Analytical sensitivity and clinical performance of "COVID-19 RT-PCR Real TM FAST (CY5) (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador)": High quality-low cost local SARS-CoV-2 tests for South America.

BACKGROUND: Dozens of commercial RT-qPCR kits for SARS-CoV-2 detection are available with or without Emergency Use Authorization (EUA) by FDA or other regulatory agencies.
OBJECTIVE: We evaluated the clinical performance of two SARS-CoV-2 RT-PCR kits designed and produced in South America, "COVID-19 RT-PCR Real TM FAST (CY5)" (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador), for RT-qPCR SARS-CoV2 detection using "TaqMan 2019-nCoV Assay Kit v1" (Thermofisher, USA) as a gold standard technique.
RESULTS: We report a great clinical performance and analytical sensitivity for the two South American kits with sensitivity values of 96.4 and 100%, specificity of 100% and limit of detection in the range of 10 copies/uL of RNA extraction.
CONCLUSIONS: "COVID-19 RT-PCR Real TM FAST (CY5)" and "ECUGEN SARS-CoV-2 RT-qPCR" kits are reliable SARS-CoV-2 tests made in South America that have been extensively used in Uruguay, Argentina, Brazil, Bolivia and Ecuador. These locally produced SARS-CoV-2 tests have contributed to overcome supply shortages and reduce diagnosis cost, while maintaining the high quality standards of FDA EUA commercially available kits. This approach could be extended for other diagnostic products to improve infectious diseases surveillance at middle and low income countries beyond COVID-19 pandemic.

Introduction

The "coronaviruses disease 2019" (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has challenged public health systems worldwide since the initial outbreak in the Chinese city of Wuhan in December 2019 [1,2]. While SARS-CoV-2 vaccination programs progress successfully at high income countries and COVID-19 pandemic is somehow under control, the challenge persists for middle and low income countries in need to improve SARS-CoV-2 testing [3-5]. A wide variety of commercial SARS-CoV-2 RT-qPCR detection kits are available in South America for clinical use, some of them with Emergency Use Authorization (EUA) from the U.S. Food & Drug Administration (FDA) or other international recognized agencies, while others even lack of their country of origin EUA and compromise a reliable diagnosis in these high burden COVID-19 settings [6-13]. Among the SARS-CoV-2 RT-qPCR commercial kits available, "TaqMan 2019-nCoV Assay Kit v1" (Thermofisher, USA) holds FDA EUA and it is considered one of the most reliable SARS-CoV-2 RT-qPCR kits [6]. All those commercial kits are based in SARS-CoV-2 detection by targeting different genes like E, S, N or the orf1ab region.

The CDC designed FDA EUA 2019-nCoV CDC kit (IDT, USA) is based on N1 and N2 gene targets to detect SARS-CoV-2 and RNase P as an RNA extraction quality control, it is considered a gold standard worldwide for SARS-CoV-2 RT-PCR detection [14-16]. The main limitation for the CDC kit is the need to run three RT-qPCR reactions per sample. To solve this inconvenience, inspired on this CDC protocol, two SARS-CoV-2 RT-qPCR multiplex assays have been designed, produced and commercialized in South America. "COVID-19 RT-PCR Real TM FAST (CY5)" (ATGen, Uruguay) is a duplex assay including N1 viral target and RNase P as an RNA extraction quality control. "ECUGEN SARS-CoV-2 RT-qPCR k" (UDLA-STARNEWCORP, Ecuador) is a triplex assay including N1 and N2 viral targets and also RNase P as an RNA extraction quality control. So, for both South American kits, a single PCR reaction is required for SARS-CoV-2 detection, reducing the time and cost of the diagnosis.

We herein present an analytical sensitivity and clinical performance evaluation of "COVID-19 RT-PCR Real TM FAST (CY5)" (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador) for SARS-CoV-2 RT-qPCR detection from nasopharyngeal samples using "TaqMan 2019-nCoV Assay Kit v1" (Thermofisher, USA) as a gold standard technique.

Materials and methods

Ethics statement

This study is part of a project approved by the IRB from the Dirección Nacional de Inteligencia de la Salud (Ministerio de Salud Publica, Ecuador) under the code 008–2020.

