PONE-D-24-05361Treatment with Oxfendazole increased levels of cardiac troponin I in pigs naturally infected with Taenia solium cysticercosis.PLOS ONE

Dear Dr. Zulu,

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Additional Editor Comments:

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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

Reviewer #3: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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: Overall comments:

The study lacks novelty, since it is already expected that anthelmintic treatment with OFZ damages cysts located in cardiac muscle, and activates the host’s immune system to produce a strong inflammatory response (several studies have demonstrated this using more precise markers such as histopathology or immunohistochemistry).

It is not known and not well detailed why studying cTn1 may help to a better understanding of the immunopathological processes that occur in CC and NC.

A positive Ag-ELISA result may give false-positives, specially in pigs with low cyst burden (pigs with only a single cyst on tongue exam. ¿Pigs where randomized according t Ag-ELISA levels?

The discussion section lacks a paragraph of limitations/strenghts of the study.

Specific comments:

Line 28: “On cardiac troponin 1 (cTn1) elevation in ……”

Lines 39-42: add P values for significance as required

Lines 42-43: ¿How you can determine this?

Line 44: “This study shows that….”

Lines 62-64: Humans are not more propense to develop NCC than pigs. This occurs because cysts in the CNS surviver for a longer time. Please, modify.

Lines 92-94: Is not clear the link for studying cTn1 for cardiac damage in the context of porcine cysticercosis. Especially, not all pigs with cysticercosis have cysts in their heart. Justify.

Line 134-135: “the role of cTnI on inflammation” or “cTnI as marker of heart damage/inflammation”??

Lines 166-175: I consider this section as unnecesary (please, remove it).

Lines 187-191: It is not described the dimensions of experimental corrals, how many animals per corral.Lines 214-215: in fasting??

Lines 246-246: ¿What happen if there were differences between duplicates (e.g. variation more than 50%)?

Lines 281-284: It would be useful to report how antigen levels were described during follow-up according experimental groups (¿mean ± SEM?).

Line 293: It would be interesting to describe cTnI levels in pigs among experimental groups at baseline (and baseline statistical comparisons).

Lines 293-298: You should emphasise the increased levels in cTn1 in the IT pigs versus the other groups.

Line 304: Figure 304: (n=9, n=9, n =…..looks very repetitive, please modify)

Lines 313-316: It is not necessary to report the F test, and degrees of freedoms for ANOVA results, just P value.

Lines 314-315: It is not clear what the modified effect is. ¿Did you see the modification effect between experimental group in hourly and weekly observations? That should be the effect, since most of the difference between IT group versus and the other groups were observed until 72 hours after treatment, but not observed during weekly observations.

Lines 317-326: Differences should emphazise that cTnI levels were higher for IT pigs (not just “different”).

Lines 329-337: In the first paragraph of the discussion section you describe again results using Mean ± SEM. It looks like results section again (in fact this should be in the results section). Re-paraphrase this section.

Lines 338-342: This section of the discussion is not described or supported in the results section. The impact of antiparasitic treatment on acute inflammation (as soon as 48h after treatment onset) has been widely described in previous studies using the NCC pig model, cite them.

Lines 358-361: the higher levels during weekly observation in the INT group compared to the IT group can also reflect differences in pre-existing cyst inflammation between groups using the natural pig model (a main drawback when using naturally infected pigs).

Lins 382-403: Conclusions are too long. You should focus on the main results and potential impact.

Reviewer #2: It is a very nice and well-prepared manuscript that reports the possible myocardial damage after treatment with oxfendazole in pigs naturally infected with Taenia solium. The paper is well organized and generally well written, though there are a few minor errors throughout the manuscript. The relevant literature is well-reviewed. Therefore, I suggest publishing this paper after revisions, primarily to address minor issues and correct a few stylistic errors.

In the Materials and Methods section, it is crucial to specify whether the infected pigs were breed or not (I assume they weren't), and the same goes for the non-infected ones. The latter came from commercial farms; I assume they were not crossbred animals. Normally, pigs from technical farms display different physiological responses compared to crossbred animals (not breeds) and free-range animals, such as stress during weaning, increased nervousness, and different feeding methods. To avoid conjecture, you must provide specific details about pigs.

