Manual for selection of molecular WHO-

recommended rapid diagnostic tests

for detection of tuberculosis and drug-resistant
tuberculosis
Manual for selection of molecular WHO-
recommended rapid diagnostic tests
for detection of tuberculosis and drug-resistant
tuberculosis
Manual for selection of molecular WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant
tuberculosis

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ISBN 978-92-4-004258-2 (print version)

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Design by Inis Communication

Contents

About this manual iv

About the Global Laboratory Initiative iv

Acknowledgements v

Abbreviations and acronyms vi

Background 1

Molecular WHO-recommended rapid diagnostic tests for selection 3

Part A. Identifying mWRDs to meet a country’s diagnostic needs 5

A1 Preselection data and analyses 5
A2 Decision pathway for identifying mWRDs for implementation 10
A3 Considerations for suitable mWRD implementation 15

Part B. Suggested reading and resources 17

B1 WHO guidelines and policies 17
B2 GLI implementation manuals 17
B3 GLI information sheets 17
B4 Stop TB Partnership information notes and GDF publications 18
B5 Global Fund to Fight AIDS, Tuberculosis and Malaria 18
B6 Diagnostic network analysis and optimization 18
B7 Specimen referral systems 18

References 19

Annex 1. Questions to consider when comparing suitable mWRDs 21

Annex 2. Test specifications 24

Annex 3. Diagnostic network optimization 27

iii
 About this manual

This manual provides practical guidance for the selection of molecular World Health Organization
(WHO)-recommended rapid diagnostic test(s) for tuberculosis (TB) and drug-resistant TB, which
countries can implement to meet the goals of their national strategic plan for TB.

The manual is designed to be suitable for use in any country; however, countries may need to modify
or customize the approach described in the guide to meet the local context of their health system.

Target audience
This manual is intended to inform those interested in implementation of molecular WHO-
recommended rapid diagnostic tests to detect TB and drug-resistant TB. The target audience
includes ministry of health officials, national TB programme managers, national TB reference
laboratory staff, donors, implementing partners, and international agencies and organizations.

About the Global Laboratory

Initiative

The Global Laboratory Initiative (GLI) is a network of international partners dedicated to accelerating
and expanding access to quality-assured TB laboratory services; GLI has been a working group of
the United Nations (UN) Stop TB Partnership since 2007. Coordinated by its core group with support
from its Secretariat at the WHO Global TB Programme, GLI’s mission is to serve as a collaborative
platform for the development and uptake of practical guidance and tools for building and sustaining
high-quality TB diagnostic networks. The GLI core group has representation from key constituencies
including national and supranational reference laboratories, programmes from countries with a high
TB and multidrug-resistant TB burden, technical partners, donors and civil society. More information
about GLI can be found on its website1 or through its Secretariat.2

1
www.stoptb.org/wg/gli.
2
[email protected].

Manual for selection of molecular

iv
WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
Acknowledgements

The development of this manual was led by Thomas Shinnick (independent consultant) under
the coordination of the UN Stop TB Partnership GLI Working Group and its Secretariat within the
WHO Global TB Programme. Lead authors of the manual included Thomas Shinnick and GLI core
group member Patricia Hall (United States Centers for Disease Control and Prevention [US CDC],
Atlanta, GA, United States of America). Technical input and critical reviews were provided by GLI
core group members Elisa Tagliani (San Raffaele Scientific Institute, Milan, Italy), Christopher Gilpin
(International Organization for Migration, Geneva, Switzerland), Sarabjit Singh Chadha (Foundation
for Innovative New Diagnostics [FIND], New Delhi, India) and Sarder Tanzir Hossain (United States
Agency for International Development [USAID] Infectious Disease Detection and Surveillance Project,
Dhaka, Bangladesh). Special thanks to Erin Rottinghaus Romano (US CDC) and Heidi Albert (FIND,
Cape Town, South Africa) for technical review and valuable feedback on the content of the manual.

GLI is a working group of the Stop TB Partnership. Development and publication of this document
were made possible with financial support from USAID.

Acknowledgements v
 Abbreviations and acronyms

AIDS acquired immunodeficiency syndrome

AMK amikacin

DR-TB drug-resistant TB

DST drug-susceptibility testing

ETO ethionamide

FQ fluoroquinolone

GDF Global Drug Facility

GLI Global Laboratory Initiative

Global Fund Global Fund to Fight AIDS, Tuberculosis and Malaria

HIV human immunodeficiency virus

Hr-TB isoniazid-resistant, rifampicin-susceptible TB

INH isoniazid

MDR-TB multidrug-resistant TB

Mycobacterium tuberculosis complex bacteria (e.g. M. tuberculosis or

MTBC
M. bovis bacteria)

mWRD molecular WHO-recommended rapid diagnostic test (for TB)

RIF rifampicin

RR-TB rifampicin-resistant TB

TB tuberculosis

WHO World Health Organization

XDR-TB extensively drug-resistant TB; that is, MDR/RR-TB that is also resistant to
a fluoroquinolone and one other Group A drug (bedaquiline or linezolid)

Manual for selection of molecular

vi
WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
Background

To meet the targets of the World Health Organization (WHO) End TB Strategy (1), WHO recommends
that :
‚ people with signs or symptoms of tuberculosis (TB) receive a molecular WHO-recommended
rapid diagnostic (mWRD) test to detect TB;
‚ people with bacteriologically confirmed TB receive a rapid molecular test to detect resistance
to at least the first-line drug rifampicin (RIF); and
‚ people with RIF-resistant TB (RR-TB) receive a rapid molecular test to detect resistance to at
least fluoroquinolones (FQs; for example, evofloxacin and moxifloxacin).3

More recently, WHO guidelines have stressed the importance of drug-susceptibility testing (DST)
before treatment. This emphasizes the need for countries to implement rapid molecular DST for the
medicines for which mWRDs are available, such as RIF, isoniazid (INH) and FQs.

There are a growing number of mWRDs to aid in the diagnosis of TB and drug-resistant TB (DR-TB).
Each of the mWRDs has good sensitivity and specificity for the detection of Mycobacterium tuberculosis
complex mycobacteria (MTBC). Most, but not all, mWRDs used to detect MTBC are also capable of
detecting DR-TB (see Table 1). All mWRDs that detect DR-TB also test for resistance to RIF, and some
mWRDs test for resistance to both RIF and INH, facilitating detection of mono-resistance to each
of these key first-line anti-TB medicines, as well as combined resistance (i.e. multidrug-resistant TB
[MDR-TB]). Lastly, mWRDs are also available to test all those with bacteriologically confirmed TB for
resistance to RIF, INH, FQs, amikacin (AMK), pyrazinamide (PZA) and ethionamide (ETO).

Importantly, the transition to rapid molecular testing does not eliminate the need for culture and
phenotypic DST. Those tests are still needed for conducting DST for drugs for which an mWRD is
not available, conducting DST to guide drug dosing determinations, monitoring the response to TB
treatment and investigating discordant results from diagnostic testing or DST. In particular, phenotypic
DST is needed for testing the new and repurposed Group A drugs used to treat RR-TB and MDR-TB,
and for detecting extensively drug-resistant TB (XDR-TB) (2). Thus, the national TB diagnostic network
will need to provide both molecular and phenotypic DST services; it will also need to have effective
referral linkages between sites conducting phenotypic DST and sites conducting mWRDs.

3
The original End TB Strategy called for the testing of all RR-TB patients for susceptibility to second-line injectable
agents (kanamycin, capreomycin and amikacin). However, WHO currently recommends that injectable medicines
be phased out as a priority in all treatment regimens and replaced by bedaquiline, which makes rapid drug-
susceptibility testing for amikacin unnecessary.

Background 1
 This manual describes a process that is designed to assist countries to identify which mWRDs may be
suitable for addressing the diagnostic needs in their specific setting. The stepwise process includes
considerations of national policies and goals, epidemiology of TB and DR-TB, diagnostic network
structure and capacity, facility and infrastructure requirements, and implementation considerations.
These factors may lead to the adoption of at least two mWRDs for use in a country, to ensure that
testing needs for all clients are met.

Successful implementation of the mWRD selected will require strong government commitment,
support from donors and implementing partners, and allocation of sufficient financial and human
resources for the implementation process. The required resources include those for annual
operating costs, performance monitoring and continuous quality improvement of the mWRDs,
and revised testing network.

Manual for selection of molecular

2
WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
Molecular WHO-recommended
rapid diagnostic tests for selection

The mWRDs discussed in this manual are listed in Table 1. They include initial diagnostic tests for
diagnosis of TB (without or with detection of drug resistance) and follow-on diagnostic tests for
detection of drug resistance.

