Evaluation of sample pooling for screening of SARS CoV-2

Andargachew Mulu; Dawit Hailu Alemayehu; Fekadu Alemu; Dessalegn Abeje Tefera; Sinknesh Wolde; Gebeyehu Aseffa; Tamrayehu Seyoum; Meseret Habtamu; Alemseged Abdissa; Abebe Genetu Bayih; Getachew Tesfaye Beyene

doi:10.1371/journal.pone.0247767

Peer Review History

Original SubmissionNovember 13, 2020
7 Dec 2020 Decision Letter - Etsuro Ito, Editor PONE-D-20-35695 Evaluation of Sample Pooling for Screening of SARS CoV-2 PLOS ONE Dear Dr. Beyene, 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 Jan 18 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. 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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: No Reviewer #2: No ****** 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: No ****** 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: Mulu A and coworkers describe sample pooling for the screening of SARS-CoV-2 in Ethiopia. The authors performed the experiments both on direct clinical sample pooling and extracted RNA pooling. Major comments: 1. One of the objectives of the study was to establish time and resource-saving method for the screening of SARS-CoV-2. But the authors did not show or discuss on the turnaround time of this strategy. 2. The RT-PCR kit describes in the Methods detected E and ORF1ab genes, but the results describe the Ct value of N and ORF1ab genes. 3. The Ct values of each pool and original sample should be demonstrated. The numbers of specimens and the numbers of pools should be shown. 4. The results described the ability of pooling strategy to detect the virus in the pool of direct clinical sample of up to eight samples. Why did the authors recommend the pooling of four samples? Also, in the extracted RNA, the pooling strategy detected the virus in the pool of 10 samples, but recommend the pool of eight. Please provide the reasons for the recommendation. 5. It might be more interesting if the authors describe that they performed the experiments in both direct clinical sample and extracted RNA in the abstract. Minor comments: 1. COVID-19 stands for coronavirus disease 2019. The full name of the disease should be corrected both in the Abstract and Background. 2. The method for calculation of pool size should be moved to the Method section. Please specify the algorithm that the authors used in the website. Does the reduction in the expected number of tests were 741% for the pool of 10 samples in Table 2? Please describe the term “testing efficiency”. 3. The manuscript should be language check. There are many typos. Also, the writing can be improved by reorganizing the content. Reviewer #2: The experimental study by Mulu et al proposed a simple and straightforward screening strategy for SARS CoV-2 using either direct clinical specimen or pooling of NA after the extraction step. Such a strategy has been demonstrated to be useful in the face of global scarcity of kits and consumables, among others, particularly in resource constrained settings. However, the manuscript requires some clarity in the methodology and findings. Specific comments to improve the manuscript include: General: Language edition needed, including revising long sentences Method: • Clearly indicate how many samples from each of Low, Medium, High Ct values categories were used in the study. Indicate their Ct values as well (for both target genes). In other words, how many different pools were made for each category of samples • How were the low, medium and High Ct cutoffs determined? • Lines 135 to 137: the sentence is not clear. • When preparing the negative pool, indicate the volume of sample taken from each individual sample (just for clarity) • Authors have stated tests were done in triplicate. It is not clear from the document that how the final result was determined and what decision were made if triplicates do not agree. Also state the degree of acceptable variation between triplicates • For the reader, authors need to clarify how they determined rates of test efficiency in the method section (though shown as table footnote) • Include a section on Quality Assurance Result • Complete the figures for the 3 cut off levels of positive samples pooled. In the result only the following figures were shown with no explanation why only those depicted (Fig 1=direct sample Low Ct, Fig 2=RNA Low Ct, Fig 3=RNA High Ct) • Accordingly revise the discussion section Discussion • Lines 230231: Instead of using the phrase “ lack of” better to state “shortage of” • Lines 230-234 needs revision; particularly the last phrase after the reference lacks continuity with the preceding statement (Lines 233-234) • There is inconsistency in the suggested RNA pooling: Lines 249-250 states 10 in 1 while in the conclusion part it is stated 8 in 1 • Pooling strategy depends on the prevalence rate of SARS Cov-2. Can you suggest a rate that pooling is not recommended? • With the increasing SARS Cov-2 prevalence, please comment on the applicability of the findings Thank you! ****** 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 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. https://doi.org/10.1371/journal.pone.0247767.r001
Revision 1