Study design

119 samples were included in this study. Those samples were leftovers of the RNA extractions of clinical specimens previously processed for SARS-CoV-2 test and stored at -80 C. Those RNA extractions were obtained from nasopharyngeal swabs collected on 0.5mL TE pH 8 buffer from community dwelling individuals attending Universidad de Las Américas for SARS-CoV-2 testing during November and December 2020. Also, negative controls (TE pH 8 buffer) were included as control for carryover contamination, one for each set of RNA extractions. The overall protocol for sample collection and SARS-CoV-2 diagnosis at our laboratory has been previously published [17-20].

All the clinical samples were processed with the same RNA extraction kit "AccuPrep Viral RNA extraction kit" (Bioneer, South Korea) and used for SARS-CoV-2 detection. Only the viral RNA extractions stored at -80 C were used for the three RT-qPCR protocols included in this study. For the RNA extractions, 200μL of TE pH8 buffer that contained the sample were used. At the end of the RNA extraction, RNA was eluted in 40μL elution buffer.

A total of 109 RNA samples were evaluated using ECUGEN SARS-CoV-2 RT-qPCR Kit (UDLA-STARNEWCORP, Ecuador) and a total of 80 RNA samples were evaluated using COVID-19 RT-PCR Real TM FAST (CY5) (ATGen, Uruguay). All the RNA samples of both sets were additionally processed with TaqMan 2019-nCoV Assay Kit v1 as gold standard SARS-CoV-2 detection method.

RT-qPCR for SARS-CoV-2 detection using TaqMan 2019-nCoV Assay Kit v1

All samples were tested following the manufacturer instructions using TaqPath 1-Step Master Mix GC in a reduced reaction volume of 15μL including 4μL of RNA sample. TaqMan 2019-nCoV Control Kit v1 was used as reaction positive control. Following the manufacturer’s manual, a viral target with Ct < 37 is considered positive and with 37 ≤ Ct < 40 is considered inconclusive. A new PCR reaction was run for inconclusive results, and a Ct < 40 is sufficient to considered that run as positive. Samples that showed positive results for at least two of the three SARS-CoV-2 genes (S, N and ORF1ab) and inconclusive samples that showed recurrent positive results for at least one viral gene target were marked as SARS-CoV-2 positive. RT-qPCR assays were performed in a CFX96 Real-Time PCR Detection System (Bio-Rad).

RT-qPCR for SARS-CoV-2 detection using ECUGEN SARS-CoV-2 RT-qPCR Kit

Samples were tested following the manufacturer instructions in a reaction volume of 15μL including 4μL of RNA sample. The criteria for positivity were Ct ≤ 40 for N1 and N2 targets simultaneously. Also, inconclusive samples where either N1 and N2 were positive, were repeated and if either N1 or N2 target amplified, the sample is considered positive. RT qPCR assays were performed in a CFX96 Real-Time PCR Detection System (Bio-Rad).

RT-qPCR for SARS-CoV-2 detection using COVID-19 RT-PCR Real TM FAST (CY5) (ATGen) kit

Samples were tested following the manufacturer instructions in a reduced reaction volume of 15μL including 3,75μL of RNA sample. Samples that presented a Ct < 35 for the viral target gene were considered positive, and samples with values of 35 ≤ Ct ≤ 40 were considered inconclusive. The latter were repeated and consistent results with the first test was sufficient to be considered positive. RT-qPCR assays were performed in a CFX96 Real-Time PCR Detection System (Bio-Rad).

Analytical sensitivity

Limit of detection (LoD) was performed using the commercially available 2019-nCoV N positive control (IDT, USA); provided at 200,000 genome equivalents/uL, it was used for calibration curves to obtain the viral loads of the samples. Viral loads can be expressed as copies/uL of RNA extraction or copies/mL of sample; the conversion factor is 200, as 0.2mL of sample is used for RNA extraction and 40uL is used as final elution volume of RNA extraction.

Statistics analysis. All data was analyzed in Excel and statistics were done using SPSS software.

