeqTB database highlights how NGS data, when
correctly curated, can be pooled to build a tool that will enable developers to customize
assay development as new genotypes for drug resistance are identified. As with all large
data collections, the information contained therein is most useful only if it is correct and
stored in a unified format where it can be easily accessed and used by all.
Automated batched PCR
The combination of excellent sensitivity and specificity, low contamination risk
and speed has made PCR technology an appealing alternative to culture- or
immunoassay-based testing methods for diagnosing TB disease.114 Automated high-
throughput screening provides the advantage for reproducible and accurate test
results, decreased risk of cross-contamination and minimal manipulation. A reduced
need for constant attention enables fewer trained staff to perform the necessary
tests. For NAATs to be considered as high throughput, the technology must perform
multiple tests within a short timeframe and at volume. There are currently three
platforms that are available and two in development. The companies are listed in
descending alphabetical order.
Abbott Molecular (USA; hereinaer Abbott) currently oers an automated DNA
extraction platform (m2000sp) performed by the m2000rt real-time PCR platform to
detect MTB from sputum, bronchial alveolar lavage or sediments derived from either
specimen type. Abbott has developed an MTB inactivation buer to reduce biohazard
risk to the test operators. Abbott MaxRatio technology analyses test data and provides
individual test results (Figure 9).115 The platforms are CE-IVD marked and can process
up to 94 samples in a single run, taking 7 hours to complete from sample preparation
(including inactivation) to interpretation of results.
Figure 9. Abbott Molecular platforms for automated sample preparation (m2000sp, le) and real-time
PCR analysis (m2000rt, right) for MTBC and first-line DST
Note: Images are not to scale.
Source: Images reproduced with permission of Abbott Molecular.
Abbott’s RealTime MTB assay for the detection of MTBC and RealTime MTB RIF/INH
Resistance assay oer genotyping of resistance alleles, both with CE marking. The
RealTime MTB assay detects MTB with a detection limit of 17 cfu/mL and a specificity
of 100% when using a panel of known mycobacteria, viruses and other organisms.116,117
Abbott reports clinical sensitivity as 99% for SSM positive/culture positive samples
and 83% with SSM negative/culture positive specimens.
The RealTime MTB RIF/INH Resistance assay can use previously extracted DNA of
MTB-positive samples in an automated reflex mode. The detection limit is 60 cfu/mL
with 100% specificity using the same 80 microorganism panel, and sensitivities of
94.8% (RIF) and 88.3% (INH); diagnostic specificity is listed as 100% (RIF) and 94.3%
(INH). The equipment requires a working temperature range of 15–30 °C and the PCR
reagents require storage at minus 25 °C to minus 15 °C. Recent peer-reviewed articles
from independent and Abbott-sponsored studies have assessed the performance of
the Abbott assays.116-121 The independent studies showed similar sensitivities and
diagnostic performance features with the MTB assay118,121 and RIF/INH resistance
assay.116 The RealTime MTB RIF/INH Resistance assay has been compared with other
commercial TB diagnostic assays and platforms with comparable results, using
both sputum and extrapulmonary specimens.116,118,120,121 While a broader range of
specimens needs to be screened, in particular for drug resistance genotyping, the
overall comments from these initial validation studies were positive with their data
typically matching the performance data supplied by Abbott.117
Akonni Biosystems (USA; hereinaer Akonni) is developing products in two areas
of the TB diagnostics space. The automated DNA/RNA extraction platform, TruTip®
Automated Sample Prep Workstation (Figure 10) can process pulmonary specimens,
in addition to all other clinical samples, including stool and tissue. The main design
features include a homogenization subcomponent for diicult to liquefy/lyse samples,
the ability to handle large viscous sample volumes, and highly competitive equipment
pricing and unit extraction costs. The platform is intended to be used for and has
been demonstrated with downstream diagnostic approaches such as microarrays,
PCR, electrophoresis, isothermal amplification methods (LAMP, helicase-dependent
amplification and transcription mediated amplification) and LPAs. The workstation
leverages o the TruTip®, an Akonni product that uses a tuneable glass capture matrix
(i.e. tuneable pore size, surface chemistry or covalent binding moieties) in a pipette tip
to host DNA/RNA extraction and purification.122,123
Figure 10. Akonni TruTip® Automated Sample Prep Workstation
Notes: HEPA, laminar flow enclosure for benchtop use (top picture, image A) or utilization in biosafety cabinet for secondary containment (bottom picture, image A).
Front shield up to display consumables, including TruTip® sample tubes for bead beating, heating tubes, reagent tray and elution tubes (all automated) (image B).
Source: Images reproduced with permission of Akonni Biosystems.
In addition, the platform has a patented rotating magnetic arm that enables noncontact
variable bead beating for diicult to liquefy/lyse samples. A heating module is also
included for traditional chemical and/or heating lysis and decontamination. The
robotic workstation runs up to eight samples in parallel to scale specimen processing.124
Greater throughput can be achieved by using the TruTip® on 96 channel heads such
as those featured on Hamilton and Tecan liquid handling platforms.124 The unit comes
in an enclosed cabinet with HEPA filtration for bench top use or is small enough to fit
within a biosafety laminar flow hood for secondary containment. The Akonni platform
is under internal design control and external design transfer to a contract manufacturer
for scaled cGMP production. The product has been successfully shipped worldwide
and is intended for use in HBCs. The platform is currently available to early adopters
for research use only and is on track to gain US FDA Class I registration and CE-IVD
certification by 2018. The product is expected to enter the WHO evaluation process
when ready.
BD (USA) is developing a multiplexed real-time PCR assay for the detection of MTB
and the BD MAXTM platform to genotype RIF and INH resistance. The platform has a
5-colour detection real-time PCR platform to expand its capacity to multiplex dierent
PCR assays in the same reaction. The BD MAX™ is a fully integrated, single platform with
CE-IVD marking and a capacity to test up to 24 sputum specimens in a single run. BD
estimates that throughput could be as high as 72 samples in an 8-hour working day. As
this product is in development, there are no performance data available yet. Equipment
pricing is to be established and regulatory approvals will be sought once the product is
readied for market release. BD anticipates the release date to be in Q3 2017.
Hain Lifescience (Germany): The NAAT assays (excluding the GenoType® LPAs) were
extensively described in the 2015 edition of this landscape report. The company’s high-
throughput platform includes a 96-well format for DNA extraction (GenoXtract® 96)
and subsequent amplification and detection via real-time PCR (FluoroCycler® 96). The
extraction platform processes sputum and extrapulmonary specimen types excluding
whole blood using the GXT extraction kit. Also oered are a lower-throughput extraction
platform and real-time PCR cycler, the GenoXtract® 12 and the the FluoroCycler® 12,
respectively. These platforms can process or amplify up to 12 samples at once.
Hain Lifescience currently oers a real-time PCR assay, the FluoroType® MTB to detect
MTB DNA. A second assay, the FluoroType® MTBDR, which can detect TB in addition to
genotyping for RIF and INH resistance alleles is to be released in Q2 2017. Both assays
use novel amplification and probe technologies, Late-Aer-The-Exponential (LATE)-
PCR coupled with “lights on/lights o” probes that tile side by side on the target
region.125-127 Fluorescent melt curve analysis is used to measure the rate by which the
probes dissociate from their target to create a highly specific and reproducible signal
that confirms the correct target. A significant advantage to this approach is that much
larger regions of target DNA can be interrogated than with the conventional method
of using a single probe yet with similar accuracy. The analytical sensitivity of the
Fluorotype® MTB assay is 15 cfu/mL, with a clinical performance of 99% specificity for
MTBC. In SSM positive/culture positive samples the sensitivity is 100% and a reported
sensitivity of 90.2% for SSM negative/culture positive specimens. There are limited
peer-reviewed data on the performance of the FluoroType® MTB assay but a direct
comparison with the Abbott RealTime MTB assay showed good concordance of results
in 97.3% of samples.116
The MTBDR assay sensitivity is 20 cfu/mL. In a low-MDR TB setting, Hain Lifescience
Figure 11. Hain Lifescience FluoroType® MTB [A] and FluoroType® MTBDR [B] processes
Source: Images reproduced with permission of Hain Lifescience.
suggests that the MTB assay be followed by the MTBDR assay as a reflex test. In regions
with high MDR TB prevalence, the direct use of the MTBDR assay may save time and
costs. Both assays are CE-IVD marked.
Hain Lifescience is developing an M(X)DR-TB assay and another assay to genotype
pyrazinamide (PZA) resistance. The M(X)DR-TB assay targets RIF, INH, aminoglycosides
(AMGs) and FLQs in a single reaction.128 The assay can screen 36 dierent alleles
associated with resistance to these drugs. The PZA assay uses lights on/lights o
probes that tile the entire pncA gene target, presenting a real-time PCR assay that can
detect mutations across a large target region, far beyond other currently used probe-
based real-time PCR methods. From screening a panel of 654 isolates with 414 hosting
pncA polymorphisms via the assay and using Sanger sequencing as a comparator, the
analytical sensitivity was 94.4% and analytical specificity was 97.5%. These assays are
planned for validation in 2017. Current pricing is not available.
Roche Diagnostics (Switzerland; hereinaer Roche) currently oers real-time PCR
assays for MTB and Mycobacterium avium infection (MAI) that are CE-IVD marked, and
are described in detail in the fourth edition of this landscape report. Currently, these
assays require a DNA extraction platform to prepare a template for PCR and are aimed
at high-income countries. Roche is developing three new high-throughput real-time
PCR assays: the COBAS® MTB Test; the COBAS® MAI; and the COBAS® RIF/INH Test. These
assays are being developed for their use with the COBAS® 6800/8800 Systems, which
are fully automated from end-to-end, designed for very high throughput, e.g. the 8800
system can process 960 tests in an 8-hour period.
