Practical Guide to Implementation of

Truenat™ Tests
for the Detection of TB
and Rifampicin Resistance

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 1
VERSION 2
 Practical Guide
to Implementation of
Truenat™ Tests
for the Detection of TB
and Rifampicin Resistance

Version 2: March 2021

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 3
Acknowledgements
Development of this document was led by Wayne van Gemert (Stop TB Partnership), Thomas
Shinnick (Independent Consultant), Amy Piatek, Kaiser Shen and Umesh Alavadi (United States
Agency for International Development). GLI core group contributors included Elisa Tagliani
(San Raffaele Scientific Institute, Italy), Khalide Azam (University of Maryland, Baltimore-
Mozambique), Sarabjit S Chadha (FIND), Kathleen England (Independent Consultant), Petra
de Haas (KNCV Tuberculosis Foundation), Sarder Tanzir Hossain (Infectious Disease Detection
and Surveillance Project, Bangladesh) and Marguerite Massinga Loembé (Africa Centres for
Disease Control and Prevention; African Society for Laboratory Medicine). Critical review was
provided by Sanjeev Saini and Shanta Achanta (Consultants, National Tuberculosis Elimination
Program, India); Melissa Sander and Cyrille Mbuli (Tuberculosis Reference Laboratory Bamenda,
Cameroon; early implementer of Truenat supported by Stop TB Partnership’s TB REACH); Praveen
K.S., Rajneesh Tripathi, Shibu Vijayan and Venkatesh Roddawar (PATH India; early implementer
of Truenat supported by Stop TB Partnership’s TB REACH); Manuela Rehr (Independent
Consultant); Inoussa Zabsonre (Infectious Disease Detection and Surveillance Project); and Jacob
Creswell, Enos Masini, Sreenivas Nair and Suvanand Sahu (Stop TB Partnership). The authors
thank Melissa Sander and Cyrille Mbuli of the Tuberculosis Reference Laboratory Bamenda,
Cameroon for sharing their SOP and maintenance log, and K.S. Sachdeva and Nishant Kumar
of the Government of India’s Central TB Division for providing the experience of the National TB
Elimination Program in adopting and rolling out Truenat.
All reasonable precautions have been taken by the authors 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 the authors be liable for damages arising from its use.
Development of this document was made possible with financial support from the United States
Agency for International Development. The views expressed herein are those of the authors
and do not necessarily reflect those of the U.S. Agency for International Development or the U.S.
Government.
Cover photo images: Molbio Diagnostics, Goa, India

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 4
Practical Guide
to Implementation of Truenat Tests
for the Detection of TB
and Rifampicin Resistance

About this guide

This guide provides practical
guidance to plan for the adoption
and implementation of the
WHO-recommended Truenat™ tests for
the rapid detection of TB and rifampicin
resistance. It includes advice on how
to translate findings from the WHO
policy guidance into an actionable
implementation plan, combined with
operational considerations on use
of the technology and initial field
experience from early implementers.
The guide also compiles as annexes a
number of resources that can be easily
adopted by programmes and sites,
including a sample SOP for running
the assays, checklists for planning and
sites assessments, and job aids. The
guide will be periodically updated to
reflect additional experiences gained
by implementers as well as to add
as annexes useful tools they have
developed that may be easily adopted
by other implementers. Please contact
any of the authoring organizations to
suggest any contributions to this guide.

Target audience
This guide is intended to inform
Ministry of Health officials,
programme managers, testing site
managers, quality assurance unit
personnel, supervisory laboratory
staff and Truenat users at national,
state/provincial and testing site
level, as well as technical partners
and donors.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 5
Abbreviations

CFU colony forming units

DST drug-susceptibility testing
DR-TB drug-resistant tuberculosis
EQA external quality assessment
FIND Foundation for Innovative New Diagnostics
FN false-negative result
FP false-positive result
GLI Global Laboratory Initiative
HIV Human Immunodeficiency Virus
HR human resources
Hr-TB isoniazid-resistant, rifampicin-susceptible TB
INH isoniazid
LAMP loop-mediated isothermal amplification
MDR-TB multidrug-resistant tuberculosis
MOH Ministry of Health
MTBC Mycobacterium tuberculosis complex bacteria
NTP National TB Programme
NTRL National TB Reference Laboratory
PCR polymerase chain reaction
PLHIV people living with HIV/AIDS
PT proficiency testing
QA quality assurance
QC quality control
RIF rifampicin
RR-TB rifampicin-resistant TB
SOP standard operating procedure
SS- sputum smear-negative
SS+ sputum smear-positive
TN true-negative result
TP true-positive result
TB tuberculosis
TWG Technical working group
WHO World Health Organization

6
 Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance

Table of Contents

Part A. Background 9
Performance of the Truenat TB tests 10
How to perform the assay: equipment, reagents and procedures 11
Part B. Implementing the Truenat TB tests 17
Placement of the Truenat TB test in the tiered structure of a laboratory network 17
Test accuracy considerations for selecting which Truenat TB test to use 18
Steps and processes for implementing the Truenat TB tests 20
1. Policies and planning 20
2. Regulatory 21
3. Equipment and site preparation 21
4. Supply chain 24
5. Procedures 25
6. Digital data 26
7. Quality assurance, control and assessment 27
8. Recording and reporting 28
9. Training and competency assessment 28
10. Monitoring and evaluation 29
Part C. Truenat TB Testing Algorithm 31
Algorithm for the use of the Truenat TB tests as the Initial Diagnostic Test for pulmonary TB 32
Decision Tree for the Truenat TB testing algorithm 34
Suggested Reading 40
Annexes 42
Annex 1: Example of an SOP for Truenat MTB Plus using a Truelab Duo analyzer 43
Annex 2: Truenat TB test implementation - high level checklist 54
Annex 3: Budget considerations for implementation 57
Annex 4. Example of a Truenat Preventive Maintenance Log 59
Annex 5. Example roadmap for implementation 60
Annex 6: Checklist to confirm suitability of a testing site 63
Annex 7: Checklist to confirm readiness of a Truenat TB testing site 68
Annex 8: Checklist to confirm readiness of a clinical site 76
Annex 9: Performance indicators for Trueprep and Truenat TB tests 78
Annex 10: Impact measures for Trueprep and Truenat TB tests 80
Annex 11: Molbio job aids for Truenat TB tests 82

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 7
PART

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 8
Background
The Truenat™ (Molbio Diagnostics, Goa, India) testing system uses portable, battery-
operated devices to rapidly detect Mycobacterium tuberculosis complex bacteria
(MTBC) and rifampicin resistance. The system involves two main devices: the Trueprep®
AUTO v2 Universal Cartridge based Sample Prep Device for the automated extraction
and purification of DNA, and the Truelab® Real Time micro PCR Analyzer for performing
real-time polymerase chain reaction (PCR), resulting in the semi-quantitative detection
of MTBC. The system uses room temperature stable reagents (Trueprep™ AUTO Sample
Pre-treatment and Prep kits) and Truenat™ micro PCR chips. The system is designed
to be operated in peripheral laboratories with minimal infrastructure and is therefore
considered to be the first molecular point-of-care test for TB recommended by the
World Health Organization (WHO).
Available Truenat chips for the detection of MTBC with the
Truelab micro PCR Analyzer include the Truenat™ MTB
chip and a more sensitive Truenat™ MTB Plus chip. WHO
recommends use of Truenat MTB or MTB Plus on sputum
specimens as the initial diagnostic test for TB rather than smear
microscopy or culture. The Truenat MTB chip amplifies a portion of
the ribonucleoside-diphosphate reductase gene, nrdB, and has a limit of
detection (LOD) of about 100 colony forming units (CFU)/ml sputum sample.
The Truenat MTB Plus chip amplifies a portion of the nrdZ gene as well as a portion
of the multicopy IS6110 element and has a LOD of about 30 CFU/ml. Extraction of DNA
and detection of MTBC takes approximately one hour.
If the Truenat MTB or MTB Plus test (hereafter ‘Truenat TB’ test refers to either the
Truenat MTB test or the Truenat MTB Plus test) result is positive, an aliquot of the already
extracted DNA may be loaded onto a Truenat™ MTB-RIF Dx chip and analyzed in the
Truelab micro PCR Analyzer. Mutations associated with rifampicin (RIF) resistance are
detected by a probe melt analysis of the real-time PCR products. In addition to the hour
required to extract DNA and detect MTBC, the detection of rifampicin resistance in an
MTBC-positive sample requires approximately an additional hour.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 9
Performance of the Truenat TB tests
Evidence reviewed by WHO on the use of the Truenat TB tests for the detection of
MTBC and RIF resistance was generated through a multicenter prospective clinical
evaluation study in 19 clinical sites (each with a microscopy center attached) and 7
reference laboratories in 4 countries, conducted by the Foundation for Innovative
New Diagnostics (FIND). The study assessed the diagnostic accuracy of the Truenat
TB assays when performed in the intended settings of use (i.e., microscopy centers),
relative to microbiological confirmation (culture) as the reference standard. Compared
to a microbiologic reference standard, key performance characteristics of these tests
among persons with signs and symptoms of pulmonary TB being evaluated at health
care facilities are reflected in Table 1.

Table 1. Diagnostic accuracy of Truenat MTB, MTB Plus and MTB-RIF Dx tests relative to culture, in
microscopy center settings, FIND evaluation study
Sensitivity Sensitivity Sensitivity Specificity
Test
(all patients) (SS+ patients) (SS- patients) (all patients)
Truenat MTB 0.73 0.91 0.37 0.98
Truenat MTB Plus 0.80 0.96 0.46 0.97
Truenat MTB-RIF Dx 0.84 0.88 0.67 0.95

The performance characteristics of the Truenat TB tests were also compared to those
of the Cepheid Xpert MTB/RIF test on the same specimens in reference laboratories as
part of this assessment. The performance characteristics of the Truenat MTB, MTB Plus
and MTB-RIF Dx tests were generally comparable to those of the performance of the
Xpert MTB/RIF test; see Table 2. Specificities of both the Truenat TB tests and the Xpert
MTB/RIF test were reduced at comparable levels in individuals who presented with a
prior history of TB disease; see Table 3.1

Table 2. Diagnostic accuracy of Truenat MTB, MTB Plus and MTB-RIF Dx tests and Xpert MTB/RIF tests
among individuals being evaluated for TB, reference laboratory settings
Sensitivity Sensitivity Sensitivity Specificity
Test
(all patients) (SS+ patients) (SS- patients) (all patients)
Truenat MTB 0.84 0.98 0.45 0.97
Truenat MTB Plus 0.87 0.99 0.55 0.95
Xpert MTB/RIF 0.85 0.99 0.48 0.97
Truenat MTB-RIF Dx 0.82 0.86 0.33 0.98
Xpert MTB/RIF 0.84 0.89 0.33 0.98

Table 3. Effect of prior treatment on specificity of Truenat MTB and MTB Plus tests and Xpert MTB/RIF
test
Test Specificity - No History of TB treatment Specificity - History of TB treatment
Truenat MTB 0.977 (0.964-0.986) 0.922 (0.830-0.966)
Xpert MTB/RIF 0.976 (0.962-0.985) 0.906 (0.810-0.956)
Truenat MTB Plus 0.959 (0.942-0.972) 0.885 (0.782-0.943)
Xpert MTB/RIF 0.975 (0.961-0.984) 0.902 (0.802-0.954)

1 WHO consolidated guidelines on tuberculosis. Module 3: diagnosis – rapid diagnostics for tuberculosis detection.
Web Annex 4. Evidence synthesis and analysis. Geneva: World Health Organization; 2020. https://apps.who.int/iris/
bitstream/handle/10665/334150/9789240010260-eng.pdf

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 10
WHO Recommendations
Based on a review of these data, WHO recommendations for the use of the Truenat
MTB, MTB Plus and MTB-RIF Dx tests are:2
• In adults and children with signs and symptoms of pulmonary TB, the Truenat MTB
or MTB Plus may be used as an initial diagnostic test for TB rather than smear
microscopy or culture
• This recommendation applies to the use of the test with sputum specimens.
There were no data available to assess the accuracy of the test in different
respiratory specimens or with extrapulmonary specimens.
• This recommendation applies to the use of the test with sputum specimens from
HIV-positive persons based on extrapolation of the data on test performance
with smear-negative sputum specimens.
• This recommendation applies to the use of the test with sputum specimens from
children based on extrapolation of the data from adults, although the test is
expected to be less sensitive in children. In the case of children, there were no
data available to assess the accuracy of the test in different specimens, and not
enough indirect evidence to extrapolate for specimens other than sputum.
• In adults and children with signs and symptoms of pulmonary TB and a Truenat
MTB or MTB Plus positive result, Truenat MTB-RIF Dx may be used as the initial
test for detection of rifampicin resistance
• There was a very low certainty of evidence for test accuracy and there is a need
for additional evidence because of the small number of RIF-resistant samples
tested and the limited spectrum of rpoB mutations represented in the tested
samples.

How to perform the assay: equipment, reagents and procedures

The assay involves three main steps using two main components of the Truelab®
Real Time micro PCR system and three reagent packs. All reagents and consumables
required for the test procedures are provided by the manufacturer, with the exception
of personal protective equipment (same level of protection as required for microscopy
or Xpert MTB/RIF), a timer, and hypochlorite-based disinfectant.

Equipment
The main components of the system are the Trueprep AUTO v2 Universal Cartridge
Based Sample Prep Device and the Truelab Real Time micro PCR Analyzer. A printer
is also available.

Trueprep AUTO Universal Truelab micro PCR Analyzer Truelab micro PCR printer
Cartridge Based Sample Prep Available with 1, 2 or 4 chip ports
Device

2 WHO consolidated guidelines on tuberculosis. Module 3: diagnosis – rapid diagnostics for tuberculosis detection.
Geneva: World Health Organization; 2020. https://www.who.int/publications/i/item/who-consolidated-guide-
lines-on-tuberculosis-module-3-diagnosis---rapid-diagnostics-for-tuberculosis-detection

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 11
The number and models of Trueprep Sample Prep Devices and Truelab micro PCR
Analyzers to be procured should be adjusted to match anticipated site-level demand. The
Truelab Analyzer is available with 1 (Uno) chip port as well as with 2 (Duo) or 4 (Quattro)
chip ports which allow for independent testing of multiple samples at once. Table 4
shows the anticipated throughput for the instruments based on the manufacturer’s
calculation and an optimized workflow.2 Early implementers have reported however
that the ‘real-world’ throughput is significantly less (about 75% of the manufacturer’s
calculation), similar to what was seen by early implementers of the Xpert MTB/RIF test.

Table 4. Device combinations and throughput

Throughput per 8-hour Estimated throughput
Devices shift with optimized with “real-world”
work flow3 conditions
1 Trueprep Device + 1 Truelab Analyzer Uno 10-12 specimens 7-9 specimens
1 Trueprep Device + 1 Truelab Analyzer Duo 20-24 specimens 15-18 specimens
2 Trueprep Devices + 1 Truelab Analyzer Quattro 40-48 specimens 30-36 specimens

Procedures
For details on procedures, see a sample SOP in Annex 1. For a sample job aid depicting
the steps visually, see Annex 11. A video demonstrating the sample preparation and DNA
extraction steps. PCR amplification and MTB detection steps can be found online here.

