Available online on 15.09.2025 at ijmspr.com

 International Journal of Medical Sciences and Pharma Research 

Open Access to Medical Science and Pharma Research

Copyright  © 2025 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

 

Performance Evaluation of Truenat rtPCR assay with COBAS -6800 in SARS COV-2 detection: A diagnostic accuracy study

Arindam Chakraborty *¹, Monica Singh ², Amod Kumar ³

¹ Associate Professor. Department of Microbiology. Autonomous state Medical College, Kaushambi, Uttar Pradesh, India 

² Associate Professor, Department of Microbiology. Motilal Nehru Medical College. Prayagraj, Uttar Pradesh. India 

³ Associate Professor. Department of Pathology. Nalanda Medical College, Patna. Bihar. India

 

 

INTRODUCTION:

The SARS-COV-2 (COVID-19) pandemic has faced significant diagnostic challenges worldwide ¹ and showed serious concern for the healthcare system. The detection of SARS-COV-2 in the laboratory by rtPCR is the gold standard method in the acute phase of infection.² At the beginning of the COVID-19 pandemic, a molecular diagnosis such as rtPCR was very limited in developing countries especially in India because of the requirement of infrastructure and trained manpower. To control the spread of the infection there was a need of high surveillance testing in the field hence there were requirements for rapid and simple assays with high sensitivity and specificity. To overcome the situation govt of India and ICMR validated and approved many newer diagnostic tools and kits for early detection. One such rapid point of care test was TrueNat assays which target the Beta CoV E gene and SARS-COV-2 Rdrp gene and can be installed in limited settings.  The study aimed to find out the performance of TrueNat assay with a highly sophisticated close system automatic rtPCR system known as cobas 6800 (Roche Molecular Systems, Pleasanton,CA, US)

SUBJECTS AND METHODS:

Study setting and population: This cross-sectional study was conducted during the period from March   2021 to May 2021 in a state-level BSL-3 reference laboratory in central India. The study was conducted after obtaining the permission from Institutional ethical committee. A total of 250 non-repeated patients of all age groups who were admitted to the triage ward with symptoms of fever breathlessness, SpO2<95%, pain in the chest, seizures, weakness, or numbness on the face were included in the study.

Sample collection and transportation to Lab: Oropharyngeal and nasopharyngeal Samples were collected in a viral transport media as per the guideline provided by ICMR. All these samples were packed and transported in triple-layered to the lab in cold chain as per guidelines provided by ICMR.³

Sample processing: All the samples were processed in the BSL3 labs with standard precaution and the sample was stored at -80 ^o c for further analysis. For TrueNat analysis, specimens are transferred into the cartridge provided by the manufacturer and processed in class II biological safety cabinet.⁴

Ribonucleic acid extraction:

COBAS 6800:A total 600 of µl of each sample was collected in a barcode label test tube provided by the manufacturer. Then the sample was transferred to the sample supply module from where it is automatically transferred to its processing module and nucleic acid extraction was performed automatically.⁵

TrueNat: For TrueNat analysis 500µl specimens are transferred into the cartridge and inserted into the extractor machine and waited for 20 min, the extracted RNA was collected in a sterile container provided by the manufacturer for further analysis.^1,4

Nucleic acid amplification: For detection of SARS-COV-2 in cobas 6800 two specific genes of the virus were targeted, containing ORF1 a non-structural region (target-1), and a conserved region in the structural protein envelope E gene (target-2). The test utilizes RNA internal control for sample preparation and PCR amplification.  

In TrueNat 6µl of extracted RNA is added to a PCR tube consisting of real-time PCR reagents and the mixture is added into a disposable microchip containing the Beta CoV E gene and SARS-CoV-2 Rdrp gene. The loaded microchip was inserted into the amplifier machine for 40 minutes, and a total of 40 cycles were run for viral RNA amplification.

 Interpretation of results: After two and half hours of process, the results were displayed in the monitor for positive results both ORF-1 (target-1) and E (target-2) should be amplified or only the ORF 1 gene amplification was considered. In the case of positivity for the E gene only (target2), the result should be reported as SARS-COV-2 presumptive positive.

RESULTS:

Of the total of 250 samples, 122 were positive by both genes (ORF and E gene) in cobas 6800. Of the 122 positive samples, 68(55%) were male and 54(44.2%) as female, and the mean age group of ±49. Among the positive patients on analysis of clinical symptoms fever with mean body temperature 100⁰c± 1 followed by dyspnoea 25%, Breathlessness (13%), Spo₂(<75%) in 25% of patients. On Hematological analysis of severe patient TLC, Neutrophil was high in 35% of the patient along with the higher value of Hb and D dimer and a decrease in lymphocytes.