Summary: The initial costs associated with using these systems are expensive in
terms of procuring equipment and preparing the appropriate infrastructure to
host them. However, these automated product systems offer advantages in terms
of test throughput, the ability to genotype drug resistance, while offering greater
consistency of test performance in a high-throughput processing algorithm. Each
of the systems has individual advantages, e.g. the sensitivity of the Abbott platform
or the potential of the Hain Lifescience assays to detect multiple drug resistance
genotypes in a single test. The BD and Roche platforms are wholly user free once
specimens are prepared for an assay run, and the throughput of the larger Roche
platforms could meet demand for large volumes of tests. Both the Abbot and Roche
platforms can be used for other disease targets thus procurement and operating
costs could be defrayed across different programmes. The Akonni platform only
addresses sample preparation and does not include detection of MTB by PCR or other
nucleic acid amplification methods. There is also the need for further validation
work to be published in order to understand not only the performance of the tools
in diagnosing TB, but also in the cost savings and associated value from offering
more rapid diagnosis than with culture when used in a centralized laboratory. None
of these products has yet been endorsed by WHO.
Autonomous NAAT reagents for use in open systems
While many of the NAAT products listed in this landscape report represent specific
assays with dedicated equipment, there are manufacturers that produce only the assay
reagents as kits that can be hosted on a variety of real-time PCR platforms in general,
on platforms recommended by the manufacturer. These reagents are manufactured
under quality conditions, have undergone validation and have some performance data
(e.g. analytical and clinical performance) and so have less risk associated than with
“in-house” PCR assays, which are prone to error and risk, creating reduced or variable
test performance.129 Also, unlike the commercial assays noted in this landscape report,
extensive performance assessment before use of the assay reagents is typically not
performed with suicient rigour. There are a large number of assay kits available on the
market, with many having CE-IVD registration, but most do not have any peer-reviewed
data to inform on their performance in clinical settings. The assay kits described in this
section were chosen because they oer scaled genotyping of MDR TB and XDR TB or
have very high sensitivity, and in most cases have regulatory approval from a national
administrative body.
Seegene Inc.
(Republic of Korea) oers several highly multiplexed NAAT assay kits that use
real-time PCR or capillary electrophoresis for amplicon detection. The Seeplex assay uses
10-colour detection using melt curve analysis aer PCR amplification. The company has
developed the proprietary soware MuDT to discriminate between the 10 channels on the
Bio-Rad Laboratories (USA) CFX96 real-time PCR platform. The viewer soware analyses
the raw data to generate test results from its assays. Sample preparation is not supplied by
the company thus a dierent product must be used to first prepare MTB DNA for real-time
PCR analysis. The Seegene Anyplex™ series of assays kits include: the Anyplex™ MTB/NTM
for MTBC and NTM; the Anyplex™ plus MTB/NTM/MDR TB for MTBC, NTM and genotyping
RIF and INH resistance; and the Anyplex™ II MTB/MDR/XDR that detects MTBC, genotypes
MDR via interrogation for INH and RIF resistance alleles in addition to a further 13 alleles
associated with XDR (seven alleles for FLQs and six alleles for injectable drugs). The time to
generate results is under 3 hours (excluding DNA extraction) and 94 samples can be tested
in one run. Sample types include sputum or bronchial washes and the assay reagents are
stable for 12 months at -20 °C.
Since the last landscape report, there have been four independent peer-reviewed articles
describing the performance of Seegene Inc. assays on either culture isolates or from
patient specimens, predominantly sputum. One study compared the Anyplex™ II MTB/
MDR/XDR assay performance to phenotypic DST and other molecular methods to address
discordant results. Overall, the Anyplex™ assay sensitivity and specificity were 93.3% and
100%, respectively.130 Two studies from South Africa have compared the performance
of the Anyplex™ MTB/NTM/MDR TB against the Xpert® MTB/RIF assay and the Hain
Lifescience MTBDRplus LPA.123,131 When the three methods were screened using qualified
culture isolates, the assays had identical performance for the detection of RIF (but the
discordances were not the same) with 96.7% sensitivity and 98.1% specificity.132 With INH,
both the LPA and Anyplex™ assay had the same sensitivity (100%) but the Anyplex™ assay
had reduced specificity (82.4%), detecting several wild-type isolates as INH resistant. The
second study compared the three assays on isolates and SSM positive and SSM negative
culture confirmed TB. All three assays had similar performance with SSM positive sputum
but the Anyplex™ assay had the lowest sensitivity at 65.5% with SSM negative samples.131
A multicentre independent evaluation of the Anyplex™ MTB/NTM/MDR TB assay was
performed in the Republic of Korea, where the researchers noted comparable performance
with the Roche COBAS® TaqMan® MTB assay when using respiratory specimens with culture
confirmation. Overall, the diagnostic sensitivity and specificity of this Anyplex™ assay was
87.5% and 98.2%, respectively; 95.2% sensitivity for SSM positive with a drop in sensitivity
to 69.2% with SSM negative specimens.133 A smaller clinical study in the United Kingdom
using the Anyplex™ MTB/NTM showed high sensitivity and specificity in the diagnosis
of MTB and NTMs using this assay.134 A recent study evaluated the performance of the
Anyplex™ II MTB/MDR/XDR assays using cultured isolates that were phenotyped for drug
resistance. The study compared the performance of this assay with two LPAs from Hain
Lifescience to detect MTB TB (GenotypeMDRTBplusv2.0) and XDR TB (Genotype MDRTBsl
v2.0). Overall, the study found that all three molecular assays had similar performance
in genotyping their targeted drug resistance alleles with the exception of INH where the
researchers noted poor specificity of 61–62% for both assays.135 All of the Seegene Inc. TB
products are CE-IVD marked and are approved by the Korean FDA. The company currently
markets in over 50 countries.
Xiamen Zeesan Biotech Co. Ltd
(China) has developed the MeltPro® Drug-Resistant TB
Molecular Diagnostics System that includes a real-time PCR instrument and a series of real-
time PCR assays to genotype drug resistance alleles (RIF, INH, EMB, streptomycin [STR],
FLQ and SLID resistance). The assays use real-time PCR and multicolour melting curve
analysis to indicate allelic variations associated with drug resistance.136 The company
notes that the assays can be used with its real-time PCR machine or with PCR machines
oered by Bio-Rad Laboratories (CFX96), Qiagen (Rotor-Gene 6000) or Roche (LC480 II),
and claims each assay has a limit of detection of 1000 cfu/mL and is 100% specific to MTBC
only. Up to 94 samples can be processed in a 96-well thermocycler and notes that results
are generated in under 4 hours. An evaluation of 1000 phenotypically scored specimens
in China noted that the INH assay (that targets 30 genotypes) showed clinical sensitivity
and specificity at 90.8% and 96.4%, respectively.137 The lowest heteroresistance (e.g. two
dierent genotypes in one sample) level that the assay can detect was 40%. A similar study
investigated the performance of the STR assay wherein clinical sensitivity and specificity
were 88.8% and 95.5%, respectively; the lowest level of heteroresistance with this assay
was 20%.138 The assays for RIF, INH, EMB, STR and FLQ have received China Food and Drug
Administration (CFDA) approval.
A prospective study assessed the performance of the MeltPro® RIF, INH, FLQ and SLID (AMK
and KAN) assays using SSM positive sputum samples that also underwent MGIT™-based
DST. 139 The RIF assay had a sensitivity and specificity of 94.2% and 97.5%, respectively,
and the FLQ assay had a sensitivity and specificity of 83.3% and 98.1%, respectively.
Data for injectable drugs varied depending on the drug, with AMK resistance detection
at 75% (sensitivity) and with 98.7% specificity. While highly specific with KAN
resistance (99.2%), the sensitivity of the assay was much less (63.5%) and considered
unacceptable. Therefore, the assay is being further developed and is anticipated for
CFDA approval in 2018. Xiamen Zeesan Biotech Co. Ltd currently markets its products
only in China. The reagents are stable for 1 year at -20 °C.
While not yet manufactured, the totally optimized PCR TB (TOP TB) assay developed
by Thisis (USA) has recently been shown to have very high performance in its first pilot
evaluation.140 The assay has an analytical sensitivity of 1–4 cfu/mL. This may be of
particular importance in the case of PLHIV where assay sensitivity is key with typically
paucibacillary samples being presented. Initial performance data were derived from
a convenience sample of 261 HIV-infected PTB suspects. Liquid culture, Xpert® MTB/
RIF, TOP TB and a composite reference PCR assay were each used to detect MTB. Using
culture as the reference, TOP TB had 100% sensitivity but only 35% specificity. Using DNA
sequencing data from the composite PCR assay as the reference standard, the sensitivity
of culture (27%) and Xpert® MTB/RIF (27%) was lower than TOP TB (99%) but each had
similar specificity (100%, 98% and 87%, respectively). The composite reference assay
targets the same gene as the TOP TB assay but in a completely dierent region. Thisis is
currently working with diagnostic companies to host its assay.
Summary: The use of assay reagent kits permits greater application of existing equipment
within a laboratory, creating cost savings as no purchase of new hardware is required.
The commercially available assay kits described above are designed with an emphasis on
genotypic identification of first- and second-line drug resistance. The primary advantage
of such kits is that they are manufactured under good manufacturing practice. Given
that these assays have a relatively low sensitivity, their application could be used for
batched screening of culture-positive isolates for first- and second-line drug resistance.
The prevalence of MDR TB and XDR TB in addition to the specific genotypes circulating
within local populations needs to be carefully considered. Logistically, the use of such
assays requires highly skilled sta and dedicated spaces to ensure the correct operation
of testing and to limit DNA/amplicon contamination. There is a limited amount of data on
test performance and there are no guidelines as to how to best incorporate this type of
test into a diagnostic programme. The WHO has not yet endorsed any of these products.