8 - 10 minutes

Step 1.
Sample preparation using the liquefaction and
lysis buffers (Trueprep AUTO MTB Sample Pre-
treatment Pack). Summary procedures:
→ Collect 2-5 ml of sputum sample
• Add 2 drops of liquefaction buffer to sputum
container containing sample
• Close the cap of the container and swirl gently
to allow buffer to mix with sample
• Incubate for 10 minutes at room temperature.
If sample is not pipettable after 10 minutes,
incubate for another 5 minutes with swirling at Trueprep AUTO MTB Sample Pre-
2 minutes intervals treatment Pack
(for 5, 20 or 50 tests)
• Transfer 0.5 ml of liquefied sample to a lysis
buffer bottle using a 1 ml graduated transfer Contents:
pipette • Graduated transfer pipettes (1 ml)
• Add 2 drops of liquefaction buffer to the lysis • Lysis buffer bottles containing 2.5 ml of
buffer bottle buffer
• Swirl gently to mix • Liquefaction buffer bottle
• Wait at least for 3-5 minutes, until sample is
completely liquefied and lysed

4 The manufacturer’s calculation for an optimized workflow assumes that while DNA amplification is being run for a
sample in the Truelab Analyzer, the next sample is being processed and undergoing DNA extraction in the Trueprep
device at the same time.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 12
 22 minutes

Step 2.
Extraction and purification of DNA using the
Trueprep AUTO v2 Universal Cartridge Based
Sample Prep Kit and the Trueprep AUTO v2
Universal Cartridge Based Sample Prep Device.
Summary procedures:
• Transfer entire contents of lysis buffer bottle to
sample chamber of cartridge using 3ml transfer
pipette
Trueprep AUTO v2 Universal Cartridge
• Load cartridge into Trueprep AUTO v2 Sample
Based Sample Prep Kit
Prep Device
(for 5, 25 or 50 tests)
• Automated extraction and purification take
Contents:
20 minutes
• Reagent pack
• After cartridge is automatically ejected from
the Trueprep AUTO v2 Device at the end of the • Transfer pipettes (3 ml)
run, use the pipette from the cartridge pouch to • Cartridge pouches, each containing:
transfer the entire volume of DNA eluate from
the cartridge into an Elute Collection Tube (ECT)4 • Cartridge
• Elute collection tube (ECT)
• Transfer pipette

Pre-treated, lysed sample being

loaded into a cartridge

Loading a cartridge into the

Trueprep AUTO v2 device

4 In a study comparing six nucleic acid extraction technologies, the Trueprep AUTO Device was found to be highly
suitable for use in resource limited settings, based on diagnostic accuracy, sample input and output volumes, total
processing time, user-required manual steps and cost estimates: Beall SG, Cantera J, Diaz MH, Winchell JM, Lillis
L, White H, et al. (2019) Performance and workflow assessment of six nucleic acid extraction technologies for use
in resource limited settings. PLoS ONE 14(4): e0215753. https://doi.org/10.1371/journal.pone.0215753. Research is still
needed however to confirm the ability to use the eluate with other testing methods, including line probe assays and
Next-Generation Sequencing.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 13
 40 minutes

Step 3.
PCR amplification and fluorescent probe-based
detection of MTB using Truenat chips, a Truepet®
6µl Precision Micropipette and the Truelab micro
PCR Analyzer. Summary procedures:
• Prepare the Truelab micro PCR Analyzer for a
TB test by using the flatscreen digital interface:
select “MTB” (or “MTB Plus”, if using an MTB Plus
chip), enter patient details and select “Sputum”
as the sample type
• Use the Truepet 6µl Precision Micropipette Truenat Chip Pack: MTB, MTB Plus or
and a filter barrier tip from the chip pouch to MTB-RIF Dx
pipette out 6µl of purified DNA eluate from the (for 5, 20 or 50 tests)
Elute Collection Tube (ECT) into the microtube
Contents:
containing freeze-dried PCR reagents. Do not
mix. • Individual chip pouches, each
containing:
• Wait 30-60 seconds
• Truenat™ chip
• Use the same tip to transfer 6µl of the
solution from the microtube to a Truenat chip. • Microtube containing freeze-dried
Note: this transfer of solution into the white PCR reagents
reaction well of a chip requires sufficient training • Filter barrier pipette tip
to ensure proficiency
• Load Truenat chip into the Truelab micro PCR
Analyzer
• Automated PCR amplification and fluorescent
probe-based detection takes 35 minutes. An
optional “Plot” view on the digital interface
allows for monitoring test progress in real
time.
• At the end of the run, the “Result” screen for
the Truenat MTB test indicates whether MTB
has been “DETECTED” or “NOT DETECTED”.
For the Truenat MTB test, if MTB is detected,
the estimated number of bacteria in terms of
colony forming units per ml (CFU/ml) in the
original sample is also reported. For the Truenat
MTB Plus test, the MTB DETECTED results are
described as high, medium, low or very low.
• Optional: Press “Print” to send the result via
Bluetooth to the Truelab micro PCR printer. Transferring the solution to a Truenat chip

Truepet 6µl Precision Micropipette

Included in instrument kits, with a free replacement every 6
months to ensure calibration

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 14
 Results screens for a Truenat MTB test and a Truenat MTB Plus test.
Note the red circle indicating that MTB has been detected.

6 0 minutes

Step 4.
PCR amplification and fluorescent probe-based detection of rifampicin resistance
using Truenat MTB-RIF Dx chips, a 6µl Truepet Precision Micropipette and the Truelab
micro PCR Analyzer.
• If MTB is detected in a sample, a portion of the same DNA eluate can be used to test
for rifampicin resistance using a Truenat MTB-RIF Dx chip. Repeat the procedures in
Step 3 above, selecting “MTB RIF” as the test type in the Truelab micro PCR Analyzer.
Automated PCR amplification and fluorescent probe-based detection of rifampicin
resistance takes an additional 60 minutes, resulting in a total runtime of ˜2 hours to
detect MTB and rifampicin resistance.

Truelab® Real Time PCR Workstation

Field Case
The Truenat TB system is also
available as part of a portable system,
which may make it particularly useful
for active case finding activities or
providing testing in hard-to-reach
areas or populations. The Truelab
Real Time PCR Workstation Field
Case contains all the instruments
(Trueprep Device, Truelab Uno Dx
micro PCR Analyzer, micro PCR printer
and Truepet SPA fixed volume (6µl)
Precision micropipette) and reagent
packs (Sample Pre-treatment Pack,
Sample Prep Kit, Truenat TB Chip
Pack) needed to conduct the Truenat
TB tests. As security measures, the
Truelab PCR analyzer includes GPS
tracking (triangulation based on
connection to cell towers when the
device is switched on) and password
protection. The field case can also be
stored in a lockable location when not
in use.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 15
PART

Image:
Practical Guide to Implementation of Truenat Tests for the Detection IRL
of TB New
and Delhi Tuberculosis
Rifampicin Resistance Centre, India NTEP 16
Implementing the
Truenat TB tests
Placement of the Truenat TB test in the tiered structure of a laboratory network
Considerations that guide the placement of any diagnostic test within the existing
laboratory network structure include established targets for expanding patient access
to rapid testing, current and planned testing algorithms, projected testing volumes,
infrastructure requirements, biosafety requirements, trained human resources (HR)
capacity, links to other laboratories for further testing, specimen referral and result
reporting systems and possibility of integration with testing for other diseases.
The Truenat TB assays use automated, battery-operated devices and are designed
to be operated in sites with minimal infrastructure, including peripheral health centers
or mobile vans. Batteries can be recharged using the main electrical system or solar
power. The devices can also be powered directly from the electrical system using the
provided AC/DC adapter and a functioning, well-grounded electrical socket.
In many countries, the intended setting for use will be the peripheral microscopy
laboratory. While countries should consider the advantages and disadvantages of
using Truenat TB tests versus other WHO-approved rapid molecular tests for detection
of MTBC, e.g., the Xpert MTB/RIF test or TB-LAMP test, a country does not necessarily
need to select only one test to meet its needs for rapid testing. Positioning the Truenat TB
test or TB-LAMP test at lower levels within the healthcare network than the Xpert MTB/
RIF test (e.g., at peripheral microscopy centers or clinical laboratories, clinic settings)
can increase patient access to rapid molecular testing for TB, decentralize testing for
rifampicin resistance, reduce the need for patient travel especially in hard-to-reach
areas, and help countries reach targets to replace sputum smear microscopy as an
initial diagnostic test. Note that adoption of any molecular test including Truenat TB
tests does not eliminate the need for microscopy, as microscopy is still required for
monitoring treatment of TB patients; needed microscopy capacity may however be
greatly reduced.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 17
 Multiplexing opportunities
The Truenat testing technology allows for multiplexing opportunities using the
same pieces of equipment for nucleic acid extraction and PCR amplification
and detection. For some testing, the same eluate of extracted nucleic acid may
also be used with various testing chips to detect multiple diseases. As of the date
of publication of this guide, the Truenat tests for Hepatitis C virus ( Truenat™
HCV) and Human Papillomavirus High Risk Types 16, 31 and 18, 45 ( Truenat™
HPV-HR) were undergoing review by the WHO Prequalification Department.
Many other Truenat tests are also available from the manufacturer, including
a test for SARS-CoV-2.

Test accuracy considerations for selecting which Truenat TB test to use

The predictive values of any test vary depending on the prevalence of TB in the population
of patients being tested. Table 5 provides population-level projections of the results of
testing with the Truenat MTB and MTB Plus tests based on sensitivity and specificity
estimates that were extracted from the WHO policy statement5 for the tests. The impact
of false-negative results may be missed opportunities to treat TB. The impact of false-
positive results may be over-treatment of patients without TB.
In deciding whether to select Truenat MTB or MTB Plus, countries will need to consider
the possible trade-offs between higher or lower sensitivity and higher or lower specificity
based on the prevalence of TB, DR-TB and TB/HIV in their country. For example, in a
population with a high prevalence of HIV, a more sensitive test (i.e., Truenat MTB Plus)
may be the more appropriate test because of its increased sensitivity for the detection
of MTBC in smear-negative samples.

Table 5. Predictive values of the Truenat TB tests in populations with different prevalences of TB

Population-level projection of
true and false results
Patient Sensitivity (Se) 2.5% 10% 30%
Truenat Test
population Specificity (Sp) prevalence prevalence prevalence
Se: 0.73 TP: 18/FN: 7 TP: 73/FN: 27 TP: 220/FN: 80
All patients MTB
Sp: 0.98 TN: 957/FP: 18 TN: 884/FP: 16 TN: 687/FP: 13
Se: 0.91 TP: 23/FN: 2 TP: 91/FN: 9 TP: 273/FN: 27
SS+ patients MTB
Sp: N/A
Se: 0.37 TP: 10/FN: 15 TP: 37/FN: 63 TP: 111/FN: 189
SS- patients MTB
Sp: 0.98 TN: 955/FP: 20 TN: 881/FP: 19 TN: 685/FP: 15
Se: 0.80 TP: 20/FN: 5 TP: 80/FN: 20 TP: 239/FN: 61
All patients MTB Plus
Sp: 0.97 TN: 941/FP: 34 TN: 868/FP: 32 TN: 675/FP: 25
Se: 0.96 TP: 24/FN: 1 TP: 96/FN: 4 TP: 288/FN: 12
SS+ patients MTB Plus
Sp: N/A
Se: 0.47 TP: 12/FN: 13 TP: 47/FN: 53 TP: 142/FN: 158
SS- patients MTB Plus
Sp: 0.97 TN: 941/FP: 34 TN: 868/FP: 32 TN: 675/FP: 25
SS+: sputum smear positive; SS-: sputum smear negative; TP: true positive; FN: false negative, TN: true negative, FP: false positive. N/A: not applicable

5 WHO consolidated guidelines on tuberculosis. Module 3: diagnosis – rapid diagnostics for tuberculosis detection. Ge-
neva: World Health Organization; 2020. https://apps.who.int/iris/bitstream/handle/10665/334150/9789240010260-
eng.pdf

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 18
Furthermore the use of chest X-ray as a screening tool to triage who should be tested
with a Truenat TB test may improve the pre-test probability for TB and, thus, the
predictive value of the Truenat TB test; such a strategy also reduces the number of
individuals requiring a Truenat TB test and the associated costs.

Adoption and roll-out of Truenat: Experience of the National TB Elimination

Programme of India
Following a successful validation study supported by the Ministry of Health
and Family Welfare (MoHFW) and conducted under programmatic settings
to evaluate operational feasibility and performance, Truenat has become
recommended in India for the detection of MTB and rifampicin resistance in
sputum samples from people with signs and symptoms of TB. The study was
conducted under the aegis of the Indian Council of Medical Research (ICMR) at
100 microscopy centers under the National TB Elimination Programme (NTEP)
in 50 districts of 10 states, identified in consultation with the Central TB Division
(CTD), MoHFW.
Based on results from 10,878 samples, the study found overall MTB positivity
rates of 13.3% for smear microscopy, 18.1% for Xpert MTB/RIF and 18.8% for
Truenat. Sensitivity of Truenat was found to be higher than Xpert MTB/RIF (84.1%
vs 81.0%, respectively); 79% of discrepant cases were resolved by PCR in favor
of Truenat. Regarding detection of rifampicin resistance, Truenat performed
similarly to Xpert MTB/RIF, without a statistically significant difference.
The study found the following operational advantages of Truenat:
• Cost-effectiveness: low equipment and test costs;
• Patient access: use of Truenat at primary healthcare level eliminates the
need for sample transport for detection of rifampicin resistance during the
first patient visit;
• Time taken for the assay: MTB detection is completed in 35 minutes and the
rifampicin assay is done only as a reflex test;
• Availability of DNA: With Truenat, DNA is available for repeat testing and
any further investigation and quality control purposes.
In order to expand access to molecular testing and detect additional cases in
line with the National Strategic Plan (2017-2025), the India MoHFW decided to
deploy Truenat machines at NTEP sites in a phased manner. In the first phase,
1,512 high workload microscopy centers have been identified for deployment
of 2-chip Truelab Duo equipment; these sites exclude facilities where 1,238
GeneXpert machines have already been deployed. The selected Truenat sites
are expected to test approximately 4,000,000 people with signs and symptoms
of TB annually across the country. The NTEP is currently considering whether
to eliminate smear microscopy as a diagnostic test with further saturation of
Truenat. The Truenat platform is also currently being used for COVID-19 testing.
From experience gained so far in use of Truenat in the field, the NTEP has found
advantages in the Truenat equipment being portable and battery-operated,
having direct connectivity with a mobile interface for data sharing, and not
requiring air conditioning. However, performing a Truenat test does require a
dedicated and skilled technician, and periodic refresher trainings are needed
to maintain adequate technical proficiency.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 19
Steps and processes for implementing the Truenat TB tests
As an initial step in implementing a new diagnostic test, countries should review WHO
policies, guidance and reports as well as any available implementation guides from
WHO6, GLI7, and implementing partners. Particular attention should be paid to WHO
policies and recommendations of the use of the test, the limitations of the test and how
to interpret test results. External technical assistance may be needed; please contact
any of the author organizations for more information on possible support.
Steps to implementing a new diagnostic test such as the Truenat TB tests are described
in detail in the WHO operational handbook on tuberculosis. Module 3.8 The section
below briefly describes aspects of implementation with an emphasis on aspects
specific to the Truenat TB tests. Annex 2 contains an example of a high-level checklist for
implementation of the Truenat TB tests. Note that the implementation steps described
below are not listed in sequential order. Many of the steps may be pursued at the same
time (see Gantt chart in Annex 5). Adopting a new technology requires a conscious
effort at change management to ensure that staff understand and are supported in
making changes. Change management is systematic, structured approach to ensure
that changes are smoothly and efficiently implemented and that the changes lead to
desired benefits.

1. Policies and planning

Establish a technical working group (TWG) to lead

the planning for the implementation, perform
a situational analysis and develop a costed Activities at a glance
operational plan with timelines and milestones. □ Establish a Technical Working Group
The TWG should lead a review of existing national (TWG)
diagnostic algorithms in light of the intended use
and placement of the Truenat TB tests, country □ Review WHO policies and available
epidemiology, existing testing algorithms, sample technical and implementation guides
referral systems, opportunities for multiplexing □ Define immediate and future purposes
by testing for other diseases on the same devices of the test
and other operational considerations, and make
recommendations to the Ministry of Health (MOH) or □ Update national diagnostic algorithm
NTP on proposed revisions to algorithms, specimen and guidelines
transport systems and linkages to referring and □ Perform a situational analysis
referral laboratories. A model algorithm for the
□ Develop a costed operational plan for
use of the Truenat TB tests is described in detail in
implementation
Section C.
The TWG should consider priorities and gaps in their
National Laboratory or TB Laboratory Strategic
Plans to maximize synergies and collaborations with other disease programmes and
improve engagement with the private sector. The TWG should also lead a review of
guidelines for the use of the Truenat TB test results in patient care decisions. Clinical
guidelines should provide clear guidance to clinicians, nurses and health care
professionals on the intended use of the new tests, target patient populations and how
to order the tests and interpret and use the results. The TWG must also consider the
revisions required to test requisition forms, registers and other recording and reporting
forms, and how to integrate the reported data into existing central surveillance systems.

6 https://www.who.int/tb/areas-of-work/laboratory/policy_statements
7 http://stoptb.org/wg/gli
8 WHO operational handbook on tuberculosis. Module 3: diagnosis - rapid diagnostics for tuberculosis detection. Ge-
neva: World Health Organization; 2020. https://www.who.int/publications/i/item/who-operational-handbook-on-tu-
berculosis-module-3-diagnosis---rapid-diagnostics-for-tuberculosis-detection

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 20
A situational analysis of the existing laboratory network and capacities should be
conducted to inform the plans for implementing the new diagnostic test. For most
tests, key elements to be assessed include: regulatory requirements; laboratory and
network infrastructure; existing sample transportation system; staff skills, expertise, and
experience; IT capabilities; diagnostics connectivity; availability and adequacy of SOPs;
supply chain; financial resources; recording and reporting forms, M&E tools and quality
assurance systems. The high-level checklist for implementation of the Truenat TB tests
in Annex 2 may be useful to guide the situational analysis.
The TWG may also be involved in the selection of sites to conduct Truenat TB testing,
although sites are usually selected by the NTP or National TB Reference Laboratory
(NTRL). Site selection should be based on factors such as TB epidemiology, geographic
considerations, testing workload, availability of qualified staff, efficiency of referral
networks and patient access to services. For the prospective testing site, assessments
will be needed of the suitability of the testing sites with respect to physical facilities,
staffing and infrastructure (see Annex 6 for a testing site assessment checklist).
Introducing Truenat TB testing at sites that previously had to refer specimens to other
sites for rapid TB testing and detection of rifampicin resistance will have implications on
the design of specimen referral networks. For resources on specimen referral planning,
implementation and monitoring, see the GLI Specimen Referral Toolkit (http://stoptb.
org/wg/gli/srt.asp).
A detailed, costed, prioritized action plan for phased implementation with targets and
timeline should be developed. Budget considerations are summarized in Annex 3.

2. Regulatory

Countries should work closely with the manufacturer

and its authorized distributors to determine
importation and registration requirements and to Activities at a glance
enable initiation of country verifications, if required.
□ Determine importation requirements
Most countries will need to only conduct small-
scale verification studies (as opposed to large- □ Conduct country verification study, if
scale validations studies) to demonstrate that 1) the required
laboratories can achieve the same performance □ Complete national regulatory processes
characteristics as indicated by the manufacturer
and 2) the method is suitable for its intended use
in the population of patients being tested. For cost
saving and efficiency, a more extensive verification
study may be done at the NTRL using 50-60 samples (e.g., leftover or frozen sputum
samples with known results) that will give a mix of results (e.g., positive and negative
results, different semi-quantitative results). Verification at each testing site with a limited
number of samples (e.g., 4-10 samples from proficiency testing panels) could serve
multiple purposes including verification, demonstration of fitness-for-purpose of the
instruments and competency assessment of users after on-site training.