Results

Clinical performance and analytical sensitivity of ECUGEN SARS-CoV-2 RT-qPCR Kit

109 samples were tested for SARS-CoV-2 with ECUGEN SARS-CoV-2 RT-qPCR Kit using TaqMan 2019-nCoV Assay Kit v1 as a gold standard. For the TaqMan 2019-nCoV Assay Kit v1, 55 samples tested positive, and 54 samples tested negative (Table 1 and S1 Data). 51 out of 54 samples tested negative for the TaqMan 2019-nCoV Assay Kit v1 were also SARS-CoV-2 negative for ECUGEN SARS-CoV-2 RT-qPCR Kit, so the specificity obtained in this study was 94.4% (95% CI = 84.6 to 98.8%). The three "false positive" samples had Ct values > 35 and viral loads of 3.1, 4.72 and 6.84 copies/uL of RNA extraction (Samples 62, 64 and 66 at S1 Data).

Table 1

Clinical performance of ECUGEN SARS-CoV-2 RT-qPCR and COVID-19 RT-PCR Real TM FAST (CY5) kits using TaqMan 2019-nCoV Assay Kit v1 as reference methodology (% values: sensitivity).

Only SARS-CoV-2 positive samples included on the study are detailed.

SARS-CoV-2 RT-qPCR kit	Positive Samples	False Negative Samples	Total SARS-CoV-2 positive samples
ECUGEN SARS-CoV-2 RT-qPCR Kit	55 (100.0%)	0	55
COVID-19 RT-PCR Real TM FAST (CY5)	53 (96.36%)	2	55

For the 55 SARS-CoV-2 positive samples for the TaqMan 2019-nCoV Assay Kit v1, 55 samples tested also positive for ECUGEN SARS-CoV-2 RT-qPCR Kit, resulting a sensitivity of 100.0% (95% CI = 93.51 to 100.00%) (Table 1 and S1 Data). Cohen’s κ was run and almost perfect agreement between results was obtained with both kits (κ = 0.945, p<0.001). In Fig 1A, the distribution of Ct values of N gene target for SARS-CoV-2 positive samples included in the study for ECUGEN SARS-CoV-2 RT-qPCR Kit and TaqMan 2019-nCoV Assay Kit v1 is show. In Fig 1B the linear regression analysis for Ct values for N gene target for both kits is presented, yielding a R2 = 0.9906.

Fig 1

A: Cycle threshold (Ct) distribution for N gene target for SARS-CoV-2 positive samples with the three commercial kits included in this study: ECUGEN SARS-CoV-2 RT-qPCR Kit (green), COVID-19 RT-PCR Real TM FAST (blue) and TaqMan 2019-nCoV Assay Kit v1 (yellow). B: Linear regression for the Ct values for N gene target for ECUGEN SARS-CoV-2 RT-qPCR Kit (green) and COVID-19 RT-PCR Real TM FAST (blue) compared to TaqMan 2019-nCoV Assay Kit v1.

The limit of detection (LoD) is defined as the lowest viral load in which all replicates are detected (100% sensitivity). As it is detailed in Table 2, after running 15 replicates for viral loads in the range from 500 to 2000 copies/mL, we could set the LoD for ECUGEN SARS-CoV-2 RT-qPCR Kit in 2000 copies/mL of sample (10 copies/μL of RNA extraction). Although N2 target fails for 1 out of 15 replicates at that viral load, as the criteria of positivity only requires the amplification of one of the viral target on the replicate, the LoD was the lowest viral load for 100% sensitivity for N1 target.

Table 2

Analytical sensitivity for ECUGEN SARS-CoV-2 RT-qPCR kit.

The ratio represents the number of positive replicates for each viral load related to the total number of replicates.

Viral load (copies/mL)	N1 replicates	N1 sensitivity	N2 replicates	N2 sensitivity
2000*	15/15	100%	14/15	93.3%
1500	14/15	93.3%	14/15	93.3%
1000	12/15	80%	12/15	80%
500	12/14	85.7%	10/14	71.4%

Clinical performance and analytical sensitivity of COVID-19 RT-PCR Real TM FAST (CY5) kit

80 samples were tested for SARS-CoV-2 with of COVID-19 RT-PCR Real TM FAST (CY5) (ATGen) kit using TaqMan 2019-nCoV Assay Kit v1 as gold standard. For the TaqMan 2019-nCoV Assay Kit v1, 55 samples tested positive, and 25 samples tested negative (Table 1 and S1 Data). 24 out of 25 samples tested negative for the TaqMan 2019-nCoV Assay Kit v1 were also SARS-CoV-2 negative for COVID-19 RT-PCR Real TM FAST Kit, so the specificity obtained in this study was 96.00% (95% CI = 79.6 to 99.9%). The sample 62 (S1 Data) had a Ct value of 34.73 for N target and also presented a positive result with ECUGEN SARS-CoV-2 RT-qPCR Kit, however, this sample tested negative with the TaqMan kit.

