Validation study of Boil & Spin Malachite Green Loop Mediated Isothermal Amplification (B&S MG-LAMP) versus microscopy for malaria detection in the Peruvian Amazon

Keare A. Barazorda; Carola J. Salas; Greys Braga; Leonila Ricopa; Julia S. Ampuero; Crystyan Siles; Juan F. Sanchez; Silvia Montano; Stephen E. Lizewski; Christie A. Joya; Danett K. Bishop; Hugo O. Valdivia

doi:10.1371/journal.pone.0258722

Peer Review History

Original SubmissionMay 5, 2021
2 Jul 2021 Decision Letter - Luzia Helena Carvalho, Editor PONE-D-21-14913 Validation study of Boil & Spin Malachite Green Loop Mediated Isothermal Amplification (B&S MG-LAMP) versus microscopy for malaria detection in the Peruvian Amazon PLOS ONE Dear Dr. Valdivia, Thank you for submitting your manuscript to PLoS ONE. After careful consideration, we felt that your manuscript requires revision, following which it can possibly be reconsidered. Although your manuscript was of interest to the reviewer, major concerns were related to study design and data presentation. According to the reviewers, the methods were not described in enough details to allow suitably skilled investigators to fully replicate and evaluate the study. Regarding the study design, the reviewer suggested that authors follow the recommendations of the STARD 2015 E QUADAS-2 tool. In addition, a significant number of issues should be clarified and/or adjust otherwise the MS’s results may be compromised. 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Thank you for stating the following in the Financial Disclosure section: "This work was supported by the US DoD Armed Forces Health Surveillance Division (AFHSD), Global Emerging Infections Surveillance (GEIS) Branch, PROMIS IDs P0106_18_N6_02 (DKB) and P0143_19_N6_02 (CAJ) from 2018 and 2019. The funders had not role in the study design, data collection and analysis, prepation of the manuscript nor decision to publish." We note that one or more of the authors are employed by a commercial company: "Vysnova Partners Inc," a) Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. 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If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. 3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section. [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: Partly 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: No 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: Yes 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: The manuscript about B&S MG-LAMP assay for the diagnosis of Malaria in Peruvian Amazon sounds interesting. The results obtained indicate that this method can be implemented in resources limited facilities in endemic settings nearing for elimination. However I identified several concerns that the AAs should evaluate carefully. I suggest that they should add more details to the methodology, mainly about of the design of study, and submit it for further evaluation. Major comments In the "Methods" section, authors need to improve writing regarding the study design. As this is an accuracy study, I suggest that the authors follow the recommendations of the STARD 2015 E QUADAS-2 tool. Including a flowchart with the study design may help. In addition, it is recommended that authors add a sample calculation. Considering the information present in line 144 ("The samples tested in this study were selected from three protocols"), this study was carried out using panel samples from 3 different studies. It is necessary to know the number of samples for each of the studies, as well as the definition of their status (cases or non-cases based on the reference test result). They must also inform the average time between collection and extraction of DNA from these samples. Apparently, the inclusion of patients was not blinded or consecutive and microscopy was used as index test and as standard reference. This can also introduce bias and may have consequences in data interpretation. I suggest a paragraph disclosing the main limitations of the present article according to the STARD 2015 E QUADAS-2 tool. Another critical point is that with the data presented by the authors, it is not possible to calculate all the values presented in the topic "Diagnostic performance of microscopy and B&S MG-LAMP". The results must be clarified by showing in the contingency table used for calculation of the statistical parameters. I also suggest including a supplementary table with all the results for each of the samples. Another suggestion is to perform the accuracy analysis including the PET-PCR test as a reference standard. Minor comments Line 247- include reference regarding the Cohen's Kappa coefficient. Lines 278-79 - In the text, the authors reported that B&S MG-LAMP detected 180 positive samples, but in Table 1 there are 179 (17 P. falciparum and 162 P. vivax). Lines 320-21 - "Eight samples diagnosed as P. vivax by the PET-PCR were found to be negative by the B&S MG-LAMP". Include in the "Discussion", the impact that false-negative LAMP results may have on malaria control programs. Table 3- indicate that the cost calculation was estimated for 8 samples. Reviewer #2: This manuscript describes interesting results that validate the simple boil and spin method for MG-LAMP based diagnosis of Plasmodium infections. Authors have covered almost all of required experiments to demonstrate performance of a new test including determination of assay turnaround time and cost evaluation. The authors claim that increasing the amount of target DNA in the reaction tube by a factor of 1.6 (ie 10 uL of sample per 25 uL reaction instead of 5 uL sample per 20 uL reaction as used in the simplified protocol) improves assay performance in cases of low density parasitaemia. However, no data is presented comparing the simplified and modified protocols within the same study population, say during the active surveillance study, to allow for assessment of cost-effectiveness of the modified protocol. Minor comments: 1) Title: replace "malaria detection" with "malaria diagnosis" 2) L38-39: Replace "detect low-parasite densities" with "detect low-density parasitaemia" 3) in L232 it is stated that tenfold dilutions were done starting from 100 000 parasites/uL whereas in L262 and Fig 2 the starting parasite density is 10 000 p/uL 4) L256: replace ".....LoD...was one parasite/uL" with "...LoD was one parasite/uL or lower". The exact LoD is not known given that 1p/uL corresponds to the lowest density tested. 