3. Equipment and site preparation

An essential step of the implementation process is the selection of instruments to fit the
needs of the testing algorithm, anticipated workload and intended setting of use the
Truenat TB tests. Laboratories should select the appropriate number and models of
Truelab micro PCR Analyzers to match anticipated demand (see Table 4 for estimation
of throughput). Note that the ability of a testing site to utilize all of the capacity of
a workstation may not be an important factor for selecting Truenat TB testing sites,

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 21
especially considering that the Truenat TB test is
designed to be placed at peripheral microscopy
centers. Activities at a glance
All instruments must be documented to be “fit for □ Select, procure, install and set-up
purpose” through testing with known positive and/ equipment
or negative material prior to commencing testing
of clinical specimens. □ Verify the instruments are fit for purpose
□ Arrange for preventive maintenance
and servicing
Planning for service and maintenance
□ Prepare site infrastructure for Truenat
Truelab and Trueprep instruments require minimal instruments and testing
preventive maintenance as per the manufacturer.
For an example of a preventive maintenance log □ Ensure a safe and functional testing
that shows routine daily and monthly activities site
as well as ad hoc activities to be conducted as
necessary, see Annex 4.
The manufacturer offers warranty packages for
equipment service and maintenance including
through its growing network of in-country service
providers. Annual warranties that provide
comprehensive service and maintenance including
on-site visits are available through the Stop TB
Partnership's Global Drug Facility (GDF); see
the GDF Diagnostics Catalog for more details.
Countries planning to implement Truenat should
inform the manufacturer in advance to allow the
manufacturer to plan as needed for expansion of
its network of service providers.

Site preparation and assessment of readiness

As part of the situational analysis, each of the
selected testing sites will have been evaluated
for suitability for conducting Truenat TB testing
using a standardized checklist (see Annex 6). Key
infrastructure and operational considerations for
site preparation include:
• Power: In-built batteries in both devices allow for
testing without power for up to 8 hours; devices Suggested specifications of a solar panel
will not allow for a cycle to start if battery power as per the manufacturer:
is too low. Batteries are expected to last five
years. Note that power is still required to charge • Panel: 150 Watts. Dimensions (LxWxH):
batteries: recharging time is approximately 4 1490 x 665 x 35 mm
hours for the micro PCR analyzers, 9 to 10 hours • Battery: 12V 18Ah Lead Acid
for the Trueprep device and 4 to 6 hours for the
micro printer. Use of instruments while charging • Solar Charge Controller + DC to DC
is possible, when the electrical socket is well- boost converter (12V to 170V, 100 Watts)
grounded. The devices are able to operate Controller and converter available from
within the 100-240 voltage range; no additional Molbio; panel, battery and installation to
voltage stabilization is required in settings with be locally sourced.
instabilities or low voltage. Solar panels may
be a solution for settings without any access to
power (see Box on specifications). Power may
also be required to possibly cool the storage
room for test chips; as described below, chips
should be stored at ≤30°C.
Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 22
• Biosafety: Truenat TB tests require the same biosafety precautions as microscopy,
Xpert MTB/RIF or TB-LAMP. A well-ventilated room minimizes the risk of infection
from aerosols during the initial specimen processing steps. None of the steps in
the Truenat TB assay procedures requires use of a biosafety cabinet. Consult the
WHO Tuberculosis Laboratory Biosafety Manual9 for more information on minimum
biosafety precautions including appropriate use of bench space and personal
protective equipment (PPE) when conducting procedures that are considered to
carry a low risk of TB infection. For countries considering use of Truenat for COVID-19
testing, consult the most recent WHO guidance on laboratory biosafety.10
• Waste management: The assay results in a significant amount of plastic waste,
which should be properly disposed of and incinerated as per national regulations.
The manufacturer recommends submerging the used Truenat chips, microtube,
microtube cap, transfer pipette, pipette tips etc. in freshly prepared 0.5% sodium
hypochlorite solution for 30 minutes before disposal as per the standard medical
waste disposal guidelines.
• Room layout: The Truelab instruments should be installed on a flat, stable surface
(preferably non-metallic) away from instruments that may cause vibrations or
electromagnetic interference and out of the path of direct sunlight or close to any
radiating or heating apparatus. A surface with dimensions of 1.2 m by 0.6 m (4-foot
by 2-foot) should be sufficient for the equipment and procedures. Exact dimensions
of equipment are: Truelab Analyzer Uno, 248 mm x 185 mm x 112 mm; Truelab
Analyzer Duo, 240 mm x 242 mm x 159 mm; Truelab Analyzer Quattro, 400 mm x
242 mm x 159 mm; Truelab micro PCR printer, 120.5 mm x 84 mm x 50.5 mm. Three
well-grounded electrical outlets are recommended for operating or charging the
instruments at once.
• Security: All laboratory equipment should be kept in a secure, lockable facility. The
portable Truelab Real Time PCR Workstation Field Case and equipment should be
stored in a secure lockable location when not in use.
• Ambient temperature: Trueprep AUTO v2 Devices and Truelab Analyzers are
designed to be used at ambient temperatures (between 15°C to 40°C). For reference,
the maximum room temperatures for use of GeneXpert (Xpert MTB/RIF) is 30°C,
and for HumaLoop (TB-LAMP) is 40°C.
• Humidity: Trueprep AUTO v2 Devices and Truelab Analyzers can be used in humid
settings (relative humidity: 10-80%)
• Dust: The Truelab Analyzer does not require air intake to allow for the PCR process,
so Truenat use may not be compromised in dusty settings. Nevertheless, the
manufacturer recommends installing the instruments in a dust-free environment
when possible.

Preparing a safe and functional testing site is an important step for many of the other
key implementation steps that follow including training, standardized procedures,
quality monitoring etc. (Sections 4-10), Following completion of all implementation steps
and prior to beginning testing of clinical specimens for patient care decisions, the site
and staff should be evaluated for readiness using a standardized checklist (see Annex
7 for an example).

Clinical site preparation

Clear clinical protocols and guidance will be needed for the selection of patients to
be tested, ordering tests, interpreting test results, and making patient care decisions.

9 https://www.who.int/tb/publications/2012/tb_biosafety/en/
10 https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance-publications

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 23
Clinical staff involved in the diagnosis and management of patients must be sensitized
on updated testing algorithms that incorporate Truenat TB testing and new protocols
and procedures prior to beginning the use of the Truenat TB test with clinical samples
(see Section 9). A checklist for assessing the readiness of a clinical site is in Annex 8.

4. Supply chain

Uninterrupted availability of reagents and

disposables at the testing site is essential to ensure
building of technical capacity in the early stages of Activities at a glance
implementation (avoiding delays between training
and availability of reagents and disposables) and □ Review forecasting, ordering and
to ensure consistent service during routine use. The distribution procedures
shelf life of reagents and their required storage □ Develop procedures to monitor reagent
conditions must be taken into consideration in the quality and shelf-life
design of procurement, distribution and storage
systems. The recommended storage conditions for
the Truenat TB chips is 2°C–30°C and for the Sample
Pre-treatment Pack and Prep Kit is 2°C–40°C. The
shelf-life of all reagents is 2 years under recommended storage conditions. Truenat TB
chips can be stored for up to 6 months at a temperature under 40°C, if conditions do not
allow for storage under 30°C.
New lot testing, also known as lot-to-lot verification ensures the quality of the testing
materials and prevents the use of test kits that generate test failures. The manufacturer
recommends running positive and negative
controls whenever a new shipment of Truenat TB
test kits is received and for each new test kit lot. For pricing of equipment, reagents
Controls can be purchased as part of the Truenat™ and service packages through Stop TB
Positive Control Kit-Panel I. Partnership's Global Drug Facility (GDF),
For countries planning an initial order, the numbers see the GDF Diagnostics Catalog.
of instruments and reagents needed per site will
depend on the expected number of tests to be
performed per day. Table 6 reflects the number of reagents to order for 1 year to match
the anticipated number of tests to be performed and the required instrumentation.

Truelab® micro PCR Analyzer Uno Dx, Duo, Quattro

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 24
Table 6. Number of reagents to order for 1 year of testing based on planned average number of tests
per day (260 working days)

Needed instruments
Average
number of 1 Truelab Analyzer Duo 1 Truelab Analyzer Quattro
1 Truelab Analyzer Uno Dx
tests per day + 1 Trueprep AUTO v2 + 2 Trueprep AUTO v2
+ 1 Trueprep AUTO v2 Device
Device Devices
11 MTB/MTB Plus kits (Pre-
treatment, Prep, Chip kits,
2
50 tests each) 3 MTB-RIF
Dx kits (50 tests each) Consider procuring a lower throughput model, unless
22 MTB/MTB Plus kits testing is expected to increase over time
4
5 MTB-RIF Dx kits
33 MTB/MTB Plus kits
6
7 MTB-RIF Dx kits
44 MTB/MTB Plus kits 44 MTB/MTB Plus kits
8
9 MTB-RIF Dx kits 9 MTB-RIF Dx kits
55 MTB/MTB Plus kits
10
11 MTB-RIF Dx kits
88 MTB/MTB Plus kits 88 MTB/MTB Plus kits
16
18 MTB-RIF Dx kits 18 MTB-RIF Dx kits
132 MTB/MTB Plus kits
24
Procure a higher throughput model to meet testing 27 MTB-RIF Dx kits
needs 175 MTB/MTB Plus kits
32
35 MTB-RIF Dx kits

Note: The number of MTB-RIF Dx kits to order should depend on the anticipated proportion of people tested that will be MTB positive, and therefore in need of a test for rifampicin resistance.
The anticipated number of tests needed should include the number of repeat rifampicin resistance tests that will be required given the need to confirm rifampicin-resistant results among
patients in whom the result is unexpected and for tests that give errors or indeterminate results (see recommended algorithm in Section C). In the table above, an estimate of 20% is used.
Tests are increased by 5% to account for potential wastage, and the resulting number of tests is rounded up to the nearest kit (50 tests per kit). Note that other kit sizes are available (kits with
5 or 20 tests).

5. Procedures

A well-defined, comprehensive set of standard-

operating procedures (SOPs) that addresses all
aspects of the Truenat testing processes from Activities at a glance
sample collection to results reporting is essential.
Some SOPs will rely on the manufacturer’s protocols □ Develop standard operating
included with the commercial kits. Other SOPs may procedures
need to be developed or modified for Truenat TB □ Update clinical procedures and
testing. An example of an SOP for Truenat TB testing strengthen the clinical-laboratory
is in Annex 1. interface
In addition to laboratory-related SOPs, clear
clinical protocols and guidance will be needed for
the selection of patients to be tested, ordering tests,
completing test requisition forms, specimen collection, specimen referral, interpreting
test results, and making patient care decisions. The availability of clear protocols and
sensitization of clinical staff are important to strengthen the clinical-laboratory interface
and ensure that clinical staff at all sites that should use the test, actually order the test.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 25
 6. Digital data

Truelab micro PCR analyzers offer the opportunity

for multiple uses of digital data through in-built
connectivity via SIM card, WIFI or Bluetooth.
Data connectivity allows for automatic and rapid Activities at a glance
transmission of test results to clinicians by email
□ Digital data and diagnostics connectivity
or SMS, as well as potential for transmission of
patient data and results to central electronic □ Develop procedures for data backup,
patient registers or LIMS (Laboratory Information security and confidentiality
Management Systems) via a customized API
(application programming interface). Data can
also be easily exported in CSV format for analyses.
Third-party connectivity softwares (e.g., GxAlert/
Aspect, DataToCare) can be configured to allow
for integration of Truenat data into those platforms.
For a discussion of the benefits and requirements of
diagnostics connectivity, see the GLI Quick Guide to
TB Diagnostics Connectivity Solutions.11 Realtime Truenat Data Dashboard
in India
Truelab analyzers can also be configured to send
data on device performance to the manufacturer’s The National TB Elimination Program
(default configuration) or local servers to allow the (NTEP) in India has worked with the
manufacturer and/or the national TB programme manufacturer to develop a customized
to monitor instrument performance on a real- online dashboard of realtime data on
time basis. This allows for the identification and Truenat use. The dashboard currently
possible prevention of instrument malfunctions includes data on numbers of Truenat and
or breakdowns, detection of user errors and Truenat Rif-Dx tests conducted, stratified by
retraining needs, and monitoring of instrument site, by time period, by age and gender, as
and test utilization across fleets of instruments. well as numbers of TB cases and rifampicin
The analyzers’ software is configured to prevent resistant cases detected. The dashboard
patient data from being sent to the manufacturer’s also provides data on inventory status to
servers. For countries interested in utilizing the facilitate procurement and supply chain
manufacturer’s server and when national data management. The NTEP is working with the
regulations permit, the manufacturer can provide manufacturer to enhance the dashboard
a free password-protected web-based dashboard to include automated analyses to facilitate
to monitor utilization of instruments and test results. monitoring, including information on
The analyzers utilize an Android operating system, malfunctioning machines and numbers
and updates can be provided via the mobile of testing hours lost, trends in machine
network or WIFI utilization, reports on errors that can guide
user action and troubleshooting, and alerts
Truelab analyzers can store up to 20,000 test when specific actions are required.
results in internal memory. Past test results can be
viewed on the device, with search options to find
results of specific patients or referring clinicians. As
with any electronic data system, there is a risk of
loss of testing data. An SOP for regular backing up
of data (e.g., to an external drive) is essential as well as an SOP for data retrieval. There
also must be policies and procedures to ensure the security of laboratory data and
confidentiality of patient data. The manufacturer is working on incorporating a remote
data wipe functionality in case the analyzer is stolen.

11 GLI Quick Guide to TB Diagnostics Connectivity Solutions. Geneva, Global Laboratory Initiative, 2016.
http://www.stoptb.org/wg/gli/assets/documents/gli_connectivity_guide.pdf

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 26
 7. Quality assurance, control and assessment

Implementation of a laboratory quality management

system including a comprehensive system of quality
assurance is needed to ensure the accuracy, reliability Activities at a glance
and reproducibility of Truenat test results. Essential □ Implement a comprehensive quality
elements of a quality assurance system include assurance program
standardized documents, use of Good Molecular
□ Establish and monitor quality controls
Biology practices, competency assessment, internal
quality controls, external quality assessment □ Develop an external quality assessment
(EQA) including proficiency testing (PT) or blinded (EQA) program
rechecking, on-site supervision and continuous □ Monitor and analyze quality indicators
quality improvement processes. Documentation (performance indicators)
of adherence or completion of each of the quality
elements is also needed. A comprehensive
discussion of the essential elements of a quality
assurance system for any rapid TB diagnostic test may be found in the GLI Practical
Guide to Implementing a Quality Assurance System for Xpert MTB/RIF Testing.12
The Truenat TB assays incorporate an internal positive control that undergoes all the
processes that the specimen undergoes, from extraction to amplification, thereby
assessing the validity of the test run from sample to result.
To ensure that the Truelab micro PCR Analyzer is working accurately, the manufacturer
recommends running positive and negative controls (which can be purchased as part
of the Truenat™ Positive Control Kit- Panel I) periodically. The positive and negative
controls can also be used for lot-to-lot verification and assessment of reagents if the
temperature of storage areas falls outside of the recommended ranges.
The Truenat system is a closed amplification system (i.e., the amplified product is sealed in
the chip) and an enzyme system is incorporated in the reaction mix to prevent previously
amplified material from getting re-amplified. Nonetheless, it is recommended that
testing sites perform negative control tests using Trueprep AUTO lysis buffer reagent and
sterile PBS monthly or when contamination is suspected (e.g., unusually high proportion
of specimens with ‘MTB detected’). Swab testing of work surfaces and both the Truelab
and Trueprep machines should be conducted monthly.
Similar to what is recommended for AFB- smear microscopy13, testing of 10–15
specimens per week is recommended to maintain proficiency of staff conducting the
Truenat TB tests. External quality assessment programmes for the Truenat TB tests are
not yet available14, but can be modeled after the proficiency testing programme used
for the Xpert MTB/RIF test. Similarly, monitoring of the quality indicators, also known
as performance indicators, used for the Xpert MTB/RIF test should also be used for
the Truenat TB test. These are described in the GLI Practical Guide to Implementing a
Quality Assurance System for Xpert MTB/RIF Testing.
Laboratories should routinely collect, analyze and report key quality indicators, also
known as performance indicators. An unexpectedly high frequency of errors may indicate
that retraining of technicians is required or that the instruments require servicing; the
Truelab analyzer manual includes a table of possible errors and their interpretations.
Annex 9 contains a list of recommended performance indicators for the Trueprep DNA
isolation; Truenat MTB and MTB Plus tests; and the Truenat MTB-RIF Dx test.

12 Practical Guide to Implementing a Quality Assurance System for Xpert MTB/RIF Testing. Global Laboratory Initiative.
2019. Geneva, Switzerland. http://www.stoptb.org/wg/gli/pgiqas.asp
13 Practical Guide to TB Laboratory Strengthening. Global Laboratory Initiative. 2017. Geneva, Switzerland. http://
www.stoptb.org/wg/gli/assets/documents/GLI_practical_guide.pdf
14 FIND will be piloting a Truenat EQA program in India in the second half of 2020

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 27
 8. Recording and reporting

Depending on the current format of the country’s

requisition (specimen examination request)
form, it may be necessary to make revisions to Activities at a glance
accommodate the Truenat TB tests. Similarly, □ Review and revise request for
laboratory and clinical registers may need to be examination and reporting forms
modified to record the results of the Truenat TB tests
and Truenat MTB-RIF Dx tests. Because the Truenat □ Review and revise laboratory and
TB tests and the Xpert MTB/RIF tests generate the clinical registers
same type of information (e.g., MTB detected or not
detected), the forms and registers being used for
the Xpert MTB/RIF test may be suitable for use with
the Truenat TB tests. As such, generic forms suitable for use with Xpert TB tests, Truenat
TB test or other NAAT are possible, although countries may decide to develop and use
test-specific forms.
The revisions to the test requisition forms, reporting forms and registers should ensure
that the relevant patient and test data are captured and that the essential information
(e.g., test result) is provided in an easy-to-read format to facilitate the interpretation of
the test results and decision making by end users (clinicians or NTP staff).