 On analysis of Ct value in cobas 6800 it is found that high Ct values (10-20) were observed in 20 patients. Similarly, in 28 patients the range of Ct value was (21-24). 74 patients have lower Ct values with the range from 25-37. ( Table 1)

In TrueNat assay out of 122 samples, 16 samples were detected High. Twenty-six samples have medium value and 6 samples that were above cutoff value were shown positive in cobas 6800. The details of Ct value in cobas and TruNnat results are summarised in table no 1.

 

 

Table 1: Comparison of COBAS 6800 positive samples with Truenat assay.

 

 

DISCUSSION:

To control the pandemic, there is a necessity for mass testing, and accurate and timely diagnosis of SARS-COV-2, for that ICMR has recommended a portable point of care  TrueNat rtPCR system for the detection of SARS-COV-2. Which can be installed and performed the test with limited manpower and setting.^6,7

 In the present study, we have found one in two patients visited in the Triage area during the pandemic were positive for SARS-CoV-2 and need hospitalization. This is in concordance with the study done by B. Ajayi et.al.⁸ where they found a higher rate of hospital admission among COVID-19 patients. 

In our study population, we found that age was an important risk factor for susceptibility to infection with SARS-COV-2. Patients with a mean age group ± 49 years were more prone to the infection. This is similar to a multicentric study done by the ICMR COVID-19 group where they reported 50-69 years age group was more prone to the diseases in the Indian population.  However, a study from London has reported elderly patients in the age group >60 years were more vulnerable to the infection and need hospitalization. We have also observed a higher proportion of male patients compared to females (55% Vs 45%). This finding is similar to other studies done by Nigam et al⁹   and Jimeno S et al.¹⁰

We are not the first to observe that fever, breathlessness, dyspnoea, and decrease SpO₂ level were some of the common symptoms in our study subject which is in concordance with the study done by Nigam et al.⁹

On analysis of hematological parameters among the study population, we have observed higher values of Hb, Neutrophil, TLC and lower values of lymphocytes and platelets which are similar to the study done by Sun s et al ¹¹, Chen et al¹², and Toledo S et al¹³. It has been found that leukocytosis, lymphopenia, and thrombocytopenia are associated with greater severity and even fatality in COVID-19 cases.¹³   

In the present study, we have found that the TrueNat system can detect various concentrations of viral load in COVID 19 patients. This finding was in accordance with the study conducted by Sadhna et al.¹⁴ In our previous study we also reported the same where we found TrueNat is   equally effective for the diagnosis of SARS-COV-2 compared to conventional rtPCR assay .¹⁵

On analysis of the cobas 6800 results with TrueNat assay, we found 95% of the cobas 6800 positive samples were also showing positive results with different viral load in TrueNat assay remaining 5%  positive samples of cobas 6800 were displayed negative with above target cutoff (ct >32) in TrueNat device.  On analysis of the sensitivity and specificity of the TrueNat device in compared to cobas 6800 we found 95.5% sensitivity and 100% specificity.

We also observed there were not many differences in the threshold level of cobas 6800 and TrueNat assay. Where we have found 38 samples with a range of Ct Value 25- 29 and 26 samples with ct value of   30-34 in cobas 6800 were shown “detected low” and “detected very low” in TrueNat assay. 

In the further analysis, we observed that nearly (5%) of positive samples in cobas 6800 with the Ct range of 35-37 were negative with the comment of " above target cut off" in TrueNat. Nevertheless, on analysis of clinical parameters, they were found positive based on clinical and radiological findings. It has been reported in the later stage of COVID-19 the viral load used to be very less in the upper respiratory tract sample due to the elimination of the viruses by the immune system. Due to this many molecular techniques were not able to detect the viruses there is a need for repeat testing.

Considering our findings we believe in TrueNat essay there is a need to increase the cut-off value (ct ≤35) so that it can be more effective for the diagnosis of COVID 19 compared to cobas 6800 which is a very sophisticated closed system RTPCR instrument with higher sensitivity and specificity.

Hence, we suggest more TrueNat machines should be installed in all the laboratories, especially in the periphery where there are limited resources for early and accurate diagnosis of covid 19 to reduce the morbidity and mortality of the disease.

Limitation of our study: Our study has a certain limitation, We consider cobas 6800 as 100% sensitive hence we have not validated cobas 6800. We believe more number of samples should be tested for further validation of the assay. 

Acknowledgement: We are grateful to the ICMR. Delhi, India, and the Government of Uttar Pradesh for providing us with the Kit and Infrastructure to conduct the study.

Source of Support: NIL

Conflicts of interest: NIL

Funding: The authors declared that this study has received no financial support.

Informed Consent Statement: Not applicable. 

Ethics approval: Not applicable.

REFERENCES:

1. Tang YW, Schmitz JE, Persing DH, Stratton CW. Laboratory Diagnosis of COVID-19: Current Issues and Challenges. J Clin Microbiol. 2020 May 26;58(6) https://doi.org/10.1128/JCM.00512-20 PMid:32245835 PMCid:PMC7269383

2. Assessing a chip based rapid RTPCR test for SARS CoV-2 detection (TrueNat assay): A diagnostic accuracy study Ghoshal U, Garg A, Vasanth S, Arya AK, Pandey A, et al. Assessing a chip based rapid RTPCR test for SARS CoV-2 detection (TrueNat assay): A diagnostic accuracy study. PLOS ONE. 2021: 16(10). https://doi.org/10.1371/journal.pone.0257834 PMid:34644333 PMCid:PMC8513858

3. Guidelines for Storage Specimens Collected for COVID-19 Diagnosis by RTPCR Platforms in Government Laboratories. Available from: http//www.icmr.gov.in/pdf/covid/labs/Govt_labs_sample_retention_advisory_25062020.pdf  

4. https://www.molbiodiagnostics.com/product_details 

5.https://diagnostics.roche.com/us/en/products/instruments/cobas-6800.html   

6. Basawarajappa SG, Rangaiah A, Padukone S, Yadav PD, Gupta N, Shankar SM. Performance evaluation of Truenat™ Beta CoV & Truenat™ SARS-CoV-2 point-of-care assays for coronavirus disease 2019. Indian J Med Res 2021;153:144 50 https://doi.org/10.4103/ijmr.IJMR_2363_20 PMid:33818471 PMCid:PMC8184085

7. Gupta N, Rana S, Singh H. Innovative point of care molecular diagnostic test for COVID 19 in India. Lancet Microbe 2020;1:e277. https://doi.org/10.1016/S2666-5247(20)30164-6 PMid:33521725

8. Ajayi B, Trompeter AJ, Umarji S, Saha P, Arnander M, Lui DF. Catching the second wave: clinical characteristics and nosocomial infection rates in major trauma and orthopaedic patients during the COVID-19 pandemic. Bone Jt Open. 2021 Aug;2(8):661-670. https://doi.org/10.1302/2633-1462.28.BJO-2021-0078.R1 PMid:34405683 PMCid:PMC8384451

9. Nigam JS, Kumar A, Sinha R, H H, Kumar N, Surabhi, Kumar T, Bharti S, Bhadani PP. Association of Peripheral Blood Parameters With Outcomes of COVID-19 Infection in a Tertiary Care Setting of Eastern India: An Institute-Based Study. Cureus. 2021 Dec 27;13(12):e20745. https://doi.org/10.7759/cureus.20745

10. Jimeno S, Ventura PS, Castellano JM, García-Adasme SI, Miranda M, Touza P, Lllana I, López-Escobar A. Prognostic implications of neutrophil-lymphocyte ratio in COVID-19. Eur J Clin Invest. 2021 Jan;51(1):e13404. https://doi.org/10.1111/eci.13404 PMid:32918295

11.Sun S., Cai X., Wang H., He G., Lin Y., Lu B., Chen C., Pan Y., Hu X. Abnormalities of peripheral blood system in patients with COVID-19 in Wenzhou, China. Clin. Chim. Acta. 2020;507:174-180. https://doi.org/10.1016/j.cca.2020.04.024 PMid:32339487 PMCid:PMC7194694

12. Chen N., Zhou M., Dong X., Qu J., Gong F., Han Y., Qiu Y., Wang J., Liu Y., Wei Y., Xia J., Yu T., Zhang X., Zhang L. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet. 2020;395(10223):507-513. https://doi.org/10.1016/S0140-6736(20)30211-7 PMid:32007143

13. tícia de Oliveira Toledo S, Sousa Nogueira L, das Graças Carvalho M, Romana Alves Rios D, de Barros Pinheiro M. COVID-19: Review and hematologic impact. Clin Chim Acta. 2020 Nov;510:170-176. https://doi.org/10.1016/j.cca.2020.07.016 PMid:32659224 PMCid:PMC7351669

14. Sadhna S, Hawaldar R. Evaluation of truenat RTPCR for diagnosis of SARS CoV2 infection -An observational study.Indian J Microbiol Res 2020;7:265-269. https://doi.org/10.18231/j.ijmr.2020.047

15. Jaiswal A, Singh M, Chakraborty A. Comparative Evaluation of Truenat Reverse Transcription Polymerase Chain Reaction with Commercially Available Reverse Transcription Polymerase Chain Reaction Kits for COVID 19 Diagnosis. Advances in Human Biology 2022;12 (1) :34-37. https://doi.org/10.4103/aihb.aihb_120_21