5) L322: Replace "seen" with "see" 6) Cost per reaction is stated as $1.2 in L408 and $9.60 in Table 3 ****** 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: Yes:** Daniel Moreira de Avelar 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. https://doi.org/10.1371/journal.pone.0258722.r001
Revision 1
17 Aug 2021 Author Response RESPONSES Comments to the Author • Have the authors made all data underlying the findings in their manuscript fully available? Reviewer #1: No Response: We do not have any restrictions to provide an unidentified database. In this regard, we are including the individual tests results from the passive surveillance component on S2 table. Regarding the active surveillance database, individual results for the positive samples are described in Table 2. The remaining 493 samples of active surveillance were negative and we do not consider necessary to detail them in another supplementary table. REVIEWER #1: Major comments 1. In the "Methods" section, authors need to improve writing regarding the study design. As this is an accuracy study, I suggest that the authors follow the recommendations of the STARD 2015 E QUADAS-2 tool. Including a flowchart with the study design may help. In addition, it is recommended that authors add a sample calculation. Considering the information present in line 144 ("The samples tested in this study were selected from three protocols"), this study was carried out using panel samples from 3 different studies. It is necessary to know the number of samples for each of the studies, as well as the definition of their status (cases or non-cases based on the reference test result). Response: We appreciate the reviewer’s suggestions. In this regard, we are providing a flowchart describing the process for the passive surveillance component on S1 fig. In addition, we included a paragraph regarding sample size calculation on the methods section. Regarding the studies and their status, the data is provided on Table 2 and S2 table. In summary, we have 258 (174 cases and 84 non-cases) from the first passive study, 25 subjects (4 cases and 21 non-cases) from the second passive study and 500 subjects (1 case and 499 non-cases) from the active surveillance. 2. They must also inform the average time between collection and extraction of DNA from these samples. Apparently, the inclusion of patients was not blinded or consecutive and microscopy was used as index test and as standard reference. This can also introduce bias and may have consequences in data interpretation. I suggest a paragraph disclosing the main limitations of the present article according to the STARD 2015 E QUADAS-2 tool. Response: The average time between sample collection and DNA extraction for the passive surveillance studies were 5 months. On the other hand, the average time between collection and DNA extraction for the active surveillance study was 2 months. We used the STARD 2015 E and QUADAS-2 tools and found that there was a low bias in three areas. For the passive surveillance study, i) samples were randomly selected, ii) the staff that performed subject enrolment was different from the one performing microscopy, iii) B&S MG-LAMP was performed in batches of 30 at the end of the week and iv) molecular technicians were blinded to the individual study files. For the active surveillance study, i) enrolment staff were different from the laboratory staff, ii) the technician doing expert microscopy was different from the one running the B&S MG-LAMP in most cases and iii) B&S MG-LAMP was performed at least one month after microscopy, iv) samples were processed in batches of 30-60 and the technician did not have access to the subject study files making very hard to remember microscopy results. A text referring the use of QUADAS is currently available in the methods section and on the discussion. 3. Another critical point is that with the data presented by the authors, it is not possible to calculate all the values presented in the topic "Diagnostic performance of microscopy and B&S MG-LAMP". The results must be clarified by showing in the contingency table used for calculation of the statistical parameters. I also suggest including a supplementary table with all the results for each of the samples. Another suggestion is to perform the accuracy analysis including the PET-PCR test as a reference standard. Response: Thank you for the suggestion. We are including S2 table with the individual results from the passive surveillance studies. For the active surveillance study, the results are presented in Table 2. We are not showing results from the remaining 493 samples since all of them were negative. Finally, table 1 now shows the results for all the laboratory tests. In this regard, calculations can be replicated using the information provided in any of both tables. 