9. Training and competency assessment

The Truenat TB test procedures require multiple

hands-on steps as well as precision micro-pipetting.
Laboratory technicians should be properly trained Activities at a glance
on all procedures and in Good Molecular Biology □ Develop and implement a training
practices. For laboratory staff conducting the curriculum and strategy
Truenat TB tests, a training curriculum may include:
□ Assess and document the competency
• Background on the scientific basis of Truenat TB of staff
testing
• The Truenat TB testing algorithm
• Use of test requisition forms, results reporting forms and laboratory registers
• Operation of testing instruments
• Standard operating procedures
• Hands-on experience with sample preparation, DNA extraction and chip processing
• Using and evaluating quality controls
• External quality assessment programmes
• Good laboratory practices including equipment maintenance and cleaning, reagent
storage, waste disposal and chemical and biological safety
• Troubleshooting, including interpretation of errors

Early implementers using the test at peripheral level health centers have not cited
significant problems in training microscopists to use the Truenat TB tests. One procedure
that requires special care in training technicians is the micropipetting/dispensing of 6µl
of DNA eluate solution into the well of the Truenat chip: a “steady hand” may also be
an asset. The flatscreen digital interface on the Truelab micro PCR analyzer has not

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 28
been cited as being problematic for technicians without prior experience with similar
technologies.
In addition to the manufacturer-provided manuals, on-site SOPs and other job aids
should be provided as reference materials for technicians. Job aids are available from
the manufacturer; see Annex 11 for examples.
Clinician training or sensitization must be done in parallel with training of laboratory
staff to ensure all clinicians involved in screening and care of TB patients understand
the benefits and limitations of the Truenat TB test, are sensitized to the Truenat TB
testing algorithm, test requisition process, specimen referral procedures, interpretation
of results and necessary follow-up testing. For clinical staff including clinicians, nurses
and other health care workers, training curricula may include:
• Background on the scientific basis of Truenat TB testing
• The diagnostic cascade and Truenat TB testing algorithms
• Guidelines for selection of patients for Truenat TB testing
• Procedures for specimen collection, labelling, storage, packaging and transport
• Use of test requisition forms and clinical registers
• Recording and reporting of results
• Guidelines for interpreting Truenat TB test results for patient care decisions
• Follow-up testing that may be needed

Competency assessments of laboratory technicians should be performed after training

and periodically (e.g., annually) thereafter and should include assessment of the
knowledge and skills for performing each of the tasks involved in a diagnostic test. The
positive and negative controls in the Truenat™ Positive Control Kit-Panel I can be used
for competency testing.

10. Monitoring and evaluation

During the initial planning phase, countries should

establish a set of key indicators and milestones that
can be used to monitor the implementation process. Activities at a glance
The high-level checklist in Annex 2 may be useful □ Monitor implementation of the Truenat
for monitoring implementation. Once launched, TB tests
utilization of the testing services should be tracked.
□ Monitor and evaluate impact of the
For example, the rate of ordering of Truenat TB tests
Truenat TB tests
could be monitored to determine if clinical staff at all
sites that should offer the test, actually order the test.
A framework for monitoring and evaluation of the
impact of a diagnostic test is essential to inform decision-making. See Annex 10 for an
example framework of indicators that can be collected to monitor and evaluate impact.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 29
PART

Image: Tuberculosis Reference Laboratory Bamenda, Cameroon

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 30
Truenat TB Testing
Algorithm
Effective and efficient TB diagnostic algorithms are key components of a diagnostic
cascade which ensures that patients with TB are accurately and rapidly diagnosed
and are promptly placed on appropriate therapy. Both laboratory and clinical staff
must be trained in the diagnostic algorithm to ensure that the testing is optimally used.
The following algorithm and decision tree describe the use of the Truenat TB tests as
the initial diagnostic test for persons being evaluated for having pulmonary TB. This
algorithm is adapted from the model WHO algorithm on use of a rapid molecular
diagnostic as the initial test for TB depicted in the 2020 WHO operational handbook on
tuberculosis, Module 3: Diagnosis (Algorithm 1)15.

15 WHO operational handbook on tuberculosis. Module 3: diagnosis - rapid diagnostics for tuberculosis detection. Ge-
neva: World Health Organization; 2020. https://www.who.int/publications/i/item/who-operational-handbook-on-tu-
berculosis-module-3-diagnosis---rapid-diagnostics-for-tuberculosis-detection

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 31
Algorithm for the use of the Truenat TB tests as the Initial Diagnostic Test for pulmonary TB

Persons to be evaluated for pulmonary TB1

Collect one specimen2, isolate DNA using Trueprep system

• Repeat Trueprep
Isolation failure or error DNA isolated
procedure3

Isolation
DNA isolated • Use 6 ul of DNA eluate for Truenat TB test4
failure

MTB not detected MTB detected5 No result, error, or invalid test

• Conduct additional testing to • Use 6 ul of DNA eluate for Truenat • Repeat Truenat TB testing8
confirm or exclude TB in accordance MTB-RIF Dx test7 • Conduct additional testing to
with national guidelines6 confirm or exclude TB in accordance
• Re-evaluate the patient clinically with national guidelines6
and use clinical judgment for
treatment decisions

RIF resistance not

RIF resistance detected Indeterminate or error
detected

• Treat with first line regimen • Evaluate the patient for • Repeat Truenat MTB-RIF Dx test12
in accordance with national MDR-TB risk factors11 • Follow this algorithm to interpret
guidelines9 • If both tests give indeterminate
• Consider DST for isoniazid if risk of results, treat with first line regimen9
isoniazid mono- or poly-resistance • Promptly conduct additional
is high10 investigations to assess resistance
to RIF13
• Review treatment based on DST
result

Patient at high Patient at low

risk of MDR-TB risk of MDR-TB

• Treat with MDR-TB regimen

in accordance with national • Repeat Truenat TB test15
guidelines14 • Follow this algorithm to interpret
• Conduct additional investigations to • Use the result of the second test for
assess resistance to other drugs in clinical decisions
the regimen13

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 32
1. Persons to be evaluated for pulmonary TB include adults and children with signs or symptoms
suggestive of TB or with a chest X-ray with abnormalities suggestive of pulmonary TB.

2. Programmes may consider collecting two specimens upfront. The first specimen should be promptly
tested using the Truenat test. The second specimen may be used for the additional testing described
in this algorithm. For persons being evaluated for pulmonary TB, sputum is the preferred specimen.

3. Repeat the DNA isolation using the same sputum specimen and a second Trueprep cartridge.
Unsuccessful isolations are typically related to cartridge errors. If both attempts fail, conduct
additional testing to confirm or exclude TB in accordance with national guidelines.

4. Truenat TB test refers to either the Truenat MTB test or MTB Plus test.

5. MTB detected includes ‘detected’ on Truenat MTB and ‘detected high’, ‘moderate’, ‘low’, or ‘very low’
on Truenat MTB Plus.

6. Further investigations for TB may include chest X-ray, additional clinical assessments, clinical
response following treatment with broad-spectrum antimicrobial agents, repeat Truenat TB testing,
testing with other WHO-approved rapid diagnostics (e.g., Xpert MTB/RIF Ultra) or culture.

7. For samples that are positive in the Truenat MTB or MTB Plus test, a portion of the DNA elute used for
the Truenat TB tests and the Truelab Real Time micro PCR Analyzer are used for the Truenat MTB-RIF
Dx test to determine RIF resistance.

8. Repeat the Truenat TB test using the same DNA eluate or a fresh sample. Tests with an Invalid result
should be repeated using a fresh specimen and processed starting with the sample preparation step.
If both tests give indeterminate inconclusive results, conduct additional testing to confirm or exclude
TB in accordance with national guidelines.

9. Patients should be initiated on a first-line regimen according to national guidelines unless the patient
is at very high risk of having MDR-TB. Such patients at very high risk should be initiated on an MDR-
TB regimen.

10. A sample may be sent for molecular (preferred) or phenotypic drug susceptibility testing (DST) for
isoniazid (INH) if there is a high prevalence of INH resistance not associated with RIF resistance
(i.e., INH mono- or poly-resistance) in this setting or if required by national guidelines. Do not delay
initiation of therapy to wait for the results of additional DST.

11. Patients at high risk for MDR-TB include previously treated patients including those who had been
lost to follow-up, relapsed, and failed a treatment regimen; non-converters (smear positive at end
of intensive phase); MDR-TB contacts; and any other MDR-TB risk groups identified in the country.

12. Repeat the Truenat MTB-RIF Dx test using the same DNA eluate or a fresh specimen. Interpret the
result of the repeat test as shown in this algorithm. Use the result of the second test for clinical
decisions. Inconclusive results are usually related to very low numbers of bacilli in the sample such
as samples with the Truenat MTB Plus test result of ‘MTB Detected very low’. Repeat testing with the
same DNA isolate only produces interpretable results in about 30% of retests.

13. Phenotypic (culture and DST) and molecular (line-probe assays, DNA sequencing, high-throughput
assays, etc.) methods are available to evaluate drug resistance. Rapid molecular methods are
preferred.

14. Patients should be promptly initiated on an MDR-TB regimen in accordance with national guidelines
and WHO recommendations.

15. Repeat the Truenat MTB-RIF Dx test using a DNA eluate from a fresh specimen. Interpret the result
of the repeat test as shown in this algorithm. Use the result of the second test for clinical decisions.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 33
Decision Tree for the Truenat TB testing algorithm
Note: ‘Truenat TB test’ designates either the Truenat MTB test or the Truenat MTB Plus.
The individual tests are named when describing test-specific features.

General considerations:
• WHO recommends the use of a Truenat TB test as the initial diagnostic test rather
than microscopy, culture and drug-susceptibility testing (DST) for all persons with
signs and symptoms of TB who are being evaluated for pulmonary TB. This includes
all newly presenting symptomatic persons and may also include patients who are
on treatment or have been previously treated if the patient is being evaluated for
possible RR-TB (e.g., non-converters at the end of the intensive phase of treatment)
or for a new or continuing episode of TB (e.g., relapse cases or previously treated
patients including those who had been lost to follow-up).

• The Truenat TB tests are recommended for use in testing adults and children with
signs and symptoms of pulmonary TB. These tests only detect MTBC. A second test
(Truenat MTB-RIF Dx test) on DNA isolated for the Truenat TB test is conducted to
assess RIF resistance. Considerations for these tests include:
• There is uncertainty about use of this test in PLHIV, because at the time of the
WHO recommendations there were no data available on the performance of
these tests in PLHIV. The indirect data on test performance in smear-negative
patients were used by WHO to extrapolate the recommendation to use in PLHIV.
Because of its increased sensitivity for detecting MTBC in smear-negative
samples, the Truenat MTB Plus test may be the better test to use in populations
with a high prevalence of HIV or for persons known to be HIV-positive.
• In children, sufficient data were available to recommend the use of these tests
with sputum samples only. There were no data on how these tests performed
with other specimens.
• The performance of these tests for the detection of extrapulmonary TB is
unknown.

• The higher sensitivity of the Truenat MTB Plus test compared to the Truenat MTB
test is accompanied by a slight loss of specificity (i.e., an increase in the number of
patients incorrectly identified as having active TB). This is because the Truenat MTB
Plus assay can detect very small numbers of bacilli which may be non-viable or
non-replicating, particularly in patients with a history of TB treatment. Such non-
viable bacteria may also be detected by the Truenat MTB test, albeit less frequently.
The Xpert Ultra test (the more sensitive test) and the Xpert MTB/RIF test (the less
sensitive test) behave similarly.16

• The Truenat TB tests are not recommended as tests to monitor treatment because
the presence of dead bacilli may generate a positive result. Microscopy and culture
should be used in accordance with national guidelines and WHO recommendations.

• The algorithm describes the collection of one initial specimen to be used for
Truenat TB testing and the collection of additional specimens as needed. For
operational issues, programmes may consider collecting two specimens (e.g., two

16 WHO consolidated guidelines on tuberculosis. Module 3: diagnosis – rapid diagnostics for tuberculosis detec-
tion. Geneva: World Health Organization; 2020. https://www.who.int/publications/i/item/who-consolidated-guide-
lines-on-tuberculosis-module-3-diagnosis---rapid-diagnostics-for-tuberculosis-detection

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 34
 spot specimens, or a spot and morning specimens) from each patient routinely
instead of only collecting a second specimen when additional testing is needed. If
so, the first specimen should be promptly tested using a Truenat TB test. The second
specimen may be used for the additional testing described in the algorithm (e.g.,
repeat testing, further DST) or for smear microscopy or culture as a baseline for
treatment monitoring.

• For the Truenat MTB test, if MTB is detected, the estimated number of bacteria in
terms of colony forming units per ml (CFU/ml) in the original sample is also reported.
For the Truenat MTB Plus test, semi-quantitative results are reported as ‘MTB not
detected’; ‘MTB detected (high, medium, low or very low)’, ‘no result’; ‘error’; or
‘invalid’. Each of the semi-quantitative categories of MTB detected is considered as
bacteriological confirmation of TB.

• The Trueprep AUTO v2 system produces approximately 100 microliters of high-

quality DNA.17 While more research is needed, the DNA preparation is likely suitable
for use in many molecular procedures including PCR and line-probe assays. Upon
confirmation of its suitability for other testing, laboratories may be able to ship the
DNA eluate rather than a specimen to other laboratories for additional testing (e.g.,
Step 6b or Step 7c below).

Decision Tree:
1. Collect a good quality specimen and transport it to the testing laboratory. For persons
being evaluated for pulmonary TB, induced or expectorated sputum samples may
be used. Little or no published data are available on the performance of the Truenat
TB test with non-sputum or extrapulmonary TB samples, and WHO did not make
a recommendation regarding the use of the Truenat TB tests with such specimens.

2. Prepare the sample (Trueprep AUTO MTB Sample Pre-treatment Pack) and isolate
DNA using the Trueprep AUTO v2 Universal Cartridge Based Sample Prep Kit and
the Trueprep AUTO v2 Universal Cartridge Based Sample Prep Device.
a. If DNA isolation is unsuccessful, repeat the DNA isolation with another portion
of the prepared sample. Unsuccessful isolations are typically associated with
cartridge errors (damaged cartridge valve, clogged cartridge, a leak in the
cartridge, heater failure, etc.), and the Trueprep AUTO v2 device instrument will
provide an error message identifying the category of error. The manufacturer
states that sputum may be stored in lysis buffer for up to 1 week at 30°C or 3 days
at 40°C with no degradation of DNA. If both tests fail, consider collecting a fresh
specimen and processing as well as conducting additional testing to confirm or
exclude TB in accordance with national guidelines.
b. If DNA isolation is successful, use 6 µL for the Truenat TB test.

3. If the Truenat TB test result is ‘MTB not detected’, re-evaluate the patient and conduct
additional testing in accordance with national guidelines.
a. Further investigations for TB may include chest X-ray, additional clinical
assessments, clinical response following treatment with broad-spectrum

17 Beall SG, Cantera J, Diaz MH, Winchell JM, Lillis L, White H, et al. (2019) Performance and workflow assessment
of six nucleic acid extraction technologies for use in resource limited settings. PLoS ONE 14(4): e0215753. https://doi.
org/10.1371/journal.pone.0215753

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 35
 antimicrobial agents, additional Truenat TB testing, testing with other WHO-
approved rapid diagnostic test (e.g., Xpert MTB/RIF) or culture.
b. Consider the possibility of clinically defined TB (i.e., TB without bacteriological
confirmation). Use clinical judgement for treatment decisions.

4. If the Truenat TB test does not give a result or gives a result of error, the Truenat
TB test should be repeated at the same testing site with a second aliquot of the
remaining DNA or with a fresh sample. Tests with an ‘invalid result’ should to be
repeated using a fresh specimen starting with the sample preparation step. In the
FIND study, repeat testing reduced inconclusive results from 6.2% to 1.7% for the
Truenat MTB test and from 9.2% to 3.9% for the Truenat MTB Plus test.
a. Follow this algorithm if the second test gives a valid result (MTB not detected or
MTB detected).
b. If both tests give inconclusive results, conduct additional testing to confirm or
exclude TB in accordance with national guidelines.

5. If the Truenat TB test result is ‘MTB detected’

a. The result of ‘MTB detected’ should be considered as bacteriological confirmation
of TB.
b. Use 6 µL of the DNA eluate and the Truelab Real Time micro PCR Analyzer for the
Truenat MTB-RIF Dx test to determine RIF resistance.
c. While any delay should be avoided, if for some reason there is a significant delay
in conducting the Truenat MTB-RIF Dx test (expected assay time is 1 hour), the
patient should be initiated on an appropriate regimen using first-line TB drugs
in accordance with national guidelines unless the patient is at very high risk of
having MDR-TB. Such patients should be initiated on an MDR-TB regimen. Note
that in most settings, a history of prior TB treatment is not sufficient to indicate
that the patient is at very high risk of having MDR-TB for the purpose of making
treatment decisions.

6. If the Truenat MTB-RIF Dx test result is ‘RIF resistance not detected’.

a. The patient should be initiated on an appropriate regimen using first-line TB
drugs in accordance with national guidelines unless the patient is at very high
risk of having MDR-TB. Such patients should be initiated on an MDR-TB regimen.
b. Programmes may request additional DST in accordance with national algorithms.
i. Molecular (genotypic) or phenotypic DST for INH is particularly indicated:
* if the patient has been previously treated with INH or is a contact of a
known Hr-TB patient;
* if there is high prevalence of INH resistance that is not associated with
RIF resistance (i.e., INH mono-resistance or poly-resistance, not MDR-
TB) in this setting;
* to determine whether there is a mutation associated with resistance
in the InhA gene (for Ethionamide) to guide decision-making around
continuation of a shorter MDR-TB regimen; or
* if national guidelines require DST for INH
ii. Molecular (genotypic) or phenotypic DST for resistance to RIF may be
requested if the patient is considered to be at risk of having RR-TB despite
the initial Truenat MTB-RIF Dx result. False RIF-susceptible results are rare

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 36
 with WHO-approved rapid diagnostics but have been observed in 1-5% of TB
cases tested in various epidemiologic settings. In contrast, phenotypic DST
for RIF, especially using liquid culture, is associated with a higher proportion
of false-susceptible results.18
7. If the Truenat MTB-RIF Dx test result is ‘RIF resistance detected’, an MDR-TB risk
assessment is needed. Patients at high risk for MDR-TB include previously treated
patients including those who had been lost to follow-up, relapsed or failed a
treatment regimen; non-converters (e.g., smear positive at end of intensive phase
of treatment for drug-susceptible TB); contacts of MDR-TB patients; and any other
MDR-TB risk groups identified in the country. In high MDR-TB burden countries,
every TB patient is considered to be at high risk of having MDR-TB.
a. If the patient is at high risk of having MDR-TB, the RIF-resistant test result is
definitive and the patient should be initiated on a regimen for RR-TB or MDR-TB
in accordance with national guidelines and WHO recommendations19.
b. If the patient is at low risk of having MDR-TB, repeat the Truenat TB test and
MTB-Rif Dx test on a fresh sample.
i. Initiate an MDR-TB regimen in accordance with national guidelines if the
second test also indicates RIF resistance.
ii. Initiate treatment with a first-line regimen in accordance with national
guidelines if the Truenat MTB-RIF Dx result for the second sample is RIF
resistance not detected. While in most situations false-positive RIF-resistant
results due to technical performance of the assay are rare, false-positive
RIF-resistant results due to laboratory or clerical errors may be more likely.
It is assumed that repeat test is performed with more caution and the result
of the second test is correct and the result of the first test may have been due
to a laboratory or clerical error.
c. For all patients with RR-TB or MDR-TB, conduct additional investigations to assess
resistance to the other drugs being used in the treatment regimen. Phenotypic
(culture and DST) and molecular (line-probe assays, DNA sequencing, centralized
molecular assays, etc.) methods are available to evaluate drug resistance.20
Rapid molecular methods are preferred.
i. For MDR-TB regimens that rely on the use fluoroquinolones (e.g., the
WHO recommended 18 to 20-month regimen with BDQ, LZD and a later
generation fluoroquinolone), a sample should be submitted for molecular
testing for fluoroquinolone resistance.
ii. Ideally, a specimen from each patient should be submitted for DST for each
of the drugs used in the regimen for which there is a reliable testing method.
However, treatment initiation should not be delayed to await DST results
(e.g., phenotypic DST can take weeks to months to provide results).
iii. Any positive culture recovered during treatment monitoring that is suggestive
of treatment failure should undergo DST for the drugs used in the treatment
regimen.

18 A. Van Deun et al. Rifampin drug resistance tests for tuberculosis: Challenging the gold standard. J Clin Microbiol.
August 2013; 51 (8): 2633-2640. DOI: http://dx.doi.org/10.1128/JCM.00553-13
19 Consolidated guidelines on drug-resistant tuberculosis treatment. WHO/CDS/TB/2019.7. Geneva: World Health Or-
ganization, 2019. https://www.who.int/tb/publications/2019/consolidated-guidelines-drug-resistant-TB-treatment/
en/
20 Technical manual for drug susceptibility testing of medicines used in the treatment of tuberculosis. WHO/CDS/
TB/2018.24. Geneva: World Health Organization, 2018. https://www.who.int/tb/publications/2018/WHO_technical_
drug_susceptibility_testing/en/

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 37
8. If the Truenat MTB-RIF Dx test gives a result of ‘RIF indeterminate’, the initial result
of ‘MTB detected’ should be considered as bacteriological confirmation of TB. The
patient should be initiated on an appropriate regimen using first-line TB drugs in
accordance with national guidelines unless the patient is at very high risk of having
MDR-TB. Such patients should be initiated on an MDR-TB regimen. Note that in
most settings, a history of prior TB treatment is not sufficient to indicate that the
patient is at very high risk of having MDR-TB for the purpose of making treatment
decisions.
a. The ‘RIF resistance indeterminate’ result with most Truenat MTB-RIF Dx tests is
usually caused by a paucibacillary TB load in the sample. In this case, repeating
the Truenat MTB-RIF Dx test using a DNA eluate from a fresh specimen or another
aliquot of the DNA eluate may be useful. However, in the FIND study, repeat
testing with the same DNA eluate produced interpretable results in only about
30% of retests. Interpret the result of the repeat test as shown in this algorithm.
Use the result of the second test for clinical decisions.
i. If the result of the second Truenat MTB-RIF Dx test is ‘RIF resistance not
detected’ follow step 6. If it is ‘RIF-resistance detected’ follow step 7.
ii. If both tests generate results of ‘RIF resistance indeterminate’, additional
investigations such as culture and phenotypic DST or genotypic DST may be
needed to confirm or exclude resistance to RIF because the indeterminate
result provides no information on resistance.
b. Culture and DST or testing with other rapid diagnostics (e.g., Xpert MTB/RIF, line
probe assays) may be performed for follow up testing to confirm or exclude RIF
resistance.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 38
Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 39
 Suggested
Reading

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 40
• WHO operational handbook on tuberculosis. Module 3: diagnosis - rapid diagnostics
for tuberculosis detection. Geneva: World Health Organization, 2020. https://
www.who.int/publications/i/item/who-operational-handbook-on-tuberculosis-
module-3-diagnosis---rapid-diagnostics-for-tuberculosis-detection
• WHO consolidated guidelines on tuberculosis. Module 3: diagnosis – rapid
diagnostics for tuberculosis detection. Geneva: World Health Organization, 2020.
https://www.who.int/publications/i/item/who-consolidated-guidelines-on-
tuberculosis-module-3-diagnosis---rapid-diagnostics-for-tuberculosis-detection
• GLI Practical Guide to TB Laboratory Strengthening. Geneva: Global Laboratory
Initiative, 2017. http://stoptb.org/wg/gli/gat.asp
• GLI Practical Guide to Implementing a Quality Assurance System for Xpert MTB/RIF
Testing. Geneva: Global Laboratory Initiative, 2019 . http://www.stoptb.org/wg/gli/
pgiqas.asp
• GLI Planning for country transition to Xpert® MTB/RIF Ultra Cartridges. Geneva:
Global Laboratory Initiative, 2017. http://www.stoptb.org/wg/gli/assets/documents/
GLI_ultra.pdf
• WHO Consolidated guidelines on drug-resistant tuberculosis treatment. WHO/
CDS/TB/2019.7. Geneva: World Health Organization, 2019. https://www.who.int/tb/
publications/2019/consolidated-guidelines-drug-resistant-TB-treatment/en/
• GLI Quick Guide to TB Diagnostics Connectivity Solutions. Geneva: Global Laboratory
Initiative, 2016. http://www.stoptb.org/wg/gli/assets/documents/gli_connectivity_
guide.pdf
• WHO Technical manual for drug susceptibility testing of medicines used in
the treatment of tuberculosis. WHO/CDS/TB/2018.24. Geneva: World Health
Organization, 2018. https://www.who.int/tb/publications/2018/WHO_technical_
drug_susceptibility_testing/en/
• Package inserts and manuals for the instruments and reagent kits are available on
the manufacturer’s website. http://www.molbiodiagnostics.com/products-listing.
php.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 41
 Annexes

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 42
Annex 1: Example of an SOP for Truenat™ MTB Plus using a Truelab® Duo
analyzer
Courtesy of the Bamenda TB Reference Laboratory, Cameroon, with minor adaptations
for this guide

Code: TN-01
Standard Operating Procedure Version: 1.0
Name of laboratory (SOP)
Truenat™ MTB Plus assay Date: X
Page: X of X

Contents
1. Scope
2. Definitions and abbreviations
3. Education and training
4. Procedure
4.1 Principle
4.2 Samples
4.3 Equipment and materials
4.4 Reagents and solutions
4.5 Detailed instructions for use
4.6 Reading and interpretation result
4.7 Storage
4.8 Quality control
5. Waste management and other safety precautions
6. Related documents
7. List of changes of SOP
8. Reading and understanding list for documents

Replace
Compiled by Examined by Approved by New version
version
Name
Date
Signature
Laboratory area: Nº of copies: 01 Reason for change:
Date of next review:

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 43
1. Scope
This SOP describes the use of the Truenat™ MTB Plus assay, a chip-based Real
Time Polymerase Chain Reaction (PCR) test, for the semi-quantitative, detection
and diagnosis of Mycobacterium tuberculosis complex bacteria (MTBC) in human
sputum samples.

2. Definitions and abbreviations

• PCR: Polymerase Chain Reaction
• DNA: Deoxyribonucleic Acid
• MTB: Mycobacterium tuberculosis complex
• IPC: Internal Positive Control
• Ct: Cycle threshold
• LCD: Liquid-crystal Display
• ECT: Elute Collection Tube

3. Education and training

All lab staff performing this procedure must have successfully completed training
in the following areas: potential risks to health (symptoms of TB disease and
transmission), precautions to be taken to minimize aerosol formation and prevent
exposure, hygiene requirements, wearing and use of protective equipment and
clothing, handling of potentially infectious materials, prevention of incidents and
steps to be taken by workers in the case of incidents (biohazard incidents, chemical,
electrical, post exposure prophylaxes and fire hazards), good laboratory practice
and good microbiological techniques, organization of work flow from clean to
dirty areas, use of chemical and biological indicators, waste management, use of
equipment (operation, identification of malfunctions, maintenance).
The training is given before a staff member takes up his/her post, strictly supervised,
adapted to take account of new or changed conditions.

4. Procedure
4.1 Principle
The Truenat™ MTB Plus works on the principle of Real Time Polymerase Chain
Reaction. A sputum specimen is first liquefied and lysed using the Trueprep™
AUTO MTB Sample Pre-treatment Pack. The DNA from the sample is then
extracted using the Trueprep™ AUTO v2 Universal Cartridge Based Sample
Prep Device and Trueprep™ AUTO v2 Universal Cartridge Based Sample Prep
Kit. The extracted DNA is then amplified by the Truelab Real Time micro-PCR
analyzer. The Truenat™ MTB Plus chip is placed on the chip tray of the Truelab™
Real Time micro PCR Analyzer. Six (6) µL of the purified DNA is then dispensed
into the reaction well of the Truenat™ MTB Plus chip and the test is started.

4.2 Sample
• Sputum samples

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 44
4.3 Equipment and Materials
4.3.1 Extraction of DNA
• Trueprep AUTO v2 Universal Cartridge Based Sample Prep Device
• Trueprep AUTO MTB Sample Pre-treatment Pack
• Trueprep AUTO v2 Universal Cartridge Based Sample Prep Kit

4.3.2 Amplification of purified DNA

• Truelab Duo Real Time Quantitative micro PCR Analyzer
• Truenat™ MTB Plus micro PCR chip
• Truelab™ micro PCR printer
• Truepet SPA fixed volume (6 µl) Precision micropipette
• DNase and RNase-free pipette tips with filter barrier

4.3.3 Others
• Truenat™ Positive Control Kit - Panel I
• Powder free disposable gloves
• Two waste disposal containers, with lids, containing bleach solutions
• Timer
• Two waste bags
• Microtube Stand
• Cartridge Holder
• Two cryovial racks

4.4 Reagents and Solutions

• Liquefaction buffer
• Lysis buffer
• Conc. bleach and 70% alcohol

4.5 Detailed procedure

4.5.1 Specimen collection
Spot and morning sputum samples are collected from each patient.

4.5.2 Sample storage and transportation

Samples collected for testing on the Truenat instruments should be
stored in the fridge between 2°C to 8°C and transported to the testing
lab. During transportation, the samples should be well parceled in a

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 45
 sample flask/sample transportation box at 2°C to
8°C containing ice packs.

4.5.3 Installation of Trueprep AUTO v2 Universal

Cartridge Based Sample Prep Kit Reagent
Pack
1. Connect a new reagent pack to the Trueprep
Auto v2 device by inserting the Plug-in
Connector into the slot provided (Figure 1).
2. One reagent pack is sufficient to conduct 50 Figure 1
extractions.
3. When the instrument is off,
press the “Power” button to
switch on the Trueprep™
AUTO v2 device. Power in use
indicator LED glows red.
Note:
□ Trueprep™ AUTO v2
device will not let you
begin a run if the battery
is low.
□ For charging or to use
Trueprep™ AUTO v2 on
direct electricity line,
connect the AC Adapter to Procedure to change Trueprep AUTO
the charging port on the v2 Reagent Pack after completion of 50
left side of the back panel extractions
of the device and the a. After completion of 50 extractions, the
other end to the lightning Trueprep AUTO v2 device will prompt
protected distributor. the user to change the reagent pack
4. When new buffer is loaded and reset the buffer count.
using a new Reagent Pack of b. Disconnect the used reagent pack by
color-coded reagent bottles, removing the Plug-in connector.
perform a buffer count reset.
When prompted to change c. Take a new reagent pack. Hold the
the Reagent Pack and reset, reagent pack's connector and remove
press ‘start’ and ‘eject’ the cap.
simultaneously to reset. d. Plug in the connector into the socket of
Trueprep AUTO v2.
4.5.4 Sample Processing procedure e. Press Eject button to open the cartridge
holder and gently pull out the door.
Prior to sample processing on the
Truelab instrument, the sputum f. Insert the Reagent card as shown
sample should be homogenized and gently push to close the cartridge
and pipettable. holder.
1. Put on personal protective g. Press start button.
equipment. h. It will display New Reagent Pack
2. Clean the working surfaces Registered and Ejects the Reagent
with freshly prepared 10% Reset Card.
bleach then with 70% alcohol. i. Remove Reagent Reset card and
proceed with further testing

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 46
3. Clean the instruments with paper towel wet with 70% alcohol.
4. Empty the two liquid waste containers and fill the two waste
containers ½ way with concentrated bleach solution.
5. Open an Trueprep AUTO MTB sample pre-treatment kit, which
contains a graduated 1 mL transfer pipette, lysis buffer bottle
and liquefaction buffer bottle. Bring all refrigerated samples or
reagents to room temperature before using.
6. Arrange the items needed to run a complete batch of 2 samples.
i. Liquefaction buffer bottle
ii. Graduated 1ml and 3ml transfer pipette
iii. Lysis buffer bottle. Visually check for any damage and that
the volume is 2.5ml. If the volume is less than 2.5ml due to
damage, do not use that lysis buffer.
iv. Cartridge pouch and the cartridge holder.
v. Result register
7. Ensure the Trueprep AUTO v2 sample prep device is ON.
8. Arrange the cryovials containing the 0.5ml pipettable sputum in
ascending order of sample numbers on the sample rack.
9. Record sample information (serial number, sample number, date
of test) in the Truenat register.
10. Label lysis buffer bottle with corresponding sample number and
date of extraction.
11. Place labelled lysis buffer bottle in front of the corresponding
sample.
12. Add 2 drops of liquefaction buffer to sputum container containing
1st sample in the batch.
13. Swirl container to allow buffer to mix with sample.
14. Incubate for 10 minutes at room temperature. If sample is not
pipettable after 10 minutes, incubate for another 5 minutes with
swirling at 2 minutes intervals.
15. Transfer 0.5 ml of liquefied sputum sample from the sample
container to the corresponding lysis buffer bottle using the 1ml
graduated transfer pipette provided.
16. Dispose the transfer pipette into the container filled with
concentrated bleach.
17. Add 2 drops of liquefaction buffer into the lysis buffer bottle.
Note: To avoid cross contamination, DO NOT bring the nozzle of
the liquefaction buffer bottle near the sample container.
18. Swirl gently to mix and incubate lysis buffer bottle
at room temperature for 3-5 minutes and observe
to ensure that the sample has completely liquified.
Note: Sputum may be stored in lysis buffer for up to 1 week at 30°C
with no degradation of DNA

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 47
4.5.5 Nucleic acid extraction
19. While incubating the sputum in the lysis buffer, tear open the
cartridge pouch. Each pouch contains a cartridge, an eluate
collection tube (ECT) and a transfer pipette.
20. Take out the cartridge and place on the cartridge stand and keep
the transfer pipette and the ECT in the pouch for later use.
21. Observe the sample chamber and visually confirm that the reddish
IPC is present. If absent, discard that cartridge and take a different
one and report this issue.
22. Label the cartridge pouch with the patient number.
23. Label the cartridge with number of the sample and the date of test.
24. Open the sample chamber of the cartridge by gently pulling the
black cap upward.
25. Transfer ALL the contents of the lysis buffer
bottle (3 ml) into the sample chamber of
the cartridge using the 3ml transfer pipette
provided.
Note: Sample should not be stored inside the
cartridge. Therefore, only load the cartridge
when ready to run the test.
26. Dispose the transfer pipette and the used
lysis buffer bottle into the waste container
filled with concentrated bleach.
Figure 2
27. Recap the cartridge sample chamber with
the black cap and put on a fresh pair of
gloves.
28. Press “Eject” button to eject cartridge holder
(Figure 2).
29. Gently pull out after ejection and insert the
cartridge into the cartridge holder of the
instrument (Figure 3).
Note: The orientation of the cartridge is to
be placed as in Figure 3. Ensure that the site
containing the sample is to the right, when
looking at the front of the instrument.
30. Gently push to close the cartridge holder. You
will hear 2 clicking sounds when the cartridge
is properly loaded in the instrument. Figure 3
Caution: Placing the cartridge in the wrong
orientation will cause the cartridge holder to
remain open, and the cartridge will not be inserted in the device.
31. Press the “Start” button to begin the DNA extraction process.
32. The reagents from the bottles in the back will be automatically
added to the cartridge based on the pre-programmed protocol.
33. While the test is in progress, prepare the next sample in the batch
as in steps 1 to 12.
34. After 18-20 minutes, the device will give a beeping sound indicating
completion of the extraction process with a displayed message.
35. The device will automatically eject the cartridge holder.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 48
 36. IMPORTANT: To avoid the elute from being evaporated by heat
generated during the extraction process, remove the cartridge as
soon as the cartridge holder is ejected.
37. Lift the cartridge up and place on the cartridge stand.
38. Inspect the tray in the cartridge holder for any spilled liquid.
Note: In the event of a spill, dispose of the tray in container filled
with concentrated bleach for 30 minutes and spray the cartridge
holder with 70% isopropyl alcohol. After 5 minutes, place a new tray
in the cartridge holder.
39. Take out the ECT tube from the test pouch, and label the tube using
the sticker provided in the pouch with patient number, age, sex
and date.
40. With the precision and filter barrier pipette tip, pierce the covering
of elute compartment in the cartridge, and aspirate the entire
amount of the elute.
41. Dispense the eluate into the labelled ECT tube and close the ECT
tube cap tightly.
42. Put the ECT tube in the labelled ECT holder.
43. If eluate is not to be amplified immediately, store in a fridge at 4°C
for up to 24 hours or at -20°C for up to 1 year.
44. Dispose the pipette tip and used cartridge into the container filled
with concentrated bleach.
45. Dispose your gloves in dedicated waste container.
46. Load the next sample in the batch into the Trueprep instrument.
47. Transfer the ECT tube containing the eluate to the Truelab micro
PCR analyzer for amplification.

