same pipette and tip and dispensed into the reaction well of the Truenat™ MTB chip

and the test is started. A positive amplification causes the dual labeled fluorescent
probe in the Truenat™ MTB chip-based Real Time PCR test to release the
MTB fluorophores in an exponential manner which is then captured by the built-in opto-
electronic sensor and displayed as amplification curve on the analyzer screen, on a
Chip-based Real Time PCR Test for Mycobacterium tuberculosis real time basis during the test run. The Cycle threshold (Ct) is defined as the number
of amplification cycles required for the fluorescent signal to cross the threshold (i.e.
1. INTENDED USE
exceed the background signal). Ct levels are inversely proportional to the amount of
Truenat™ MTB (REF 601030005/601030020/601030050) is a chip-based Real target nucleic acid in the sample. (i.e. the lower the Ct level the greater is the amount
Time Polymerase Chain Reaction (PCR) test for the quantitative detection and of target nucleic acid in the sample). In the case of negative samples, amplification
diagnosis of Mycobacterium tuberculosis (MTB) in human pulmonary and EPTB does not occur and a horizontal amplification curve is displayed on the screen during
specimen and aids in the diagnosis of infection with MTB. Truenat™ MTB runs on the the test run. At the end of the test run, a MTB “DETECTED” or “NOT DETECTED”
Truelab™ Real Time micro PCR Analyzers. result is displayed and in positive cases, quantitative values is also displayed on the
screen. Based on the Ct of the internal positive control (IPC), the validity of the test
2. INTRODUCTION run is also displayed. The IPC is a full process control that undergoes all the
Tuberculosis (TB) is an infectious disease caused predominantly by the bacillus processes the specimen undergoes - from extraction to amplification thereby
Mycobacterium tuberculosis. It typically affects the lungs (pulmonary TB) but can validating the test run from sample to result. Absence of or shift of IPC Ct beyond a
affect other sites as well (extra pulmonary TB). Tuberculosis (TB) is the second pre-set range in case of negative samples invalidates the test run. While IPC will co-
largest killer worldwide, after HIV and is the leading cause of death in HIV patients. amplify in most positive cases also, in some specimen having a high target load, the
Pulmonary TB spreads through air and is highly contagious. Over 80% of TB IPC may not amplify, however the test run is still considered valid. The results can be
infections are pulmonary and if left untreated, a pulmonary TB patient can infect up to printed via Bluetooth using the Truelab™ micro PCR printer or transferred to the lab
10-15 other people through close contact over the course of a year.Due to the highly computer/or any remote computer via Wifi network or 3G/GPRS network. Upto 5000
infectious nature of pulmonary TB, it is important to diagnose and treat the disease results in Truelab TM Uno to 20,000 results in Truelab TM Uno Dx/ Duo/Quattro can be
very early. Despite the availability of highly effective treatment for decades, TB stored on the analyzer for future recall and reference.
remains a major global health problem mainly because of poor case detection. The
most common method for diagnosing pulmonary TB worldwide is sputum smear 4. TARGET SELECTION
microscopy. However sensitivity of direct smear microscopy is low and estimates
The target sequence for Truenat™ MTB is nrdZ gene which codes for
range from 30% to 70%. It is even lower in case of HIV-infected patients. Culture is
ribonucleoside- diphosphate reductase large subunit.
more sensitive than microscopy and is considered the current gold standard. Culture
requires specialized and controlled laboratory facility and highly skilled manpower
5. CONTENTS OF THE Truenat™ MTB KIT
and takes 3 to 6 weeks to provide the result. Molecular techniques such as
A. Individually sealed pouches, each containing
polymerase chain reaction (PCR) or Real Time PCR are much more sensitive than
1. Truenat™ MTB micro PCR chip.
microscopy and culture. However PCR or Real Time PCR tests have so far been
2. Microtube with freeze dried PCR reagents.
restricted to centralized reference laboratories as they require skilled manpower and
3. DNase & RNase free pipette tip.
elaborate infrastructure. Also the turnaround time for results could take a few days.