LPAs are a relatively low-cost NAAT and a variety of assays have been developed for detection
of mycobacterial species, TB diagnosis, speciation of NTMs and to genotype common alleles
to first- or second-line drug resistance alleles (Table 2). Due to their relatively low sensitivity,
the use of LPAs is restricted to either with SSM positive specimens or to culture isolates to
confirm TB infection, and in most cases rapid genotyping for RIF/INH and possibly other
drugs for MDR TB. While a relatively simple assay in principle, the appropriate use of LPAs
needs dedicated areas and equipment for the various processes used in LPA methodology
(DNA extraction, PCR amplification, hybridization and data interpretation). Based on the
comments above, LPAs are used only in the upper- and middle-tier facilities where the
conditions can be met and there are trained sta for their use.
Table 2. Current LPA products and associated equipment marketed for MTBC diagnosis, mycobacterial
speciation and genotypic DST
Myco, all mycobacteria; MTBC, MTBC only; Spec, speciation of mycobacteria other than MTBC; N/A, not available; Y, yes; N, no. For other abbreviations, see the list
at the start of this landscape report.
Developer Test name Myco/
DST CE-IVD Automated
AID TB Resistance Module
Q2 2013
AID TB Resistance Module
Q2 2014
AID TB Resistance Module
Q2 2015
GenoQuick® MTB N/Y/N No Y GT-Blot 48 GenoScan® 2010
Y/Y/ N RIF/INH Y GT-Blot 48 GenoScan® 2012
Y GT-Blot 48 GenoScan® 2012
Y/Y/ N FLQ/AMG Y GT-Blot 48 GenoScan® 2015
Mycobacterium CM
Y/Y/ Y No Y GT-Blot 48 GenoScan® 2004
Mycobacterium AS
Y/Y/ Y No Y GT-Blot 48 GenoScan® 2004
LG Life Sciences AdvanSure
Mycobacteria Assay
Y/Y/ Y No N/A Genoline
Genoblot 2006
LG Life Sciences AdvanSure MDR TB
Y/Y/ N RIF/INH N/A Genoline
Genoblot 2006
YD Diagnostics MolecuTech REBA
YD Diagnostics MolecuTech REBA
YD Diagnostics MolecuTech REBA MTB
YD Diagnostics MolecuTech REBA MTB
YD Diagnostics MolecuTech REBA MTB
Improvements to test sites and protocols have reduced concerns over amplicon
contamination of the test area and user variation in result determination. Throughput
of LPA testing can be increased via using automated instrumentation for DNA
extraction (generic or developer specific, e.g. Hain Lifescience GenoLyse® platforms).
Most developers now oer associated equipment for use with the assays, including
PCR machines, wash/hybridization platforms, digital scanners and the soware to
score test results from processed strips. These advances in processing have improved
test throughput and also simplified the implementation of LPA testing in many upper-
tier laboratories and reduced risks around incorrect use or interpretation. The time to
process up to 48 samples aer DNA extraction is typically under 4 hours if using the
automated systems described in Table 2.
As noted earlier in this document, WHO has approved the use of several LPAs in 2016;
two for the diagnosis of TB in addition to the genotyping of RIF and INH,7 and a third
LPA to genotype resistance to SLIDs and FLQs for XDR TB.8 Specifically, the Hain
Lifescience GenoType® MTBDRplusv2.0 assay and the NIPRO Corporation NTM+MDRTB
detection kit can be used to genotype RIF/INH. FIND recently published the data from
a multicentre non-inferiority study where the performance for both of the new assays
was compared to the original GenoType® MTBDRplusv1.0, which had been previously
endorsed by WHO in 2008, and concluded that both of the new assays had comparable
performance to the existing test.141
An independent study in South Africa assessed the performance of the MTBDRplusv2.0
and the GenoType® MTBDRsl as a follow-on rapid screening test for MDR TB and XDR
TB directly for sputum specimens. This study indicated that the test performed
well with SSM positive/culture positive sputum but that performance significantly
dropped in SSM negative/culture positive samples.142 The pricing of the NIPRO
Corporation assay is not known but FIND has negotiated preferential pricing for the
MTBDRplusv2.0 for HBCs. The MTBDRplusv2.0 assay retails at €720 (96 tests) and the
other ancillary equipment needed to perform this assay are also available under this
pricing initiative.20
Hain Lifescience has two versions of the GenoType® MTBDRsl assay. Both assays
detect MTBC and have internal controls to inform on adequate amplification of the
sample by PCR and for hybridization. The first version was developed to genotype
resistance to FLQ via gryA, SLID resistance (SLID including KAN, AMK and CAP) via
rrs and ethambutol (EMB) resistance via embB. The MTBDRslv2.0 assay targets gyrA
but includes assays for gyrB mutations that are also associated with FLQ resistance.
Furthermore, the assay incorporates further SLID resistance genotypes via the eis
promoter region. The embB resistance component is not used in the v2.0 of the assay.
The WHO endorsement of the MTBDRsl assays is significant in terms of the application
of new shorter treatment regimens for MDR TB for both adults and children older than
4 years of age as there is a need to first screen RR TB patients for resistance to these
second-line drugs.15,18 Patients can be screened in a matter of days and those not
resistant to second-line drugs are eligible for short-term treatments. WHO also noted
that among patients prescribed a conventional MDR TB regimen, these tests would
also help decide from the start who would benefit from adding one of the new drugs
to strengthen the regimen.8 Performance data from a variety of evaluations are now
available, including a multicentre evaluation study,143 and performance evaluations
in both European and South African laboratories using both phenotyped culture
isolates and clinical specimens.142,144,145 This assay uses the same processing equipment
oered by Hain Lifescience and currently, while negotiated pricing is available for the
hardware,20 the price per test is €7.50 with volume discounts available.
The MolecuTech REBA MTB-MDR® LPA oered by YD Diagnostics has undergone
evaluation in a study led by FIND and a manuscript describing its performance is being
Summary: The ability to rapidly detect MDR TB or XDR TB has a great impact on patient
care and in selecting the correct treatment from the start. LPAs can oer actionable
results within 1-2 days and can be applied to SSM positive sputum samples. Allied
with culture methodology, LPAs oer a much faster time to result for DST than by
using subsequent phenotypic testing. Test sensitivity is more challenging with SSM
negative/culture positive samples but Hain Lifescience has been improving its PCR
assay reagents to increase sensitivity using SSM negative/culture positive samples
with the MTBDRslv2.0 assay. In 2015, WHO noted that the increase in reported MDR TB
cases was due to expansion of molecular genotypic tests such as LPAs and Xpert ® MTB/
RIF.11 Further key benefits to the use of these tests are that genotyping can be scaled
and at a lower cost than compared to phenotypic testing. It should be noted that
the complexity of using LPAs does limit their application to laboratories in reference
laboratories or tertiary or secondary hospitals that are at the upper tiers of health-care
systems. LPAs are also challenged by the limited number of allelic targets that can be
screened on a single strip. This is demonstrated with PZA due to the higher number of
alleles associated with resistance that led to a 2-strip method using 49 probes to be
investigated or with INH where other rare alleles associated with INH resistance are
not included in the assay.146,147 Recently, there were concerns around false-positive
FLQ results with the MTBDRsl assay due to amplified target DNA not binding to either
the wild-type or to the resistant allele probes leading to the assumption that as
neither the wild type nor mutant were not positive, a novel mutation may be present
and thus likely to be FLQ resistant.148 A detailed analysis of this problem using DNA
sequencing and phenotypic testing for FLQ resistance concluded that the absence of
wild-type probe hybridization without mutation probe hybridization was the result of
a failure of the mutation probe not hybridizing correctly and not the result of novel or
rare mutations within the target sequence.149
Microarrays may be used as an alternative method to rapidly diagnose and speciate TB
and genotype for MDR TB. Microarrays require the purification and amplification of TB
DNA with target-specific primers to enable asymmetric PCR to generate labelled DNAs.
As with LPAs, this now labelled ssDNA is subsequently bound to their complementary
capture probe that represents a specific allele (e.g. wild type or drug resistant) via
hybridization. The principle dierence is that each probe is printed in a small discrete
spot, as opposed to a comparatively large stripe with LPA. This decrease in area allows
more probes to be printed in a geometric array oering two advantages. First, more
controls or probes to other resistance alleles may be incorporated into a test. Second,
probes are typically printed in duplicate or triplicate on the array thereby providing
greater accuracy when scoring a test result. However, the interpretation of microarray
data does require a dedicated instrument as the probe spots are not visible to the naked
eye and typically detection uses fluorophores or electrochemical detection. Microarray
kits oer primers to amplify the target DNA regions in addition to the arrays and other
materials required for performing the test. There are several microarray products on the
market or in development.
Beijing Capital Biotechnology Ltd
(China) oers two microarray products (GeneChip)
directly related to TB: the Mycobacterial Species Identification Array Kit; and the
M. tuberculosis Drug Resistance Detection Array Kit. The first product speciates MTB
and other NTMs from a specimen, while the second genotypes for resistance to RIF via
rpoB and INH via katG and the inhA promoter region. There are a significant number
of operator steps to extract and purify DNA, prepare and perform PCR followed by
hybridization of PCR product to the array and the subsequent washing before reading
each array on a microarray scanner. Many of these processes are automated but the
user is required to transfer the test materials between the necessary equipment.
Published evaluation reports noted the entire process takes 6 hours.150,151 The company
oers the ancillary equipment related to these tests. As with LPAs, assay sensitivity
is more limited than most NAAT diagnostics and the company notes that the limit of
detection for either assay is 1000 cfu/mL.
MTB identified through the use of microarrays was identified using clinical specimens
and culture-positive samples, with 100% specificity.150 Recently, one study also showed
good performance in the correct identification of culture isolates, while using sputum
with 100% concordance with culture (18/18) but more limited with pleural fluid (16.7%;
2/12) and bronchiolar lavages (10%; 1/10).151 The initial evaluation of the assay compared
phenotypic culture testing with the MDR TB assay where the researchers noted 91.8% for
isolates and 94.6% for sputum samples for RMP resistance, and 70.2% for isolates and
78.1% for sputum samples for INH resistance.152 A multicentre evaluation of the assay
versus phenotypic cultured-based testing gave similar data with the sensitivity and
specificity of the GeneChip of 87.56% and 97.95%, respectively, for RIF resistance and
80.34% and 95.82%, respectively, for INH resistance.153 The GeneChip was also used to
investigate MDR TB in naive cases and with patients already enrolled on treatment. This
study was in a city in China where there is a high prevalence of MDR TB. In an analysis,
the mean cost of diagnosing a single case of MDR TB using the GeneChip test, in China,
was US$ 22.38 as opposed to US$ 53.03 using the phenotypic algorithm. The company
concluded that a decrease in market price and improvements in the performance of
the GeneChip would influence its wider application. Both arrays have received CFDA
approval for use in China and have CE-IVD marking. Prices in 2015 were US$ 25 per
MTB/NTM array with the MDR TB assay listed at US$ 21.