Similarly, the use of naturally infected T. solium-infected pigs makes it crucial to determine the approximate age of the animals. This would indirectly help to determine the age of the cysts. Remember that older pigs are more likely to have degenerated cysts.

This last observation prompts me to question why they didn't perform necropsies on the pigs to assess the status of the cysts in the untreated pigs. You would even have been able to evaluate myocardial damage through histopathology. You could potentially include this as one of the "limitations of the study" in the Discussion section.

Minor comments

Title: replace “Oxfendazole” with “oxfendazole”

Line 44: replace “Taenia solium” with “T. solium”

Line 60: "metacestode larval stages" is redundant; it uses only "metacestode"

Line 61: humans are “accidental intermediate hosts”

Line 70: replace “Oxfendazole” with “oxfendazole”

Line 82: replace “ELISA” with “enzyme linked immunosorbent assay (ELISA)”

Line 108: to define “CK-MB”

Lines 126-132: An animal ethics protocol (IACUC), not an IRB, is typically required for conducting experiments with animals. If authors have an IACUC protocol, they should include it.

Line 136: replace “Oxfendazole” with “oxfendazole”

Lines 156-161: add a reference or references that support the techniques.

Line 181: write “ad libitum” in italics

Line 214: Add oxfendazole information (oncentration, brand and country of origin)

Figure 1: replace “T. Solium” with “T. solium”

Figure 1: replace “n= 18” with “n = 18”, idem in others numbers

Reviewer #3: ABSTRACT

- Line 39 – Please show the numbers (mean, sd) of the levels of cTnI in each group

- Is there any additional information of necropsy? What was the appearance of cyst after treatment? Any inflammatory response in their heart? How did the authors confirm the presence of cysts in the hearts?

INTRODUCTION

- Line 108 – What does CK-MB stand for?

M&M

- Line 149 – Could the authors give an estimated prevalence of porcine cysticercosis in the area where pigs came from?

- Line 155 – I am kind of confused with the source of pigs. Authors sought for 18 tongue positive pigs and 18 tongue negative ones from that market; could the authors confirm this? In addition, they said that another group of pigs were purchased from a commercial farm. Please clarify this point.

- Line 166 – what was the purpose of washing and dipping the pigs? If this is not crucial for the development of the trial then it can be removed from the manuscript.

- Line 195 – whare did the numbers 10 and 20 come from? I would have calculated the sample size using the mean difference of cTnI that was expected in each group.

- What was the mean age of the pigs?

- Line 244 / line 262 – Is there any references for the described technique? I suggest including them as authors might have followed that protocol

- Did the authors perform a necropsy to verify the pig heart infection? It might be possible that the number of cysts in the hearts influence the levels of cTnI

RESULTS

- Please provide some numbers (means) in this section to be able to visualize those differences

- Line 301 – was the “slightly higher” significant? If not, please say it

- Line 311 – Again, please include some means here to see the differences

DISCUSSION

- First paragraph – this is what I would like to see in Results. Instead of SEM add the numerical value

- Line 346 – I think the way it is written is not the appropriate style … “as reported by (17)”

- Line 359 – was that difference statistically significant compared to non-infected pigs? If not be cautious to rise a conclusion

- Line 338-357 – it is a very long paragraph, it would be better to split into at least two

- It would be interesting to see what limitations this study presented. For instance, I do not see the necropsy results, number of cyst in the pig hearts, level of inflammation, etc?

REF

- There is a research group which has published several paper on OXF use in pigs, however, I do not see any reference from it. I would suggest to include some of them in the intro or discussion sections.

- Pharmacokinetics, Safety, and Tolerability of Oxfendazole in Healthy Volunteers: a Randomized, Placebo-Controlled First-in-Human Single-Dose Escalation Study.