WHO recommendations and policy guidance for each of these tests are available in the WHO
consolidated guidelines on tuberculosis Module 3: Diagnosis – rapid diagnostics for tuberculosis detection,
2021 update (3). Implementation guidance is available in the WHO operational handbook on tuberculosis
Module 3: Diagnosis – rapid diagnostics for tuberculosis detection, 2021 update (4) and, for some mWRDs,
in Global Laboratory Initiative (GLI) implementation manuals and information sheets.4

Table 1. Molecular WHO-recommended rapid diagnostic tests to detect TB and DR-TB

Typea of Resistance
Test Manufacturer Description
approval detected

Initial diagnostic tests for diagnosis of TB without detection of drug resistance

Loopamp™ MTBC detection Eiken Chemical, Tokyo, Manual or automated Individual None
kit Japan NAAT

FluoroType® MTB Bruker/Hain Lifescience, Automated NAAT MC-aNAAT None

Nehren, Germany

Initial diagnostic tests for diagnosis of TB with detection of drug resistance

Xpert® MTB/RIF Cepheid, Sunnyvale, CA, Automated NAAT Individual RIF

USA

Xpert MTB/RIF Ultra Cepheid, Sunnyvale, CA, Automated NAAT Individual RIF
USA

Truenat® MTB or MTB Plus Molbio Diagnostics, Goa, Automated NAAT Individual RIF
for TB detection, reflexingb India
to Truenat MTB-RIF-Dx for
DR-TB detection

RealTime MTB for TB Abbott Molecular, Des Automated NAAT MC-aNAAT RIF, INHc
detection, reflexingb to Plaines, IL, USA
RealTime MTB RIF/INH for
DR-TB detection

BD MAX™ MDR-TB Becton Dickinson, Automated NAAT MC-aNAAT RIF, INHc

Sparks, MD, USA

FluoroType MTBDR Bruker/Hain Lifescience, Automated NAAT MC-aNAAT RIF, INHc

Nehren, Germany

4
Individual implementation manuals are available for some of the mWRDs on the GLI website: https://www.stoptb.
org/wg/gli/gat.asp.

Molecular WHO-recommended rapid diagnostic tests for selection 3

 Typea of Resistance
Test Manufacturer Description
approval detected

cobas® MTB for TB Roche Molecular Automated NAAT MC-aNAAT RIF, INHc
detection, reflexingb to Diagnostics, Pleasanton,
cobas MTB RIF/INH for CA, USA
DR-TB detection

Follow-on diagnostic tests for detection of drug resistance

Xpert MTB/XDR Cepheid, Sunnyvale, USA Automated NAAT LC-aNAAT INH, FQ, ETO,
AMK

GenoType MTBDRplus Bruker/Hain Lifescience, Manual reverse FL-LPA RIF, INH, ETO
Nehren, Germany hybridization assay

Genoscholar™ NTM + NIPRO Corporation, Manual reverse FL-LPA RIF, INHb

MDRTB Detection Kit Osaka, Japan hybridization assay

GenoType MTBDRsl Bruker/Hain Lifescience, Manual reverse Individual FQ, AMK

Nehren, Germany hybridization assay

Genoscholar PZA-TB NIPRO Corporation, Manual reverse HC-rNAAT PZA

Osaka, Japan hybridization assay

AMK: amikacin; DR-TB: drug-resistant TB; ETO: ethionamide; FL-LPA: first-line line-probe assay; FQ: fluoroquinolone; HC-rNAAT: high
complexity reverse hybridization nucleic acid amplification test; INH: isoniazid; LC-aNAAT: low complexity automated nucleic acid
amplification test (for isoniazid and second-line drugs); MC-aNAAT: moderate complexity automated nucleic acid amplification test;
MTBC: Mycobacterium tuberculosis complex bacteria; PTO: prothionamide; PZA: pyrazinamide; RIF: rifampicin; TB: tuberculosis; USA:
United States of America.
a
Only WHO-recommended tests are listed. WHO approval is based on the review of evidence for an individual test or class of tests;
the classes include MC-aNAAT, LC-aNAAT, FL-LPAs and HC-rNAAT.
b
These assays are designed as a two-step process with two separate amplification reactions. The first step is detection of MTBC;
the second step is detection of drug resistance.
c
This assay detects mutations in the inh promoter region that confer resistance to INH and ETO/PTO; however, the performance
for the test for detecting resistance to ETO/PTO resistance has not yet been reviewed.

In the past, WHO issued policies and recommendations for individual tests based on reviews –
undertaken by guideline development groups (GDGs) – of test diagnostic accuracy, feasibility,
balance of benefits and harms, cost considerations and acceptability. However, in 2021, WHO
developed a class-based scheme for mWRD test classification (3). The classes were defined by the
type of test technology, the complexity of test implementation and the target conditions for use.
Diagnostic accuracy of the individual members of the class were combined and reviewed, then used
to establish class-wide recommendations and design mWRD-inclusive algorithms (4).

Manual for selection of molecular

4
WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
Part A. Identifying mWRDs to
meet a country’s diagnostic
needs

This section of the manual describes a stepwise process designed to assist countries to identify one
or more mWRDs that are suitable for addressing the diagnostic testing needs for their specific setting.
‚ Part A1 describes the collection and analysis of information needed for selecting one or more
mWRDs. It outlines the main factors that influence the identification of the testing instruments
and mWRDs that best address a country’s diagnostic testing needs.
‚ Part A2 describes how the information collected in Part A1 can be used to identify mWRDs
that are suitable to meet the diagnostic needs of the country and ensure patient access to
diagnostic services.
‚ Part 3 describes factors to consider when determining which suitable mWRDs could be
implemented; such factors include issues related to registration and importation of instruments
and commodities (including customs clearance), supply chain requirements, service and
maintenance requirements, and availability of in-country support from the manufacturer,
distributors and authorized local service providers.

A1 Preselection data and analyses

The suitability of an mWRD to meet a country’s diagnostic testing or DST needs is determined by:
‚ national policies, goals and targets, which inform testing priorities;
‚ subnational epidemiology of TB and DR-TB in the country, which informs diagnostic service
needs; and
‚ the existing TB diagnostic network structure and components, which are reviewed to identify
successes and gaps in current testing services and may be used to inform strategies and
opportunities for providing and improving diagnostic services.

Part A. Identifying mWRDs to meet a country’s diagnostic needs 5

 6
Fig. 1. Preselection data and analyses

1 2 3 4
STEP 1 National Strategic Plan, National TB and DR TB National and Subnational Testing Site Infrastructure
Guidance, and Policy Epidemiology Records Records and Master Lists and Human Resource
Collect Data for Documents for TB Patient, Testing, and Records
• DS TB
Sample Referral Networks
Diagnostic Network • Strategic Plan Goals and • Physical facilities
• RR/MDR TB
Assessment Targets • Number and location of
• Electrical supply
• Hr TB persons needing testing
• TB and DR TB Testing

Manual for selection of molecular

• Environmental controls
Algorithms • FQR TB • Number and location
of testing sites, • Reagent storage
• TB and DR TB • Pre-XDR/ XDR TB
instruments and capabilities
Treatment Regimens • HIV/TB and Pediatric modules (as relevant) • Diagnostics
TB
• Specimen Referral connectivity
System Linkages • Human resources

STEP 2 Identify the primary Identify the types and Measure the potential Identify testing sites with
use(s) of mWRDs across distribution of TB and DR test demand at mWRD adequate infrastructure
Analyze all and priority patient TB patients to be tested testing sites and and human resources
Collected Data populations with mWRDs identify opportunities to
strengthen patient access
to mWRD testing services

STEP 3 Estimated Testing Demand at mWRD Sites and Availability of Specimen Referral System Suitability of testing site
Linkages to Support Timely Referral of DS and DR TB Samples infrastructure and human
resources for mWRD:
Use Outputs to Inform
mWRD Selection • Low complexity testing
• Moderate complexity

WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
Identified mWRD Capabilities Required to Meet Patient and testing
TB Program Needs for Detection of TB Across Resistance Types • High complexity testing
and Patient Populations

DR-TB: drug-resistant TB; DS-TB: drug-susceptible TB; FQR-TB: fluoroquinolone-resistant TB; HIV: human immunodeficiency virus; HIV/TB: HIV-associated TB; Hr-TB: isoniazid-resistant, rifampicin-
susceptible TB; MDR/RR-TB: multidrug-resistant TB or rifampicin-resistant TB; mWRD: molecular WHO-recommended rapid diagnostic; TB: tuberculosis; WHO: World Health Organization; XDR-TB:
extensively drug-resistant TB.
The first step in the process for evaluating the suitability of the various mWRDs for use in the different
geographical and epidemiological settings in a country is to collect and analyse information needed
to inform key decisions. Relevant information includes the country’s national policies and strategic
plans, epidemiology of drug-susceptible TB and DR-TB, intended uses of the tests, anticipated
testing volumes, coverage and accessibility (Fig. 1).