Other array developers are developing microarray platforms that have more
integrated steps to automate more key processes and, therefore, limit the need for
user input to complete a test. The technologies also combine MTB diagnosis and
genotyping of drug resistance within a single-test unit. Sample preparation is still
required to extract and purify TB DNA from sputum specimens. Greater automation
may permit their use at the intermediate level due to reduced need for skilled user
input but the equipment still requires reliable main power and reagents stored
under cold chain.
continues to develop the microarray platform (TruDx®2000) based on its
proprietary gel array drops (TruArray®), which are claimed to improve DNA/RNA
hybridization kinetics (Figure 12). This is achieved by encapsulating the capture probes
in a porous gel matrix that is 99% water rather than simply printed on a solid surface
that can compromise hybridization kinetics and eiciency due to the immediate
proximity of printed probes to the surface. The assay is designed to be used with DNA
from sputum that is prepared via the TruTip® extraction tool (Figure 12), a pipette tip
that contains a nucleic acid binding matrix with volume transfer and reagent mixing/
washing mixing being performed via a hand-held battery-powered automatic pipettor
that hosts the TruTip®. In developing its array, Akonni has employed several innovative
components in the design of the chip, including an all plastic microfluidic design that
is valve-less and pulls the sample and other reagents into the reaction detection
chamber via capillary flow.
Figure 12. Akonni TruDx®2000 platform with TruTip® extraction pipette, TruArray® processor and
TruArray® scanner (le); TruArray® test, microfluidic valve-less design for simultaneous on-slide PCR and
microarray hybridization in a closed format (right)
Source: Images reproduced with permission of Akonni Biosystems.
MTB PCR and
Asymmetric PCR amplification of targets is simultaneously coupled to probe-hybridization
events on the array in a single chamber so that the array “sees” the total sample (no
sample splitting).154,155 Waste wash fluids are wicked into an absorbent chamber, therefore
limiting the risk for amplicon contamination of the test area (Figure 12). The all plastic
design and plastic film-based substrate significantly reduce manufacturing costs versus
traditional glass, silicon wafers or microelectronic chips with surface functionalization
coatings. Akonni is pursuing a very low-cost reel-to-reel manufacturing approach to
making the TruArray® tests. The company labels amplified DNA with a fluorescent dye
that is used to detect the bound probe to its respective complementary target probe and
then detected by a charged-coupled device reader that employs a light-emitting diode to
excite the fluorophore. The processor and application are hosted on the reader so that
an attendant computer to operate the process and analyse the raw data is not necessary.
The prototype array is designed to identify MTB and M. avium complexes via
6110 and
1245 in addition to genotyping resistance alleles to RIF, INH, EMB and STR.156,157 An
internal control to indicate the presence of confounding materials is also included to
qualify each assay’s performance. The analytical sensitivity limit of detection is estimated
to be 25–110 genome copies per amplification reaction. In a blinded analysis of 153 clinical
isolates, microarray sensitivity for first-line drugs relative to phenotypic DST was 100%
for RIF (14/14), 90.0% for INH (36/40), 70% for EMB (7/10), and 89.1% (57/64) combined.
Overall, microarray specificity for RIF, INH, and EMB combined was 97.2% (384/395).
For the second-line drug STR, overall sensitivity was 34.8% (8/23) because of limited
microarray coverage for STR-conferring mutations, and specificity was 99.2% (129/130).
All false-susceptible discrepant results were a consequence of DNA mutations that are not
represented by a specific probe on the microarray. There were zero invalid results from 220
total tests. The test was performed in 6 hours and can be run in batches of 1–16 samples
at a time. The simplified microarray system is suitable for detecting resistance-conferring
mutations in clinical MTB isolates and can now be used for prospective trials or integrated
into an all-in-one, closed-amplicon consumable.156,157
The combined platform (TruTip® workstation and TruDx®2000) has shown promising
results with sediment and raw sputum under National Institutes of Health (NIH)
programmes with Harvard University and the University California San Diego
(manuscripts pending). The platform is currently undergoing retrospective and
prospective trials in China, Mexico, Peru and the Republic of Moldova. Preliminary
data on non-sputum samples such as stool and gastric aspirate are promising. Studies
done in Peru resulted in an analytical limit of detection of 32 cfu/mL. Also, the TruTip®
workstation was retrospectively tested with MTB-positive paired raw sputum and
sediment samples (extracts tested via real-time PCR) resulting in 99.2% MTB detection
(114/114 SSM positive and 9/10 SSM negative). The TruTip® microarray specificity is
91.3% (63/69) for RIF and 98.2% (56/57) for INH. Sequencing analysis of discordants is in
progress for discrepant analysis. Two new MDR arrays are in production and prototype
testing: a PZA mini-sequencing array; and an XDR array inclusive of markers for FLQs
and SLIDs in addition to broadening the coverage for RIF and INH resistance. The release
date for this product is also unknown but the developer is intending to submit the tool
for CE-IVD marking in addition to aiming for WHO endorsement in the coming years.
Akonni intends to oer the tool globally once these are obtained and in production.
Veredus Laboratories
(Singapore) market the VerePLEXTM Biosystem platform, developed
ST Microelectronics
(Switzerland). This platform can host several disease-specific
microarrays, including the VereMTB™ Detection chip. The VereMTB™ chip is designed
to identify MTB as well as speciate common NTMs.158 The array can also genotype for
resistance to both RIF and INH. The system first involves independent extraction of TB
DNA from samples. The extracted DNA solution is then added to two chambers on the
array chip that is inserted into a processing module on the VerePLEX™ Biosystem where
target amplification occurs via PCR with subsequent binding of amplified DNAs to their
complementary probes. This platform can independently host up to five test arrays for
PCR at one time (Figure 13). Aer PCR, each chip is individually read in a reader and
the raw data are analysed by VereID soware to generate results on speciation and
resistance to RIF and/or INH. The time to result is around 2 hours and the assay may be
used with culture or SSM positive samples.
One evaluation study has examined the performance of the assay in both low- and high-
burden settings. The assay correctly identified MTBC isolates from negative samples.
Drug resistance was assessed via culture DST, the MTBDRplusv1.0 LPA, the Xpert®
MTB/RIF assay, DNA sequencing and the VereMTB™ Detection chip. The sensitivity and
specificity of the chip for genotyping resistance to RIF were both 100% as compared to
MTBDRplus and DST. With INH, the specificity was lower depending on the target gene
or the qualifying assay used.158 In the fourth edition of this landscape report, Veredus
Laboratories had noted that the product had been on the market as a research use only
product since 2012 and that it would be entered for CE-IVD certification in 2016. It has
not been possible to confirm if this has occurred.
Figure 13. Veredus Laboratories VerePLEX™ Biosystem and VereMTB™ Detection Kit
Notes: Both PCR amplification and microarray hybridization are performed in the VereMTB™ Detection chip (image A). The VerePLEX™ Biosystem and reader includes
a five-unit processing station for the on-chip PCR and the subsequent hybridization and washing of the chip (image B). For analysis each chip is manually inserted
into the reader that interrogates the array (far right).
Source: Images reproduced with permission of Veredus Laboratories.
Figure 14. Hydra 1K hand-held platform (le) and chip (centre)
Notes: Disposable arrays (centre) are inserted into the reader (le) and PCR amplification is measured in real-time on each detector pad of the CMOS biochip (centre)
that contains integrated heating elements and digital imaging at each spot throughout to enable PCR and also melt curve analysis post-amplification (right). Test
data are downloaded from the instrument via a USB port (le).
Source: Images reproduced with permission of InSilixa Inc.
InSilixa Inc
(USA) and
Stanford University
(USA) have developed the Hydra-1K microarray
platform. This technology employs complementary metal-oxide semiconductor (CMOS)
technology for lens free digital imaging and uses heating within 1024 individual spots
on the array to enable on-chip PCR of the specific MTB target amplicons (Figure 14). The
array can detect the binding of complementary DNAs to their arrayed probes on DNA
sensor pixels and, in addition, the assay uses melt curve analyses aer amplification and
hybridization to measure the rate of dissociation of amplicons from their capture probes
as the temperature is increased. This creates specific fingerprints that can more accurately
interrogate specific genotypes as compared to LPAs or other microarrays. InSilixa Inc. also
claims to have improved the detection of alleles that are challenging to detect due to
complex secondary structure of these targets preventing hybridization.159,160 The company
has completed, tested and validated the multiplex PCR assay and capture probes as these
pertain to the Hydra-1K CMOS biochip for the detection of MTB and 120 mutations that
confer resistance to INH, RIF and FLQs. InSilixa Inc. is currently working to incorporate a
DNA extraction component into the device with a prototype completed in Q1 2017 and
projects that a functional prototype will be ready for pilot testing in field settings in 2 years.
Summary: Microarrays can oer rapid genotyping and with greater confidence than LPAs
based upon multiple probes per array targeting the same allele to confirm a binding event.