4. Desiccant pouch.
The Truelab™ Real Time micro PCR System enables decentralization and near
B. Package Insert.
patient diagnosis of MTB by making real time PCR technology rapid, simple, robust
and user friendly and offering “sample to result” capability even at resource limited 601030005 601030020 601030050
settings. This is achieved through a combination of light weight, portable,
mains/battery operated Truelab™ Real Time micro PCR Analyzer and Trueprep®
MAG/AUTO Sample Prep Device and room temperature stable Truenat™ micro 6. CONTENTS OF Trueprep® AUTO MTB Sample pre-treatment pack (only for
PCR chip and Trueprep® MAG/AUTO Sample Prep kits so that even the peripheral Trueprep®AUTO users)
laboratories with minimal infrastructure and minimally trained technicians can easily A. Liquefaction buffer
perform these tests routinely in their facilities and report PCR results in less than an B. Lysis buffer
hour. Moreover, with these devices PCR testing can also be initiated in the field level,
on site. C. Disposable transfer pipette (graduated)
Truenat™ MTB is a disposable, room temperature stable, chip-
60204AS05 60204AS20 60204AS50
based Real Time PCR test with dried MgCl2 in reaction well and
freeze dried PCR reagents in microtube for performing Real
Time PCR test for detection of Mycobacterium tuberculosis and
7. STORAGE AND STABILITY
runs on the Truelab™ Real Time micro PCR Analyzer. It
TruenatTM MTB chip is stable for two (2) years from the date of manufacture if stored
requires only six (6) μL of purified DNA to be added to the
between 2-30oC. It is also stable for upto six (6) months at temperatures up to 40o C
reaction well for the analysis.
and one (1) month at temperatures up to 45o C. Avoid exposure to light or elevated
The intelligent chip also carries test and batch related information including standard
temperatures (above recommended levels). Do not freeze.
values for quantitation. The TruenatTM MTB chip-based Real Time PCR test also
Trueprep® AUTO MTB Sample pretreatment pack is stable for two (2) years from the
stores information of used test to prevent any accidental re-use of the test.
date of manufacture if stored between 2-40oC. It is also stable for one (1) month at
NOTE :Truelab™/ Truenat™ / Trueprep® / Truepet® are all trademarks of Molbio
temperatures up to 45oC. Do not freeze.
Diagnostics (P) Limited.
The Truelab™ Real Time micro PCR Analyzer is protected by the following
8. MATERIALS REQUIRED BUT NOT PROVIDED WITH THE KIT
patents and patents granted: IN 2313/CHE/2007 (Patent No. 281573),
Truelab™ Real Time micro PCR Workstation (REF 603010001/62301001/
WO2009/047804 and corresponding claims of any foreign counterpart(s)
633010001/64301001) consisting of
thereof.
1. Trueprep® MAG / AUTO Sample Prep Device (REF 603040001/603041001).
The Truenat™ micro PCR chip is protected by the following patents and patents
2. Truelab™ Uno / Truelab™ Uno Dx / Truelab™ Duo / Truelab™ Quattro
pending: IN 2312/CHE/2007, WO 2009/047805 and corresponding claims of any
Real Time micro PCR Analyzer (REF603020001/603021001/603022001/
foreign counterpart(s) thereof. The Truenat™ MTB chip-based Real Time PCR
603023001).
test is protected by the following patents and patents pending: IN
3. Truelab™ micro PCR Printer (REF 603050001).
796/CHE/2012 and corresponding claims of any foreign counterpart(s) thereof.
4. Truepet® SPA fixed volume precision micropipette - 6 µl (REF 604070006).
5. Truelab™ Microtube Stand (REF 603070001).
3. PRINCIPLE OF THE TEST
Also required additionally are:Trueprep® MAG Sputum Sample Prep Kit (REF
Truenat™ MTB works on the principle of Real Time Polymerase Chain Reaction
602020005/REF 602020050)/Trueprep® AUTO Universal Cartridge Based Sample
based on Taqman chemistry. The DNA from the patient sample is first extracted using
Prep Kit (REF 60203AR05/REF 60203AR25/REF 60203AR50), Truenat™
Trueprep® MAG Sample Prep Device and Trueprep® MAG sputum Sample Prep kit
Universal Control Kit (REF 601100008), DNase and RNase-free pipette tips with filter
or using Trueprep® AUTO Universal Cartridge Based Sample Prep Device and
barrier, which may also be procured from Molbio, Powder free disposable gloves,
Trueprep® AUTO Universal Cartridge Based Sample Prep Kit. The Truenat™ MTB
waste disposal container with lid.