From the 55 samples that tested positive with TaqMan 2019-nCoV Assay Kit v1, 53 samples were positive for COVID-19 RT-PCR Real TM FAST Kit, resulting a sensitivity of 96.4% (95% CI = 87.5 to 99.6%) (Table 1 and S1 Data). Samples 59 and 68 amplified for at least one SARS-CoV-2 target in different TaqMan RT-qPCR reactions, therefore, these samples were marked as positive. Cohen’s κ was run and almost perfect agreement between results obtained with both kits was found (κ = 0.914, p<0.001). In Fig 1A, the distribution of Ct values of N gene target for SARS-CoV-2 positive samples included in the study for COVID-19 RT-PCR Real TM FAST Kit and TaqMan 2019-nCoV Assay Kit v1 is show. In Fig 1B the linear regression analysis for Ct values for N gene target for both kits is presented, yielding a R2 = 0.9811.

As the limit of detection (LoD) is defined as the lowest viral load in which all replicates are detected (100% sensitivity), our data indicates that the LoD for COVID-19 RT-PCR Real TM FAST (CY5) Kit should be in the range of 5–10 copies/μL of RNA extraction (1000–2000 viral RNA copies/mL of sample), as the 4 samples with viral loads within that range were detected (S1 Data), and positive samples that failed for this kit were below 5 copies/uL.

Discussion

Although the main limitation of our study is the sample size (119 specimens), our results support that "COVID-19 RT-PCR Real TM FAST (CY5) (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador) kits had a great clinical performance with sensitivity values of 96.4% and and 100%, respectively. Moreover, although a reduction of specificity was found for "COVID-19 RT-PCR Real TM FAST (CY5) (96%) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-Starnewcorp, Ecuador)" (94.4%) kits, we believe that the 3 "false positive" samples would actually be true positives samples as the Ct values obtained indicated that those samples had really low viral loads on the threshold for detection of the gold standard method used. Actually, one of the false positive samples was positive for both "COVID-19 RT-PCR Real TM FAST (CY5) and "ECUGEN SARS-CoV-2 RT-qPCR" kits. Additionally, both kits use the same N viral targets than the CDC protocol that do not have cross reactivity with other respiratory virus [14,15], and those 3 samples were reported as positive for the regular CDC protocol used for clinical diagnosis in our laboratory [14,15]. So, the specificity for "COVID-19 RT-PCR Real TM FAST (CY5)" and "ECUGEN SARS-CoV-2 RT-qPCR" kits could be considered 100%.

We could calculate the LoD for "ECUGEN SARS-CoV-2 RT-qPCR" at a really low viral load of 10 viral copies/uL of RNA extraction (2000 viral RNA copies/mL of sample) that it is equivalent to LoDs of high quality commercial RT-qPCR SARS-CoV-2 kits. Also, a similar LoD was estimated for "COVID-19 RT-PCR Real TM FAST (CY5). Moreover, this LoD is extremely reliable for SARS-CoV-2 diagnosis considering the viral load frequency population distributions [21,22].

In the Table 3, analytical parameters and other features for "COVID-19 RT-PCR Real TM FAST (CY5) and "ECUGEN SARS-CoV-2 RT-qPCR" RT-PCR kits are summarized. Considering the great clinical performance and analytical sensitivity for those locally designed and produced SARS-CoV-2 tests, compared to a high quality commercial kit like TaqMan 2019-nCoV Assay Kit v1 (Thermo Fisher), the current study endorses the use of these kits as a reliable alternative to expensive imported commercial kits. This would potentially allow to increase SARS-CoV-2 testing capacities by two main reasons: a) SARS-CoV-2 testing cost reduction as this locally produced RT-qPCR kits are substantially cheaper than high quality imported ones (less than 10 USD per reaction); b) supplies shortages would not affect SARS-CoV-2 testing capacities as local production is guaranteed.

Table 3

Description of ECUGEN SARS-CoV-2 RT-qPCR (UDLA-Starnewcorp, Ecuador) and COVID-19 RT-PCR Real TM FAST (CY5) (ATGen, Uruguay) features (LoD means limit of detection in copies/uL of RNA extraction elution).