In principle, arrays can also interrogate more allellic targets than LPAs in a single test but
they have to be first amplified by multiplexed PCR, which may be a rate-limiting step if
many dierent gene targets are required. While the processing of LPAs can be automated to
enable higher throughput, each of the steps is independent. With arrays, some developers
are focusing their eorts on creating chips where PCR, and other subsequent steps, are
performed without user intervention, essentially creating platforms that maintain a high
test quality but with limited user input. Historically, microarrays have been considered
relatively expensive but developers are looking into using novel substrates to significantly
reduce manufacturing costs (e.g. Akonni) and in some cases using the substrate for
measurement of probe binding (e.g. InSilixa Inc. or Veredus Laboratories). In addition,
new detection methods permit low-cost instrumentation due to reduced complexity as
opposed to traditional instruments using confocal microscopy and fluorescence detection
of bound probe spots. There are very limited peer-reviewed data on these products and
performance needs to be more rigorously assessed, especially by independent groups
since several pilot studies in this section were performed by the developers.
Modular, cartridge-based, fully automated NAATs
The scaled adoption of the Cepheid Inc. Xpert® MTB/RIF assay across many HBCs has led
to other technology developers following its lead in developing cartridge-based assays
for the diagnosis of MTB and for genotyping of drug resistance. The advantage to such
platforms is that a high-quality test can be oered but without requiring significant skilled
user input. In principle, the result can be obtained on the same day. A further key advantage
to most cartridge-based platforms is that diagnostic tests for other key diseases can be
hosted on the same platform, e.g. HIV or hepatitis C virus. In this section, the Cepheid Inc.
Xpert® and the Tosoh Bioscience (a subsidiary of Tosoh Corporation, hereinaer Tosoh)
TRC.80 instruments and associated assays are described.
The Xpert® system fully integrates and automates sample extraction, amplification
and detection in one cartridge. The sample reagent is supplied with the test so sputum
specimens are liquefied and MTB cells inactivated. The processed sample is then transferred
to the test cartridge. Aer the cartridge barcode is scanned, it is placed into the Xpert®
system. The processing of the sample, DNA extraction, semi-nested PCR amplification
and determination of real-time data analysis are all fully automated in a process that
takes less than 2 hours. The Xpert® system is available in a one-, two-, four- or up to a
16-module configuration. The Infinity is an automated, multimode molecular diagnostic
analyser that uses exclusive “load and go” technology for total walkaway operation with
complete random access availability. Infinity systems allow configurations in sets of eight
modules from 16 to 80 modules. All instrument configurations use the same patented
cartridge technology for every Xpert® test. Most laboratories in the emerging markets use
the GX-4 with four independent test modules for processing test cartridges (Figure 15). In
2012, Unitaid, the President’s Emergency Plan for AIDS Relief (PEPFAR), the United States
Agency for International Development (USAID) and the Bill & Melinda Gates Foundation
finalized an agreement with Cepheid Inc. to further reduce the negotiated price of the
Xpert® MTB/RIF test for eligible customers on the FIND country list to US$ 9.98 per test, ex-
works and prepaid. As of December 2016, eligible procurers bought 6.9 million cartridges
at the concessional price. A recent publication reviews experiences with implementation
of the Xpert® MTB/RIF assay.21
A growing number of countries have already adopted national algorithms positioning the
Xpert® MTB/RIF as the initial diagnostic test for all people with signs and symptoms of
pulmonary TB.1 Nigeria, with the fourth largest TB burden in the world, has become the
latest country to issue a national directive calling for the use of Xpert® MTB/RIF as the
initial diagnostic test for all presumptive TB cases. During phased implementation of this
directive, smear microscopy will remain the initial diagnostic with referral for Xpert® MTB/
RIF testing for high-risk groups in areas where access to Xpert® remains a challenge. The
directive was issued on 16 March 2016 by the Nigeria National TB and Leprosy Control
Uptake by countries appears to be accelerating with over 23 million Xpert® MTB/
RIF cartridges and almost 30 000 Xpert® systems procured between 2010 and 2016.
Significantly, India recently announced the procurement of 500 Xpert® systems. However,
with the exception of South Africa, the number of procured cartridges in 2015 compared
to the total number of instrument modules reflected an average ratio of only 1.0 test per
module per working day.1 A recent review of the Xpert® MTB/RIF assay noted that more
than 16 million tests have been performed in 122 countries since 2011.21 However, work
remains to be done to realise the full potential of the installed capacity. New data suggest
that Xpert® is very highly priced with mark-ups of three to four times purchasing costs
at the point of care primarily driven by factors such as transport, custom fees and local
distribution. In the private sector in high-burden countries access is quite limited, with
notable exceptions, such as the Initiative for Promoting Aordable and Quality TB Tests
(IPAQT) programme in India.
Figure 15. Cepheid Inc. GeneXpert® IV System (GX-4) with four independent modules for processing test
cartridges (le) and the Xpert® MTB/RIF cartridge (right)
Source: Images reproduced with permission of Cepheid Inc.
Cepheid Inc. has been developing two new TB cartridges to be hosted on the current
GeneXpert® platform. The design goal of the Ultra MTB/RIF assay is that it is as sensitive
as culture-based methods and can also genotype RIF resistance as a surrogate for MDR TB
diagnosis (Figure 16).163 The key modifications include a larger reaction volume, nested
PCR, faster PCR cycling, multicopy MTB specific targets (IS6110 and IS1081), and melt
curve analysis to more accurately discriminate alleles in rpoB that are associated with RIF
resistance. Whilst both Ultra MTB/RIF and Xpert® MTB/RIF assays had similar performance
in identifying RIF resistance, as compared with sequencing of rpoB, the use of melt curve
analysis to genotype RIF resistance in the Ultra MTB/RIF allows for better discrimination
of silent and active mutations in the rpoB target. The current Xpert® MTB/RIF assay cannot
discriminate between silent and active mutations in rpoB and therefore any mutation is
flagged as being RIF resistant.
Initial analytical data collected using Ultra MTB/RIF method on an open platform (i.e. not
in the cartridge) showed high sensitivity and specificity for both RIF-resistant and sensitive
strains. The Ultra MTB/RIF assay was also able to detect mixed alleles (heteroresistance)
when one of the alleles represented 40% of the total MTB DNA.
An MTB positive sample is identified when either or both IS targets are detected before 37
cycle thresholds (Cts) of amplification and at least two of the rpoB targets are amplified
before 40 Cts. Conversely MTB is reported as not detected if neither of the multi-copy
target probes are positive and the sample processing control is positive with a Ct of less
than 35 cycles. A further result definition, the “trace result”, has been created for this
platform. A “trace result” is defined as when either or both IS targets are amplified before
37 cycle thresholds (Cts) and only one of the rpoB targets are amplified before 40 Cts. If
MTB is detected by a “trace call” then no result can be determined for RIF resistance and
the result is reported as MTB detected, trace, RIF indeterminate.
Figure 16. Cepheid Inc. Xpert® MTB/RIF Ultra cartridge
Source: Images reproduced with permission of Cepheid Inc.
WHO conducted a technical expert meeting in January 2017 in order to assess the results
of the primary multicentre clinical evaluation study. The conclusion was that Ultra MTB/
RIF is non-inferior to Xpert® MTB/RIF. The greater sensitivity of the Ultra MTB/RIF assay is
anticipated to improve diagnosis in cases where paucibacillary specimens are typical or
clients challenging to diagnose, e.g. in PLHIV, paediatric and extra pulmonary TB (notably
cerebrospinal fluid). Cepheid Inc. projects that the Ultra MTB/RIF assay will be on the
market as a CE-IVD registered diagnostic test in 2017. The Ultra MTB/RIF assay will be
available to eligible customers at US$ 9.98 per test, ex-works and prepaid, the same price
as the current Xpert® MTB/RIF assay.
The second assay in development is intended to detect XDR TB as a reflex assay to
Xpert® MTB/RIF or Ultra MTB/RIF positive tests for TB. The XDR TB assay is designed to
detect mutations for resistance to INH, FLQ and AMG with further use of asymmetric PCR
in conjunction with sloppy molecular beacons and fluorescent melt curve analysis to
genotype for resistance alleles to these drugs. A further development with this product
is the expansion to a 10-channel fluorescent detection spectrum rather than the current
6-channel spectrum. The expansion of the range of fluorophores in a multiplexed
reaction permits the use of further sloppy molecular beacon designs to interrogate yet
more alleles in a single-test reaction.163 The XDR TB assay is currently undergoing pilot
studies and the release date and price is not known. While these assays utilize novel
attributes as compared with the Xpert® MTB/RIF cartridge, existing Xpert® systems can
be modified by optical recalibration and soware updates to operate the new XDR
TB assay. Cepheid Inc. was recently acquired by the Danaher Corporation but it is not
expected to aect the current role of Cepheid Inc. in production and supply of Xpert®
systems and Xpert® assays to the global TB community.
Tosoh Bioscience
(Japan) has developed a standalone device for integrated molecular
diagnostics, the TRCReady® 80 that can process up to eight samples. The platform
requires a computer for operation and two test units can be used. Tosoh markets the
TRCRapid® M.TB kit for use on this platform to permit fully automated sample purification,
amplification and detection. Sample preparation uses liquefied sputum to which
lysis buer is added, with heating to inactivate MTB cells and to release 16S RNA. The
assay uses transcription reverse-transcription concerted reaction (TRCR) amplification
technology to amplify the MTBC-specific target within 16S RNA and also uses an internal
control. The amplification reactions are measured in real-time via total fluorescence and
take only 30 minutes to complete. The internal control is used to indicate the presence of
confounders that may inhibit TRCR amplification. The entire process takes 40 minutes if
sample preparation is included. One multicentre evaluation of an earlier TRCR MTB assay
noted an overall sensitivity and specificity of 87% and 98%, respectively, with a detection
limit of 30–50 cfu/mL.164 Currently, there are no data available on reagent stability, cost,
regulatory certification or intended markets for this device. However, the device has been
described in clinical settings in both Europe and Japan.164-166Summary: Although there are
other standalone automated NAAT platforms, there are currently few developers other
than Cepheid Inc. in this space who are oering products now or in the near future in
terms of suicient evidence to enable WHO recommendations as to their performance
and use. Xpert® has changed approaches to TB diagnosis, especially with PLHIV and by
improving the rates of early diagnosis of MDR TB. The recent recommendation of the
more sensitive Xpert® Ultra MTB/RIF cartridge will permit the rapid testing for MTB but
with the a similar sensitivity to culture and also inform on RR TB.167-169 Similarly, the use
of the XDR TB assay will provide more rapid diagnosis of XDR TB and, in addition, as
this assay also detects FLQs and SLIDs, it may be used for screening eligible patients for
shorter-term MDR TB treatment.15 Since the Xpert® MTB/RIF assay was approved over 6
years ago it has seen a dramatic uptake in global terms, a process that has highlighted
the gaps and challenges that are described in a 2016 review.21 A primary challenge is to
better measure the impact of Xpert® in terms of its use in improving patient outcomes;
while it is clearly evident that the assay is more sensitive than SSM and can identify
MDR TB, other gaps in the TB test and treat continuum have been shown to obscure the
impact of improved diagnosis. These include the empiric treatment of TB cases that
were Xpert® or SSM negative or that studies on improved outcomes were statistically
underpowered to detect important eects on mortality.21,169,170 The cost of Xpert® remains
a challenge to widespread use and there currently appears to be limited competition in
terms of other automated platforms. Tosoh described above, has yet to be submitted
for WHO review and release dates are unknown.