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 using the provided micropipette and tip into
9. SPECIMEN PREPARATION FOR EXTRACTION WITH Trueprep® MAG
the microtube containing freeze dried PCR reagents and allowed to stand for 30-60
Truenat™ MTB requires purified nucleic acids from pulmonary and EPTB specimen
seconds to get a clear solution. No mixing by tapping, shaking or by reverse
that are extracted using the Trueprep® MAG Sample Prep Device and Trueprep®
pipetting should be done. Six (6) µL of this clear solution is then pipetted out using the
MAG Sputum Sample Prep Kit (Refer to the User Manual of Trueprep® MAG Sample
 Prep Device and the package insert of Trueprep® MAG Sputum Sample Prep Kit for Bay (Idle1/2) for Duo and (Idle1/2/3/4) for Quattro from the Status Screen to
details). view the Profiles Screen. Select the test profile for “MTB” to be run from the
Profiles Screen, on the Analyzer screen.
10. SPECIMEN PREPARATION FOR EXTRACTION WITH Trueprep®AUTO 5. Enter the patient details as prompted in the Truelab™ Analyzer screen.
Truenat™ MTB requires purified nucleic acids from pulmonary and EPTB specimen 6. Press Start Reaction.
that are extracted using the Trueprep® AUTO Universal Cartridge Based Sample 7. For TruelabTM Uno/Uno Dx, Press the eject button to open the chip tray. For
Prep Device and Trueprep® AUTO Universal Cartridge Based Sample Prep Kit. TruelabTM Duo/Quattro, the chip tray opens automatically on tapping the “Start
Samples must be liquefied and pre-treated using the Trueprep® AUTO MTB sample Reaction” button.
pre-treatment pack provided (Refer to the package insert of Trueprep® AUTO MTB 8. Open a pouch of Truenat™ MTB and retrieve the micro PCR chip, microtube
Sample Pre-treatment Pack for details) before proceeding for extraction. and DNase & RNase free pipette tip.
Sample Storage and Transportation: 9. Label the chip with the patient ID using a marker pen at the space provided on the
Sample pre-treatment decontaminates the specimen and makes it ready for back side of the chip.
extraction. Sample in this form is stable for 3 days at upto 40°C and 1 week at 30°C . 10. Place the Truenat™ MTB chip on the chip tray without touching the white
Nucleic acid extraction: Follow Extraction procedure (section-13) of Trueprep® reaction well. The reaction well should be facing up and away from the Analyzer.
AUTO Universal Cartridge Based Sample Prep Kit package insert. (Refer to the Gently press the chip to ensure that it is seated in the chip tray properly.
User Manual of Trueprep® AUTO Universal Cartridge Based Sample Prep Device 11. Place the microtube containing freeze dried PCR reagents in the microtube
and the package insert of Trueprep® AUTO Universal Cartridge Based Sample Prep stand provided along with the TruelabTM Real Time micro PCR workstation after
Kit for details). Dispose off lysis buffer tube and transfer pipette after use, as per the ensuring that white pellet of dried PCR reagents remains at the bottom of
section on “Disposal and Destruction” (Section 18). the microtube. Remove the microtube cap and dispose it off as per the section
on “Disposal and Destruction” (Section 18). Using the filter barrier tip provided in
11. SAFETY PRECAUTIONS the pouch, pipette out six (6) µL of the purified DNA from the Elute Collected Tube
1. For in vitro diagnostic use only. into the microtube. Allow it to stand for 30-60 seconds to get a clear solution.
2. Bring all reagents and specimen to room temperature (20 - 300C) before use. Do not mix it by tapping, shaking or by reverse pipetting. Using the same filter
3. Do not use kit beyond expiry date. barrier tip, pipette out six (6) µL of this clear solution and dispense into the centre
4. Carefully read the User Manuals and package inserts of all the components of of the white reaction well of the Truenat™ MTB chip. Take care not to scratch the
the Truelab™ Real Time micro PCR System before use. internal well surface and not to spill elute on the outside of the well. Dispose off
5. All materials of human origin should be handled as though potentially infectious. the microtip as per the section on “Disposal and Destruction” (Section 18).
6. Do not pipette any material by mouth. 12. For TruelabTM Uno/Uno Dx, slide the chip tray containing the Truenat™ MTB
7. Do not eat, drink, smoke, apply cosmetics or handle contact lenses in the area chip-based Real Time PCR test loaded with the sample into the TruelabTM
where testing is done. Analyzer. Press Done on the “Please Load Sample” Alert message. For
8. Use protective clothing and wear disposable gloves when handling samples and TruelabTM Duo/Quattro, select “YES” at the Please load Sample prompt. Chip
while performing sample extraction. tray will close automatically and the reaction will start.