30 Jan 2021 Author Response Responses Reviewer Reviewer #1: Mulu A and co-workers describe sample pooling for the screening of SARS-CoV-2 in Ethiopia. The authors performed the experiments both on direct clinical sample pooling and extracted RNA pooling. Major comments: 1. One of the objectives of the study was to establish time and resource-saving method for the screening of SARS-CoV-2. But the authors did not show or discuss on the turnaround time of this strategy. Response: Thanks a lot! this has now been included both in the result (last paragraph) and discussion section. 2. The RT-PCR kit describes in the Methods detected E and ORF1ab genes, but the results describe the Ct value of N and ORF1ab genes. Response: It was my mistake that we wrote “E gene”, now corrected to N gene in all sections. 3. The Ct values of each pool and original sample should be demonstrated. The numbers of specimens and the numbers of pools should be shown. Response: we included supplementary (S1-S4) Tables with the Ct value of the original positive samples and Ct values of the pools for both direct clinical samples and RNA samples 4. The results described the ability of pooling strategy to detect the virus in the pool of direct clinical sample of up to eight samples. Why did the authors recommend the pooling of four samples? Also, in the extracted RNA, the pooling strategy detected the virus in the pool of 10 samples, but recommend the pool of eight. Please provide the reasons for the recommendation. Response: Indeed, the pooling strategy using the specified NA extraction and RT-PCR protocol we can detect the virus in pool of 8 for direct clinical sample and in pool of 10 for RNA. However, considering the uncertainty in quality of samples, the efficiency of RNA extraction protocols, sensitivity of RT-PCR and possible human error in preparing pool of large samples, we recommended pool of 4/5 and 8. Furthermore, reports show that the gold standard test (detection of nucleic acids of SARS-CoV2 by RT-PCR) has up to 30% False negative reports. 5. It might be more interesting if the authors describe that they performed the experiments in both direct clinical sample and extracted RNA in the abstract. Response: Thanks again: We included pool of RNA in the abstract section too. Minor comments: 1. COVID-19 stands for coronavirus disease 2019. The full name of the disease should be corrected both in the Abstract and Background. Response: this is now corrected! 2. The method for calculation of pool size should be moved to the Method section. Please specify the algorithm that the authors used in the website. Does the reduction in the expected number of tests were 741% for the pool of 10 samples in Table 2? Please describe the term “testing efficiency”. Response: Thank you very much, this question has helped us to re-analyse the calculation. We ask apologies that our calculation of the figures in the previous Table 2 were wrong. Now we have correctly calculated the most optimal testing configuration pool size, expected number of tests reduced, and testing efficiency using a Shiny App for pooled testing by Hierarchical algorithm. the previous Table 2 now is Table 3. By using disease prevalence rate 0.05, the calculated optimal pool size is 5 and the expected is number of tests reduced is 58%, with test efficiency of 137%. 3. The manuscript should be language check. There are many typos. Also, the writing can be improved by reorganizing the content. Response: Thanks again, now the MS is read by native speaker. Thanks a lot for your comments and suggestions! Reviewer #2: The experimental study by Mulu et al proposed a simple and straightforward screening strategy for SARS CoV-2 using either direct clinical specimen or pooling of NA after the extraction step. Such a strategy has been demonstrated to be useful in the face of global scarcity of kits and consumables, among others, particularly in resource constrained settings. However, the manuscript requires some clarity in the methodology and findings. Specific comments to improve the manuscript include: General: Language edition needed, including revising long sentences Response: we revised and edited the Language Method: • Clearly indicate how many samples from each of Low, Medium, High Ct values categories were used in the study. Indicate their Ct values as well (for both target genes). In other words, how many different pools were made for each category of samples Response: Thank you, now we included in the method section that the total number of pools done on direct clinical sample were 54 using two positive and 18 negative samples. For the nucleic acid arm, a total of 18 pools were conducted using two positive and 16 negative samples. • How were the low, medium and High Ct cutoffs determined? Response: This is really a very nice comment. As there could be subjectivity, and we could not come across any literatures that categorise Ct values in to Low, Medium and High, we just determined the cut-off values based on convince. Now we have modified the range of categories Ct values that is, we used the Ct value of the internal positive control as a reference. That is, Ct values less than or equal to the Ct value of the internal control (32) was considered as low ct value, and Ct values between >32 and < 34 were considered as medium and Ct value