A1.1 National strategic plan, guidance and policies

The national strategic plan or national TB laboratory strategic plan should define which mWRD
capabilities are needed to inform the mWRD targets.

Key questions to
consider:
A1.2 National TB and DR-TB epidemiology
‚ What are the objectives of
Estimating future testing demand (i.e. number of
national testing algorithms?
samples for each test type) requires information about
‚ What is the intended primary
the epidemiology of TB and DR-TB in the various settings use of national testing with
in the country, to understand the types and distribution mWRDs (e.g. detection of
of tests required to meet patient needs. Important TB, detection of RR-TB or
detection of TB and resistance
information includes the number of estimated, tested to RIF and INH), and what
and notified cases of TB, paediatric TB, TB/HIV and DR-TB, will the testing be used for as
and the proportion of cases with resistance to key anti-TB capacity grows?
medicines (i.e. RIF, INH and FQs). Where possible, the ‚ Which groups are priority
populations for testing with
epidemiological data should be stratified by district, region mWRDs?
or other subnational geographical area. Much of this ‚ Which anti-TB medicines are
information should be available in annual and quarterly used in current treatment
reports or from prevalence and drug-resistance surveys. regimens for which molecular
or phenotypic DST is needed?

A1.3 National and subnational TB patient population, testing and sample

referral networks
The capacities and capabilities of the TB diagnostic network will be important for determining
which mWRDs could be implemented in which settings and at which tiers of the network. Key
aspects include the structure of the existing network and its relationship to the clinical service
delivery network, such as the diagnostic services and facilities available in each tier of the network
(centralized, decentralized and mixed-model services); molecular testing platforms currently in use
at different levels of the laboratory network; linkages between laboratories and health care facilities
(public sector and private sector); and processes and pathways for referring specimens from health
care facilities to centralized or decentralized mWRD testing facilities.

The diagnostic network structure and referral linkages should be assessed, to understand how
diagnostic services are organized in a country, identify gaps in access to diagnostic testing services, and
identify opportunities to optimize coverage and turnaround times of diagnostic services for all clients.
The assessment may be conducted by compiling country-specific, national health facility and testing
site data, including health facility presumptive and confirmed TB patient volumes; linkages to on-site or
referral-based testing sites; testing site instruments, tests and testing capacity; and testing turnaround

Part A. Identifying mWRDs to meet a country’s diagnostic needs 7

 times for clients or health facilities. These data are then
analysed to identify gaps in testing coverage by geography Potential sources
or patient population, and gaps in test service turnaround of data for the
assessment:
time that limit patient access to diagnostic testing services.
Identified gaps that could be closed by addressing minor ‚ Population distribution –
or localized system challenges should be prioritized for www.worldpop.org
resolution, with activities monitored for their impact on gaps ‚ Distribution of TB –
prevalence, DR-TB surveys or
or for further action. However, if significant gaps in testing
reports of notified cases
services are identified that cannot be completely addressed
‚ Number and locations of
through activities that strengthen existing or new networks, health facilities – master
or if new testing needs are identified that cannot be met by facility list, public databases
the existing network, a full diagnostic network optimization or survey
‚ Number and locations of
exercise may be conducted (see Annex 3).
testing sites – master facility
list or survey
‚ Capabilities and capacities of
individual sites – annual or
quarterly reports
‚ Specimen referral linkages –
survey or network map

Box 1. Key definitions for mWRD site and instrument assessment

Capacity: The number of tests that can be completed by a defined site, instrument or testing
unit (i.e. module) in a defined period.
Example: One module of a Cepheid GeneXpert IV instrument in Country X runs three tests in a workday
that includes ≤8 hours for testing. Thus, the instrument’s capacity, given the availability of four testing
modules, is 12 tests per day (3 tests per module × 4 modules = 12), although minimum, average and
maximum capacities may differ.
Example: One module of a Cepheid GeneXpert IV instrument in Country X can run a minimum of one
test, an average of two tests and a maximum of four tests in the same 8-hour workday.
Given the differences between these examples, it is important to define and specify the type of capacity
that is being calculated and used in network analyses.

Utilization: The percentage of maximum site, instrument or testing unit (i.e. module) capacity
that is used for testing in a defined period.
Example: 24 BD MAX tests are run in an 8-hour workday, for which the BD MAX has a maximum
capacity of 48 tests. The BD MAX instrument therefore has a utilization rate of 50% (24/48).
Note that “optimal” utilization of testing site and instrument capacity is often less than 100%, to ensure that
sufficient surge capacity is available in the event of increased workload or testing equipment service needs.

Coverage: The percentage of patients that needed an mWRD and received mWRD testing.
Example: Of 200 patients presenting with presumptive TB at health facility X, 150 received
mWRD diagnostic and RIF resistance testing, representing a coverage of 75% (150/200).
Note that coverage may be calculated at the site level or the above-site (regional or national) level.
Similarly, coverage may be calculated for all eligible patients or patient subpopulations (e.g. paediatric
patients and patients living with HIV).

Manual for selection of molecular

8
WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
A1.4 Testing site infrastructure and human resources

A1.4.1 Anticipated workload

The epidemiology of TB in the local area should provide an estimation of test-specific volumes by
assessing the number of people that need an mWRD test, as well as the types of diagnostic and
drug-susceptibility tests needed for each anti-TB medicine. The diagnostic network analysis that
maps people needing testing alongside existing or needed mWRD sites should enable estimation
of the anticipated workload at each mWRD site. Using these data, the WHO TB Diagnostics Capacity
Calculator (5)5 may be modified to calculate the number of mWRDs needed at each individual mWRD
testing site. Also, current workload information for existing sites should be available from quarterly
or annual reports, and can be combined with network analysis data to confirm or refine estimates
of the anticipated workload. Anticipated workload is important for the following reasons:
‚ The workload must be supported by sufficient mWRD test and instrument capacity. Although
some instruments can process a maximum of 10 samples per day, others can process 50–100 or
even more samples per day (see Annex 2). Both overuse and underuse of instrument capacity
should be avoided.
‚ Staffing at the testing site should support completion of the required number of tests per day
at each testing site. Necessary modifications to staffing plans can be determined by considering
the current number of staff, workhours per day, standard and shifted start and end times,
workdays per week and “real-world” throughput of the mWRD platform. For example, this
might be the theoretical throughput adjusted for the actual number of test cycles possible
in a workday that is inclusive of sample preparation and result reporting time, as well as any
instrument downtime for cleaning and maintenance.
‚ The anticipated workload should also consider the possibility of multidisease testing. For
instruments that are shared with other disease testing (e.g. HIV or COVID-19), the total number
of samples to be tested per day should be considered when determining the efficient use
of the instrument. There are potential cost advantages to sharing an instrument used for
multidisease testing (e.g. sharing of purchase and maintenance costs) provided sufficient
capacity for each programme’s testing demand is ensured. Also, having laboratory staff testing
samples for multiple diseases may improve testing efficiencies and staff proficiencies, and
facilitate scheduling and shift creation.

A1.4.2 Resource availability

The selection of an mWRD will be influenced by the availability of a testing facility that has the
infrastructure needed to support its use. Important considerations for mWRD testing site
infrastructure include:
‚ reliability of the electricity supply;
‚ availability of temperature and humidity control;
‚ manufacturer recommendations for sample, equipment and reagent storage;
‚ access to the required waste disposal supplies and methods;
‚ biosafety requirements;
‚ space and security requirements for physical facilities;

5
See Microsoft Excel spreadsheet at http://www.who.int/entity/tb/publications/calculations_of_lab_capacity.xls?ua=1.

Part A. Identifying mWRDs to meet a country’s diagnostic needs 9

 ‚ number and technical skill of laboratory staff (e.g. precision pipetting and computer skills);
‚ availability of internet; and
‚ diagnostic connectivity for health care provider and programme reporting.
‚ Guidance for assessing the suitability of testing sites is available in the implementation guides
for the various mWRDs available on the Stop TB Partnership website (6).

In addition to the categories above, this preselection phase should also include reviewing relevant
WHO policies and guidance (3, 4), to become familiar with the recommended uses and operational
characteristics of the various mWRDs.

A2 Decision pathway for identifying mWRDs for

implementation
Collated and analysed preselection data (see Part A1) will provide information on where persons
seek care (e.g. specimen collection sites), linkages to testing (e.g. on-site testing or specimen referral)
and testing site demands and capabilities (e.g. estimated workload). The decision pathway (Fig. 2)
uses this information to identify which mWRDs could be used at each individual mWRD testing site
to address the molecular diagnostic needs of the patient population being served. The results of
the site analyses can be combined to identify the type and potential number of mWRDs needed to
meet the molecular diagnostic needs of an epidemiological or geographical setting, and ultimately
of the country and the patients being served.