13. Read the result from the screen.
12. PROCEDURAL PRECAUTIONS 14. After the reaction is completed, for TruelabTM Uno/Uno Dx, push the Eject button
1. Check all packages before using the kit. Damage to the packaging does not to eject the chip tray. For TruelabTM Duo/Quattro, tap the “Open/Close Tray”
prevent the contents of the kit from being used. However, if the outer packaging button to eject the chip tray.
is damaged the user must confirm that individual components of the kit are intact 15. Take out the Truenat™ MTB micro PCR chip at end of the test and dispose it off
before using them. as per the section on “Disposal and Destruction” (Section 18).
2. Do not perform the test in the presence of reactive vapours (e.g. from sodium 16. Turn on Truelab™ micro PCR printer and select print on the screen for printing
hypochlorite, acids, alkalis or aldehydes) or dust. out hard copy of the results. Test results are automatically stored and can be
3. While retrieving the Truenat™ MTB micro PCR chip, microtube and the DNase retrieved any time later. (Refer to Truelab™ Analyzer manual).
& RNase free pipette tip from the pouch, ensure that neither bare hands nor 17. Switch off the Truelab™ Analyzer.
gloves that have been used for previous tests run are used.
16. RESULTS & INTERPRETATIONS
13. PROCEDURAL LIMITATIONS Two amplification curves are displayed on the Truelab™ Analyzer screen when
1. Optimal performance of this test requires appropriate specimen collection, optical plot is selected to indicate the progress of the test. Both the target and the
handling, storage and transport to the test site. internal positive control (IPC)* curves will take a steep, exponential path when the
2. Though very rare, mutations within the highly conserved regions of the target fluorescence crosses the threshold value in case of positive samples. The Ct will
genome where the Truenat™ assay primers and/or probe bind may result in the depend on the number of bacterial genomes in the sample. The target curve will
under-quantitation of or a failure to detect the presence of the concerned remain horizontal throughout the test duration and the IPC curve will take an
pathogen. exponential path in case of negative samples. In case the IPC curve remains
3. The instruments and assay procedures are designed to minimize the risk of horizontal in a negative sample, the test is considered as Invalid. At the end of the test
contamination by PCR amplification products. However, it is essential to follow run, the results screen will display “DETECTED” for Positive result or “NOT
good laboratory practices and ensure careful adherence to the procedures DETECTED” for Negative result. The result screen would also display the Ct value
specified in this package insert for avoiding nucleic acid contamination from and the colony forming units per milliliter (CFU/ml) for positive specimen. The result
previous amplifications, positive controls or specimens. screen also displays the validity of the test run as “VALID” or “INVALID”. Invalid
4. A specimen for which the Truenat™ assay reports “Not Detected” cannot be samples have to be repeated with fresh specimen from the sample preparation stage.
concluded to be negative for the concerned pathogen. As with any diagnostic *Note: IPC will co-amplify in most positive cases also, in some specimen having a
test, results from the Truenat™ assay should be interpreted in the context of high target load, the IPC may not amplify, however the test run is still considered valid.
other clinical and laboratory findings.
17. QUALITY CONTROL PROCEDURES
14. CLEANING AND DECONTAMINATION To ensure that the Truelab™ Analyzer is working accurately, run positive and
1. Spills of potentially infectious material should be cleaned up immediately with negative controls from time to time. The Truenat™ Universal Control Kit containing
absorbent paper tissue and the contaminated area should be decontaminated Positive Control and Negative Control must be ordered separately. It is advisable to
with disinfectants such as 0.5% freshly prepared sodium hypochlorite [10 times run controls under the following circumstances:
dilution of 5% sodium hypochlorite (household bleach)] before continuing work. ● Whenever a new shipment of test kits is received. ● When opening a new test kit lot.
2. Sodium hypochlorite should not be used on an acid-containing spill unless the ● If the temperature of the storage area falls outside of 2-30o C. ● By each new user
spill-area is wiped dry first. Materials used to clean spills, including gloves, prior to performing testing on clinical specimen.
should be disposed off as potentially bio-hazardous waste e.g. in a biohazard
waste container. 18. DISPOSAL AND DESTRUCTION
1. Submerge the used Truenat™ MTB chip, microtube, microtube cap, transfer
15. TEST PROCEDURE pipette, pipette tips etc. in freshly prepared 0.5% sodium hypochlorite
(Please also refer the Truelab™ Real Time micro PCR Analyzer user manual) solution for 30 minutes before disposal as per the standard medical waste
1. Switch on the Truelab™ Analyzer. disposal guidelines.
2. If using the TruelabTM Uno device, also switch on the touch screen. If using the 2. Disinfect the solutions and/or solid waste containing biological samples before
TruelabTM Uno Dx/Duo/Quattro proceed to step 3. discarding them according to local regulations.
3. Select user and enter password. 3. Samples and reagents of human and animal origin, as well as contaminated
4. For TruelabTM Uno/Uno Dx, select the test profile for “MTB” to be run from the materials, disposables, neutralized acids and other waste materials must be
Profiles Screen on the Analyzer screen. For TruelabTM Duo/Quattro, select the
 discarded according to local regulations after decontamination by immersion in a Interfering Substances
freshly prepared 0.5% of sodium hypochlorite for 30 minutes (1 volume of 5% The purpose of this study is to determine the effect of potentially interfering
sodium hypochlorite for 10 volumes of water). substances on the Truenat™ MTB assay. For this study low load sample has been
4. Do not autoclave materials or solutions containing sodium hypochlorite. used. To the sputum sample, 10% and 30% blood was spiked and then the sample
5. Chemicals should be handled in accordance with Good Laboratory Practice and was extracted on Trueprep® AUTO Universal Cartridge Based Sample Prep Device
disposed off according to the local regulations. and PCR was performed on TruelabTM Uno Dx Real Time Quantitative micro PCR
analyzer using TruenatTM MTB assay. The presence of blood till 30% did not interfere
19. SPECIFIC PERFORMANCE CHARACTERISTICS with the performance of TruenatTM MTB assay.
Analytical Exclusivity(Primer Specificity):
Genomic DNA sequence of following microorganisms were evaluated in silico from Precision of TruenatTM MTB assay:
the the NCBI database using the NCBI nucleotide blast and primer blast tools to Precision was tested by performing Truenat™ MTB assay with extracted DNA from
determine for potential cross-reactivity in the Truenat™ MTB assay. No cross sputum of High (2.00E+06 copies/mL), Medium (2.00E+04 copies/mL) and Low
reactivity in the performance of the Truenat™ MTB assay was observed with the (2.00E+02 copies/mL) for five consecutive days. Every day PCR for each titre DNA
below listed microorganisms. was run in duplicates. The %CV values obtained for High titre (2.9), Medium titre
(1.55) and low titre (2.1) were within the accepted range of ≤15% CV for TruenatTM
Bacteria Bacteria
Escherichia coli Acinetobacter anitratus MTB assay.
Enterobacter cloacae Chlamydia trachomatis
Enterococcus faecalis Gardeneralla vaginalis Clinical Validations:
Candida albicans Streptococcus mutans a) Clinical validation 1:
Trichomonas vaginalis Salmonella enterica A pilot study was conducted at P. D. Hinduja National Hospital and Medical Research
Staphylococcus aureus Neisseria gonorrheae Centre (Nikam, Chaitali, et al. “Rapid diagnosis of Mycobacterium tuberculosis with
Virus Virus TruenatTM MTB: a near-care approach.” PLOS One 8.1 (2013): e51121). 226 sputum
Adenovirus Human Immunodeficiency Virus specimens from suspected TB patients were analyzed using smear microscopy,
Epstein-Barr virus Herpes Simplex virus culture, in-house nested PCR and Truenat™ MTB. Pelleted sputum specimens were
Simian virus Cytomegalovirus re-suspended in lysis buffer from the Trueprep® MAG Sputum kit and processed
Hepatitis B virus Hepatitis C Virus using the Trueprep® MAG Sample Prep Device followed by PCR on Truenat™ MTB
chip-based Real Time PCR test. Results were compared with a Composite
Specificity of the Truenat™ MTB assay was evaluated by testing Non tuberculosis Reference Standard (CRS) comprising microbiological tests, clinical and radiological
mycobacterium (NTM) strains as given in the below table. No cross reactivity in the findings and patient history. The results are tabulated below:
performance of the Truenat™ MTB assay was observed with the below listed NTM Smear Culture In-house Nested PCR Truenat™ MTB
strains. +ve -ve +ve -ve +ve -ve +ve -ve
CRS +ve 120 71 141 50 173 18 174 17
Bacteria Bacteria
CRS -ve 00 35 00 35 03 32 00 35
M. malmoense M.simiae Sensitivity % 62.83 73.82 90.58 91.10
M. intracellulare M.trivale Specificity % 100 100 91.43 100
M. scrofulaeium M.terrae PPV% 100 100 98.30 100
M. ulcerance M.flavescens NPV% 33.02 41.18 64.00 67.31
M. abscessus M.haemophilum
M.fortuitum M.thermoresistibile CRS - Composite Reference Standard, PPV - Positive Predictive Value, NPV -