NAATs for use at peripheral centres
While the majority of the above technologies represents some advances in the diagnosis
and treatment of TB, these are typically for centralized facilities and usually not available
to the majority of the population initially seeking care. The reality is that most access
to diagnosis and care for TB infection is via a vast network of decentralized, peripheral
microscopy centres oen located within primary or community health centres.171 There
are a variety of clinical, analytical and logistical challenges associated with this thus
while microscopy remains the lowest cost assay for the diagnosis of PTB, there is a need
to significantly improve the rates of diagnosis, especially with populations challenging
to diagnose via SSM e.g. PLHIV and paediatric groups. There have been eorts to better
define microscopy centres in terms of the infrastructure, sta skills and resources available
in HBCs,97 in conjunction with eorts to estimate the cost per SSM event in order to better
define the potential available markets in BRICS and HBCs so that test developers can better
understand the market.65,172 A survey of microscopy centres in HBCs and BRICS revealed
that most countries faced challenges in terms of extreme environmental conditions (e.g.
humidity and dust), infrastructure needs (mains electrical power), available equipment and
skills.97 These factors, and others such as cost, create significant challenges to developers as
tools for use in these settings must be robust, operate independently of mains power, easy
to use, scalable, more accurate than SSM and oered at an acceptable cost.95,97,173 Allied to
this is the need for a low-cost molecular diagnostic test to inform on drug resistance as most
current methods are oen inadequate in terms of a rapid turnaround time.174
There are currently some products that have been marketed for several years with an
intended use in peripheral settings. However, of these only one technology has been
suiciently evaluated in a variety of settings in order to now be approved by WHO, the
Loopamp™ MTBC Detection kit from
Eiken Chemical Corp.
(Japan).6 Since 2012, this test has
undergone 20 evaluation studies in 17 countries. This is the first NAAT product specifically
designed for use in microscopy-level facilities to receive WHO endorsement, which had
two conditional recommendations for the use of the assay instead of conventional SSM
where patients present with symptoms of TB or as a follow-on test where follow-on testing
of conventional SSM results is necessary.
The first version of the assay was released in 2011 by the company and has undergone
some modifications since in terms of specimen and reaction volumes and the protocol
used for the assay. The assay is well suited to resource-limited settings as the equipment is
relatively simple and several user steps are added to reduce instrumentation complexity,
including sample preparation and the interpretation of test results (Figure 17). The assays
can be batched and up to 14 samples can be screened in a single run with control reactions.
The assay uses 60 μL of raw sputum that is transferred to the sample preparation tube
(Figure 17). MTB cells are inactivated and lysed via a combination exposure to highly
alkaline conditions and temperature (90 °C) for 5 minutes. The sample preparation
tube interlocks with a sample neutralization tube where the pH of the heated sample is
neutralized. The final step is the addition of an applicator tube whereby ~30 μL of the
liquid treated sputum contents can be expressed into a reaction tube. The assay reagents
are stored as a glassified pellet inside each reaction tube lid. The DNA extracts are added
to the tube strip, the caps are then closed and the tube strips inverted for 2 minutes to wet
the reagents and then mixed to permit their introduction to the samples.
Figure 17. HumaLoopT instrument (from HUMAN Diagnostics Worldwide, top le) and the Pure DNA
Extraction kit
Notes: The images show the Extraction kit components that are sequentially integrated during the sample preparation process (right top to lower) and the fluorescent
signal generated by MTB DNA positive reactions (bottom le).
Source: Images reproduced with permission of HUMAN Diagnostics Worldwide.
The assay uses LAMP, an isothermal manual DNA amplification method that takes 40
minutes to perform at 67 °C in this assay. Aer incubation, the reactions are terminated
by briefly heating at an elevated temperature. The results of each reaction are scored
visually by the user via fluorescence, which is generated when DNA is amplified by the TB-
LAMP reaction (Figure 17). The light source is supplied with the instrument and the user
compares the green fluorescence of the positive control to each test. A negative control
provides further user input to score any negative tests. The summary data from the 2016
WHO policy guidance document noted the pooled sensitivity of TB-LAMP was higher than
for SSM, ranging from 77.7% to 80.3%. The pooled sensitivity for the TB-LAMP among SSM
positive patients ranged from 95.2% to 96.6% across studies, depending on the reference
standard used.6 The pooled specificity of the assays was also slightly variant depending
on the reference methods used to qualify results with ranges from 97.7% to 98.1%. Eiken
Chemical Corp. has partnered with HUMAN Diagnostics Worldwide (Germany) to globally
distribute and market the assay and instrumentation necessary to perform the Loopamp™
MTBC Detection kit. Training requirements for LAMP is similar to the amount of training
for smear microscopy.175 These products have been available since Q4 2016 and eligible
countries and negotiated pricing can be accessed on the FIND website.20 The Loopamp™
Pure DNA Extraction kit costs €298.20 (90 extractions), the Loopamp™ MTB Detection Kit
is €352.50 (2 x 48 reactions) and the instrument is €2450.00.
Epistem Ltd
(UK) oers the Genedrive® MTB/RIF assay for use with its Genedrive® PCR
instrument. This assay can test one sputum sample at a time and requires some manual
preparation of the test material. The Genedrive® MTB/RIF cartridge assays can screen for
three individual components: an MTBC assay; a genotyping assay targeting rpoB to indicate
RIF resistance; and a control assay to indicate inhibitory compounds in the sample. The
assay reagents are provided pre-dried in each reaction tube and the user rehydrates these
to 20 µL with sterile water. Small volumes of sputum samples (e.g. 20–50 µL) that are
smeared onto a paper-based substrate, le to dry and then 1 mm discs are removed via a
circular punch and one disc is transferred to each rehydrated test reaction well. The test
cartridge is then inserted into the Genedrive® PCR instrument for PCR analysis, and the
results automatically scored aer melt curve analysis. Excluding cartridge preparation,
the process cartridge insertion to results delivery is approximately a 1-hour process. The
technology has been CE-IVD certified and in April 2015 the Drug Controller General of India
(DCGI) issued a 3-year import license to the company’s distributor, Xcelris Labs. The MTB/
RIF product was released in May 2016 in India only.
Peer-reviewed data on the performance of this product are very limited. However, one
independent study has reported analytical and clinical performance of the assay to detect
MTB but did not investigate the RIF resistance component.96 The analytical sensitivity of
the MTB assay was 100 genome equivalents when using MTB DNA. Using quantified MTB
cells spiked into distilled water or non-infected sputum, the limit of detection increased
to 2.5 x 104 and 2.5 x 105 cfu/mL, respectively. In screening specificity with NTMs and other
bacterial species, all were negative via the MTB-specific assay but several NTMs were
occasionally positive via the rpoB assay and indeed several negative controls (9.4%) also
gave a false-positive result upon rpoB melt curve analysis.
To evaluate clinical performance the Genedrive® MTB/RIF assay was compared to culture,
SSM and the Xpert® MTB/RIF assay. The sensitivity and specificity of the assay were
assessed using pre- and post-homogenized sputum and from a second sputum sediment
pellet. With SSM positive/culture positive samples, the sensitivity of the Genedriv
sequentially improved from pre-homogenization (58.7%), post-homogenization (74.7%)
and then with pellets (85.1%); specificity remained similar for all at ~97.5% on all
three specimen types. With the SSM negative/culture positive samples prepared in the
same way, sensitivity was very low at either 0% pre-homogenized or ~4.5% for post-
homogenized and sediment samples. The sensitivity of Xpert® on SSM positive/culture
positive and SSM negative/culture positive samples was 98.4% and 68.2%, respectively.
The sensitivity and specificity of SSM for the first sputum sample (used for the pre- and
post-homogenized samples) was 77.3% and 100%, respectively. Therefore, in this study,
the researchers noted that sensitivity of the Genedrive® was inferior to Xpert® and also
poorer than SSM, the assay it is intended to replace. If samples are first concentrated,
then sensitivity increases but the need for liquefaction and sedimentation detract from
the inherent simplicity intended with this assay. The study also highlighted that MTB cells
are not fully inactivated on the sample preparation paper and so the need for biosafety
also aects the use setting of the assay. The researchers recommended a biosafety level
2 laboratory in addition to a safety cabinet as a minimum requirement for use of the
assay. As a result of these findings, civil society groups have written to the Drug Controller
General of India to ask that this technology not be used in India until the performance of
the test is significantly improved.176
Epistem Ltd is marketing the product in India but it has noted that it is recommending
its use in appropriate biosafety settings and it is working to redevelop the assay sample
preparation to increase sensitivity. The company is generating more performance data
to better inform on the assays performance regarding the concerns of civil society
groups. Since the recent assessment of its technology, Epistem Ltd has noted that the
lack of specificity of the rpoB assay when challenged by some NTMs has been resolved
via a subsequent refinement to the data logic used in processing test results. It also oers
post-market data on test performance in a white paper located on its website.177
Figure 18. Molbio Diagnostics technologies: current and pending products for NAAT-based detection of
MTBC and drug resistance
Notes: Trueprep™ AUTO, an automated and cartridge-based sample preparation extraction tool (image A). Truelab™ Uno and Truelab™ Quattro, single and 4x scaled
test reactors for amplification and analysis of test data (image B) from the Truenat™ reaction chip (image C).