4. Another suggestion is to perform the accuracy analysis including the PET-PCR test as a reference Response: We appreciate the suggestion. However, the aim of the current work was to present the scientific community with a B&S MG-LAMP field deployable test and therefore it should be compared with other field deployable tests. It is important to notice that we are planning to test the modified B&S MG-LAMP employed in the active study using PET PCR and nested PCR as reference. However, this work is still ongoing and samples are still pending to be collected. Minor comments 5. Line 247- include reference regarding the Cohen's Kappa coefficient. Response: Reference was included following your suggestion. 6. Lines 278-79 - In the text, the authors reported that B&S MG-LAMP detected 180 positive samples, but in Table 1 there are 179 (17 P. falciparum and 162 P. vivax). Response: Thank you for the suggestion; we have updated table 1 and the text on this section for accuracy. 7. “Line 292 :The species-specific B&S MG-LAMP identified 179 positives (17 P. falciparum, 162 P. vivax) , the genus B&S MG-LAMP identified 1 Plasmodium spp and 103 negatives." Response: Thank you for the suggestion; we have corrected the information for clarity. 8. Lines 320-21 - "Eight samples diagnosed as P. vivax by the PET-PCR were found to be negative by the B&S MG-LAMP". Include in the "Discussion", the impact that false-negative LAMP results may have on malaria control programs. Response: Thank you for the suggestion; we have included a paragraph in the discussion section. 9. Table 3-indicate that the cost calculation was estimated for 8 samples. Response: Thank you for the suggestion, the title in Table 3 was updated indicating in the equivalence “8 samples = 1 strip of tubes”. REVIEWER #2: Major comments 1. The authors claim that increasing the amount of target DNA in the reaction tube by a factor of 1.6 (ie 10 uL of sample per 25 uL reaction instead of 5 uL sample per 20 uL reaction as used in the simplified protocol) improves assay performance in cases of low density parasitaemia. However, no data is presented comparing the simplified and modified protocols within the same study population, say during the active surveillance study, to allow for assessment of cost-effectiveness of the modified protocol. Response: Thank you for the insight provided for this manuscript. Regarding the modified B&S LAMP, we run a standard curve to document the limit of detection (LOD) and the estimated LOD was 0.5 parasites/µL for the genus and species-specific assays. Therefore, this result supports that this slight modification in master mix volumes and target DNA could potentially improve the assay performance for low parasitemia detection compared to the simplified protocol. However, we are aware that a more rigorous evaluation of this modified mixed is required and we are stating that in the discussion section of manuscript. In this regard, the comparison of the original MG-LAMP master mix versus the new master mix with increased target DNA that you kindly suggested goes beyond the scope of this manuscript. Furthermore, a bigger sample size of low parasitemia/submicroscopic cases is needed for an accurate comparison both methods. We are aiming to obtain sufficient samples from upcoming follow-ups of the active cohort study. Minor comments 2. Title: replace "malaria detection"with "malaria diagnosis" Response: This change was performed as suggested. 3. L38-39: Replace "detect low-parasite densities" with "detect low-density parasitaemia" Response: Thank you for the suggestion, this change was updated as suggested. 4. in L232 it is stated that tenfold dilutions were done starting from 100 000 parasites/uL whereas in L262 and Fig 2 the starting parasite density is 10 000 p/uL Response: Thank you for finding the error, we have corrected it to 10,000 p/uL. 5. L256: replace ".....LoD...was one parasite/uL" with "...LoD was one parasite/uL or lower". The exact LoD is not known given that 1p/uL corresponds to the lowest density tested. Response: This change was corrected as suggested. 6. L322: Replace "seen" with "see" Response: Corrected as suggested. 7. Cost per reaction is stated as $1.2 in L408 and $9.60 in Table 3 Response: This is because 9.6 showed in table 3 is the cost per a strip of 8 samples. We have added a text in the legend for clarification. Attachments Attachment Submitted filename: PLOSone _Comments to the author.pdf https://doi.org/10.1371/journal.pone.0258722.r002
5 Oct 2021 Decision Letter - Luzia Helena Carvalho, Editor Validation study of Boil & Spin Malachite Green Loop Mediated Isothermal Amplification (B&S MG-LAMP) versus microscopy for malaria detection in the Peruvian Amazon PONE-D-21-14913R1 Dear Dr. Valdivia, 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, Luzia Helena Carvalho, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: The authors adequately addressed my comments raised in a previous round of review. Recommendation: Accept. ****** 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 https://doi.org/10.1371/journal.pone.0258722.r003
Formally Accepted
14 Oct 2021 Acceptance Letter - Luzia Helena Carvalho, Editor PONE-D-21-14913R1 Validation study of Boil & Spin Malachite Green Loop Mediated Isothermal Amplification (B&S MG-LAMP) versus microscopy for malaria detection in the Peruvian Amazon Dear Dr. Valdivia: 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 Dr. Luzia Helena Carvalho Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0258722.r004

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