Although the focus is on the individual mWRD testing site, the decision pathway begins with a
consideration of the specimen collection sites that are linked or are expected to be linked in the
future to an mWRD testing site. This is important for determining the anticipated workload for a
testing site, and for patient access to mWRD testing. It may also identify gaps in coverage.

For specimen collection sites that are not currently linked to an mWRD testing site, access to
mWRD testing can be provided by either implementing such testing at that specimen collection
site or by establishing a linkage (e.g. specimen referral system) to an existing or planned mWRD
testing site. The decision pathway can be used to determine which mWRDs would be suitable for
implementation at a new mWRD testing site. The decision pathway can also be used to reevaluate an
mWRD testing site when the linkage of an additional specimen collection site (or sites) significantly
alters the anticipated workload.

Manual for selection of molecular

10
WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
 Fig. 2. Decision pathway

Specimen Collection Site

Pre-Selection Data
3
Diagnostic Is there a timely and efficient specimen referral system connecting the collection site with an mWRD site?
Network Analysis
Improve and/or Establish Specimen Referral System OR
No Yes
Establish mWRD Testing On-Site

mWRD Testing Site (Combine Data from all Referring Collection Sites)
1
NSP Targets
Detection of
2 TB only1 Detection of TB and RR-TB Detection of TB, RR-TB, MDR-TB, Hr-TB

Epidemiology
Low Throughput Low Throughput
Needed High Throughput Needed Needed High Throughput Needed
3
Diagnostic
Network Analysis 2
Facilities suitable Facilities suitable Facilities suitable Facilities suitable Facilities suitable Facilities suitable Facilities suitable
for LC testing for LC testing for LC testing for MC testing for LC testing for LC testing for MC testing
4
Testing Site
Resources 3,4
Eiken Loopamp Molbio Truenat Molbio Truenat BD MAX Molbio Truenat Molbio Truenat BD MAX Roche
Bruker/ Hain Cepheid Xpert Cepheid Xpert Roche cobas Cepheid Xpert Cepheid Xpert cobas
Fluorotype Bruker/ Hain Bruker/ Hain
Fluorotype Fluorotype
Abbott RealTime Abbott RealTime
Molbio Truenat Molbio Truenat
Cepheid Xpert Cepheid Xpert

Refer TB-positive samples for additional drug susceptibility testing (DST) as needed: a) molecular DST for RIF for TB-positive Loopamp
samples; b) molecular DST for INH if needed, c) molecular DST for FQ for all RR-TB, MDR-TB, and Hr-TB and d) phenotypic DST as needed
(e.g., Group A drugs).

DR-TB: drug-resistant TB; DST: drug-susceptibility testing; FQ: fluoroquinolone; Hr-TB: INH-resistant, RIF-susceptible TB; INH: isoniazid; LC: low complexity; MC: moderate complexity; MC-aNAAT: moderate
complexity automated nucleic acid amplification test; MDR-TB: multidrug-resistant TB; MTBC: Mycobacterium tuberculosis complex bacteria; mWRD: molecular WHO-recommended rapid diagnostic; NSP:
national strategic plan for TB; RIF: rifampicin; RR-TB: RIF-resistant TB; SRS: specimen referral system; TB: tuberculosis; WHO: World Health Organization.
a
Any of the tests for detection of TB and DR-TB could also be used in a situation where only the detection of TB is needed. If so, follow the pathway for “Detection of TB and RR-TB”.
b
Complexity of testing relates to the requirements of infrastructure, equipment and the technical skills of laboratory staff.
c
The Eiken Loopamp MTBC assay and the Bruker/Hain FluoroType MTB assay are designed as tests for the detection of MTBC only. Although the Roche cobas MTB, RealTime MTB and Truenat MTB
tests could be used for the detection of MTBC only, they are designed for use as initial assays in a two-test procedure that includes resistance tests. If used for detection of MTBC only, the decision

Part A. Identifying mWRDs to meet a country’s diagnostic needs

pathway for “Detection of TB and RR-TB” can be used to evaluate their suitability. However, programmes are strongly encouraged to use these tests to detect MTBC and drug resistance, to meet the
goal of universal testing for rifampicin resistance.
d
Tests listed are described in Table 1 and compared in Annex 2. The mWRD abbreviations are defined as follows: Eiken Loopamp (Loopamp MTBC Detection Kit); Bruker/Hain FluoroType (FluoroType
MTB); Molbio Truenat (Truenat MTB, MTB Plus and MTB-RIF-Dx); Cepheid Xpert (Xpert MTB/RIF or Xpert MTB/RIF Ultra); BD MAX (BD MAX MDR-TB); Roche cobas (cobas MTB and cobas MTB RIF/INH);

11
and Abbott RealTime (RealTime MTB and RealTime MTB RIF/INH).
 A2.1 Steps for identifying suitable mWRDs for use as the initial diagnostic
test at a testing site
The steps for identifying suitable mWRDs are outlined below.

Step 1

Identify specimen collection sites with acceptable turnaround times

The diagnostic network analysis in Part A1 should identify the specimen collection sites (and
hence the patients) that are linked to an mWRD site by a specimen referral system6 that supports
an overall, acceptable turnaround time (≤48 hours) from specimen collection to result reporting.
a. The projected test demand from all linked collection sites should be combined to inform the
anticipated workload of the mWRD testing site.
b. The network analysis may identify sites that are not currently linked to an mWRD testing
site but could be linked, and where a specimen referral system could be established or
strengthened to achieve a turnaround time of 48 hours or less. The projected test from any
newly linked collection sites should be added to the anticipated workload of the newly linked
mWRD testing site.
c. For some specimen collection sites, it may be necessary to implement molecular testing at
the collection site to provide access to timely molecular results for all those needing testing.
The decision tree can be used to determine which tests would be suitable to establish at
such a standalone site.

Step 2

Identify the categories of tests needed

For established or new mWRD sites that provide the diagnostic testing at the specimen collection
sites, national policies and goals for testing using mWRDs, along with the epidemiology of
TB and DR-TB in the population to be tested, should identify the category of test needed (i.e.
detection of TB, or detection of resistance to RIF or any other anti-TB medicines). For example,
in a setting with a high prevalence of MDR/RR-TB and a national algorithm indicating that all
patients with presumptive TB should receive DST for RIF, an mWRD that detects TB and assesses
RIF resistance (and possibly INH resistance) would be preferred over one that only detects TB.
Also, mWRD sites that serve different geographical and epidemiological settings in the country
may require different classes of mWRDs or different mWRDs within a class, to provide tailored
services to their local clients.

6
Specimen referral systems include a policy and governance framework, standard operating procedures (SOPs),
mechanisms and equipment to move specimens safely and to report results promptly, logistics, trained personnel,
data management, financing, and monitoring and evaluation. See the GLI Guide to TB specimen referral systems
and integrated networks, which can be found on the Stop TB Partnership website (7).

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WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
Step 3

Identify the instrument capacity needed at each testing site

The estimation of the anticipated workload at each testing site should identify whether low-
or high-throughput systems will be needed to adequately complete the volume of testing and
ensure result reporting within 48 hours of specimen collection.
a. mWRD sites that anticipate high volumes of samples may best meet testing demand using an
instrument with a high throughput (e.g. cobas 6800 or 8800) or by using multiple instruments
with a low throughput (e.g. multiple Truelab Quatro microPCRs).
b. Some mWRD instruments are available in modular formats to accommodate a wide range
of tests per day. For example, Xpert MTB/RIF Ultra tests can be conducted on GeneXpert
instruments with 1, 2, 4, 8, 16 or more modules, and more than one instrument can be
connected to run on a single computer.

Step 4

Identify infrastructure improvements that may be needed at testing sites

The availability of suitable testing facilities may influence the choice of test or may indicate what
infrastructure improvements will be needed to implement a specific test. For example:
a. In a setting with an unreliable electrical supply, an mWRD that uses a battery-operated
instrument may be preferable to making significant investments in infrastructure upgrades
(e.g. installation of solar power, generators or uninterruptible power supply [UPS]).
b. The choice of mWRD may be limited by the number of rooms available for testing, the potential
for establishment of molecular testing unidirectional workflow across multiple rooms, and
the presence of environmental conditions (e.g. temperature, humidity and dust):
i. number of rooms required for mWRD testing is 1–3 rooms;
ii. mWRD testing instruments vary in size from under 0.5 m wide to over 4 m wide and
from under 10 kg to over 2400 kg in weight;
iii. the maximum operating temperature of mWRD testing instruments ranges from 28 °C
to 40 °C; and
iv. mWRD reagent storage conditions vary from –20 °C to 30 °C.

c. If a molecular testing platform is currently in use by another disease programme (e.g. HIV
or COVID-19) at a testing facility in the TB diagnostic network, or accessible by that network,
implementing the corresponding mWRD for TB would be logical. Platforms that use random
access approaches (e.g. GeneXpert) or that allow different types of tests to be performed in the
same batch (e.g. cobas 6800/8800 and BD MAX) would be preferable, to ensure that the needs
of all patients and programmes can be met. Careful planning will be needed to ensure cross-
disease equitable access to testing instrumentation and resources, predefined prioritization of
sample or test types, cross-programme implementation and optimization of the multidisease
testing strategy, and use of non-duplicative data management and reporting solutions.