4.5.6 Nucleic acid amplification

Important note: The Truelab Duo device can run a maximum of 2 tests
at a time.
1. Clean the working surfaces with 10% beach followed by 70% alcohol.
2. Clean the instruments with paper towel wet with 70% alcohol.
3. Put on a fresh pair of gloves
4. Before starting the amplification process for a maximum of 2
samples, ensure you have the following arranged on your work
station:
i. PCR chip set which contains the chip, micro tube with freeze
dried PCR reagent micropipette tip and a blue desiccant
ii. The white, fixed-volume micropipette
iii. The microtube stands to hold the microtubes
iv. ECT tubes containing the extracted DNA
5. Switch on the Truelab Duo device by pressing the red button in the
back left corner for 2 seconds. The power/ in use indicator will glow
green. In 30-50 seconds, the boot screen will appear followed by
the home screen. Ignore the insert sim pop up message.
6. Click on Molbio and select a user name from the drop-down menu.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 49
7. Tap on the password text box to pull up the on-screen keyboard.
8. Enter password and press “sign in “to log in’’ the selected user.
9. Inspect the two-chip bay to ensure there are no used chip in the
instrument by clicking on each bay. Select ‘open/close’ to close the
bay.
10. Choose any of the test bay 1 or 2 and select MTB plus.
11. A pop up will appear. Confirm selection by pressing “PROCEED”.
12. Enter the information required (referred by, patient ID, patient
name, age and gender).
13. Select the sample type SPUTUM.
14. Press ‘start test’ and the cartridge bay selected will automatically
open.
Note: When the “please load sample” prompt appears, DO NOT
press “YES” until the chip is loaded.
15. Tear open the chip pouch.
16. Pull out the desiccant pouch and confirm that it is blue.
Note: If the desiccant pouch is white or pink in color, do not use the
contents of that pouch. Take another one. (This means the chip has
been exposed to excess moisture.)
17. Pull out the chip enclosed in the chip sleeve.
Note:
-NEVER touch the white reagent well
-Minimize the exposure of the chip to light by preparing and
running the test immediately after opening
18. Label the chip with the participant ID using a marker at the space
provided on the back side of the chip. Avoid writing on the QR
code.
19. Place the chip on the tray by aligning the registration holes with
the tray pins.
Note: The white reaction well should face upward and away from
the device
20. Take out the microtube containing the freeze-dried PCR reagent
and remove the lid
21. Place the microtube containing the freeze-dried PCR reagents in
the microtube stand provided.
22. Inspect to be sure the PCR reagent is at the bottom of the tube.
23. Take the 6µl precision micropipette and attach the micropipette
filter barrier tip enclosed in the chip pouch.
24. Pipette out 6µl of purified DNA from the ECT and put into the
microtube. Confirm visually that the pipetted solution is 6µl.
Note: DO NOT mix it by tapping, shaking or by reverse pipetting.
25. Do not dispose the pipette tip. Keep it attached to the fixed volume
micropipette but ensure the tip is retain into the sleeve.
26. Cap the remaining extracted DNA in the ECT tube and move it one
step behind.
27. Allow mixture of eluate and PCR reagent to stand for 30 to 60
seconds to get a clear solution.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 50
 28. Pipette out 6µl of treated DNA from the microtube and put into the
reaction well of the chip.
Note: DO NOT spill eluate on the outsides of the well. Take care not
to scratch the internal well surface
29. On the instrument screen, select ‘‘YES’’ when the “please load
sample’’ pops up.
30. Chip tray will close automatically and reaction will start.
31. Dispose the microtube, microtip and used gloves into waste
container containing concentrated bleach.
32. Truelab will verify chip and commence test.
Note:
-If desired, press ‘’PLOT’’ to view test progress in real time. No user
intervention or interpretation is required. Amplification profile is
visible in ‘’optical’’ view.
-DO NOT touch or shake the instrument while the test is in progress
33. Follow steps 9-29 to load the second sample in the batch but this
time selecting the other test bay.
34. At the end of the run (35 minutes), press ‘’RESULT’’ to go to the
result screen.
35. Possible result:
i. Valid/invalid and
ii. MTB result Detected/not detected/Errors
36. Record the result in the lab register.
37. If result is MTB detected, test same eluate for rifampin resistance
using MTB-RIF chip. In this case, select the MTB Rif assay.
38. If test gives an invalid or error result, record result and repeat the
amplification using the same extracted DNA and a different chip. If
valid result cannot still be obtained, run test with a different sample
and eluate.
39. Press ‘’print’’ to print results.
Note: Test results are automatically stored and can be retrieved
any time later.
40. Lift the chip from the instrument tray and directly dispose into
waste container filled with concentrated bleach.
Caution! DO NOT put the chip down on table or any other place. Do
not discard chip anywhere else. The amplicons may contaminate
another test and give a false positive result.
41. Dispose your gloves in the dedicated waste container.
42. Switch off the Truelab analyzer and Trueprep device at the end
day.
43. Cover each of these instruments with the instrument plastic covers.
44. Clean the surfaces using bleach, at the end of the day

4.6 Reading and interpretation results

At the end of the test run, the result screen will display “DETECTED” for Positive
result or “NOT DETECTED” for Negative results. The result screen will also
display the MTB load as “HIGH”, “MEDIUM”, “LOW” or “VERY LOW” for positive

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 51
 specimens. The result screen also displays the validity of the test run as “VALID”
or “INVALID”.
1. Two amplification curves are displayed on the Truelab Analyzer
screen when optical plot is selected to indicate the progress of the
test.
2. The target and the internal positive control (IPC)* curves will take
a steep, exponential path when the fluorescence crosses the
threshold value in case of POSITIVE samples.
3. The target curve will remain horizontal throughout the test duration
and the IPC curve will take an exponential path in case of NEGATIVE
samples.
4. If the IPC curve remains horizontal in a negative sample, the test is
considered as INVALID. This may be due to inhibitors in the sample
or issues with the reagents used. Tests with an Invalid result should
to be repeated using a fresh specimen and processed starting
with the sample preparation step.
Note: IPC will co-amplify in most positive cases. In some specimens
having a high target load, the IPC may not amplify, however the test
run is still considered valid.

4.7 Storage of DNA

Store the rest of the eluate after extraction and amplification in the ECT tube
at -20oC.

4.8 Quality control

To ensure that the Truelab Analyzer is working accurately, positive and
negative controls may be run one time per month. The Truenat Positive Control
Kit- Panel I containing Positive Control and Negative Control may be used in
running these controls; alternatively, PBS may be used as a negative control
and a known positive sample (e.g., from culture) as a positive control.
• Quality control will also be performed if the temperature of the storage
area falls outside of 2-30oC.
Acceptable criteria: The result will be acceptable if the positive controls give
positive results while negative controls give negative results.
Corrective action: Repeat the control and/or inform the Lab Director.
Documentation: Controls should be recorded in the result register.

5. Waste management and other safety precautions

• Submerge the used cartridges, replaceable trays, reagent bottles and other
consumables in freshly prepared 10% bleach for 30 minutes before disposal as
per the standard medical waste disposal guidelines.
• Samples and reagents of human origin, as well as contaminated materials,
disposables, neutralized acids and other waste materials must be discarded
after decontamination by immersion in a freshly prepared 0.5% of sodium
hypochlorite for 30 minutes (1 volume of 5% sodium hypochlorite for 10 volumes
of contaminated fluid or water).
• Do not autoclave materials or solutions containing bleach.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 52
 • Chemicals should be handled in accordance with good laboratory practice and
disposed according to the Biosafety Manual.
• Check all packages before using the kit. Damage to the packaging does not
prevent the contents of the kit from being used. However, if the outer packaging
is damaged the user must confirm that individual components of the kit are
intact before using them.
• Do not perform the test in the presence of reactive vapours (e.g., from sodium
hypochlorite, acids, alkalis or aldehydes) or dust.
• While retrieving the Truenat™ MTB micro PCR chip and the DNase & RNase free
pipette tip from the pouch, ensure that neither bare hands nor gloves that have
been used for previous tests run are used.
• All pipetting steps should be performed with utmost care and accuracy in order
to prevent cross-contamination between reagents and samples which may
lead to invalid results.

6. References
• Truenat MTB Plus package insert version 5.
• The Trueprep™ AUTO v2 Universal Cartridge Based Sample Prep Device user
manual.
• TBRL Bamenda Biosafety manual, Version 4.0, section 10.
• Truenat™-A Point-of-care Real Time PCR Test for Tuberculosis, video by Molbio
available at https://youtu.be/ydR2I5S2v3c

7. List of changes to SOP

Date Change(s) made Page number Initial(s)

8. Reading and understanding list for documents

I acknowledge having read and understood this SOP

Date Change(s) made Page number

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 53
Annex 2: Truenat TB test implementation - high level checklist

1. Policy and planning

• Have roles and responsibilities for coordinating the Truenat adoption process
been clearly defined?
• Which national guidelines, policies and other materials will need to be updated
to include the Truenat TB tests (e.g., NTP policies and guidelines, diagnostic
algorithm, TB-HIV policies and guidelines, case definitions, etc.)?
• Has a stakeholder mapping process been conducted, including all key internal
(within government) and external stakeholders (local and international)?
• What support can partners provide for the implementation process?
• Has the intended use and setting of the Truenat TB tests been decided? Have
projections been made for the number of samples to be tested per year or per
site?
• Has a costed implementation plan been developed?
• Have adequate financial resources for capital investments, implementation and
projected on-going costs been secured?

2. Regulatory
• What are the importation requirements for instruments, reagents and supplies
for the Truenat TB tests?
• What is the regulatory process required for the Truenat TB tests?
• Is country verification of the Truenat TB tests needed for regulatory approval?
• If so, what type of protocol and number of samples are required? Timeline?
Where will verification studies be conducted?
• Is the designated authority (NTP, procurement agency or partners) engaged
with manufacturers to support regulatory processes?

3. Site readiness
• Are adequate laboratory facilities, space and infrastructure available?
• Do facilities, equipment, policies and practices meet TB biosafety standards?
• Are appropriately trained and competent staff available to conduct the Truenat
TB tests?
• Are adequate specimen referral and results reporting systems available?

4. Procurement and supply chain

• Which partners procure instruments and consumables?
• Has the manufacturer engaged a country-level distributor or other service
provider to support implementation, equipment maintenance (warranties or
service contracts) and commodity importation?
• Is a procurement system available to ensure the availability of reagents and
supplies that takes into account procurement times, consumption rates and
shelf-life of reagents?
• What is the planned procurement by MOH and partners for this year?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 54
5. Procedures
• Which SOPs, forms and registers will need to be updated or developed?

6. Data
• What applications of electronic data connectivity will be pursued? (e.g., results
to clinicians by SMS/email, results to electronic patient registers or other data
systems, results to third party connectivity systems, remote monitoring of data
on manufacturer-provided dashboard)
• What modifications or upgrades to existing data systems (e.g., electronic patient
registers, laboratory information management systems, third party connectivity
systems) are required to make use of Truenat data?
• Are procedures in place to define data sharing protocols and ensure the
confidentiality?

7. Quality assurance
• Are the essential elements of a quality assurance system in place at the testing
site?
• Are protocols in place to conduct and document quality checks of each step of
the Truenat TB test process and ensure the use of positive and negative controls?
• Is an external quality assessment programme in place?
• Which partners can assist with proficiency testing, supervisory visits and re-
checking of samples?
• Have quality (performance) indicators been defined and appropriate data
collection tools developed?

8. Recording and reporting

• Is a national request form in use? If no, review all request forms being used to
Truenat TB tests and revise as needed.
• Is revision of the current request for examination form required for introduction
of Truenat TB tests?
• Is revision of reporting forms needed?
• Is a revision of laboratory or clinical registers needed?
• If an electronic laboratory information system is in use, what updates will be
required?
• If an electronic recording and reporting system is in place, what updates will be
required? If not, what system will be used to inform clinicians promptly about
Truenat TB test results?

9. Training
• Is a national approved training curriculum available?
• Who is responsible and what is the process for updating training materials for
laboratory and clinical staff?
• Is the approved curriculum used for all trainings, including those delivered by
partners?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 55
 • Are standard procedures used to assess and document the competence of all staff involved in
Truenat TB testing?

10. Monitoring the transition

• What changes to M&E tools and processes would be required to enable monitoring of additional
indicators (i.e., progress indicators, laboratory indicators and clinical impact indicators)?
• What support can partners provide in monitoring of new algorithms and adherence to guidelines
at sites?
• What support can partners provide for operational research to monitor the impact of Truenat TB
testing?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 56
Annex 3: Budget considerations for implementation

Budgetary considerations
• Workshop for stakeholder engagement and planning
• Technical workshop for guideline and algorithm update
• Situational analysis cost – HR, travel and report writing
Policy and Planning • Printing and distribution costs for revised guidelines and
algorithms
• Development of a costed operational plan
• Cost of external technical assistance, if needed
• Regulatory submission costs, if applicable (borne by
manufacturer)
Regulatory • Local travel costs to regulatory authority
• Importation processes and costs
• Verification study – samples, reagents, HR
• Workshop and HR for the development of SOPs
• Printing and dissemination of revised procedures
Procedures • Development, printing and dissemination of revised clinical
protocols and guidance for the selection of patients to be tested,
ordering tests, interpreting test results, and making patient care
decisions
• Costs of assessing site readiness – travel, HR
Truenat TB testing laboratory
• Costs of upgrading laboratory facilities and infrastructure
• Purchase (or lease) of instruments and ancillary equipment
• Delivery and importation costs
Purchase and installation of • Installation by manufacturer or authorized service provider
Truelab instruments • Training
• Extended warranty or service contract
• Costs of routine preventive and annual maintenance
• Costs of data transmission (e.g., 3G data transfer, SMS)
• HR and costs of remote monitoring

Diagnostics connectivity • Cost of technical assistance

• Optional: purchase or acquisition (e.g., subscription fee) of
third-party diagnostics connectivity solution to complement
manufacturer’s system
• Workshop for stakeholders involved in procurement planning
• Cost of maintaining centralized stores and costs of distribution
• Material cost per test, including but not limited to test reagents,
Procurement and supply chain consumables, sample collection items, printing paper, etc.
Additional equipment costs that include additional equipment
requirements (printer, computer, printer cartridges), shipping
and courier costs
• Costs of new lot testing

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 57
 Budgetary considerations
• Workshop and HR to update recording and reporting forms,
Recording and reporting registers
• Printing and distribution of updated materials
• Workshop and HR to update training packages
• Training of training workshop, on-site trainings/sensitization
Training meetings for laboratory technicians and for clinicians
• Printing and distribution of updated training manuals and
clinician sensitization materials
• Preparation and regular review of all testing and quality
assurance documents (SOPs, checklists, etc.) based on national
requirements
• Cost of conducting quality controls (e.g., testing known positives
or negatives)
• Costs of HR for routinely collecting and analysing quality
indicators
• Costs of conducting on-site visits – travel, HR, preparation of
Quality assurance, control and checklists and reports
assessment • Costs associated with hosting an on-site visit and preparation of
documents
• Costs associated with providing PT panels and overseeing PT,
reporting results and corrective actions and costs associated
with testing PT panels at each site
• Costs associated with re-testing of samples at a higher-level
laboratory (e.g., shipment of samples, testing, reporting, etc.), if
applicable
• Costs associated with annual competency testing of staff
• Meetings to update M&E system and regular meetings to review
impact of transition and re-plan
Monitoring
• M&E refresher training
• Operational research study to measure clinical impact
• Consumables
• Human resources
• Equipment maintenance and servicing
Annual on-going costs
• Digital data systems and connectivity solutions
• Specimen referral and results reporting system
• External quality assessment