An G, Murry DJ, Gajurel K, Bach T, Deye G, Stebounova LV, Codd EE, Horton J, Gonzalez AE, Garcia HH, Ince D, Hodgson-Zingman D, Nomicos EYH, Conrad T, Kennedy J, Jones W, Gilman RH, Winokur P. Antimicrob Agents Chemother. 2019 Mar 27;63(4):e02255-18. doi: 10.1128/AAC.02255-18

Oxfendazole: a promising agent for the treatment and control of helminth infections in humans. Gonzalez AE, Codd EE, Horton J, Garcia HH, Gilman RH. Expert Rev Anti Infect Ther. 2019 Jan;17(1):51-56. doi: 10.1080/14787210.2018.1555241.

Preclinical studies on the pharmacokinetics, safety, and toxicology of oxfendazole: toward first in human studies. Codd EE, Ng HH, McFarlane C, Riccio ES, Doppalapudi R, Mirsalis JC, Horton RJ, Gonzalez AE, Garcia HH, Gilman RH; Cysticercosis Working Group in Peru. Int J Toxicol. 2015 Mar-Apr;34(2):129-37. doi: 10.1177/1091581815569582.

A high oxfendazole dose to control porcine cysticercosis: pharmacokinetics and tissue residue profiles. Moreno L, Lopez-Urbina MT, Farias C, Domingue G, Donadeu M, Dungu B, García HH, Gomez-Puerta LA, Lanusse C, González AE. Food Chem Toxicol. 2012 Oct;50(10):3819-25. doi: 10.1016/j.fct.2012.07.023.

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Reviewer #3: Yes:  Cesar M. Gavidia

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PONE-D-24-05361R1Treatment with Oxfendazole increased levels of cardiac troponin I in pigs naturally infected with Taenia solium cysticercosis.PLOS ONE

Dear Dr. Zulu,

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 Feb 09 2025 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: https://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,

Chengming Fan, MD, PhD

Academic Editor

PLOS ONE

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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 #2: All comments have been addressed

Reviewer #4: (No Response)

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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 #2: Yes

Reviewer #4: Partly

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

Reviewer #2: Yes

Reviewer #4: N/A

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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 #2: Yes

Reviewer #4: No

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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 #2: Yes

Reviewer #4: Yes

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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 #2: Congratulations to the authors on their commendable effort in enhancing the quality of their manuscript.

Reviewer #4: The manuscript is well written and is significantly improved based on the reviewers’ concerns.

It is important for the authors to know and note that cardiac troponin cannot be used as a specific marker for cysticercosis infection in pigs. Cardiac troponin IS a biomarker for any form of cardiac injury and non-specific for injury related to cysticercosis. Therefore, any stress related cardiac injury, or other underlying diseases that cause myocardial injury in pigs will cause the release of cardia troponin. Given this, the fundamental premise of the study is flawed.

Evaluation of cardiac troponin is not specific, as other cardiac diseases or infection that targets cardiac muscles will induce myocardial cell damage that will release in the release of cardiac troponin, so elevation of cardiac troponin level doesn’t necessarily translate to cardiac cysticercosis.

So, there are no gross pictures of cyst-infested tissues from the necropsied animals; even on the last of experiment? Not even the heart that is the basis of this study? No histopathologic evaluation of tissues to support their findings of presence of cysts, inflammation, and mineralization? No evaluation of cytokine levels? No immunohistochemistry?

The response of the authors to the reviewers about these questions is unacceptable.

Does the number of cysts correlate with an increase in cardiac troponin?

“Carcass dissections at the end of the study also confirmed presence of cysts in the heart.”

No pictures to document this? Do the number of cysts in tissues increase overtime?

Line 440-442: Of course. The pigs are not specific pathogen free animals; therefore, they potentially could have an underlying condition that increased stress and thus higher levels of cardiac troponin.

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Reviewer #2: No

Reviewer #4: Yes:  Shakirat Adetunji

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Treatment with oxfendazole increased levels of cardiac troponin I in pigs naturally infected with Taenia solium cysticercosis.

PONE-D-24-05361R2

Dear Dr. Zulu,

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.

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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,

Chengming Fan, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All the comments were well addressed

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 #2: All comments have been addressed

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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 #2: Yes

**********

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

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 #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 #2: Yes

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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 #2: I congratulate the authors on their commendable work in enhancing the quality of their manuscript...

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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 #2: No

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