M. avium M.marinum Negative Predictive Value.
M.gordanae M.xenopi The results show that Truenat™ MTB was the most sensitive (91.10%) and specific
M.szulgai M.vaccae (100%) test compared with the Composite Reference Standard. Truenat™ MTB
M.kansasii M.chelonae also showed high sensitivities of 99.12% among smear positive and culture positive
M.asiaticum M.smegmatis specimen and 75.86% among smear negative and culture positive specimen.
M.celatum Another study evaluating the Truenat™ MTB test was performed using a
characterized 100 sample panel from suspected TB patients referred to a hospital in
ASSAY RANGE AND LIMIT OF DETECTION South East Asia. The study involved processing of 500µl of each sputum specimen
The MTB strain H37Rv from Zeptometrix was used for LoD determination. The LoD using the Trueprep® MAG Sputum Sample Prep Kit on the Trueprep® MAG
was determined by making dilutions of 50,100, 250, 500 and 1000 CFU/ml of H37Rv Sample Prep Device. The purified nucleic acids were tested using Truenat™ MTB
strain in negative sputum samples. Further, the MTB DNA from each of these chip-based Real Time PCR test and MTB specific primers and probe on a
dilutions were extracted using Trueprep® AUTO Universal Cartridge Based Sample commercial real-time PCR machine.
Prep Device and PCR performed on TruelabTM Uno Dx Real Time micro PCR
Analyzer using TruenatTM MTB chips. LoD was determined to be 100 CFU/ml sputum Sample Type Commercial real-time PCR machine result Truenat™ MTB
sample. S+C+ 40/40 (100%) 40/40 (100%)
S-C+ 30/40 (75%) 30/40 (75%)
Percentage Positivity Vs CFU/ml for MTB
CFU/ml % Positivity 120 S-C- 0/20 (nil detected) 0/20 (nil detected)
50 50 100
(S: Smear, C: Culture)
Percentage Positivity

80
100 100
60
The Truenat™ MTB chip-based Real Time PCR test was able to detect 100% of the
250 100 40
S+C+ samples (40/40), 75% of S-C+ samples (30/40) and gave a negative result for
20
100% of the S-C- samples (20/20).
500 100
0

1000 100
0 100 200 300 400 500 600
CFU/ml
700 800 900 1000 1100
b) Clinical validation 2: validation of EPTB Samples
A retrospective study was conducted at P. D. Hinduja National Hospital and Medical
Robustness: Research Centre, Mumbai. A total of 266 specimens from suspected EPTB patients
To determine whether the Truenat™ MTB chip-based Real Time PCR test showed were analyzed using culture and Truenat™ MTB. EPTB Specimens
any signs of carryover between the runs, alternate positive and negatives sputum (Pleural/Peritoneal fluid, lymph node aspirate, Abscess, Bronchoalveolar lavage,
samples were extracted and further tested the same by PCR. 20 positive samples Biopsy and Tissues) obtained from patients were processed using Trueprep® MAG
and 20 negative samples were used for the study. The Truenat™ MTB test did not Sample Prep Device followed by PCR on Truelab™ Uno real time micro PCR
exhibit detectable carryover from positive to negative samples. analyzer using Truenat™ MTB chip-based Real Time PCR test. Sensitivity and
specificity for each sample type was calculated taking Culture as ‘Gold Standard’. The
Reproducibility: results are tabulated below:
The purpose of this study is to compare the functional performance of the TruenatTM
Sample Lymph Tissue
MTB assay using three different titres of samples on Truelab™ Uno Dx real time type/No.of BAL Pleural/
Abscess Biopsy node
Peritoneal Fluid
micro PCR analyzer. High, Medium and low titre samples were extracted on Samples (n=27) (n=57) (n=81) (n=49) (n=32) (n=20)
Trueprep® AUTO sample prep device and tested among three different users(Inter
user), on three different devices(Inter device) and on 5 consecutive days(Inter day) Sensitivity 84.62% 44.44% 82.22% 100% 71.43% 83.33%
to check the variability. Mean %CV values for all titres has been calculated for Inter
User (1.53), Inter day(1.74) and Inter Device (1.02) which were in the accepted range
Specificity 71.43% 89.58% 69.44% 93.33% 77.78% 92.86%
of ≤15% CV for TruenatTM MTB assay.