Source: Images reproduced with permission of Molbio Diagnostics.
Molbio Diagnostics
(India) represents a joint venture of the Tulip Group and Bigtec Labs
(both India), which has developed several platforms for the rapid diagnosis of infectious
diseases, including MTB using the Truenat™, an innovative on-chip real-time PCR assay
(Figure 18). The chips are hosted by the Truelab™ Uno RealTime micro PCR System, a battery-
powered processing unit that is portable and has data storage, processing power, a global
positioning system, Bluetooth connectivity and WiFi by virtue of incorporating an android
phone to operate the technology. Two newer Truelab™ devices are soon to be available:
the Truelab™ Uno Dx in which the system has increased fluorescence detection from two
to three channels for further multiplexing of assays (release scheduled for Q2 2017); and
the Truelab™ Quattro where four PCR chips can be independently processed rather than
one with the Uno systems (release scheduled for Q3 2017, Figure 18). Molbio Diagnostics
also oers an independent and semi-automated device to prepare DNA for analysis, the
Trueprep™ MAG, which is also battery-powered and the company oers extraction kits for
both PTB and extrapulmonary TB using this device (Figure 18). MTB DNA is detected via
real-time PCR on the Truenat™ MTB chip which detects MTB and also an internal control to
indicate adequate sample integrity for PCR. A second assay, the Truenat™ MTB/RIF, has been
developed to genotype for RIF resistance in addition to detecting MTB. As noted previously,
the company is developing a fully automated extraction platform, the Trueprep™ AUTO,
which can process up to 48 samples in an 8-hour period. The time from sample to result
using these platforms is 1 hour and its release is anticipated in Q2 2017.
Evidence of acceptable performance via the independent evaluation of the Molbio
Diagnostics TB platforms is still needed with only two peer-reviewed articles regarding the
performance of the Truenat™ platform for diagnosis of TB.178,179 The first study noted that
the MTB assay had high sensitivity and specificity with SSM positive/culture positive of 99%
and 100%, respectively. With SSM negative/culture positive specimens the sensitivity and
specificity was found to be of 76% and 100%.179 The second study also included the Xpert®
MTB/RIF assay as a comparator and both assays had very similar performance in terms of
specificity and sensitivity with both SSM positive/culture positive and SSM negative/culture
positive specimens.178
In 2016, six TB-related Molbio Diagnostics products received CE-IVD marking, including
devices (Truenat™ Uno and Trueprep™ MAG), sample preparation kits (Trueprep™ MAG
sputum and Trueprep™ MAG EPTB) and diagnostic assays (Truenat™ MTB and Truenat™
MTB-RIF). In addition, these products have also been approved for use in India. Ongoing
evaluations include a multicentric study for including the Truelab™ platform in the Revised
National Tuberculosis Control Program led by the Indian Council of Medical Research is
nearing completion. If the performance metrics from this study are successful, then the
products may be procured by the Indian public sector. The Molbio Diagnostics TB products
are currently being evaluated by FIND, which intends to perform an accuracy study at three
sites, and is planning an operational assessment of the technology at the microscopy level.
Current prices oered by the company for the private market to procure the Trueprep™ and
Uno Systems are US$ 7000 and US$ 14 per assay and it is intended that public sectors will
receive a further discount.
Ustar Biotechnologies
(China) is producing the EasyNAT™ TB assay to detect MTB DNA
from sputum (Figure 19). Unlike the earlier described technologies, the company does not
provide a platform but instead relies upon manual processing for the extraction of DNA
and testing of sputum samples with a requirement for a uniform heat source to amplify
DNA via a water bath, PCR machine or similar. The assay uses an isothermal amplification
method, cross-priming amplification, to amplify and label the
6110 target of MTBC. The
cross-priming amplification amplicons are then detected by an immunochromatographic
strip that detects the hapten labelled amplicons but within a sealed cassette to prevent
amplicon contamination of the test site. Using batched processing the company estimates
up to 40 samples can be tested in an 8-hour working day. Independent data on the
performance of the assay are limited to two studies in China and the United Republic
of Tanzania, respectively.180,181 A multicentre evaluation of the EasyNAT™ TB assay in
China had sensitivity and specificity with SSM positive/culture positive samples of 84.1%
and 97.8%, respectively. For SSM negative/culture positive samples, the sensitivity was
reduced to 59.8%. In the much smaller cohort assessed in the United Republic of Tanzania,
the assay performance was assessed on a population with a high HIV comorbidity (46.2%).
The sensitivity and specificity of the assay were 81.6% and 100% with SSM positive/culture
positive samples, respectively. However, with SSM negative/culture positive samples from
PLHIV the sensitivity was reported to be only 10% (only 10 patients were SSM negative/
culture positive). The assay has received CE-IVD marking and was approved by the CFDA
in 2014. The approvals have also been granted in Indonesia and the Philippines. Approvals
for use in India were expected in December 2016. Ustar Biotechnologies is targeting
markets in both private and public systems with a price point of US$ 6–8 per test. The
company is currently also in the early development phase of a fully integrated diagnostic
tool for MTB diagnosis.
Other NAAT-based platforms in early or mid-development: In earlier landscape reports, this
area was shown as being very promising with 13 groups noted as developing products
Figure 19. Ustar Biotechnologies EasyNAT™ TB assay
Source: Images reproduced with permission of Ustar Biotechnologies.
targeting the microscopy centre as their primary area of use. Since the 2015 landscape
report, several companies have stopped development, postponed development or
have been removed from this list due to a lack of any development information.
Tangen Biosciences
(USA) is developing a small modular platform to diagnosis TB
and other infectious diseases but they have currently stopped development of an
MTB assay in order to focus development eorts on other disease targets. The North
Western Global Health Foundation has developed a modular assay to be integrated
onto the Savannah platform being developed by Quidel. Currently, Quidel is reviewing
its development of this platform and, once again, development work in terms of a TB
assay has been delayed to de-risk the business case by focusing on more incentivized
products. Similarly, Qiagen is considering if there is a business case to develop its point-
of-need technology for TB diagnosis. Fluorosentric Inc. (USA), GenePOC™ (Canada) and
Wave80 Biosciences (USA) have platforms on which they claim to be developing MTB
assays but there is no available evidence to support this. Updates on the products and
development work by Tosoh Bioscience and Thisis are noted earlier in this landscape
report. Development work under way by Cepheid Inc., QuantuMDx (UK), KGI (USA) and
Scanogen (USA) is described below.
Cepheid Inc.
(USA), in 2015, announced they are developing a true POC molecular
system, the GeneXpert® Omni (hereinaer Omni) (Figure 20). This is a single standalone
system that is capable of processing Xpert® cartridges in more austere settings than
the current Xpert® instruments, which are not designed for extreme conditions such
as elevated temperatures and humidity above 30 °C.181,182 This system will run both the
current Xpert® MTB/RIF assay and Xpert® Ultra MTB/RIF, in addition to Xpert® HIV-1 Qual,
Xpert® HIV-1 Viral Load and Xpert® HCV Viral Load assays. These assays will be available
aer Q3 2017. Over time, it is intended that the majority of the Xpert® menu will be
available on the Omni, which will utilize the same fully automated sample preparation
and processing as the original Xpert® systems. The Omni is small (23.1 x 7.6 x 10.6
centimetres) and portable, weighing only 1.0 kilograms. The system can be powered via
mains electricity but also includes an internal rechargeable battery to prevent a test run
being aborted due to a sudden power outage. Paired with an external battery, the Xpert®
Omni will have 16 hours of power (i.e. 2 days of operation), without mains electricity – a
common feature in many microscopy centres.97
While the Omni uses the same core technology as the modular Xpert® systems,
Cepheid Inc. has replaced key components with solid state technology that enables
miniaturization and also adds robustness to delicate electrical systems. The Omni is
computer independent and instead houses an internal processor to operate the system
via a dedicated mobile device, thus no computer interface is required for operation. The
mobile device is designed to permit secure connectivity that integrates real-time data
streams for improved monitoring of productivity and performance for external quality
assurance purposes. The device can store data for more than 20 000 tests if connectivity
is absent or sporadic. The projected release of the Omni in emerging markets is aer Q2
2018. At the original press release (July 2015) the price per unit was listed as US$ 2895
but it is not known if this has changed.
Figure 20. GeneXpert® Omni
Notes: The Omni is a true POC molecular system, currently in development, for the independent processing of the Xpert® test cartridges for use in resource-limited
Source: Image reproduced with permission of Cepheid Inc.
QuantuMDx Group
(UK) is developing the Q-POCTM platform. The device is presently in a
small benchtop format and at alpha prototype stage. The company intends on moving
towards a portable hand-held, battery-powered device, although this may be a v2
product. The Q-POC™ is intended to provide extremely sensitive detection of MTB, similar
to that of culture, and achieves this through a novel sample preparation technology that
concentrates MTB from sputum samples from at least 1 mL (essentially harvesting the
MTB cells from as much sputum as the patient can produce). The concentrated MTB cells
are then lysed, amplified and detected by the Q-POC™. The initial assays will use real-
time PCR for DNA amplification and detection with future assays, including subsequent
microarray detection for a comprehensive and integrated DST. All reagents are on board
with a target shelf life of 18 months, the Capture-XT™ assay is projected to take 45 minutes
from raw sputum, while the molecular assay (including many drug susceptibility markers)
takes <20 minutes. Both devices will be battery operated and will be able to operate for
8 hours on a single charge. As the instrument is fully automated, user training will be
less than 1 day. Soware updates will be updated via USB and units replaced if a fault
occurs. The company is working with strategic partners and third party manufacturers to
determine the costs with rigour to ensure that meeting cost expectations of donors and
NTPs match the pricing. The technology entered the clinical testing phase in Q4 2016 and
the estimated release date for the Q-POC™ MTB assay is Q4 2017–Q1 2018. The Q-POC™
device has a target price of ~US$ 2000, with the molecular test at US$ 4–7 with distribution
to the global TB market. QuantuMDx Group recently received funding from the Bill &
Melinda Gates Foundation to further develop this product.
