Performance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Validation Report er’ sy oS) & 3 g { 5 ‘ 3 e, 3 & INDIAN COUNCIL OF MEDICAL RESEARCH (2 Yo, Validation performed by: Dr. Shantala G.B. Principal Investigator, State Level Virus Research & Diagnostic Laboratory, Bangalore Medical College & Research Institute, KR. Road, Fort, Bengaluru -560002. India State Level VRDL, BMCRI, Bengaluru Page 1Performance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Contents Background... Study Details uw. Test name & manufacturer. Description of test and devices.. Objective 1: Evaluation of analytical sensitivity of test ‘A. Trueprep Auto extraction and serial dilutions. (Ct values of control and target). B. Linearity of assay (Trueprep Auto extract). C. Qiagen extraction and serial dilutions (Ct values of Control and Target) Objective 2: Evaluation of repeatability of test (Precision) Objective 3: Clinical Sensitivity sms Clinical sensitivity. Objective 4: Clinical specificity. A. Cross reactivity to H1N1 samples. B. Cross reactivity to Severe Acute Respiratory Illness (SARI) samples... 1 C. Evaluation of blood samples. Objective 5: Evaluation of efficiency of extraction system (Trueprep Auto). Objective 6: Parallel runs to VRDL on Covid-19 suspected samples, Summary of analysis: Clinical sensitivity and Specificity. Analysis templat Diagnostic performance: lin Overall concordance: Annexure I: Representative amplification plots of Truenat Beta coV Annexure Il: Raw data (Truenat BETA CoV) Annexure Ill: Raw data Comparison of Truenat BETA CoV with Gold standard... Recommendatio1 21 SaaS State Level VRDL, BMCRI, BengaluruPerformance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Background In the backdrop of a surge in cases of Corona Virus Disease -19 (COVID-19) in India and with over 150 affected countries across the globe, the rapid diagnosis of cases is being considered as a major tool towards the containment of cases. Till date Real time Polymerase Chain Reaction (RT-PCR) has been globally accepted as the gold standard for detection of SARS CoV-2, the causative pathogen of COVID-19. Currently, Real time RT-PCR is being performed in testing sites identified by Indian Council of Medical Research (ICMR) across the country using kits supplied by National Institute of Virology (NIV), Pune. A rapid and sensitive molecular assay is the need of the hour. ICMR-NIV accepted this challenge and took up the task of validation of a point of care (POC) assay for the rapid detection of SARS CoV-2 virus. A rapid point of care assay for the diagnosis of COVID-19 has been developed by Molbio Diagnostics Private Limited; the Truenat Beta CoV, which is expected to shorten the turnaround time of reporting of results and also can be used for field investigations of covID-19. Benefits of rapid PoC Test: = Case confirmation in field settings would guide early public health interventions like contact tracing, isolation of case, prophylaxis, etc. On the spot case confirmation of SARS- CoV-2 would improve case management. = Testing of a single sample starting with RNA extraction till getting Real time results takes only about 15 hrs time = No need to prepare the Master mix prep, No need of clean hood requirement = Minimal training required for field testing, Light and portable - ideal for field settings. Objective: To validate the performance of Truenat Beta CoV (Molbio) point of care diagnostic