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 58
Annex 4. Example of a Truenat Preventive Maintenance Log
(Courtesy of the Bamenda TB Reference Laboratory, Cameroon)

Version: 1.1
Truelab and Trueprep maintenance log
Date:
Compiled by:            Reviewed by:            Approved by:
Month:
Days of the month
Maintenance 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
activity
Daily maintenance
Clean work area
Discard used
chips and
cartridges
Monthly maintenance
Disinfect Date:
instrument Initials:
surfaces
Clean Truelab Date:

bays Initials:

Temperature Date:

calibration Initials:

Verification of Date:
the fixed 6µl Initials:
pipette
As necessary
Flush protocol
for the Trueprep
instrument
Spillage tray or
linear motion
guide tray
replacement
Slider glass
replacement -
indicate bay

Notes:
Indicate completion of an activity by writing your initials in the corresponding date box.
• Slider glass should be replaced after running at most 50 tests and/or when related errors occur.
• Temperature calibration of the Truelab should be done on a monthly basis and/or when error related to temperature occurs
and/or when temperature curve is abnormal, i.e having blips. Normal values: 3.39-3.42.
• Flush protocol for the Trueprep instrument should be done if no test will be run for the next 10 days and/or when errors
relating to extraction process occur.
• Spillage tray and linear motion guide trays should be replaced when there is sample spillage on the trays during extraction
or when cross contamination is suspected.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 59
Annex 5. Example roadmap for implementation

Preparation,
Planning and
installation and Testing
Assessing
Training

Policy and planning

Establish and define roles and

responsibilities of a TWG
Review WHO policies and any available
technical or implementation guide
Define immediate and future purposes
of Truenat TB testing
Update national diagnostic algorithm
and guidelines

Perform a situational analysis

Identify laboratories that will perform

Truenat TB testing

Assess Truenat TB testing site suitability

Develop a costed operational plan for

implementation

Regulatory

Determine importation requirements

Conduct country verification if required

Complete national regulatory processes

Equipment

Equipment selection

Procure and Install equipment

Perform necessary upgrades to testing

site infrastructure

Assess site readiness

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 60
 Preparation,
Planning and
installation and Testing
Assessing
Training

Supplies

Review forecasting, ordering and

distribution procedures
Develop procedures to monitor reagent
quality and shelf-life

Procedures

Develop standard operating procedures

Update clinical procedures and

strengthen the clinical-laboratory
interface

Data

Plan for use of digital data and

diagnostics connectivity
Acquire and install any necessary
hardware and/or SIM cards to allow for
diagnostics connectivity

Training in use of digital data

Establish data monitoring dashboards

and systems
Develop procedures for data backup,
security and confidentiality

Quality assurance

Strengthen quality assurance

programmes in Truenat TB testing
laboratories
Develop policies and procedures for
quality control of Truenat TB testing
Develop and implement an external
quality assessment (EQA) programme
Develop tools and system for routine
monitoring of quality indicators

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 61
 Preparation,
Planning and
installation and Testing
Assessing
Training

Recording and reporting

Revise and disseminate test requisition,

recording and reporting forms
Modify and disseminate laboratory and
clinical registers as needed

Training and competency assessment

Develop training curricula for different

staff cadres

Conduct training

Conduct competency assessments after

training and periodically thereafter

Monitoring and evaluation

Develop key indicators or milestones for

implementation

Monitoring implementation

Develop a framework to monitor and

evaluate impact

Monitor and evaluate impact

Begin testing

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 62
Annex 6: Checklist to confirm suitability of a testing site
This checklist is used to assess the suitability of a laboratory to conduct Truenat TB testing. The checklist
focuses on the key operational and environmental requirements for conducting Truenat TB testing as well
as basic laboratory procedures for ensuring quality. Once selected, preparations for Truenat TB testing
should be implemented as described in the Stop TB / USAID / GLI Practical Guide to Implementation
of Truenat Tests for the Detection of TB and Rifampicin Resistance. A separate checklist is available to
assess the readiness of a site to begin testing clinical samples for patient care. Most questions are to be
answered with a ‘Yes’, ‘No’ or ‘Partial’. Space is provided to provide comments for the responses for each
question.

Name of laboratory

Location of site (City/town, District, State)

( ) National
Laboratory Level ( ) Intermediate (supervisory)
( ) Peripheral
( ) Collect specimens. Average number per
How does the laboratory receive specimens for month: ____________
testing? ( ) Receive referred specimens. Average
number per month: ____________
TB tests performed at this site Average number of tests
(check all that apply) conducted per month

( ) AFB Smear-microscopy for diagnosis ( )

( ) Xpert MTB/RIF or
( )
( ) Xpert MTB/RIF Ultra

( ) Culture ( )

( ) DST (FL or SL) ( )

( ) LPA (FL or SL) ( )

( ) LF-LAM ( )

( ) Other: ____________ ( )

Anticipated monthly number of Truenat TB tests

based on planned algorithm and number of
( )
specimens expected to be received including
from referring labs
Persons interviewed
Name Position and contact details

Assessor name:
Assessor contact details:
Date of assessment:

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 63
 Partial
Yes
Comments

No
Human resources
a. Are a sufficient number of qualified staff
available for the anticipated Truenat TB
testing workload?
b. Is a staff member (part-time or full-time)
available to oversee the implementation of
Truenat TB testing?

A safe and functional testing site

a. Does the laboratory have a lockable door and
secure windows
b. Is there adequate space for receiving and
processing specimens?
c. Is there adequate amount and quality of
bench space for the Truelab instruments and
ancillary equipment?
• flat, stable surfaces?
• vibration-free or vibration-dampened?
• away from instruments that cause
electromagnetic interference? (note:
Molbio recommends placing micro PCR
instruments at least one meter away from
other instruments or equipment)
• out of direct sunlight?
• away from to any radiating or heating
apparatus?
d. Does the testing site provide sufficient
ventilation and biosafety for Truenat TB
testing procedures?
e. Does the testing site ensure an optimal
working temperature (15°C to 40°C) and
environment (humidity [10-80%], dust-free) for
the Truelab instruments?
f. Are existing workstations clean, free of clutter,
and organized for efficient operation?
g. Is available electricity adequate for Truenat
TB testing and battery charging? Note: For
sites that would rely on Truelab equipment in-
built batteries, up to 10 hours of electricity is
required to recharge the batteries, in order to
be able to run tests for 8 hours.
h. Would a solar panel system need to be
procured to provide the required electricity?
i. Are there 3 available sockets on the lab bench
for the Trueprep and Truelab instruments?
j. Is there sufficient, secured, and organized
storage space for chips (2°C–30°C) and
reagent kits (2°C–40°C)?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 64
 Partial
Yes
Comments

No
k. Is there documented monitoring of
environmental temperatures at the testing
and storage areas?
l. Does the laboratory have a refrigerator for
storing sputum samples?
m. Does the testing site use appropriate
disinfectants and are they prepared correctly?
n. Does the testing site use appropriate
disinfectants and are they prepared correctly?
o. Is suitable personal protective equipment
(PPE) provided at the testing site and are staff
trained in its correct use?
p. Does the testing site segregate waste and
dispose of it by incineration or as per national
regulations or guidelines?
q. Does the testing site have adequate capacity
for safely and properly disposing of the
anticipated significant amount of plastic
waste generated by Truenat testing?

Equipment service and maintenance

a. Are routine maintenance (daily, weekly, and
monthly) procedures performed and recorded
for existing instruments?
b. Is there an SOP in place to obtain repairs or
service for existing instruments?

Supply chain
a. Would the current supply chain be adequate
to continuously supply Truenat reagents?
b. How long is the average lead time from
ordering to receiving stock
c. Does the testing site monitor consumption of
consumables?
d. Are testing supplies available, in-date, labeled
with receive date, organized and stored at
recommended conditions?
e. Is quality control testing performed on new
lots of reagents prior to their use for testing
patient samples?
f. Are supplies inventoried (physical count) at
least monthly?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 65
 Partial
Yes
Comments

No
Procedures
a. Are all of the needed standardized forms
related to Truenat TB testing readily accessible
to all staff?
• TB test requisition form
• Sample collection and transport forms
• Laboratory register
• Instrument maintenance log
• Stock cards
• Temperature monitoring records
• Result reporting form

b. Are the following standard operating

procedures (SOPs) approved and accessible
at the testing site?
• Specimen collection
• Specimen processing and storage
• Specimen referral
• Specimen receipt and accessioning
• Laboratory testing
• Recording and reporting
• External quality assessment
• Quality indicator monitoring and data
analysis
• Waste management
• Spill management
c. Is there evidence that all SOPs, documents
and forms have been read by the personnel?

Digital data and diagnostics connectivity

a. Is an electronic laboratory management
system in use? If yes, which?
b. Does the testing site have WIFI or at least 3G
coverage, to allow for data transfer?
c. Are any diagnostics connectivity solutions in
use? If yes, which?
d. Are procedures in place that ensure the
confidentiality of patient information?
e. Is suitable secure storage available for
laboratory test data?

Quality Assurance
a. Are protocols in place that ensure the routine
use of positive and negative controls in
laboratory testing?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 66
 Partial
Yes
Comments

No
b. Is an external quality assessment programme
in place? If yes, describe
• Proficiency testing (PT)?
• Re-checking of samples?
• On-site supervisory visits?
c. Are laboratory statistics and performance
indicators are currently reported to the TB
programme and how?

Recording and reporting

a. How are results currently returned to
clinicians?
b. If an electronic laboratory information system
is in use, can Truenat TB testing results be
entered into it?
c. If an electronic recording and reporting
system is in place, can Truenat TB testing
results be recorded and reported using it?

Training
a. What are the anticipated training needs for
the laboratory staff?

Partner support (optional)

a. Are any partners available that could support
Truenat TB implementation testing and how
can they contribute?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 67
Annex 7: Checklist to confirm readiness of a Truenat TB testing site
This checklist is used to assess the readiness of a site to conduct Truenat TB testing of clinical specimens
and for clinicians to use the results for patient care. The checklist may also be used at the beginning of
the implementation process to identify areas in need of improvement. The numbering of the sections in
this checklist corresponds with the numbering of the sections in the Stop TB / USAID / GLI Practical Guide
to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance. Most questions are
to be answered with a ‘Yes’, ‘No’ or ‘Partial’. Some questions will have text answers. Space is provided to
provide comments for the responses for each question.
Note: not all sections or questions must be answered yes for a site to be ready to begin testing. For
example, developing systems to transmit digital data and use the data to maximum potential may require
a phased approach that would not be expected to be fully executed before testing begins. Likewise, a
fully functional EQA programme is not a requirement to initiate testing.

Name of laboratory

Location of site (City/town, District, State)

( ) National
Laboratory Level ( ) Intermediate (supervisory)
( ) Peripheral
( ) Collect specimens. Nº. per month:
How does the laboratory receive specimens for ____________
testing? ( ) Receive referred specimens. Nº. per
month: ____________
TB tests performed at this site Average number of tests
(check all that apply) conducted per month

( ) AFB Smear-microscopy for diagnosis ( )

( ) Xpert MTB/RIF or
( )
( ) Xpert MTB/RIF Ultra

( ) Culture ( )

( ) DST (FL or SL) ( )

( ) LPA (FL or SL) ( )

( ) LF-LAM ( )

( ) Other: ____________ ( )

Anticipated monthly number of Truenat TB tests

based on planned algorithm and number of
( )
specimens expected to be received including
from referring labs
Persons interviewed
Name Position and contact details

Assessor name:
Assessor contact details:
Date of assessment:

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 68
 Partial
Yes
Comments

No
1. Policies and planning
a. Are the following national guidelines, policies
and plans accessible at the testing site:
• National TB diagnostic algorithm that
includes the use of Truenat TB testing?
• National plan for Truenat TB test
implementation?
b. Has a staff member (part-time or full-time)
been appointed in the laboratory to oversee
the start-up of implementation of Truenat TB
testing?
c. Are partners available that are supporting
readiness for Truenat TB testing and how are
they contributing?
d. Are adequate resources (e.g., funding, staff,
laboratory infrastructures, etc.) available to
support on-going costs of Truenat TB testing
including:
• Equipment maintenance and service
contracts?
• Diagnostics connectivity costs?
• On-going training and competency
assessments?
• Quality assurance (proficiency testing,
monitoring indicators etc.)?
• Projected on-going costs related to Truenat
TB testing (staff, reagents, consumables,
etc.)?
e. Are a sufficient number of qualified staff
available for the anticipated Truenat TB
testing workload?

2. Equipment, service and maintenance

a. Are copies of the manuals and package
inserts for Truelab instruments and reagents
readily available and accessible at the testing
site?
b. bWere the Truenat TB instruments verified on
site prior to routine use for patient testing?
c. Is a routine maintenance log available
indicating daily, weekly, and monthly tasks?
d. Is there an SOP in place to obtain repairs or
service for all Truenat TB instruments?
e. Is a service contract in place to provide
comprehensive service and maintenance?

3. Site readiness
a. Does the laboratory have a lockable door and
secure windows

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 69
 Partial
Yes
Comments

No
b. Is there adequate space for receiving and
processing specimens?
c. Is there adequate amount and quality of
bench space for the Trueprep and Truelab
instruments and ancillary equipment?
• flat, stable surfaces
• vibration-free or vibration-dampened
• away from instruments that cause
electromagnetic interference (note: Molbio
recommends placing the micro PCR
instruments at least one metre away from
other instruments or equipment)
• out of direct sunlight
• away from to any radiating or heating
apparatus
d. Are the Truenat TB instruments placed
correctly in the laboratory?
e. Are Truenat TB workstations clean, free of
clutter, and organized for efficient operation?
f. Does the testing site provide sufficient
ventilation and biosafety for Truenat TB
testing procedures?
g. Does the testing site ensure an optimal
working temperature (15°C to 40°C) and
environment (humidity [10-80%], dust-free) for
the Truelab instruments?
h. Is available electricity adequate for Truenat
TB testing and battery charging? Note: For
sites that would rely on Truenat equipment
in-built batteries, up to 10 hours of electricity is
required to recharge the batteries in order to
be able to run tests for 8 hours.
i. Are there 3 available sockets on the lab bench
for the Truenat instruments?
j. If needed, is a solar powered generator
available for charging of Truelab instruments?
k. Is there sufficient, secured, and organized
storage space for chips (2°C–30°C) and
reagent kits (2°C–40°C)?
l. Is there documented monitoring and review
of environmental temperatures at the testing
and storage areas?
m. Does the laboratory have a refrigerator for
storing sputum samples?
n. Does the testing site use appropriate
disinfectants and are they prepared correctly?
o. Is suitable personal protective equipment
(PPE) provided at the testing site and are staff
trained in its correct use?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 70
 Partial
Yes
Comments

No
p. Does the testing site segregate waste and
dispose of it by incineration or as per national
regulations or guidelines?
q. Does the testing site have adequate capacity
for safely and properly disposing of the
anticipated significant amount of plastic
waste generated by Truenat testing?

4. Supply chain
a. Is procurement system adequate for Truenat
TB reagents and consumables?
b. Will the testing site monitor consumption of
Truenat TB reagents and monitor inventory
(physical count)?
c. Are Truenat TB testing supplies available at
the testing site, in-date, labeled with receive
date, organized and stored at recommended
storage conditions?
d. Is quality control testing (QC) performed on
new lots of Truenat TB reagents prior to their
use for testing patient samples to ensure that
they perform as expected?
e. Does the testing site adequately store Truenat
TB reagents?

5. Procedures
a. Are all of the needed standardized
documents, records and forms related to
Truenat TB testing readily accessible to all
staff?
• Truenat TB test requisition form
• Sample collection and transport forms
• Laboratory register
• Truenat TB instrument maintenance log
• Stock cards
• Temperature monitoring records
• Truenat TB performance indicator reporting
form
• Truenat TB test reporting form
• Truenat TB PT results form & failure follow-
up form

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 71
 Partial
Yes
Comments

No
b. Are the following Truenat TB-related standard
operating procedures (SOPs) approved and
accessible at the testing site?
• Truenat TB test requisition
• Specimen collection
• Specimen processing and storage
• Specimen referral
• Specimen receipt and accessioning
• Sample processing for DNA extraction
• DNA extraction (Trueprep)
• PCR amplification (Truenat micro PCR)
• Recording and reporting
• External quality assessment
• Quality indicator monitoring and data
analysis
• Waste management
• Spill management
c. Is there evidence that all SOPs, documents
and forms have been read by the personnel?
d. Is there an established sample transportation
system from clinical sites to the Truenat TB
testing laboratory? if yes, describe the current
system, its adequacy, efficiency and coverage
e. Is a sample referral system in place for
additional testing for TB samples at referral
laboratories (e.g., second-line DST)? If yes,
describe the current system, its adequacy,
efficiency and coverage

6. Digital data and diagnostics connectivity

a. Are laboratory staff familiar with use of the
digital flatscreen interface of the Truelab
analyzer?
b. Is an electronic laboratory management
system in use? If yes, which?
c. Are any third-party diagnostics connectivity
solutions in use? If yes, which?
d. Is a mechanism in place for data transfer (via
3G or WiFi)?
e. Is an online dashboard available from the
manufacturer to allow for monitoring of
testing?
f. What support are partners able to provide
for implementing diagnostics connectivity
applications?
g. Are procedures in place to define data sharing
protocols and ensure the confidentiality of
patient information?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 72
 Partial
Yes
Comments

No
h. Is suitable secure storage available for the
archived or back-up copies of Truenat TB test
data?
i. Are adequate resources available for
projected on-going costs of data transfer,
storage and analysis?