greater than 34 were considered as high. Please note that Ct value for negativity is 40 and above. • Lines 135 to 137: the sentence is not clear. Response: We have revised this section • When preparing the negative pool, indicate the volume of sample taken from each individual sample (just for clarity) Response: The sum volume of negative samples taken for dilution (pooling) indicated, for direct clinical samples in Table 1, and Table 2 for RNA. • Authors have stated tests were done in triplicate. It is not clear from the document that how the final result was determined and what decision were made if triplicates do not agree. Also state the degree of acceptable variation between triplicates Response: We agree on the importance of showing the statistical variation of the experiments. We have used One-way ANOVA and analysed the average variance of pools Ct values between experiments and within, this is now indicated it result section. • For the reader, authors need to clarify how they determined rates of test efficiency in the method section (though shown as table footnote) Response: Thanks, now we clearly stated in method section how test efficiency is calculated. We obtained test efficiency by dividing expected number of tests reduced by expected number of tests per individual. • Include a section on Quality Assurance Response: We included quality assurance section in this revised version, at the end of method section. We also indicated that our laboratory has been participating in an external quality assessment program, and given the approval that results we produce are reliable. Result • Complete the figures for the 3 cut off levels of positive samples pooled. In the result only the following figures were shown with no explanation why only those depicted (Fig 1=direct sample Low Ct, Fig 2=RNA Low Ct, Fig 3=RNA High Ct) • Accordingly revise the discussion section Response: we have revised this part accordingly. Discussion • Lines 230-231: Instead of using the phrase “lack of” better to state “shortage of” Response: we revised this section and we replace the “lack of” by “shortage of” • Lines 230-234 needs revision; particularly the last phrase after the reference lacks continuity with the preceding statement (Lines 233-234) Response: thank you, we revised this statement • There is inconsistency in the suggested RNA pooling: Lines 249-250 states 10 in 1 while in the conclusion part it is stated 8 in 1 Response: thanks again, what we indicated in Lines 249-250 is the highest number of sample pool (9 negative and 1 positive samples) that we can get positive test. However, in the conclusion part, considering the uncertainties in the quality of samples, the efficiency of RNA extraction protocols, sensitivity of RT-PCR, and the possible human error in preparing pools of large samples, we recommended pooling of 8 samples in 1 for RNA. • Pooling strategy depends on the prevalence rate of SARS Cov-2. Can you suggest a rate that pooling is not recommended? Response: Although we did not see the impacts of pooling in community-based samples, we suggest using pooling in a community samples with estimated prevalence of less than or equal to 7%. However, we don’t advise pooling of clinical samples when disease prevalence is greater than 7%; particularly when sample size is large. Because, a disease prevalence of greater than 7% has a benefit of less than 50% in terms of the expected number of tests reduced. • With the increasing SARS Cov-2 prevalence, please comment on the applicability of the findings. Response: with the increasing SARS Cov-2 prevalence the benefit of pooling is very minimal especially, when processing a very large volume sample. For example, suppose that the disease prevalence is 8%, if you are processing 1000 samples in a pooling of 4 samples in 1, you will have 250 pools. Out the 250 pools, potentially 80 of your pools will have positive signal. Then, in the subsequent step, you have to process 4x80 = 320 samples separately to find out which of the sample/s from each pool is truly positive. From this we can conclude that using sample pooling in such situation is a waste of time and resource. Thank you very much for your comments and suggestions! Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0247767.r002
15 Feb 2021 Decision Letter - Etsuro Ito, Editor Evaluation of Sample Pooling for Screening of SARS CoV-2 PONE-D-20-35695R1 Dear Dr. Beyene, 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, Etsuro Ito Academic Editor PLOS ONE Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. 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 ****** 5. 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: Yes Reviewer #2: Yes ****** 6. 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: (No Response) Reviewer #2: (No Response) ****** 7. 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 https://doi.org/10.1371/journal.pone.0247767.r003
Formally Accepted
17 Feb 2021 Acceptance Letter - Etsuro Ito, Editor PONE-D-20-35695R1 Evaluation of sample pooling for screening of SARS CoV-2 Dear Dr. Beyene: 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. Etsuro Ito Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0247767.r004

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