kit for detection of SARS CoV-2 infected cases with respect to the following: * Sensitivity * Specificity * Cross reactivity * Inter-machine variation Methodolo; The rapid point of care assay devised by MolBio was prospectively validated at State Level Virus Research & Diagnostic Laboratory (VRDL), Department of Microbiology, Bangalore Medical College & Research Institute, Bengaluru under technical supervision by National Institute of Virology, Pune. Real Time PCR was considered as the gold standard against which the rapid assay was validated for all samples tested based on the following diagnostic parameters: reproducibility, precision, sensitivity & specificity. The diagnostic parameters are explained in the subsequent pages. State Level VRDL, BMCRI, Bengaluru Page 3 QkPerformance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Study Details Validated by :Dr. Shantala G.B, Bangalore Medical College & Research Institute Protocol prepared by _ : State Level VRDL, BMCRI, Bengaluru Protocol approved by _ : Indian Council for Medical Research & NIV, Pune Date +34 April, 2020 Test name & manufacturer Test name: Truenat™ Beta CoV Product code: REF 601410005/601410020/60141 Test license MFG/TL/IVD/2020/000046 Lot number ECVO2 Manufacturer: Molbio diagnostics Pvt Ltd, Verna, Goa. Devices used: ‘Truelab Quattro [TLQU 001] Truelab Duo [TLDU 381] Truelab Duo [TLDU 431] Truelab Duo [TLDU 404] ‘Trueprep Auto [17100493, 16100206, 16100173] State Level VRDL, BMCRI, BengaluruPerformance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Description of test and devices Truenat™ Beta CoV is a chip-based Real Time Reverse Transcription Polymerase Chain Reaction (RT PCR) test for the semi- quantitative detection of Beta Coronavirus RNA. Intended use includes detection and screening of Beta Coronavirus (Sarbeco taxonomy), using nucleic acids purified from human specimens- nasopharyngeal and oropharyngeal swabs. The Truelab™ Real Time micro PCR System is achieved through a combination of lightweight, portable, mains / battery operated Truelab™ Real Time micro PCR Analyzers and Trueprep® AUTO Universal Cartridge based Sample Prep Device and room temperature stable Truenat™micro PCR chips and Trueprep® AUTO Sample Prep kits. 1. RNA Extraction system: Trueprep AUTO: The system consists of a portable RNA extraction machine which uses a universal cartridge based sample Prep kit. One sample can be extracted at a time. The extraction process takes approximately 20 minutes, 2. Real time RT-PCR system [Truelab]: The system consists of a portable real time RT-PCR device which uses beta coronavirus detection specific chips. The Teaction can be performed in approximately 45 minutes. Three types of machines are available: . Truelab Uno Dx: Only one sample can be processed ata given time . Truelab Duo: Two samples can be processed simultaneously . Truelab Quattro: Four samples can be processed simultaneously Ofthese, Truelab Duo & Quattro was validated at State Level VRDL, BMCRI, Bengaluru. State Level VRDL, BMCRI, Bengaluru Page 5Performance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Objective 1: Evaluation of analytical sensitivity of test Evaluation of analytical sensitivity of True Nat SARS CoV-2 qPCR assay, in comparison to ‘TaqMan SARS CoV-2 qRT-PCR VRDL assay was performed. Sample with low Ct value (ID 613) was used for this study. An aliquot of VTM of sample ID 613 was extracted using Trueprep Auto (As per manufacturer protocol) and also by manual RNA extraction kit (QIAGEN QiAmp viral RNA extraction kit - mini prep). RNA was diluted 10 fold from 10 -1 to 10 -66 dilutions (1:10) were made from both Trueprep elute and Qiagen elute. Note: Strong Positive: Low Ct value: 20 +/- 1.5; Medium Positive: Medium Ct value: 29+/- 1.5; Weak Positive: High Ct value: 32 +/- 1.5 VIM sample ies, i cae | ! | Extract by Extract by Trueprep Auto Qiagen | Make 6 serial cilutions Make 6 serial dilutions lee T~ Ha) Run on Run on Runon Run on Truenat Beta CoV VRDLrealtime PCR Truenat Beta CoV —_VRDL real time PCR Figure 1: Analytical sensitivity study method Both dilution series were run on Truenat Beta CoV chips as well as TaqMan SARS CoV-2 RT-PCR system in parallel. Observed Ct values are given in below tables. A. Trueprep Auto extraction and serial dilutions. (Ct values of control and target)\ B. Table 1: Serial dilutions from Trueprep Auto extraction ‘ ‘Truenat BETA CoV ‘SARS CoV 2 real-time PCR Dilutions RNAseP Egene RNAseP Egene 613 Neat 21.33 154 2894 21.45 613 D1 25.14 188 31.65 22.91 61302 28.14 21.8 3528 26.63 6133 31 25.75 "39.19 (ve) *36.39(-ve) 6134 ND 29.33 ND ND 61305 ND 31.33(+ve) ND. ND 613 D6 ND ND ND. ND —_—————————— State Level VRDL, BMCRI, BengaluruPerformance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Conclusion: Truenat Beta CoV detected E gene target up to dilution 10 (D5) from undiluted sample, with valid Ct value while real-time RT-PCR could detect up to dilution 103 (D3) as per criteria for E and Rnase P gene. Sensitivity of Truanat Beta CoV detected E gene two logs more. C. Linearity of assay (Trueprep Auto extract) Using the dilution series from Trueprep Auto elutes run on Truenat Beta CoV, loglinear curve was plotted to check the linearity of Ct values on Truenat Beta CoV test. © Serigst,4, ‘$f assert, 2, 2933seriest, 3, 27 5seriest, 4, = A8Seriest, 5, @8Seriest, 6, 15.4 y=-3.2911x+35,254 RE=0,9944 ctValue Dilutions (log) Figure 2: Linearity and PCR efficiency on Truenat Beta CoV. Y axis indicate Ct values and X axis is arbitrary log numbers indicating dilutions. Conclusion: Slope of the curve is -3.298, Assay was observed to be linear over the range of dilutions tested and PCR efficiency was found to be 101.3%. D. Manual extraction and serial dilutions (Ct values of Control and Target) Table 2: Serial dilutions from manual extraction ‘Truenat BETA CoV SARS CoV? real-time PCR itions: RNAseP | Egene RNAseP | Egene 613NeatBMG | _ND. ND 27.37 21.01 613 D1 BMC 23.5 1633 3112 21.58 613 D2. BMC 33 | 20.17 35.63 25.21 613 D3 BMC 43 245 39.21 31.68 eispspmc | 33:38 | 2829 | *39.38(-ve) ND 613 D5 BMC ND 316 ND ND 613 D6 BMC ND ND ND ND State Level VRDL, BMCRI, Bengaluru Page 7Performance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Truenat Beta CoV detected E gene target upto dilution 10- (D4), with valid Ct value. Neat sample could not be detected in this case, for both target and control (repeated once). IVT RNA Dilutions IVT RNA received from ICMR-NIV, Pune was given with dilution of 10°, From this, serial dilutions of IVT RNA was done and compared on both Truenat Beta CoV and Realtime PCR. Table 3: Serial dilutions of IVT RNA Truenat BETACoVgene | Realtime Egene (cy ( Dilutions Runt Run2 Runt Run2 10° (105 Copies) 21.29 21.75 26.98 26.90 10° (10* Copies) 25 25.5 35.67 36.08 107 (108 Copies) 284 28.67 ND ND 10° (100 Copies) 326 335 ND. ND 10° (10 Copies) ND ND ND ND Conclusion: The In-vitro transcript RNA was diluted and used for E gene testing and showed better sensitivity with MolbioTruenat BETA CoV E gene in comparison of Real Time RT-PCR assay. Objective 2: Evaluation of repeatability of test (Precision) /Inter-machine variations Repeatability of PCR test is essential to ensure assay reproducibility & reliability. Three clinical elutes representing High, Medium and Low Ct values {Sample IDs: 522 (Ct: 30.24), 1304 (Ct: 24.19), 1342 (Ct: 20.97)} were run on all 4 PCR devices used in this evaluation. Following table depicts the Precision analysis. Ct values for E gene are given in table below, with observed standard deviation and % CV. Table 4: Precision of Truenat BETA CoV test. ‘Truenat BETA CoVE gene Equipment ID (ct) 522 | 1304 13420 | TLDU 431. 15.5 20.83 | 22.43 Tipu 404 | 15.33 | 206 22.33 | __ TLDU 381 15.6 | 21.17 22.6 TLQU 001 16.4 22 22.43 | Mean 15.71 | 2115 22.45 STDEV 047 0.61 O11 wov 3.0% | 2.9% 0.5% State Level VRDL, BMCRI, Bengaluru Page 8Performance evaluation of Truenat™ BETA CoV test on Truelab™ workstation The test was found to be reproducible with percent coefficient of variation in Ct values significantly less than 10%, across samples. Objective 3: Clinical sensitivity Clinical sensitivity was tested by running confirmed positives samples of SARS-COV-2; representing high, medium and low Ct value sam| ples for testing and comparison with both the system Table 5: Comparison of clinical positives ‘Truenat BETA CoVE gene SARS CoV 2 Egene (ct) real-time PCR Sample 1D | Date RNaseP |Egene | Date | RNAseP | Egene 1301_| 31-03-2020 | 25.33 | fisg | 16-03-2020 | 3114 | i998 c834___[ 31-03-2020 | 21.33 30-03-2020 | 28.67 | 19.34 1342 | 31-03-2020 | 24.33 17-03-2020 | 2620 | 2097 c3e3__ [31-03-2020 | 26.29 21-03-2020 | 31.36 c375_ | 31-03-2020 | 29.33 22-03-2020 | 34.48 coaa | 31-03-2020 | 23.33 27-03-2020 | 33.09 csz1___| 31-03-2020 | 25.29 26-03-2020 | 35.14 522 | 31-03-2020 | 295 26-03-2020 | 32.33, co33__| 31-03-2020 | _27 27-03-2020 |_34.77 cesi__| 31-03-2020 | 24.17 27-03-2020 | 29.60 Conclusion: 100 % concordance was noted with real time RT-PCR and Truenat BETA CoV method of screening for positive samples. State Level VRDL, BMCRI, Bengaluru Page 9Performance evaluation of Truenat™ BETA CoV test on Truelal Objective 4: Clinical specificity. b™ workstation To evaluate clinical specificity, known positive samples of H1N1, Severe Acute Respiratory Illness (SARI), and also negative blood samples were used. A. Cross reactivity in suspected H1N1 samples Cross reactivity was evaluated by testing 15 clinical specimens RNA which was previously tested for H1N1 at VRDL lab. This panel includes 5 (H1N1) positives and 10 (H1N1) negatives. Data on testing these samples are given in table below: Table 6: Data on analysis of HiN1 positive and negative elutes. ‘Truenat BETA CoV ‘SARS CoV 2 real-time PCR NAS ie Sample | mans resue | Hand RWseP en Ran 1 Run 2 1D Rwaser | Waser | cP | gene asap tees] RNASE gene H1167 ND 22.29 ND 27.24 ND 27.77 ND mais [ND 25 ND__| 3002 ND 3101 ND #1187, ND 26.2 ND 31.19 ND 32.92 ND wizse | _ND 2133 | ND] 2604 ND 26.62 ND mazes | Petesed Tosa | np 2a ND 20.04 ND [aiaei_ [xp a4 ND 3a ND 35a ND 1202 | __ND 1929 | _No_| 2685 ND 2425 ND 1298, ND 27.14 ND 32.25 ND 33.23 ND i319 |__Np 2513 | ND] 3095 ND 3179 ND 1343 | __ND 25 ND_| 2975 ND 2055 ND. #1348 ND 23.4 ND 28.04 ND 29.38 ND ucazo | Peteted | ag xo | 3053 xo | 3007 ND vee | Peteted | 256 np | 3225 xp | 2894 xp Hoag | Deed | sss ND 30.83 ND 299 ND Detected oan | Petect 236 | np | 2653 xp | 2923 ND 776 ND 26.8 ND 3850” ND 35.45 ND 77_| ND 286 ND_| 3433 ND. 3125 ND 7@2_[ ND 268 ND 3524 ND 27.88 ND 783 ND 25.5 ND. 31.25 ND 32.09 ND 784 ND 248 ND 27.88 ND 30.41 ND State Level VRDL, BMCRI, Bengaluru Page 10Performance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Conclusion: No cross reactivity was observed with H1N1 positive and negative clinical specimens. B. Cross reactivity to Severe Acute Respiratory Illness (SARI) samples, This panel includes clinical samples from patients presented with severe acute respiratory illness. These were earlier tested and confirmed negative for SARS CoV-2 and H1N1 diseases, ‘Table 7: Cross reactivity data on SARI samples Truenat BETA CoV E and SARS Cov 2 reaktime Pom nee 4 gene cont, Run2 Semple Date ae Egene oe : ES Date RNAseP | Egene e vos | 28082000 | 9475 a 3099 | ND | o/o4ra02 a oe coe |24082020] 5, a 30.50 | ND eee per) NB can_[3#082000 | 4, ia 3a02 | ND | o1/047202 pa Ht m2_|[2#082000| ong = 27.17 | ND 1/047 = in Conclusion: 1. _ No cross reactivity was observed with SARI suspected clinical specimens but RNAseP worked with both the assay. HL Egene was not detected in any of the H1N1 positive/negative (n=15) and SARI samples (n=4) by both methods, C. Evaluation of blood samples Six blood samples (3 samples from SARCOV-2 positive cases and 3 from SARCoV-2 negative cases) total RNA were extracted using Trueprep Auto and run on Truenat Beta CoV. Results are compared to Ct values from manual extraction and real-time PCR assay. State Level VRDL, BMCRI, Bengaluru P;Performance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Table 8: Cross reactivity data on blood specimens Truenat BETA CoV E and | RNAse P gene SARS CoV 2 real-time RT- (ct) _ PCR Sample ID Date RNAseP | Egene Date RNAseP | Egene 31-03- 01-04- 904569 zo20 | 2014 | no | 2020 | 2646 | ND 31-03- 01-04- 904581 2020 20.29 ND. 2020 25.34 ND 31-03- 01-04- 904608 2020 19.6 ND 2020 26.26 ND 31-03. 01-04- SL 2020 23.2 ND 2020 28.45 ND 31-03- 01-04. S2 2020 21.5 ND 2020 26.19 ND 31-03- 01-04- $3 2020 22 ND 2020 27.59 ND Conclusion: E gene was not detected in all the samples by both methods which show 100% concordance. Objective 5: Evaluation of efficiency of extraction system (Trueprep Auto) Five positive and negative Throat swab samples were extracted using Trueprep Auto system and these RNA elutes were run on Truenat Beta CoV. Results are compared with that obtained from manual extraction method and real-time PCR assay. Table 9: Cross reactivity data on SARI samples Truenat BETA CoV CoV Eand i RNAse P gene (ct) SARS CoV 2 real-time PCR Sample 1 Date RNAseP_| Egene | Date RNAseP_| Egene C883 (-Vesample)_| 31-03-2020 | _ 22.33 np__| 01-04-2020 | 27.46 ND 884 (-Vesample)_| 31-03-2020 | 224 np__| 02-04-2020 | 28.42 ND. 01-04- 885 (+Ve sample) | 34-03-2020] 9, 67 17 2020 2886 | 19.32 886 (-Ve sample) | 31-03-2020 | 26 no__| 01-04-2020 [32.11 ND. C887 (-Vesampley | 31-03-2020 | 27.29 np | 01-04-2020 | 32.22 ND C869 (-Vesample)_| 31-03-2020 28 Np__| 01-04-2020 | 34.05 ND. 834 (+Ve sample) | 31-03-2020] 23.14 | 17.6 | 30-03-2020 | 2867 | 1934 1304 (+Ve sample) | 31-03-2020| 27.17 | 20.6 | 16-03-2020 | 34.56 | 24.19 1342 (+Ve sample) | 31-03-2020 [ 24 2243 | 17-03-2020 | 2620 _|_20.97 C450 (+Ve sample) | 31-03-2020 