7.1. Quality Assurance

a. Are the essential elements of a quality
assurance system in place?
• SOPs, training and competence assessment
• Quality control (QC)
• Lot testing (also known as incoming quality
control or new batch testing)
• External quality assessment (EQA)
• Quality indicator monitoring

7.2. Establish and monitor quality controls

a. Are protocols in place that ensure the use of
positive and negative controls?

7.3. External quality assessment

a. Is an external quality assessment programme
in place?
• Proficiency testing (PT)?
• Re-checking of samples?
• On-site supervisory visits?
b. Will the site participate in a PT programme for
Truenat TB testing if yes,
• who will provide the PT panels?
• who will oversee the PT programme and
provide feedback on the performance of
testing site?
c. Does the laboratory participate in inter-
laboratory comparisons (re-checking of
samples)? If yes, with which laboratories?
d. Does the laboratory receive on-site
supervisory visits? If yes,
• who conducts the supervisory visits?
• is feedback provided to the testing site
following a supervisory visit?
• when was the last supervisory visit and
what was the feedback?

7.4. Monitor and analyze quality indicators

a. Are appropriate data collection tools for
Truenat TB test quality indicators available?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 73
 Partial
Yes
Comments

No
b. Which laboratory statistics and performance
indicators are currently reported to the TB
programme and how?
c. Are the following quality indicators monitored
and analysed by the Truenat TB testing site
and reported:
• Number of specimens tested
(Disaggregated by HIV status, MDR risk,
extra-pulmonary TB, pediatric)
• Number and proportion of Truenat TB tests
that generated a result of MTBC detected
• Number and proportion of Truenat TB
tests that generated a result of MTBC not
detected
• Number and proportion of Truenat TB tests
that generated non-determinate results
• Number and proportion of Truenat MTB-RIF
Dx tests that generated a result of RIF-
resistance detected
• Number and proportion of Truenat MTB-
RIF Dx tests that generated a result of RIF-
resistance not detected
• Number and proportion of Truenat MTB-RIF
Dx tests that generated non-determinate
results
• Number and proportion of specimens for
which a Truenat TB test result was reported
within the target turnaround time (i.e., time
from receipt of specimen to reporting of
results)

8. Recording and reporting

a. How will results be returned to clinicians? Via
paper and/or electronically?
• Are laboratory staff aware of how to send
results to clinicians electronically via SMS
and/or email?
b. Is an approved request for examination form
available to request Truenat TB testing?
c. Is an approved reporting form available to
report results of Truenat TB testing?
d. Are the laboratory and clinical registers
suitable for recording the results of Truenat TB
testing?
e. If an electronic laboratory information system
is in use, can Truenat TB testing results be
entered into it?
f. If an electronic recording and reporting
system is in place, can Truenat TB testing
results be recorded and reported using it?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 74
 Partial
Yes
Comments

No
9. Training and competency assessment
a. Are terms of reference and competency
-based job descriptions available for key staff
involved in Truenat TB testing?
b. Are records in place documenting that all
staff have been trained on assigned work
processes, procedures, and tasks?
c. Are standard procedures used to assess and
document the competence of all staff involved
in Truenat TB testing?
d. Are competency assessments for Truenat TB
users conducted annually?
e. Are records in place documenting that all
Truenat TB users have been assessed for
competency?
f. Has the site provided Truenat TB training for
on-site clinicians and healthcare workers on:
• Diagnostic algorithm
• Ordering tests?
• Sample requirements for testing?
• Sample transport?
• Interpretation of test results?
g. Have clinicians and healthcare workers in sites
that will be referring specimens to this site
been trained on all of the above components?
h. Are there any additional training needs for
clinical staff?

10.1. Monitoring and evaluation of implementation of Truenat TB testing

a. Will the accomplishment of key indicators and
milestones for Truenat TB implementation be
reported to the national programme?

10.2. Monitoring and evaluation of impact of Truenat TB testing

a. Will the following indicators be monitored and
reported to the national programme at least
annually
• Number and proportion of notified
bacteriologically confirmed TB cases with
reported Truenat TB test results
• Number and proportion of notified
bacteriologically confirmed TB cases with
reported Truenat MTB-RIF Dx test results for
rifampicin susceptibility

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 75
Annex 8: Checklist to confirm readiness of a clinical site
This checklist is used to assess the readiness of a clinical site to participate in a Truenat TB testing
programme and use the Truenat TB test results for patient care decisions. Clinical sites to assess should
include all sites with Truenat instruments on the premises as well as all sites that will be referring samples
to other health care facilities with Truenat instruments. The checklist may also be used at the beginning of
the implementation process to identify areas in need of improvement. Most questions are to be answered
with a ‘Yes’, ‘No’ or ‘Partial’. Some questions will have text answers. Space is provided to provide comments
for the responses for each question.

Name of laboratory

Location of site (City/town, District, State)

( ) Central hospital
( ) Regional hospital
Type of Facility ( ) District hospital
( ) Primary health facility
Other: ____________
TB activities performed at this site Average number of procedures
(check all that apply) conducted per month

( ) Screen presumptive TB patients ( )

( ) Chest X-ray ( )

( ) Refer patients to testing laboratory ( )

( ) Collect specimens ( )

( ) Package and ship samples to testing

( )
laboratory

( ) Initiate patients on TB therapy ( )

Other: ____________ ( )

Persons interviewed
Name Position and contact details

Assessor name:
Assessor contact details:
Date of assessment:

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 76
 Partial
Yes
Comments

No
a. Is the National TB diagnostic algorithm that
includes the use of Truenat TB testing available at
the clinical site?
• Have clinical staff been trained on the national
TB testing algorithm?
• Are clinical staff aware of intended uses of the
Truenat TB tests?
• Are clinical staff aware of criteria for selecting
which patients should get a Truenat TB?
b. Are all of the needed standardized forms related
to Truenat TB testing readily accessible to all staff
at the clinical site?
• An approved request for examination form for
requesting Truenat TB testing?
• Sample collection and transport forms?
• An approved reporting form to report results of
Truenat TB testing?
c. Are the following Truenat TB-related standard
operating procedures approved and accessible at
the clinical site?
• How to order a Truenat TB test?
• How to complete the Truenat TB test requisition
form?
• How to collect specimens suitable for Truenat
TB testing?
• How to store specimens if testing cannot be
done immediately?
• How to package TB specimens for safe
transport?
• How to arrange for transport to the Truenat TB
testing site?
• How to record Truenat TB test in clinical
registers?
• How to interpret Truenat TB tests and use
results for patient care decisions?
d. Have all clinicians and healthcare workers at
this clinical site been trained in all procedures in
question ‘c’? If no, describe training needs.
e. Is there an established sample transportation
system from clinical sites to the Truenat TB testing
laboratory?
• if yes, describe the current system, its adequacy,
efficiency and coverage
• Are appropriate packaging materials available?
f. How will results be returned to clinicians? Via
paper and/or electronically?
g. Are the clinical registers suitable for recording the
results of Truenat TB testing?
h. If an electronic patient management system is in
use, can Truenat TB testing results be entered into
it?

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 77
Annex 9: Performance indicators for Trueprep and Truenat TB tests

General laboratory performance indicators*

Indicator Target

Number of tests performed, by type of test -

Service interruptions No interruptions

Stock outs No stock outs leading to service interruption

No equipment downtime leading to service

Equipment down time
interruption

Turnaround time (TAT) 90% of results meet test-specific TAT

Test statistics (quality indicator) report 100% reports completed by defined due date

EQA results >90% EQA panels are passed

QC results >90% QC results meet expected criteria

Specimen rejection <1% specimens rejected

Customer satisfaction >80% surveyed customers are satisfied

Report average number of tests performed per

Technician productivity
month per technician
*General indicators are from the GLI Practical Guide to TB Laboratory Strengthening. Geneva, Global Laboratory Initiative, 2017. http://stoptb.org/wg/gli/gat.asp

Performance indicators for Truenat TB tests

The performance indicators are modeled after the GLI-recommended performance indicators for
Xpert MTB/RIF testing that should be monitored monthly by each testing site. For some indicators (e.g.,
proportion of specimens that are rifampicin resistant), targets are setting-specific. Laboratories should
monitor indicators and establish local targets and acceptable ranges. Deviations from expected values
should be investigated.

Indicator Description Target

Trueprep
Number of specimens for which
DNA could not be extracted
Number and proportion of Initial test: <3%
/ Total number of specimens
specimens for which DNA
processed Repeat test: <1%
extraction was unsuccessful
Errors should be stratified by
type, to enable troubleshooting

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 78
 Indicator Description Target

Truenat TB
Number of specimens with
Dependent on population tested
Number and proportion of MTBC detected/ Total number of
and country drug-resistance
specimens with MTBC detected specimens tested with successful
prevalence and patterns
results
Number of specimens with
Number and proportion of Dependent on population tested
MTBC not detected / Total
specimens with MTBC not and country drug resistance
number of specimens tested
detected prevalence and patterns
with successful results
Number of specimens with
Number and proportion of unsuccessful results/ Total <3%
specimens with unsuccessful number of specimens tested. Initial test: <10%
results (errors, invalid, no results) Errors should be stratified by Repeat test: <3%
type, to enable troubleshooting.

Truenat MTB-RIF Dx
Number of specimens with
Number and proportion of rifampicin resistance not Dependent on population tested
specimens with rifampicin detected / Total number of and country drug-resistance
resistance not detected specimens tested with successful prevalence and patterns
results
Number of specimens with
Number and proportion of Dependent on population tested
rifampicin resistance detected
specimens with rifampicin and country drug-resistance
/ Total number of specimens
resistance detected prevalence and patterns
tested with successful results
Number of specimens with
Number and proportion of Dependent on population tested
rifampicin indeterminate / Total
specimens with rifampicin (e.g., proportion of patients with
number of specimens tested for
indeterminate smear-negative TB)
rifampicin resistance
<3% for Truenat MTB or MTB
Number of specimens with
Plus test
Number and proportion of unsuccessful results / Total
Initial RIF-Dx test: <7% if reflexed
specimens with unsuccessful number of specimens tested.
from Truenat MTB
results (errors, invalid, no result) Errors should be stratified by
Initial RIF-Dx test: <15% if
type, to enable troubleshooting.
reflexed from Truenat MTB Plus

Laboratory turnaround time

Time between receipt of
Laboratory turnaround time specimen at the laboratory and 2-24hrs
result reporting

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 79
Annex 10: Impact measures for Trueprep and Truenat TB tests
A framework for monitoring and evaluation of the impact of a diagnostic test is essential to inform decision-
making. New or improved TB diagnostic tests often have the objective to improve the laboratory confirmation
of TB or the detection of drug resistance. For each objective of a test, indicators should be developed to
assess its impact. For example, implementation of the Truenat TB tests should increase the ability to detect
TB and RIF-resistant TB as measured by the WHO targets for laboratory strengthening under the End TB
Strategy. The WHO indicators described in the Table below reflect the use of any WRD. Programmes will
need to assess the contribution of Truenat TB tests, Xpert MTB/RIF tests and any other WRD used in the
country to determine the impact of Truenat TB testing on the accomplishment of these indicators.

WHO indicators for Laboratory Strengthening WHO Target

Percentage of notified new and relapse TB cases
tested with a WHO-approved rapid diagnostic
80% (2020)
test (WRD) as the initial diagnostic test (End TB
Strategy Laboratory Indicator 2)21
Percentage of notified new and relapse TB cases
80% [relapse: 90%] (2020)
with bacteriological confirmation (Indicator 3)
Percentage of testing sites using a WRD at which
a data connectivity system has been established
that transmits results electronically to clinicians 100% (2020)
and to an information management system
(Indicator 4)
Percentage of notified bacteriologically confirmed
TB cases with DST results for rifampicin (Indicator 100% (2020)
7)
Percentage of notified rifampicin-resistant TB
cases with DST results for fluoroquinolones and 100% (2020)
second-line injectable agents (Indicator 8)

The implementation of Truenat TB tests at facilities closer to point-of-care may lead to improvements in
the entire patient care cascade from screening to initiation of appropriate treatment. In addition to WHO
indicators 2 and 3 above, possible impact measures may include:
• Number and proportion of presumptive TB patients that have been tested with a WRD
• Proportion of the population that has access to WRD within a 5-kilometer distance
• Number and proportion of estimated TB patients that are evaluated for TB (i.e., reach a diagnostic
center)
• Number and proportion of presumptive TB patients that reach a diagnostic center and for whom
a TB test is ordered
• Number and proportion of presumptive TB patients for whom a test is ordered and who provide
a specimen for testing
• Number and proportion of presumptive TB patients for whom a specimen is collected and whose
specimen is received at the testing laboratory
• Number and proportion of presumptive TB patients whose specimen is received at the testing
laboratory and for whom a test is conducted
• Number and proportion of presumptive TB patients for whom a test is conducted and for whom
test results are reported to the clinician

21 The indicator number in parentheses refers to the number of the global indicators in the WHO Framework of indicators and targets for
laboratory strengthening under the End TB Strategy. http://www.who.int/tb/publications/labindicators/en/

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 80
 • Number and proportion of presumptive TB patients for whom test results are reported to the
clinician and that are notified of the result
• Proportion of specimens collected for WRD testing for which a result was received by the ordering
clinician within the specified target time (i.e., time from collection of a specimen to receipt of
results). Note that the ‘specified time’ should be determined for each laboratory taking into account
testing schedules and specimen transport schedules.

It should be noted that a detailed analysis of the impact measures (time and completion rate) for each
step in the patient care pathway is an intensive process that may require data from laboratory, clinical
and programme personnel. Such information may be collected using a once a year survey or through
operational research. Programmes should establish baseline performance and monitor the impact of
Truenat TB tests to improvements in performance over time.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 81
Annex 11: Molbio job aids for Truenat TB tests

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 82
Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 83
 Troubleshooting, Alerts and Errors
Trueprep® AUTO v2 Universal Cartridge based Sample Prep Device

E1 E2 E3 E6 E9 E10 E11&12
Cartridge Cartridge Cartridge Cartridge Cartridge
valve is
E1:CARTRIDGE valve is
E2:CARTRIDGE valve is
E3:CARTRIDGE valve is
E6:CARTRIDGE valve is
RESET CARD
READ ERROR
E10: INVALID E11:RTD-
L ERROR
VALVE ERROR ERROR CLOGGED NOT LOADED RESET CARD
damaged damaged damaged damaged damaged
Press Eject Press Eject Press Eject Press Eject Press Eject Press Eject E12:RTD-
E11:RTD-
to exit to exit to exit to exit to exit to exit E ERROR

Cartridge Pressure Sample/ Cartridge not Problem with the Reset card Device
valve is drop error specimen is detected or QR code reader heater plates
damaged too thick not working

SOLUTION
E1/E2*: Please start afresh by processing remainder of sample in lysis buffer and load into new cartridge.
E3*: Ensure sample is liquefied and pipettable. Repeat extraction with new cartridge/request for new sample.
E6*: Ensure cartirdge is loaded properly in correct orientation
E9/E10/E11/E12: Contact Molbio support *If error persists, contact Molbio support

Truelab® Real Time Quantitative micro PCR Analyzer

ERROR 1 ERROR 2 ERROR 3 ERROR 4 ERROR 5 INVALID

Thermal Test stopped Incorrect Runtime Error Probe check Internal Control did not
cycling error manually optical profile Error amplify in PCR or improper
sample extraction

SOLUTION
ERROR1/2/3/4/5**: Repeat the run using a fresh chip and re-load*** the elute by pressing Repeat button

INVALID**: Re-run the same elute using another chip. If Invalid repeats, process the sample again and run
elute using another chip.

**Contact the Molbio support team if the problem persists

***Follow User Guidelines for proper loading of elute onto the white reaction well of the chip
Truelab® Alert Messages
“Unable to read chip “Could not initialize. Please try “Chip is already used”
information” again” OR
“Chip loaded is expired”
Analyzer was unable to read chip The system was unable to establish User loaded a used chip/expired
memory. Check if chip was loaded an internal connection. chip in the Tray.
properly into the tray. Remove the chip
attempt the test again by using a Use a fresh chip and re-load the
and re-select the profile from Status
Screen and repeat the steps. new chip and re-loading the elute elute
If message reappears, load a new chip again
and re-load the elute again.

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 84
Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 85
 Do’s and Dont’s
Trueprep® AUTO v2 Universal Cartridge Based Sample Prep Device

ü Place the device in a dust free environment × Do not leave a used cartridge inside the
device. Remove the cartridge after extraction
and collect the elute in the ECT
Wipe the surface of the device regularly
ü with 1% Sodium hypochlorite solution
followed by distilled water × Do not keep used material on the work bench
and on the device

ü
Replace the cartridge holder
tray in case of a spillage in
the device
× ×
× Do not tilt or shake the device while
connecting charger, during extraction or
while removing cartridge from device

ü Ifdays,
the device is kept idle for more than 10
carry out the flushing protocol (Refer
manual section 8.2.3) before resuming
operation
× Do not touch the nozzle of
the liquefaction buffer bottle
to the lysis buffer tube during ×
sample preparation

Truelab® Uno Dx/Duo/Quattro Real Time Quantitative micro PCR Analyzer

Place the analyzer on a flat surface in a

ü dust free environment, away from direct × Do not move the analyzer when a test is in
progress

×
sunlight and vibration causing instruments
Do not spill water or any other solution on
the surface of the analyzer

× Do not touch the white reaction well or the

connector pads of the chip. Hold the chip
by the edges only

×
Wipe the surface of the device regularly with
ü 1% Sodium hypochlorite solution followed
by distilled water
üü
ü Ensure that the elute is loaded in the center
of the reaction well × Do not leave a used chip inside the device

× Do not keep used material on the work bench

and on the device

Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance 86