SARS-CoV-2 RT-PCR kit	Gene Targets	Estimated LoD (copies/uL)	countries of distribution
ECUGEN SARS-CoV-2 RT-qPCR (Ecuador)	N1, N2 (virals) RNaseP (control)	10	Ecuador
COVID-19 RT-PCR Real TM FAST (CY5) (Uruguay)	N (viral) RNaseP (control)	5–10	Uruguay, Argentina, Bolivia, Brasil, Ecuador.

Finally, we point out a common feature for both South American kits evaluated in this study: they were designed and produced by a consortium between universities and private companies. "COVID-19 RT-PCR Real TM FAST (CY5)" was created by collaboration of "Universidad de La República" (public university), "Instituto Pasteur de Montevideo" (research center) and ATGen (private company); "ECUGEN SARS-CoV-2 RT-qPCR" was created by collaboration of "Universidad de Las Américas" (private university) and STARNEWCORP (private company). In both cases, the role of the Academia has been crucial to improve good quality SARS-CoV-2 testing, as it has been suggested even for USA [23]. Moreover, we describe two cases of cross talk and knowledge transference among the Academia and the private sector, common on high incomes countries but not as usual in the context of South America. We hope that these two successful stories will inspire similar biotechnological developments in the future to improve South American public health systems and reduce the regional overall technological dependency beyond COVID-19 pandemic.

Ct values for all samples included in this study for all the gene targets included in the three commercial SARS-CoV-2 RT-qPCR kits tested.

(XLSX)

Click here for additional data file.

21 Jul 2021

Dear Dr Garcia-Bereguiain,

Thank you very much for submitting your manuscript "Analytical sensitivity and clinical performance of "COVID-19 RT-PCR Real TM FAST (CY5) (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-Starnewcorp, Ecuador)": high quality-low cost local SARS-CoV-2 tests for South America." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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Sincerely,

Susanna Kar Pui Lau, M.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Susanna Kar Pui Lau

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: I have problems with the limit of detection (LOD) calculations made by the authors. It appears that no replicates of the samples were tested to determine the LOD. I would expect a minimum of 4 (but ideally more) for a statistically robust analysis.

More information on the 119 clinical samples is required. When were they collected? From whom? Had they already been tested? How were they stored?

Why does the number of samples tested per kit differ (i.e. 109 for the ECUGEN and 80 for the ATGen kit)?

Page 5: line 87. What do you mean by “reduced reaction volume”? Did you reduce the volume? If so, why?

Page 5 line 90-91 and 102 to 103. Please rewrite this sentence for clarity. What do you mean by “consistent results with the first test were considered positive”?

Page 5 line 97. How was the criteria for positivity (i.e. Ct ≤40) calculated? Please provide details.

Please identify the statistical programmes used for analyses.

Reviewer #2: The methods used in this study are the methods described by the different kits' manufacters assessed in this study.

Nevertheless one point was unclear to me : how was the Positive / inconclusive Cycle Threshold set...this seemed a little arbitrary and could change drastically the conclusions (i.e. samples positives and negatives) . It would strengthen the study if the authors could explain how they chose CT37 for Taq man, Ct 40 for Ecugen and Ct 35 for Tim Fast

Moreover in the case of the Tim Fast kit only 3.75uL of samples was used (instead of 4uL fort the other kits...this also needs explanation )

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: The results are clearly presented and the "raw" data are in supplemental material. Nevertheless for the samples where a duplicate experiment was necessary (for exemple samples 59 and 68 ) to draw a conclusion on the samples it would be nice to show the result of each replicate. When reading that supplemental figure it was not clear why 59 and 68 were called positive whereas 85 and 98 were called negative (Taq Man kit)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The conclusions of this manuscript are fully supported by the datas presented here . This study is of general interest as it shows that alternative methods for SARS-COV-2 detection are as performant as the CDC approved ones and can be use for general testing.The number of samples (#100 ) is good enough to be able to draw solid conclusions.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: The English is sufficient but could be improved.

A brief description of the SARS-CoV-2 genome describing all of the target genes would be helpful.

Last paragraph can be removed Page 9 line 182-193 as I do not think it is relevant to the study.