Keck Graduate Institute
(USA; hereinaer KGI) is developing the TBDx system
(Figure 21), in collaboration with this fully integrated nucleic acid testing device is
designed to be compact, simple, inexpensive and robust to enable TB diagnosis in
peripheral sites of HBCs. Proof of principle has been established through an alpha
prototype cartridge and instrument that can perform all processing steps in the analysis
of liquefied and disinfected sputum on board a single disposable cartridge. MTBC cells
are lysed in a microbead beater, followed by DNA capture on the beads, wash and
elution using a derivative of Claremont BioSolutions’s PureLyse® technology. This novel
solid-phase extraction method does not require chaotropic salts or organic solvents,
therefore significantly simplifying nucleic acid preparation.
The DNA-containing eluate solubilizes dry thermostable master-mix reagents that
are uniquely packaged and integrated into the cartridge. In this system, isothermal
DNA amplification is performed either with LAMP or cross-priming amplification.
Aer amplification, the test reaction is interrogated for MTBC amplicons via an
immunochromatographic strip with a visual readout for the alpha prototype, and
electronic readout for future instrument iterations. The cartridge contains all reagents
on board, and liquid handling processes within the cartridge are operated through
inexpensive electrolytic pumps (epumps). The alpha prototype can detect MTB spiked
into sputum with a limit of detection of ~4800 cfu/mL based on PROBIT analysis. Further
optimization is ongoing to improve the sensitivity. A preliminary clinical evaluation
with sputum specimens from TB patients and controls revealed a sensitivity of 90%
and specificity of 96% relative to qPCR. Eorts are ongoing to refine the system into
a beta prototype that is suitable for scale-up and comprehensive validation. KGI and
its partners intend to complete development of this beta prototype by Q2 2018. The
technology will not require onsite calibration and a swap-out system is envisaged to
replace faulty instrumentation. Current cost estimates are US$ 400 for the instrument
and US$ 8 per test cartridge. The test is targeted at HBC markets.
Figure 21. KGI TBDx system
Notes: The alpha prototype instrument (image A) and cartridge (image B). Design concept for the beta prototype (image C).
Source: Images reproduced with permission of Keck Graduate Institute.
Sample In
Answer Out
Figure 22. Scanogen reader and test cartridge for use in microscopy centres
Source: Images reproduced with permission of Scanogen.
(USA) are developing a product that uses DNA to detect the presence of MTB
in a sample but does this directly from the available DNA rather than via its specific
amplification and so, in principle, oers a faster time to result. Its core technology uses
single-molecule detection to identify target molecules under force of DNA-tethered
micrometer beads.183,184 Scanogen claims that this technology enables single-molecule
detection with extremely low background noise and high sequence specificity. In the
context of limited-resource setting TB diagnosis, single-molecule detection may oer
important advantages over the available amplification-based molecular platforms in
that the core reagents are low cost and are less prone to stability issues outside of
cold chain (e.g. proteins and oligonucleotides). In addition, the equipment consists
of inexpensive and low-power equipment comprised of light-emitting diode ring
illumination, a lens and a digital complementary metal-oxide semiconductor camera.
A further advantage of this technology is that the assay does not require temperature
cycling and so instrumentation complexity and the need for electrical power is greatly
reduced. The device will be hand held and the intended use is by a health-care worker
in a microscopy centre setting or similar (Figure 22). The assay will detect TB cells in
a sputum sample collected directly in the disposable cartridge with no manual step
for sample preparation required. Sputum digestion as well as TB cell inactivation
and lysis will take place inside the disposable cartridge. The test is aimed to take 30
minutes to perform and preliminary performance targets are a limit of detection of 25
cfu/mL with >98% specificity. The intended cost per test is US$ 6 and US$ 2000 for the
instrument. The time to product release has been projected to be in 2019.
Summary: It is disappointing to note the reduction in peripheral diagnostic technologies
in development since the 2015 landscape report. However, the development challenges
facing diagnostics intended for use in such settings are great and varied. While eorts
have been made to identify the value of these markets (e.g. SSM in BRICS), some
companies still cite insuicient commercial incentive or lack of market visibility as
deterrents to commercialisation. The cost of developing an existing platform and new
assay can be too high. For example, despite significant grant funding, the AlereTM q MTB
and DST assay was unable to be commercialized at an aordable price to the end user. A
challenge observed with the Eiken Chemical Corp. and Genedrive® tools is the relatively
small amount of sputum sample applied to an assay, which will have a bearing on
sensitivity in screening SSM negative samples where the number of MTB cells may be
very low and likely undetectable by either method.
The Eiken Chemical Corp. Loopamp™ MTBC assay is the first NAAT product intended for
use in microscopy centres that has received WHO endorsement. Further information
is needed to better understand the performance of this assay and where it oers the
greatest benefit. Inclusion in WHO guidelines may trigger country uptake, and thus,
experience and data with implementation. Additionally, the Molbio Diagnostics products
are undergoing field evaluation. The Cepheid Inc. and Molbio Diagnostics products can
oer screening for MDR TB and, with the Cepheid Inc. Omni, possibly XDR TB at the
microscopy-centre level. While most TB programmes remain as separate components
within the public health-care systems in many HBCs, the Cepheid Inc. and Molbio
Diagnostics platforms can also host other disease-specific assays, many of which are
highly relevant to the burden of other diseases, including HIV-1, dengue, chikungunya
and hepatitis C viruses. In addition to these, the Eiken Chemical Corp. also oers a
malarial diagnostic assay using its Loopamp™ platform. Further performance data are
needed to realise the potential of new technologies in development.
Developer Technology
Product name Stage in pipeline CE or CE-IVD
release date
Abbott Platform m2000sp/
In production Yes On market No
Abbott Diagnostic
RealTime MTB In production Yes On market No
Abbott DST Assay RealTime MTB-DR In production Yes On market No
Akonni Platform TruDx®2000 Late development No Unknown No
Akonni Diagnostic
TruDx® array Late development No Unknown No
BD Platform BD MAX™ In production Yes On market No
BD Diagnostic/
DST assay
Unnamed assay Early development No Unknown No
Cepheid Inc. Platform GeneXpert® In production Yes On market Yes
Cepheid Inc. Diagnostic/
DST assay
Xpert® MTB/RIF
In production Yes On market Yes
Cepheid Inc. Diagnostic/
DST assay
Xpert® Ultra MTB/
Validation No Q3 2017 Yes
Cepheid Inc. DST Assay Xpert® XDR Late development No 2017 No
FluoroType® MTB In production Yes On market No
DST assay
In production Yes Q1 2017 No
Platform FluoroCycler® 96 In production Yes Q1 2017 No
Platform GenoXtract® 96 In production Yes On market No
Platform FluoroCycler® 12 In production Yes On market No
DST assay
In production Yes On market Yes
DST assay
In production Yes On market Yes
NIPRO Corp. Diagnostic/
DST assay
NTM+MDRTB Kit 2 In production Yes On market Yes
Platform Savanna Paused No Unknown No
Status update on NAAT-based technologies
for reference and intermediate laboratories
Developer Technology
Product name Stage in pipeline CE or CE-IVD
release date
Not named Paused No Unknown No
Roche Platform COBAS® In production Yes On market No
Roche Diagnostic
COBAS® TaqMan®
In production Yes On market No
Roche Diagnostic
COBAS® TaqMan®
In production Yes On market No
Roche Diagnostic
COBAS® TaqMan®
Early development No Unknown No
Roche Diagnostic
COBAS® TaqMan®
Early development No Unknown No
Tosoh Diagnostic
TRC-80 In production Unknown Unknown No
Tosoh Diagnostic
TRCRapid® M.TB In production Unknown Unknown No
Platform VerePLEX™
In production In process Unknown No
DST assay
VereMTB™ In production Q2 2017 2016 (RUO) No
Platform VerePLEX™
In production In process Unknown No
DST assay
VereMTB™ In production Q2 2017 2016 (RUO) No
RUO; research use only
Developer Technology
Product name Stage in pipeline CE or CE-IVD
release date
Cepheid Inc. Platform Omni Late development No Q2 2018 No
Platform Truelab™ Uno 2
In production Yes On market No
Dx assay Truenat™ MTB In production Yes On market No
DST assay Truenat™ RIF In production Yes On market No
Platform Trueprep™ Mag In production Yes On market No
Platform Truelab™ Uno Dx
3 channel
In production No Q2 2017 No
Platform Trueprep™ Auto In production No Q2 2017 No
Platform Truelab™ Quattro Ready for
No Q3 2017 No
Eiken Chemical
Dx assay Loopamp™ MTBC In production Yes On market Yes
Eiken Chemical
Platform LF-120 In production Yes On market Yes
Dx assay EasyNAT In production Yes On market No
InSilixa Inc. Platform HYDRA Early development No 2018 No
InSilixa inc. Dx/DST assay Not named Early development No 2018 No
KGI Platform TBDx system Mid-development No Unknown No
KGI Dx assay TBDx system Mid-development No Unknown No
Qiagen Platform PON (point of
Paused No Unknown No
Qiagen Dx assay PON (point of
Paused No Unknown No
QuantuMDx Platform Q-POC™ Early development No Unknown No
QuantuMDx Dx/DST assay Not named Early development No Unknown No
Scanogen Platform Not named Early development No 2019 No
Scanogen Dx assay Not named Early development No 2019 No
Platform Not named Paused No Unknown No
Dx assay Not named Paused No Unknown No
Update on technology status of NAAT-based
technologies intended for use in microscopy centres
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