Part A. Identifying mWRDs to meet a country’s diagnostic needs 13

 d. mWRD sites may be able to accommodate low, moderate or high complexity mWRDs, as
indicated by their WHO class assignment. Complexity of the testing is based on each test’s
requirements for infrastructure, equipment and laboratory staff technical skill:
i.  Low complexity – Facilities suitable for low complexity testing are typically found in the
lower tiers of TB laboratory network (e.g. peripheral and district laboratories) and have
few or no special laboratory infrastructure requirements (e.g. reliable supply of electricity
or low dust environment) and laboratory staff with basic technical skills (e.g. basic
pipetting, and precision not critical). Instruments required for the mWRD used in such
facilities are relatively easy to install, operate and maintain. The mWRDs suitable for such
facilities include Xpert MTB/RIF, Xpert MTB/RIF Ultra and Xpert MTB/XDR; Truenat MTB,
Truenat MTB Plus, and Truenat MTB-RIF-Dx; and Loopamp MTBC tests. Low complexity
testing can be performed in facilities suitable for moderate or high complexity testing.
ii.  Moderate complexity – Facilities suitable for moderate complexity tests are typically
found in the intermediate or central tiers of the TB laboratory network (e.g. regional,
intermediate or national laboratories), have specific laboratory infrastructure
requirements (e.g. multiple rooms), and have qualified laboratory staff with specific
computer and moderate complexity testing skills, such as the ability to successfully
complete multi-step procedures with precision. Required instruments for moderate
complexity mWRDs have more extensive installation, operational and maintenance
requirements. The mWRDs suitable for such facilities include RealTime MTB and RealTime
MTB RIF/INH; BD MAX MDR-TB; FluoroType MTB and FluoroType MTBDR; and cobas MTB
and cobas MTB RIF/INH tests. Moderate complexity testing can also be performed in
facilities suitable for high complexity testing, but not in low complexity settings.

iii.  High complexity – Facilities suitable for high complexity tests are typically found in the
intermediate or central tiers of the TB laboratory network, and their requirements are
similar to those of moderate complexity laboratories. The key difference is that a higher
level of technical skills is required to perform the assay and interpret results. The mWRDs
suitable for such facilities include the follow-on Genoscholar PZA-TB line-probe assay for
the detection of PZA resistance. Low and moderate complexity testing can be performed
in facilities suitable for high complexity testing.

Step 5

Consider the need for follow-up testing

The diagnostic pathway does not always stop with mWRD testing; it can include necessary
follow-up testing, such as testing for resistance to additional anti-TB medicines. Thus, the
following are important:
a. Consider whether multiple mWRDs can be used in combination to meet testing demand. For
example, in certain circumstances, tests listed as “initial diagnostic tests for diagnosis of TB
with drug-resistance detection” can also be used as follow-on tests to detect drug resistance.
The BD MAX MDR-TB, for example, can be used as a follow-on test to detect resistance to INH
and RIF for TB-positive samples identified by the Loopamp MTBC detection test.
b. Consider WHO and national recommendations for comprehensive diagnostic and DST
services. For example, WHO recommends that all persons with MDR/RR-TB or INH-resistant,

Manual for selection of molecular

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WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
 RIF-susceptible TB (Hr-TB) receive a drug-susceptibility test for FQ resistance. This can be
achieved by having an mWRD for FQ resistance at the mWRD site or by referring a sample to
a second mWRD site that offers an mWRD for FQ resistance. The decision process described
here can be applied to choosing follow-on tests to detect resistance to additional drugs. It
may also be necessary to refer samples for phenotypic DST, particularly for the new and
repurposed drugs (e.g. bedaquiline, delamanid and linezolid) for which an mWRD is not
available, or for drugs for which clinical dosing may benefit from determination of critical
concentrations (e.g. moxifloxacin). Lastly, DST should be available for all drugs included in a
treatment regimen for which there is a reliable DST method (8).

A2.2 Steps for identifying suitable mWRDs for use as follow-on tests at a
testing site
A similar process is employed for selecting a suitable mWRD to use as a follow-on test to detect drug
resistance. A critical step is to determine which drug resistances to assess. This should be guided
by national policies and testing algorithms, along with the epidemiology of DR-TB in the population
to be tested. Patients with resistance detected at initial testing (e.g. RIF) can be referred to specific
treatment centres and testing at laboratories linked to these sites; alternatively, provision of on-site
testing at the treatment site needs to be considered. A low complexity follow-on test (Xpert MTB/XDR)
is available to assess resistance to INH, FQ, AMK and ETO. Follow-on tests of increased complexity
are available that assess resistances to RIF and INH (GenoType MTBDRplus and Genoscholar NTM
+ MDRTB Detection Kit) and to FQ and AMK (GenoType MTBDRsl). A high complexity follow-on test
(Genoscholar PZA-TB) is available to assess resistance to PZA. Each of these tests can be configured
to be used in a low- or high-throughput setting.

A3 Considerations for suitable mWRD implementation

The decision process described above should help to identify which mWRDs are suitable to address
the diagnostic needs of the country. The decision regarding which of the suitable mWRDs to
implement in the various epidemiological and geographical settings will involve country-specific
considerations such as:
‚ existing molecular testing platforms and capacity for TB and other diseases;
‚ financial aspects (e.g. available budget, cost of instruments and commodities, availability of
Global Drug Facility [GDF] pricing, implementation costs and annual operating costs);
‚ instrument capabilities, service and maintenance requirements and the availability of authorized
local service providers, extended warranties and service agreements;
‚ sample referral network and its ability to deliver timely results in different areas;
‚ procurement issues (e.g. supply chain, shelf life, storage conditions, importation formalities,
customs regulations and in-country distributors);
– shelf lives range from 9 months to 24 months;
– storage conditions for reagents range from –15 °C to –25 °C, to 2 °C to 8 °C and 2 °C
to 30 °C;
‚ need for upgrading facilities to meet infrastructure requirements and operating conditions;
‚ availability of enough staff with the appropriate skills; and
‚ availability of in-country technical support and assistance.

Part A. Identifying mWRDs to meet a country’s diagnostic needs 15

 Questions to consider when comparing suitable mWRDs are listed in Annex 1.

Finally, the decision process focuses on individual mWRD testing sites. The results of the analyses
of individual sites in an epidemiological or geographical setting should be combined to obtain an
overview of the mWRDs appropriate for use in the setting. When considering the setting as a whole,
issues related to procurement, equipment maintenance and quality assurance (for example) may
influence which mWRDs will be feasible to implement and maintain over the long term. A mixture
of different options that fit the needs of each localized setting may be needed, to provide the best
overall solution. Programmes will also need to take into account plans for future expansion of
mWRD testing in the country.

Manual for selection of molecular

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WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
Part B. Suggested reading and
resources

B1 WHO guidelines and policies7

‚ WHO consolidated guidelines on tuberculosis Module 3: Diagnosis – rapid diagnostics for
tuberculosis detection, 2021 update (3).
‚ WHO operational handbook on tuberculosis Module 3: Diagnosis – rapid diagnostics for tuberculosis
detention, 2021 update (4).
– The narrative summarizes the individual mWRDs, their performance characteristics and
recommended uses.
– Information sheets in Annex 2 provide overviews of the Abbott RealTime MTB and MTB
RIF/INH tests, Becton Dickinson BD MAX MDR-TB test, Roche cobas MTB and cobas
MTB-RIF/INH tests, Bruker/Hain Lifescience FluoroType MTB and FluoroType MTBDR,
Cepheid Xpert MTB/XDR and Nipro Genoscholar PZA-TB II tests.

B2 GLI implementation manuals8

‚ Implementing a quality assurance system for Xpert MTB/RIF testing.
‚ GLI planning for country transition to Xpert MTB/RIF Ultra cartridges.
‚ Practical guide to implementation of Truenat tests.
‚ GLI guide for the interpretation and reporting of line probe assays.

B3 GLI information sheets9

‚ Practical considerations for implementation of the Abbott RealTime MTB and Abbott RealTime
MTB RIF/INH tests.
‚ Practical considerations for implementation of the BD MAX MDR-TB test.
‚ Practical considerations for implementation of the Roche cobas MTB and cobas MTB-RIF/INH
assays.
‚ Practical considerations for implementation of the Bruker/Hain Lifescience FluoroType MTB and
FluoroType MTBDR.