23.17 18.33 _| 23-03-2020 26.90 22.20 Conclusion: The results were found to be concordant. State Level VRDL, BMCRI, Bengaluru Page 12Performance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Objective 6: Parallel runs to VRDL on Covid-19 suspected samples. ‘Ten throat samples in virus transport medium were extracted by Qiagen RNA extraction method and this RNA was compared at both the platform of Truenat Beta CoV and real-time PCR assay. ‘Table 10: Data on parallel runs to VDRL - Qiagen RNA extracted Truenat BETACOVE | SARs Cov2 real-time Sample and RNAse P PCR 1D gene (Ct) Fae | see | RMAseP | Egene 830 22.14 ND. 32.26 ND 831 274 ND 30.08 ND 837 27.8 ND 34.86, ND 838 23.6 ND 30.01 ND 339 | 2343 | ND | 3013 ND. 840 23 ND 29.79 ND ga | 22.29 | np | 29.24 ND 842 20.6 ND 2742 ND 843 25.17 ND 3118 ND sea | 2417 | np | 2934 ND Conclusion: Ten freshly received Covid-19 suspected were run in parallel to VRDL. These were extracted on Qiagen RNA extraction kit and run on Truenat Beta CoV chips. Both showed RNAse P data in concordance and no reactivity for E gene. ‘Table 11: Data on parallel runs to VDRL - Trueprep AUTO extracted ‘Truenat BETA CoV E and an RNASE P gene (60 SARS CoV 2 real-time PCR D Date RNAseP | Egene Date RNAseP_ | Egene [03 [31-03-2020 | 22.33 np __| 01-04-2020 | 27.46 ND ge4_| 31-03-2020 | 224 np___| 01-04-2020 | 28.42 ND gg6 | 31-03-2020 26 np__| 01-04-2020 | 32.11 ND g7_|_31-03-2020 | 27.29 np | 01-04-2020 | 32.22 ND caso | 31-03-2020 | 28 np__| 01-04-2020 | _34.05 ND Conclusion: Five freshly received samples were extracted on Trueprep AUTO and run on ‘Truenat Beta CoV chips. Both showed RNAse P data in concordance and no rea gene. State Level VRDL, BMCRI, BengaluruPerformance evaluation of Truenat™ BETA CoV test on Truelab™ workstation Summary of analysis: Clinical Sensitivity and Specificity In this study totally 18 confirmed SARS CoV-2 positives and 41 confirmed negatives were tested. Comparison of results from Real Time PCR (BioRad CFX96) and Truenat Beta CoV is summarized in 2x2 matrix table below. Diagnostic performance: ‘SARS CoV 2 real-time PCR (n=64) Positive Negative Total Positive |_18 (TP) | __o(FP) 18 a coves! a CoV T Negative | O(N) | 46 (TN) 46 Total 18 46 64 Sensitivity: 100%; Specificity: 100%; PPV = 100.00%; NPV=100.00% (n=64) ‘TP=True positive, TN= True negative, FP = False positive, FN = False negative, PPV=Positive Predictive Value, NPV = Negative Predictive Value Clinical Sensitivity: 100%; Clinical Specificity: 100%; Overall concordance: 100% Recommendation: ‘© Truenat BETA CoV test, a rapid Point of Care Real Time RT-PCR developed by Molbio Diagnostic Private Limited was evaluated for SARS CoV-2 at State Level VRDL, BMCRI, Bengaluru as directed by ICMR, following the protocol approved by NIV & ICMR. © The test results were found to be 100% concordant with the results of the gold standard (Real Time PCR used at State Level VRDL, BMCRI). © The clinical sensitivity, clinical specificity and overall concordance were determined to be 100%. The PoC test is herewith recommended for screening of SARS CoV 2 using E gene and RNAseP gene for suspected cases. ‘© The test can be deployed at the COVID19 testing laboratories for screening of the suspected cases. However, as per ICMR guideline further confirmation would be needed for screening SARS-CoV-2 by testing RdRp(1), R4Rp(2) and orf1b gene to confirm the case, State Level VRDL, BMCRI, Bengaluru