 Sensitivity and specificity for all sample types combined was calculated against (2013). Rapid Diagnosis of Mycobacterium tuberculosis with Truenat MTB: A Near-
culture and found to be 78.00% and 84.07% respectively. The results are tabulated Care Approach, PLoS ONE, 8(1): e51121.doi:10.1371/journal.pone. 0051121.
below: (10) Nikam C, Kaz Mi, Nair C, Jaggannath M, Manoj M, et al. (2014). Evaluation of the
Overall (All Sample Types) Indian TrueNAT micro RT-PCR device with GeneXpert for case detection of
pulmonary tuberculosis. International Journal of Mycobacteriology, 3(3):205-210,
Culture +Ve Culture -Ve doi: org/10.1016/j.ijmyco.2014.04.003.
Truenat MTB +Ve 71 28 (11) Lee DJ, Kumarasamy N, Resch SC, Sivaramakrishnan GN, Mayer KH, Tripathy
S, et al. (2019). Rapid, point-of-care diagnosis of tuberculosis with novel Truenat
Truenat MTB -Ve 20 147 assay: Cost-effectiveness analysis for India’s public sector. PLoS ONE 14(7):
e0218890. https://doi.org/10.1371/journal.pone.0218890
c) Clinical validation 3:
A panel of 30 samples comprising of 10 known positives and 20 known negative
sputum samples were tested on three different manufacturing lots of TruenatTM
MTB assay at National Institute for Research in Tuberculosis, Chennai against
WHO approved system as comparator. DNA from 30 sputum samples were
extracted using Trueprep® AUTO Universal Cartridge Based Sample Prep Device.
The elutes were run in parallel on three lots of TruenatTM MTB chips.
Specificity: All 20 negative samples by comparator assay were also found to be
negative by the three lots of TruenatTM MTB assay, showing 100% specificity.
Sensitivity: All 10 positive sample results were correlated between the method
giving a sensitivity of 100% for the three lots of TruenatTM MTB assay.
Concordance: The results obtained by TruenatTM MTB assay on the10 positive
samples were compared with the results obtained by the comparator assay. This
showed good performance and 100% concordance with the WHO approved
comparator test for all three lots on the tested panel of samples. Mean Standard
deviation of Ct values across the 3 lots for MTB target was 0.45 well within
acceptable Ct variation of 1.66 indicates (0.5 log) showed good performance and no
significant lot to lot variation.

d) Clinical validation 4:
A study evaluating the performance of TruenatTM MTB assay was conducted at
National Reference Laboratory of Mycobacteriology, Tunisia. A total of 114
specimens (91 sputum and 23 extrapulmonary specimens) were collected from
suspected TB patients were analysed. Out of 91 sputum samples, 25 sputum
specimens from patients with other lung infections were taken as negative controls.
All collected specimens were analysed using smear microscopy, culture,
comparator PCR assay and TruenatTM MTB and TruenatTM MTB-RIF assay.
Results were compared with a composite reference standard (CRS) comprising
microbiological test results, clinical and radiological findings and patient history to
assess the performance of the TruenatTM MTB assay.
Performance (% of cases detected) of molecular tests in various specimen
categories:

Test S+ (N=46) C+ (N=49) S+C+ (N=46) S-C+ (N=3)

Comparator assay 100 95.9 97.8 66.6

Truenat MTB 100 100 100 100

(S: Smear, C: Culture)
Sensitivity: The TruenatTM MTB chip-based Real Time PCR Test was able to detect
all of the S+C+ specimens (46/46) and S-C+ specimens (3/3), showing 100%
sensitivity.
Specificity: All 48 CRS negative specimens were also found to be negative by
TruenatTM MTB assay, showing 100% specificity.
Rifampicin Resistance: 4 strains are resistant to rifampicin by proportion method
and comparator assay, 3 strains are resistant by TruenatTM MTB-RIF and for one
strain, the test is indeterminate as quantity of specimen was not enough.

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