Reviewer #2: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: In their manuscript the authors have evaluated the performance of two commercial diagnostic RT-qPCR kits for the detection of SARS-CoV-2. Evaluation of kits is important but it must be done in a systematic and statistically robust way. Accordingly, this manuscript requires revision.

Reviewer #2: This study compares the efficacity of 2 novel commercial kits to the CDC approved one for SARS-Cov-2 detection . This study is straight forward and well performed. It is important for each country to be able to implement wide testing with its own ressources to avoid problems of shortage.

The datas are presented clearly and the conclusions are strongly supported by the results.

--------------------

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Reviewer #1: Yes: William G. Dundon

Reviewer #2: No

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8 Sep 2021

Submitted filename: responseletterECUGENPNTD.docx

Click here for additional data file.

11 Oct 2021

Dear Dr Garcia-Bereguiain,

Thank you very much for submitting your manuscript "Analytical sensitivity and clinical performance of "COVID-19 RT-PCR Real TM FAST (CY5) (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-Starnewcorp, Ecuador)": high quality-low cost local SARS-CoV-2 tests for South America." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Susanna Kar Pui Lau, M.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Susanna Kar Pui Lau

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: no more comments. The authors addressed my comments from the first round of review

I just found a typo page 2 line 24 "with our" ???

Reviewer #3: Straightforward methodology with manufacturer provided protocols. Details have been provided accordingly as previous reviewer's suggestion. The sample limitation seems to be a problem but unsolvable. The authors have addressed this concern in the manuscript.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: yes

Reviewer #3: The supplementary data is weird. For sample ID 20414, there are Ct values for TaqMan 2019-nCoV Assay Kit v1 but no result indication for this sample. Is this sample tested or not?

The LOD range calculation is a pure deduction from the clinical samples. It is not wrong but the evidence is relatively weak.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: yes

Reviewer #3: The aim of this manuscript fits the Plos NTD in terms of focusing the low income countries and their respective situation of molecular diagnosis of COVID-19. The evaluation of these local produced kits may be of interest to relevant countries which have difficulties in accessing the golden commercial kits. However, the need of molecular diagnosis is necessary to global prevention of COVID-19. Such simple and direct testing may provide alternatives for those countries' public health systems.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have satisfactorily answered all of the questions put to them. Unfortunately it seems that some of the shortcomings of their study highlighted by this reviewer were due to a lack of ATGen kits for a complete and robust evaluation. Even though this is not the fault of the authors, it still does not justify the publication of an incomplete evaluation study. I therefore recommend that the authors revise their manuscript and concentrate on the evaluation of the ECUGEN kit alone.

Reviewer #2: (No Response)

Reviewer #3: A straightforward and simple diagnostic kit evaluation from respective company. This finding can provide alternatives for low income countries to support their need of COVID-19 detection kits. In order to better commercialize the kits, a larger sampling size is definitely needed for displaying confident kit performance. Yet, this paper can still provide preliminary performance quality for the public section to consider.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

8 Nov 2021

Submitted filename: responseletterECUGENPNTDV2.docx

Click here for additional data file.

7 Dec 2021

Dear Dr Garcia-Bereguiain,

We are pleased to inform you that your manuscript 'Analytical sensitivity and clinical performance of "COVID-19 RT-PCR Real TM FAST (CY5) (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador)": high quality-low cost local SARS-CoV-2 tests for South America.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Susanna Kar Pui Lau, M.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Susanna Kar Pui Lau

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #3: This part has been addressed by the authors.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #3: The supp. result has been appropriately modified.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #3: Appropriate conclusion.

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Language revisions are recommended to improve clarity.

Reviewer #3: Nil

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have understood the limitations of their study and have tried to resolve them. Nevertheless, limitations still exist in the number of samples tested and the lack of standard LOD calculations for one of the kits.

Reviewer #3: The question is addressed with further elaboration on the performance of ECUGEN kit. Limitation is appropriately stated in the content.

**********

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.

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Reviewer #1: No

Reviewer #3: No

8 Apr 2022

Dear Dr Garcia-Bereguiain,

We are delighted to inform you that your manuscript, "Analytical sensitivity and clinical performance of "COVID-19 RT-PCR Real TM FAST (CY5) (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador)": high quality-low cost local SARS-CoV-2 tests for South America," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases