Assessing a chip based rapid RTPCR test for SARS CoV-2 detection (TrueNat assay): A diagnostic accuracy study.

COVID-19 testing is required before admission of a patient in the hospitals, invasive procedures, major and minor surgeries etc. Real Time Polymerase chain reaction is the gold standard test for the diagnosis, but requires well equipped biosafety laboratory along with trained manpower. In this study we have evaluated the diagnostic accuracy of novel TrueNat molecular assay for detecting SARS CoV-2. TrueNat is a chip-based real time PCR test and works on portable, light weight, battery powered equipment and can be used in remote areas with poor infrastructure. In this study 1807 patients samples were collected for both TrueNat and RTPCR COVID-19 testing during study period. Of these 174 (9.7%) and 174 (15%) were positive by RTPCR and TrueNat respectively and taking results of RTPCR as gold standard TrueNat test showed a sensitivity, specificity and diagnostic accuracy of 69.5, 90.9% and 89.2% respectively. It can be concluded that TrueNat is a simple, easy to use, good rapid molecular diagnostic test for diagnosis of COVID-19 especially in resource limited settings and will prove to be a game changer of molecular diagnostics in future.

Introduction

The on-going COVID19 pandemic; caused by severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) was declared as pandemic on 11.03.2020 by World Health Organization (WHO) [1]. Since then till 20.02.2020 it has affected globally approximately 110 million cases and 2.5 million deaths.

COVID-19 testing is required before admission of a patient in the hospitals, invasive procedures, major and minor surgeries etc. Real Time Polymerase chain reaction (RTPCR) is the gold standard test for the diagnosis, however its turnaround time is 6–8 hours and requires well equipped biosafety laboratory level-II along with trained manpower. In cases where early and rapid diagnosis of COVID-19 is warranted RTPCR testing leads to delay in diagnosis along with tension and anxiety both among patient and treating health care workers. So a rapid, cheap molecular diagnostic test with high sensitivity and specificity is urgently required for detecting SARS CoV-2, especially in developing countries and in rural area where there is poor infrastructure and lack of well-equipped labs [2].

In this study we have evaluated the diagnostic accuracy of TrueNat assay, a chip based rapid molecular diagnostic test for detecting SARS CoV-2. This technology is based on portable, light weight, battery powered, TaqMan probe based Real time polymerase chain reaction technology developed and manufactured by Molbio Diagnostics Private Limited, Goa, India. The TrueNat device can be used for detection of more than 25 pathogens like malaria, tuberculosis, hepatitis B, HIV, dengue, H1N1 influenza, chikungunya, Rabies, Influenza, SARS Cov-2 etc [3]. In 2018 TrueNat technology was adopted by Revised National Tuberculosis Control Programme (RNTCP) for tuberculosis diagnosis in India and in 2020 it was also endorsed by World health organization for diagnosis of Multi drug resistant tuberculosis [4].

This equipment is a laboratory in a suitcase and can be used in remote areas with poor power supply and connectivity. The device has an automated reporting system and is GPRS/Bluetooth enabled, to aid in result data transfer. The TrueNat machine is available in three different models UnoDx, Duo, and Quattro, with capacity to test one, two, and four samples per run, respectively [5]. At our center we have used the Quattro machine, this equipment set cost approximately USD 18,000 and running cost per test is USD 15 only.

In April, 2020 the TrueNat test for diagnosis of SARS CoV-2 was launched by Molbio Diagnostics and was subsequently approved by Indian apex medical research organization—Indian Council of Medical Research (ICMR), New Delhi [6]. The manufacturer claims a sensitivity and specificity of 100% and 98.8% respectively but currently there are no data on diagnostic accuracy in field setting [5]. Thus this study was planned with aims to evaluate the diagnostic accuracy of TrueNat test for the diagnosis of SARS CoV-2 as compared to RTPCR; the gold standard reference test.

Material and methods

Study site and population

This retrospective observational study was designed and conducted at the Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India. This study was approved by the Institutional Ethics Committee (IEC) and being retrospective in nature the IEC waived the requirement for patient consent by approval number 2020-279-IP-EXP-31. All study data was retrieved from hospital information System of SGPGIMS.

The samples were collected at triage of a dedicated COVID-19 tertiary care center with 180 beds including 30 ICU ventilator beds. For TrueNat testing single oropharyngeal swab was collected in viral lysis media (VLM) provided by Molbio diagnostics Ltd, whereas for RTPCR both oropharyngeal and nasopharyngeal swabs were collected in viral transport media (HI media Labs, India) and transported to the COVID-19 laboratory in a cold chain. At time of COVID-19 pandemic peak (August–October 2020) any patient requiring treatment at SGPGIMS, Lucknow had to undergo mandatory COVID-19 testing and all cases in which both samples for RTPCR and TrueNat were collected within time gap of less than 24 hours by treating physician were included in this study.

TrueNat workstation

The TrueNat workstation consist of a nucleic acid extraction device (Trueprep AUTO V2) and a real time polymerase chain reaction analyzer (Truelab Uno Dx/Uno/Quattro), along with accessories such as a RNA cartridges, TrueNat Chips, micro tip holding stand etc. Both devices are portable, powered by a rechargeable batteries and can run continuously for ≥ 6 hours on single charge. Trueprep AUTO V2 is fully automated RNA extractor and uses a disposable fluidic cartridge to extract RNA from VLM within 15 minutes. 6 μL of extracted RNA is added to a PCR tube consisting of room temperature stabilized real time PCR reagents and the mixture is added onto a disposable microchip. The chip is loaded in Truelab analyzer and programme is selected for appropriate assay. COVID -19 testing was done using two step strategy; all samples are initially tested by E gene assay and all positive samples with cut off threshold of <32 are confirmed by RNA dependent RNA polymerase (RdRp) gene assay [6]. All samples that are positive by RdRP assay with Ct value < 32 are considered as true positives. We used Truelab Quattro model in this study. Four samples can be processed at a time taking about 1 hour, including sample preparation. One device can process 64 samples per day in a 24 hour working laboratory.[Figs 1–3]

Fig 1

Heading: Images of consumables used in the TrueNat assay.

Legend: (1a) Viral Lysis media and the sample buffer. (1b) RNA extraction cartridge. (1c) Micro PCR chip.

Fig 3

Heading: Shows the RTPCR graph generated by TrueNat machine.

Legend: 3a shows a negative report with only fluorescence in the Control gene RNase P; 3b shows the fluorescence in the E-gene and control gene; 3c shows fluorescence above the cutoff to RdRP and control gene; 3d shows an invalid test result with no amplification in RNase gene.

Heading: Images of TrueNat equipment.

Legend: (2a) Trueprep AUTO v2 Sample Prep Device. (2b) Quattro Real Time micro PCR Truelab Analyzer.

COVID-19 detection by real time PCR

RNA extraction

RNA extraction was performed on 200 μl of viral transport media using a QIAamp RNA mini kit (Qiagen, Inc., Valencia, Calif.) as per manufacturer’s instructions.

Qualitative real time PCR

A 25 μL reaction was prepared for detection of SARS CoV-2 by RTPCR utilizing 5 μL of extracted RNA, 12.5 μL of 2X PCR buffer, 1 μL of Primer and TaqMan probe sequences targeting E genes, RdRP and RnaseP as per W.H.O protocol [7]. The thermal cycling was performed at 55°C for10 min for reverse transcription, followed by 95°C for 3 min and then 40 cycles of 95°C for 15 s, 58°C for 30s using Quant Studio 5 Real Time PCR system (Thermo Fisher Scientific, Massachusetts USA). All samples were screened for E gene and positive samples were confirmed by detection of specific RdRP gene. Cut off threshold (Ct value) <40 were considered as positive.

Statistical analysis

The sensitivity, specificity, positive predictive value, negative predictive value, diagnostic accuracy and 95% Confidence Intervals were calculated SPSS software version 23.0 (SPSS, Inc., Chicago, USA)

Results and discussion

Among 1807 patients samples for both TrueNat and RTPCR COVID-19 testing were collected during study period. Of these 174 (9.7%) and 174 (15%) were positive by RTPCR and TrueNat respectively. A total of 121 (6.7%) samples were positive by both RTPCR and TrueNat test; 149 (8.2%) samples were TrueNat positive and RTPCR negative, 53 (3.0%) samples were TrueNat negative and RTPCR positive and 1484 (82.1%) were negative. (Table 1) Taking results of RTPCR as gold standard TrueNat test showed a sensitivity, Specificity and diagnostic accuracy of 69.5, 90.9% and 89.2% respectively. (Table 2)

Table 1

Comparison of the positivity rates of TrueNat with RTPCR results.

	RTPCR positive	RTPCR negative	Total
TrueNat Positive	121	149	270
TrueNat negative	53	1484	1537
Total	174	1633	1807

Table 2

Showing statistical analysis of study data.

S No	Statistic	Value	95% CI
1	Sensitivity	69.5%	59.2% - 76.5%
2	Specificity	90.88%	89.3% to 92.3%
3	Positive Predictive Value (PPV)	39.8%	35.6% to 44.3%
4	Negative Predictive Value (NPV)	97.2%	96.5% to 97.7%
5	Diagnostic Accuracy	89.2%	64% to 90.5%

Detailed analysis of 149 samples that were TrueNat positive and RTPCR negative revealed that the mean cut off threshold (Ct value) of E gene among TrueNat positive samples was 27 and in 50 (33%) samples Ct value was high (>30). Similarly detailed analysis of 53 samples that were TrueNat negative and RTPCR positive showed that of these 18 samples were TrueNat positive for E gene with CT value more than 32, but were considered negative as per TrueNat interpretation guidelines. (S1 Data) Overall wastage in TrueNat processing was also calculated and it was found that while processing 1807 samples; 50 RNA cartridges (2.7%) and 18 TrueNat Chips (1%) were wasted.

Detection of SARS CoV-2 by RTPCR is the current gold standard for diagnosis of COVID-19. World health organization has repeatedly stressed the importance of the molecular diagnosis of COVID-19 for prompt management of patients, isolation and contact tracing and limit its spread. Point of care test has been identified by a WHO expert group as the first of eight research priorities in response to the COVID-19 outbreak [8] and play an important role in medical emergencies like myocardial infarction, acute abdomen, emergency surgeries and other medical emergencies where urgent intervention is required.

Accurate and timely results are back bone of decision making, both in the inpatient and OPD settings. Quick turnaround time of test reports is also critical for prudent use of resources, such as the availability of emergency and Triage area beds, isolation rooms and real-time cohorting decisions. However for performing RTPCR testing a fully functional air-conditioned biosafety level-2 microbiology laboratory is required; equipped with specialized instruments like biosafety cabinets class II, automated RNA extractors, Real time PCR machine and trained manpower to process the samples while ensuring biosafety and bio security [9]. It is challenging to set up a fully functional molecular testing laboratory in resource limited developing countries where availability of high end equipments, trained manpower, uninterrupted power supply are a big problem and at such places newer options should be explored.

There is an urgent need for a nucleic acid-based COVID-19 test that is highly sensitive and specific, and can be used at point-of-care in resource-limited settings. In April 2020 Molbio Diagnostics Ltd, Goa, India introduced TrueNat COVID-19 testing; It is portable, battery-operated point of care molecular diagnostic test especially designed for areas with low resources. The device has an automated reporting system and is GPRS/Bluetooth enabled, to aid in result data transfer. The TrueNat manufacturer claims a sensitivity and specificity of 100% with lower limit of detection at 407 copies. Till date there are no field trials of SARS CoV-2 molecular detection by TrueNat; thus this study was planned to evaluate the diagnostic accuracy of TrueNat test for the diagnosis of SARS CoV-2 as compared to gold standard test RT-PCR.

The results of this study showed a sensitivity of 69.5%, Specificity of 90.9%, NPV- 97.2% and diagnostic accuracy of 89.2%. In another recent laboratory based study performed on pre characterized archived samples study by ICMR, New Delhi; 75 samples (30 positives and 45 negatives) were tested with TrueNat for SARS Cov-2 and were found to be 100% sensitive and specific with 100 copies as lower limit of detection [10]. However in this study the researchers used viral transport media consisting of both nasopharyngeal and oropharyngeal swabs instead of recommended viral lysis media containing only oral swab. Further only 30 positive samples were tested and these reasons might have contributed to high sensitivity and specificity of 100%. TrueNat has also been evaluated for other infectious diseases. In a study on human papilloma virus detection in cervical samples TrueNat showed sensitivity and specificity of 97.7% and 98.9%, respectively compared to conventional test. In another study targeting malaria parasite TrueNat was 99% sensitive compared to gold standard microscopy. Recently it has been approved by WHO for testing drug resistant tuberculosis [11-14].

As per Ministry of Health and family welfare, India; currently TrueNat COVID-19 testing is performed at 886 centers across India, 632 in government sector and 254 in private sector. Of these more than 50% machines are installed in remote distant places with poor infrastructure; TrueNat COVID-19 testing is available at remote places like karavati Island, Lakshadweep (500 Kms away from Mainland) and 23 machines are installed in distant north east Indian state of Arunachal Pradesh having a rough hilly terrain [14]. The extensive infrastructure of TrueNat testing has played a key role in control of community transmission of SARS CoV-2. The operational cost of TrueNat (USD 15/Sample) is less compared to Real time PCR and it can provide report in 1 hour compared to 10–12 hour time required in RTPCR. Further the sample is collected in virus lysis media which immediately lyses the infective virus and makes the sample non infectious and removing the requirement of biosafety cabinet and makes it a excellent point of care test having minimal facilities.

There are few limitations of this study. Ideally for evaluating a new diagnostic test it should be performed on same clinical specimens that is tested by reference method; however in this study as per manufacturer’s instructions and ICMR, New Delhi, India guidelines TrueNat testing was performed on virus lysis media containing only Oropharyngeal swab while RTPCR was performed on viral transport media containing both oro pharyngeal and nasopharyngeal swabs. In most of the cases both samples were collected simultaneously however in some cases the time gap of two sample collection was 12–24 hour.

Laboratory testing with real time PCR has the advantage of high throughput processing that cannot be achieved by the TrueNat platform. In RTPCR 96 samples can be processed simultaneously; whereas in TrueNat each RNA extraction unit can process only one cartridge at a time, and maximum 4 samples can be tested simultaneously. However, judicious use of TrueNat testing could relieve the burden on central molecular laboratories and increase overall testing capacity, complementing existing approaches. The TrueNat plays a strategic role in places where results can affect real time decision making such as screening trauma victims requiring emergency surgeries, triaging admissions, maternity labor rooms and screening elective admissions or staff (eg, before dialysis or chemotherapy) etc.

Conclusion

Based on study results it can be concluded that TrueNat is a simple, easy to use, good rapid molecular diagnostic test for diagnosis of COVID-19 and will prove a game changer in molecular diagnostics of infectious disease in future especially in areas with poor Infrastructure.

(XLSX)

Click here for additional data file.

27 May 2021

PONE-D-21-10427

Assessing a chip based rapid  RTPCR test for SARS C0V-2 detection (TrueNat assay): a diagnostic accuracy study.

PLOS ONE

Dear Dr. Garg,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 11 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Seyed Ehtesham Hasnain, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

and

2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

The name of the colleague or the details of the professional service that edited your manuscript

A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

3. We note that Figure 1 in your submission contains copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (a) present written permission from the copyright holder to publish this figure specifically under the CC BY 4.0 license, or (b) remove the figure from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b.  If you are unable to obtain permission from the original copyright holder to publish this figure under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

Additional Editor Comments:

Major Revision

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments:

Present study of Ghoshal U et al. is based on diagnostic approach to encounter present pandemic globally. It is a comparative study between to molecular diagnostics and more informative while testing COVID 19 virus. However, manuscript is written in a very casual approach.

Abstract:

• Abbreviation of PCR should write at proper sections (Line no. 30, 31, 33, 35).

• Author can modify the Conclusion section.

Introduction:

• Advised to write the current global as we as India epidemiological data (Line no. 46, 47).

• It is advised to authors to modify the sentence, as it seems duplicated from abstract section (COVID-19 testing is required before admission of a patient…..).

• Advised to write the updated name of RNTCP.

Material and Methods:

• Author should write the abbreviation RTPCR trough out the manuscript (WHO, OPD etc).

• It is advised to write abbreviation in a single way (line no. 46, 47, 129, 130).

• Write NPV and PPV in statistical analysis section also.

Results and Discussion:

• It is advised to author that please recheck the percentage as well as sample number before writing the result.

• Don’t use full stop sign (.) within a single sentence (Line no. 166-169).

• It is advised to avoid the duplication of sentences (Line no. 181- 190).

• Modify the sentence (Further only 30 positive samples were tested and these reasons might have…..).

• Reference should write as per the journal guideline.

• Reference required for actual Operational cost for TrueNat test and RTPCR.

Reviewer #2: This study is in time and address issue of quick and handy detection of the samples which can be helpful in accessing the infection rate and frequency. However i have one major concern which need clarification from author .

Whether author compared the fidelity of this assay with the RAT ( rapid antigen test ) , how correlative and reliable is this assay. Whether the author used simple PCR or modified nested one. Can we identify mutated strain also in the patients.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

19 Jul 2021

POINT WISE ANSWERS TO REVIEWER COMMENTS

Reviewer 1:

1. Abbreviation of PCR should BE written at proper sections (Line no. 30, 31, 33, 35).

Answer: We have corrected the manuscript as suggested

2. Author can modify the Conclusion section of abstract.

Answer: We have modified the conclusion in abstract.

3. Advised to write the current global as well as India epidemiological data (Line no. 46, 47).

Answer: We have updated the global data and added Indian data as per your suggestion.

4. It is advised to authors to modify the sentence, as it seems duplicated from abstract section (COVID-19 testing is required before admission of a patient…..).

Answer: We have modified the sentence in revised manuscript.

5. Advised to write the updated name of RNTCP.

Ans: Name updated as National Tuberculosis Elimination Programme.

6. Author should write the abbreviation RTPCR trough out the manuscript (WHO, OPD etc).

Answer: We have incorporated this change in revised manuscript.

7. It is advised to write abbreviation in a single way (line no. 46, 47, 129, 130).

Answer: We have taken care to write abbreviation in single way in revised manuscript.

8. Write NPV and PPV in statistical analysis section also.

Answer: We have incorporated this change in revised manuscript.

9. It is advised to author that please recheck the percentage as well as sample number before writing the result.

Answer: Thank you for pointing out the typing mistake. We have corrected the data in revised manuscript.

10. Don’t use full stop sign (.) within a single sentence (Line no. 166-169).

Answer: We have incorporated this change in revised manuscript.

11. It is advised to avoid the duplication of sentences (Line no. 181- 190).

Answer: While revising the manuscript we have removed the duplicate sentences.

12. Modify the sentence (Further only 30 positive samples were tested and these reasons might have…..).

Answer: We have revised the sentence in revised manuscript.

13. Reference should be written as per the journal guideline.

Answer: Reference formatted as per Journal guidelines.

14. Reference required for actual Operational cost for TrueNat test and RTPCR.

Answer: Reference added in revised manuscript.

Reviewer 2:

This study is in time and address issue of quick and handy detection of the samples which can be helpful in accessing the infection rate and frequency. However, one major concern which need clarification from author.

1. Whether author compared the fidelity of this assay with the RAT (rapid antigen test), how correlative and reliable is this assay.

Answer: In this study in small subset of patients (n=30) all three test i.e. antigen detection by STANDARD Q COVID-19 Ag Test (SD Biosensor, South Korea), Truenat test and RTPCR was done. Taking RTPCR results as gold standard; Antigen detection and Truenat test showed sensitivity of 55% and 70 % respectively. The specificity in each test was 100%. Antigen detection missed 2 RTPCR positive cases (Ct value 29 and 31) but were detected by Truenat test. However, as the sample size is small this data is not included in manuscript.

2. Whether the author used simple PCR or modified nested one.

Answer: We used simple RTPCR protocol suggested by Indian council of medical research, India. All samples were tested for E gene and RdRP gene and cut off threshold <40 was considered as positive.

3. Can we identify mutated strain also in the patients.

Answer: Mutated strains of SARS Cov-2 cannot be identified by Truenat testing or routine RTPCR test.

Submitted filename: PLOS POINT WISE ANSWERS TO REVIEWER COMMENTS.docx

Click here for additional data file.

13 Sep 2021

Assessing a chip based rapid  RTPCR test for SARS C0V-2 detection (TrueNat assay): a diagnostic accuracy study.

PONE-D-21-10427R1

Dear Dr. Garg,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Seyed Ehtesham Hasnain

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I have gone through the revised manuscript and also the author's response to the comments of the reviewers. The manuscript was sent for revision and Authors have modified the manuscript keeping in mind the comments of the Reviewers. Conclusion section of the Abstract has been modified and Authors have updated the global data and added Indian data in the manuscript. Reference part has been updated. All grammatical and spelling errors have been taken care off.

In my view, the authors have satisfactorily addressed all the comments made by the reviewers and added all required information, and have revised the manuscript accordingly. I recommend this manuscript for publication.

Reviewers' comments:

5 Oct 2021

PONE-D-21-10427R1

Assessing a chip based rapid  RTPCR test for SARS C0V-2 detection (TrueNat assay): a diagnostic accuracy study.

Dear Dr. Garg:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Seyed Ehtesham Hasnain

Academic Editor

PLOS ONE