7
The most up-to-date WHO policy guidance on TB diagnostics and laboratory strengthening can be found on the
WHO Global TB Programme website (https://www.who.int/teams/global-tuberculosis-programme).
8
See the Stop TB Partnership website (7).
9
See the Stop TB Partnership website (7).

Part B. Suggested reading and resources 17

 ‚ Practical considerations for implementation of the Cepheid Xpert MTB/XDR test.
‚ Practical considerations for implementation of the Nipro Genoscholar PZA-TB II assay.
‚ Practical considerations for implementation of the loop-mediated isothermal amplification
(TB-LAMP) test.

B4 Stop TB Partnership information notes and GDF publications

‚ Xpert® MTB/RIF and Ultra: technical information note (9).
‚ Automated rapid nucleic acid amplification tests (NAATs) for detection of TB and resistance to
rifampicin and isoniazid: Stop TB information note (10).
‚ Practical considerations for implementation of Truenat (11).
‚ Diagnostics, medical devices & other health products catalog (12) – Describes test specifications,
ordering information, shelf lives and storage conditions for tests eligible for purchase using
funds from the Global Fund to Fight AIDS, Tuberculosis and Malaria (Global Fund).

B5 Global Fund to Fight AIDS, Tuberculosis and Malaria

List of TB diagnostic test kits and equipment classified according to the Global Fund quality assurance
policy (13) – Describes prices of equipment, consumables and warranties available through the
Global Fund to Fight AIDS, Tuberculosis and Malaria (Global Fund).

B6 Diagnostic network analysis and optimization

‚ Bringing data analytics to the design of optimized diagnostic networks in low- and middle-income
countries: process, terms and definitions. Nichols et al. (2020) (14).
‚ Designing an optimized diagnostic network to improve access to TB diagnosis and treatment in
Lesotho. Albert et al. (2020) (15).
‚ Laboratory mapping program (LabMaP) – what we do. African Society for Laboratory Medicine
(2022) (16).

B7 Specimen referral systems

‚ Guide to TB specimen referral systems and integrated networks – available from the Stop TB
Partnership website (6).

Manual for selection of molecular

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WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
References

1. The End TB Strategy. Geneva: World Health Organization; 2015 (https://www.who.int/

publications/i/item/WHO-HTM-TB-2015.19, accessed January 2022).

2. Meeting report of the WHO expert consultation on the definition of extensively drug-resistant
tuberculosis, 27–29 October 2020. Geneva: World Health Organization; 2020 (https://apps.
who.int/iris/handle/10665/338776, accessed January 2022).

3. WHO consolidated guidelines on tuberculosis Module 3: Diagnosis rapid diagnostics for

tuberculosis detection, 2021 update. Geneva: World Health Organization; 2021 (https://apps.
who.int/iris/handle/10665/342331, accessed January 2022).

4. WHO operational handbook on tuberculosis Module 3: Diagnosis – rapid diagnostics for

tuberculosis detection, 2021 update. Geneva: World Health Organization; 2021 (https://apps.
who.int/iris/handle/10665/342369, accessed January 2022).

5. Framework of indicators and targets for laboratory strengthening under the End TB Strategy.
Geneva: World Health Organization; 2016 (https://apps.who.int/iris/handle/10665/250307,
accessed January 2022).

6. Guidance and tools [website]. Stop TB Partnership; (https://stoptb.org/wg/gli/gat.asp, accessed

January 2022).

7. GLI specimen referral toolkit. Stop TB Partnership; 2020 (https://stoptb.org/wg/gli/srt.asp,

accessed January 2022).

8. Technical manual for drug susceptibility testing of medicines used in the treatment
of tuberculosis. Geneva: World Health Organization; 2018 (https://apps.who.int/iris/
handle/10665/275469, accessed January 2022).

9. Xpert® MTB/RIF and Ultra. Stop TB Partnership; 2019 (https://stoptb.org/assets/documents/

gdf/drugsupply/Xpert_info_note.pdf, accessed January 2022).

10. Automated rapid nucleic acid amplification tests (NAATs) for detection of TB and resistance
to rifampicin and isoniazid – Stop TB information note. Geneva: Stop TB Partnership; 2021
(https://stoptb.org/assets/documents/resources/publications/sd/RIH_INH_NAATs.pdf, accessed
January 2022).

11. Practical considerations for implementation of Truenat. Geneva: Stop TB Partnership;(https://

stoptb.org/assets/documents/resources/wd/Practical%20Considerations%20for%20
Implementation%20of%20Truenat.pdf, accessed January 2022).

References 19
 12. Diagnostics, medical devices & other health products catalog. Geneva: Stop TB Partnership;
2021 (https://stoptb.org/assets/documents/gdf/drugsupply/GDFDiagnosticsCatalog.pdf,
accessed January 2022).

13. List of TB diagnostic test kits and equipments classified according to the Global Fund quality
assurance policy. Geneva: The Global Fund; 2020 (https://www.theglobalfund.org/media/9461/
psm_productsdiagnosticstb_list_en.pdf, accessed January 2022).

14. Nichols K, Girdwood SJ, Inglis A, Ondoa P, Sy KTL, Benade M et al. Bringing data analytics to the
design of optimized diagnostic networks in low- and middle-income countries: process, terms
and definitions. Diagnostics (Basel). 2020;11(1) (https://pubmed.ncbi.nlm.nih.gov/33374315/,
accessed January 2022).

15. Albert H, Purcell R, Wang YY, Kao K, Mareka M, Katz Z et al. Designing an optimized
diagnostic network to improve access to TB diagnosis and treatment in Lesotho. PLoS One.
2020;15(6):e0233620 (https://pubmed.ncbi.nlm.nih.gov/32492022/, accessed January 2022).

16. Laboratory mapping program (LabMaP). What we do. [website]. African Society for Laboratory
Medicine; (https://aslm.org/what-we-do/labmap/, accessed January 2022).

Manual for selection of molecular

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WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
Annex 1. Questions to consider
when comparing suitable mWRDs

The questions in this annex are designed to help countries to decide which of the “suitable” molecular
World Health Organization (WHO)-recommended rapid diagnostic tests (mWRDs) for tuberculosis
(TB) identified by the decision pathway meet the diagnostic testing needs of respective mWRD sites
selected for test introduction by the TB diagnostic network analysis. The included questions are
not exhaustive; instead, they are designed to highlight the different characteristics of mWRDs that
have the same level of complexity and similar throughputs. Annex 2 contains tables that compare
mWRD test specifications, for reference.

The questions given here were identified by consensus among members of the Global Laboratory
Initiative (GLI) Core Group. Country-specific factors will affect the priority of the questions and the
relative importance of the answers in determining which mWRD best meets the needs of a particular
epidemiological and geographical setting.

mWRD site testing demand

‚ Is the test needed for initial detection of TB alone, or for initial or follow-on detection of resistance
to TB specific medicines (if so, which medicines?) at the selected mWRD site or sites?
‚ Which level of mWRD test class complexity and operational requirements can be accommodated
by existing site infrastructure and human resources; also, if upgrades are needed, are they feasible?
‚ What level of throughput (i.e. tests per day) is needed to meet the anticipated demand for
testing at the mWRD site?

WHO recommendations for mWRD use and performance (1)

‚ Do the WHO recommendations for test use, target populations (e.g. adults, children, people
living with HIV) and specimen types match the needs of the patient population served by the
mWRD site? Note: all currently recommended mWRDs are approved for use with sputum
and bronchial alveolar lavage samples, but only some mWRDs approved for use with various
extrapulmonary samples.
‚ At the mWRD site’s TB prevalence rate, what are the performance parameters of the test (e.g.
sensitivity, specificity, positive and negative predictive value, and error rates) in the population
served by the mWRD testing site?
‚ How flexible and adaptable are the test and test platform? Can the test and test platform be
used to address current and planned diagnostic targets of the national strategic plan?

Annex 1. Questions to consider when comparing suitable mWRDs 21

 Costs
‚ What commodity-specific costs are associated with the mWRDs (e.g. test, supplies, reagents,
instruments and equipment)?
‚ What annual operating costs are associated with the mWRDs (e.g. consumables, instruments,
service and maintenance, human resources and external quality assurance)?
‚ Is Global Drug Facility (GDF) pricing needed for the mWRDs; if so, is it available?
‚ What are the costs to support the introduction of each mWRD and how do they differ (e.g.
facility upgrades, national and site-level documentation revisions, clinical and laboratory
trainings, diagnostic connectivity solutions and external quality assurance)?10
‚ Does the mWRD instrument have the capacity to test for multiple diseases – TB, HIV, COVID-
19 or other diseases – if so, can costs for introduction, implementation and maintenance be
shared across disease programmes?

Procurement and supply chain

‚ Does the test have regulatory approval to be used in the country and can customs clearance
be achieved? What are the importation requirements for instruments, reagents and supplies?
‚ Is a cold chain required for distribution of reagents and commodities?
‚ What is the shelf life of the required reagents and commodities?

Instrumentations, maintenance and service

‚ Is a molecular testing instrument in use at a current or planned mWRD testing site? If so, does
it have capacity available to accommodate the anticipated TB testing workload, either alone
or in combination with testing for other diseases?
‚ What are the service and maintenance requirements for required instruments?
‚ What are the calibration requirements for necessary ancillary equipment?
‚ Is support from the manufacturer or authorized local service providers for installation,
implementation and equipment maintenance (warranties or service contracts) available
in-country?
‚ Is there in-built or potential diagnostic connectivity, to allow for rapid transfer of results and
remote monitoring?

Facility requirements
‚ What biosafety precautions are needed? Is a biological safety cabinet required and available?
‚ Is the main electricity supply reliable? Is there a need for alternative energy sources, generators
or uninterruptible power supply (UPS)? Can the test be conducted with a battery-powered
instrument, if necessary?

10
For a discussion of budgetary needs for implementation, see the Global Laboratory Initiative Planning and
Budgeting Tool for TB and Drug Resistant TB Testing on the Stop TB Partnership website (2).

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WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
 ‚ Will upgrades to the facility be needed to meet the operating environment requirements (e.g.
temperature, humidity and dust free), room and bench space requirements, and security
requirements?
‚ What are the recommended storage conditions for the components of the test, and can the
selected mWRD sites accommodate any necessary freezers and refrigerators?

Human resources
‚ What is the complexity of instrument use and site-level maintenance, and the degree of
automation available? Is the level of complexity suitable for testers at the mWRD testing site?
‚ What is the required hands-on time and the complexity of the hands-on steps? Does the assay
require precision pipetting?
‚ Is detection of Mycobacterium tuberculosis complex bacteria (MTBC) and drug resistance
accomplished in a one-step process or is it a two-step process that requires separate assays
for detection of MTBC and detection of resistance?
‚ How many staff will be required to handle the anticipated workload?
‚ Where multidisease testing is conducted, could staff be supported by multiple disease
programmes?
‚ What is the total time required for a test (hands-on time plus instrument run time) and how
can it fit into the workflow of the laboratory?

References for Annex 1

1. WHO consolidated guidelines on tuberculosis Module 3: Diagnosis – rapid diagnostics for

tuberculosis detection, 2021 update. Geneva: World Health Organization; 2021 (https://apps.
who.int/iris/handle/10665/342331, accessed January 2022).

2. Guidance and tools [website]. Geneva: Stop TB Partnership; 2022 (https://stoptb.org/wg/gli/

gat.asp, accessed January 2022).

Annex 1. Questions to consider when comparing suitable mWRDs 23

 24
Annex 2. Test specifications

Manual for selection of molecular

Initial molecular diagnostic tests for detection of TB

Without detection of drug

With detection of drug resistance
resistance

Cepheid Molbio Abbott BD MAX™ Bruker/Hain Roche Bruker/Hain Eiken

Xpert® MTB/ Truenat® RealTime MDR-TB FluoroType® cobas® MTB FluoroType Loopamp™
RIF and MTB, MTB MTB and MTBDR and MTB MTB MTBC
Ultra Plus and MTB RIF/ RIF/INH Detection
MTB-RIF-Dx INH (TB-LAMP)

WHO class N/Aa N/A MC-aNAATb MC-aNAAT MC-aNAAT MC-aNAAT MC-aNAAT N/A

Drug resistance detected RIF RIF RIF, INH RIF, INH RIF, INH RIF, INH None None

Maximum capacity 12 (GX-IV) to 9 to 36 94 48 Up to 288 384 to 1056d Up to 288 70

(maximum no. of tests/ 48 (GX-XVI)c
workday)

Batch size 4 (GX-IV) to 1 (Uno) to 4 Up to 94 Up to 24 Up to 94 Up to 94 Up to 94 Up to 14

16 (GX-XVI) (Quatro)

Run time 2 hours 1 hour 7 hours 4.5 hours 2.5 hours 3.5 hours 2.5 hours 90 minutes
(MTB/RIF) (detection) (detection) (detection)
90 minutes plus 1 hour plus plus

WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
(Ultra) (resistance) 3.5 hours 3.5 hours
(resistance) (resistance)

Required 6-color Trueprep m2000sp and BD MAX GXT96 and cobas GXT96 and HumaLoop T
instrumentation GeneXpert sample prep m2000rt Fluorocycler 6800/8800 Fluorocycler or HumaTurb
device and microplate XT XT system
Truelab centrifuge
microPCR
analyzer
 Initial molecular diagnostic tests for detection of TB

Without detection of drug

With detection of drug resistance
resistance

Dimensions GX-IV Trueprep m2000sp BD MAX GXT96 112.3 cobas 6800 GXT96 112.3 HumaLoop
27.94 cm device 21.5 145 × 79.4 94 × 75.4 × 77.4 × 292 × 216 × 77.4 × T 25 × 30.6
× 30.48 × × 23.5 × × 217.5 cm, × 72.4 cm, 82.5 cm, × 129 cm, 82.5 cm, × 18.2 cm,
29.72 cm, 11.5 cm, 314.4 kg 113.4 kg 140 kg 1624 kg 140 kg 9.5 kg
11.8 kg 2.5 kg m2000rt 34 FluoroCycler cobas 8800 FluoroCycler
GX-XVI: Truelab Uno × 49 × 45 cm, XT 43 × 57 × 429 × 216 XT 43 × 57 ×
57.8 × 65.6 PCR 24 × 18.5 34.1 kg 73 cm, 65 kg × 129 cm, 73 cm, 65 kg
× 33.7 cm, × 112 cm, 2405 kg
21.5 kg 1.5 kg

Molecular workflow No No Yes No Yes No Yes No

required

Operating conditions <30 °C 15 °C to 15 °C to 18 °C to 28 °C - - - <30 °C

40 °C, with 28 °C, with with 20–80%
10–80% 30–80% relative
relative relative humidity
humidity humidity

Reagent storage 2 °C to 28 °C 2 °C to 30 °C –15 °C to 2 °C to 28 °C –20 °C to 2 °C to 8 °C –20 °C to 2 °C to 30 °C
–25 °C –18 °C –18 °C

Reagent shelf life 9 months 2 years 12 months 9 months On request 16 months On request 14 months
(MTB RIF/ (MTB RIF/
INH) or INH) or
18 months 18 months
(MTB) (MTB)

Connectivity Yes Yes Yes Yes Yes Yes Yes No

INH: isoniazid; MC-aNAAT: moderate complexity automated nucleic acid amplification test; N/A: not applicable; PCR: polymerase chain reaction; RIF: rifampicin; TB: tuberculosis; WHO: World Health
Organization.
a
Not applicable: The test has not been assigned to a class. WHO recommendations were made following a review of the performance of the individual test.
b
Detailed operational characteristics of the individual moderate complexity (MC-aNAATs) are available (1)
c
Individual GeneXpert instruments can be connected to run on a single computer and thereby increase throughput. Also, the GeneXpert infinity system can process more than 2000 samples per day.

Annex 2. Test specifications

d
Maximum throughput for the cobas instruments reflects the number of liquefied, lysed and inactivated samples that can be processed in one day.

25
 Follow-on mWRDs for detection of drug resistance

Cepheid Xpert Nipro Bruker/Hain Bruker/Hain

MTB/XDR Genoscholar™ MTBDRplus MTBDRsl
PZA-TB II

WHO class LC-aNAAT HC-rNAAT FL-LPA SL-LPA

Drugs tested INH, FQ, AMK, ETO PZA RIF, INH, ETO FQ, AMK

Sample type Sputum, BAL Cultures Sputum or cultures Sputum or cultures

Maximum tests 16 (GX4) to 62 12 or 48 12 or 48 12 or 48

per day (GX16)

Batch size Up to 4 with GX4 or Up to 12 with Up to 12 with Up to 12 with

up to 16 with GX16 TwinCubator or up TwinCubator or up TwinCubator or up
to 48 with Multi-Blot to 48 with GT-Blot 48 to 48 with GT-Blot 48
NS-4800

Run time 90 minutes 1–2 days plus time 1–2 days 1–2 days

required for culture

DNA extraction Integrated into assay Manual Manual or separate Manual or separate
instrument instrument

Testing process Low complexity High complexity Manual reverse Manual reverse
automated NAAT reverse hybridization hybridization test hybridization test

Required GeneXpert (10 color) Multi-Blot NS-4800 TwinCubator or TwinCubator

instrumentation or TwinCubator, GT-Blot 48, or GT-Blot 48,
Thermocycler Thermocycler Thermocycler

Molecular No Yes Yes Yes

workflow required

Operating <30 °C - Ambient to 55 °C Ambient to 55 °C

conditions

Reagent storage 2 °C to 28 °C 2 °C to 10 °C Kit 1: 2 °C to 8 °C Kit 1: 2 °C to 8 °C
Kit 2: –20 °C Kit 2: –20 °C

Reagent shelf life - 12 months 18 months 18 months

Connectivity Yes - No No

Multiplexing Yes - No No

AMK: amikacin; BAL: bronchoalveolar lavage; ETO: ethionamide; FL-LPA: first-line line probe assay; FQ: fluoroquinolone; HC-rNAAT: high
complexity reverse hybridization NAAT; INH: isoniazid; LC-aNAAT: low complexity automated NAAT; MC-aNAAT: moderate complexity
automated NAAT; mWRD: molecular WHO-recommended rapid diagnostic; N/A: not applicable; NAAT: nucleic acid amplification test;
RIF: rifampicin; SL-LPA: second-line line probe assay; WHO: World Health Organization.

Reference for Annex 2

1. FIND cDST. WHO supplement. Geneva: World Health Organization; 2019 (https://www.finddx.
org/wp-content/uploads/2019/08/FIND_cDST_WHO_Supplement.xlsx, accessed January 2022).

Manual for selection of molecular

26
WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
Annex 3. Diagnostic network
optimization

Diagnostic network optimization is a three-step process (1):

Step 1: Geographical mapping and baseline scenario model creation.

Step 2: Alternative scenario creation and analysis.

Step 3: Network optimization (i.e. comparison of the scenarios to identify the optimal network
design).

The first step relies on information that may already be available in the ministry of health, national TB
programme (NTP) or national TB laboratory, or from simple surveys, and it can provide important
information. In contrast, a full analysis and network optimization exercise requires considerable human
resources and time (3–6 months), and countries may require expert technical assistance. Optimal
network configurations may differ between different geographical and epidemiological settings.

Step 1: Geographical mapping and

Baseline scenario
baseline scenario model creation
The first step involves mapping – that is, spatial analysis ‚ What is the potential demand
using geographic information system (GIS) coordinates – for testing?
of the populations that require testing; the number and ‚ Where are specimens
locations of health facilities where people seek care; the collected for testing?

number, locations, capabilities and capacities of testing ‚ Where is testing done?

‚ How do specimens get from
sites; and referral linkages.
collection sites to testing sites?

Guidance on collecting the information needed for a

spatial analysis can be found in these resources:
‚ Laboratory mapping program (LabMaP) – what we do (2);
‚ Master facility list resource package: guidance for countries wanting to strengthen their master
facility list (3); and
‚ How to include laboratories in a master facility list: preliminary guidance (4).

Also, various databases of geocoded health facilities in sub-Saharan Africa are available (5, 6).

The main purpose of this step is to generate a baseline model of the diagnostic network for use in
the network optimization process. However, the inventory of GIS-mapped health facilities and GIS-
mapped TB laboratories (including current inventory of diagnostic tests and instruments) should

Annex 3. Diagnostic network optimization 27

 be useful to the NTP for strategic planning, allocating resources and planning for continuation of
TB services in case of service disruptions. For example:

‚ the inventory of laboratories and current mWRD instruments and test volumes may identify
underused and overused instruments, and opportunities to redistribute instruments to
improve the efficiency of testing;
‚ overlaying the maps of specimen collection sites, testing sites and transportation routes might
identify potential specimen referral linkages and inform the design of specimen transport
routes; and
‚ comparison of the TB network baseline model with other network models (e.g. for HIV) may
identify opportunities to collaborate or cost-share services while providing clients with one-
stop-shop testing services.

Step 2: Alternative scenario creation and analysis

The next step is to develop alternative scenarios to the baseline model, in consultation with key
stakeholders. These scenarios should reflect decision points such as the following:
‚ Where can new sites for mWRDs be established to increase detection rates of TB or drug-
resistant (DR-TB), or to address national strategic plan goals and priorities for improving TB
testing in populations that are underserved or a priority?
‚ Are there opportunities for linking a cluster of specimen collection sites to generate sufficient
test demand to justify establishing a molecular testing site with high throughput? Sites that
could form a cluster include those within 40–50 km of an mWRD site (which is a feasible
daily driving distance for a courier) or those that can be linked by a specimen referral system
that allows the testing laboratory to report results within the recommended turnaround time
(≤48 hours from specimen collection).
‚ How can molecular testing services be provided for difficult-to-reach areas or areas where
specimen referral systems with short turnaround times are not currently feasible?
‚ Are there opportunities for using existing molecular testing platforms (e.g. a Roche cobas
8800 or Abbott m2000 instrument, used for HIV testing) for TB testing?
‚ How does changing the acceptable turnaround time affect access to molecular testing?

Although the next step is formal – software-driven evaluation of the alternative scenarios –
programmes will be able to generate useful insights from a less formal interim analysis. For example,
a visual analysis of a map that overlays population distribution and existing mWRD sites may be
able to quickly identify areas that would benefit from the establishment of a new mWRD site and
thus help programmes to decide where to place new mWRD instruments.

Step 3: Network optimization

The third step, network optimization, relies on specialized software and modelling approaches to
evaluate baseline and alternative network configurations using a set of predefined outputs (7).
The aim of network optimization is to increase patient access to testing services and optimize the
delivery of those services. The predefined outputs assess the impact of various diagnostic placement,

Manual for selection of molecular

28
WHO-recommended rapid diagnostic tests for detection of tuberculosis and drug-resistant tuberculosis
number and use scenarios on the effectiveness, efficiency and adaptability of the diagnostic network.
Possible outputs include improved:
‚ availability of molecular diagnostic testing services for TB – that is, the proportion of specimen
collection sites that are linked to an mWRD site;
‚ accessibility of molecular diagnostic testing for TB – that is, the proportion of the population
that live within walking distance11 of a specimen collection site that has onsite mWRD testing
or is linked to an mWRD site by a specimen referral system that enables an overall turnaround
time for mWRD testing of 48 hours or less (i.e. from specimen collection to return of results);
‚ promptness of molecular diagnostic testing for TB – that is, the proportion of mWRD sites that
achieve the target turnaround time of 48 hours or less; and
‚ quality of molecular diagnostic testing for TB – that is, the proportion of mWRD sites that
have sufficient trained and competent staff, and that meet GLI key performance indicators
(e.g. expected rates of error, failures and specimen rejection) and standards for internal and
external quality assurance.

References for Annex 3

1. Nichols K, Girdwood SJ, Inglis A, Ondoa P, Sy KTL, Benade M et al. Bringing data analytics to the
design of optimized diagnostic networks in low- and middle-income countries: process, terms
and definitions. Diagnostics (Basel). 2020;11(1) (https://pubmed.ncbi.nlm.nih.gov/33374315/,
accessed January 2022).

2. Laboratory mapping program (LabMaP) – what we do [website]. Addis Ababa: African Society
for Laboratory Medicine; 2022 (https://aslm.org/what-we-do/labmap/, accessed January 2022).

3. Master facility list resource package: guidance for countries wanting to strengthen their
master facility list. Geneva: World Health Organization; 2018 (https://apps.who.int/iris/
handle/10665/326848, accessed January 2022).

4. How to include laboratories in a master facility list: preliminary guidance. Chapel Hill: MEASURE
Evaluation; 2020 (https://www.measureevaluation.org/resources/publications/ms-20-196.html,
accessed January 2022).

5. Health facilities in sub-Saharan Africa [website]. The Hague: Humanitarian Data Exchange;
OCHA: United Nations Office for the Coordination of Humanitarian Affairs; 2022 (https://data.
humdata.org/dataset/health-facilities-in-sub-saharan-africa?force_layout=desktop, accessed
January 2022).

6. LabMap dashboard [website]. Addis Ababa: African Society for Laboratory Medicine; 2022
(https://aslm.org/labmap-dashboard/, accessed January 2022).

7. https://www.finddx.org/wp-content/uploads/2021/11/Guide-to-Diagnostic-Network-
Optimization_15.11.2021.pdf, accessed January 2022.

11
For accessing primary health care, a walking distance of 5 km is often considered acceptable. Terrain-specific
accessibility algorithms are available that consider geographical variability in determining acceptable walking
distances.

Annex 3. Diagnostic network optimization 29

For further information, please contact:

Global TB Programme
World Health Organization
20, Avenue Appia CH-1211 Geneva 27 Switzerland
Web site: www.who.int/tb