Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Evaluation of Oral Supplementation with a Casein Hydrolysate-based Formula to Favor the Clearance of HR-HPV Infections and their Derived Lesions

Version 1 : Received: 2 December 2019 / Approved: 3 December 2019 / Online: 3 December 2019 (12:07:59 CET)

How to cite: Pingarrón, C.; Duque, A.; López, A.I.; Ferragud, J. Evaluation of Oral Supplementation with a Casein Hydrolysate-based Formula to Favor the Clearance of HR-HPV Infections and their Derived Lesions. Preprints 2019, 2019120029 (doi: 10.20944/preprints201912.0029.v1). Pingarrón, C.; Duque, A.; López, A.I.; Ferragud, J. Evaluation of Oral Supplementation with a Casein Hydrolysate-based Formula to Favor the Clearance of HR-HPV Infections and their Derived Lesions. Preprints 2019, 2019120029 (doi: 10.20944/preprints201912.0029.v1).

Abstract

Cervical cancer screening systems aim to detect established HPV infections early. However, if there are no high-grade lesions, the intervention choice is basically limited to observational follow-up with recommendations on life habits like diet, to favor infection control. Therefore, it is important to establish specific feeding guidelines that provide clear evidence about the benefit they may bring against HPV infections. The present study evaluates the benefits of nutritional supplementation with a casein hydrolysate-based formula in patients with HR-HPV infection, compared with a non-supplemented control group. A total of 118 patients completed 6 months of follow-up. Significant differences between supplemented and control groups were observed for total or partial HR-HPV clearance at 6 months (74.6% vs 35.6%). Supplemented patients also suffered a lower occurrence of new intracervical lesions (0% vs 28.6%), a significantly greater resolution (67.4% vs 41.9%) and less progression of pre-existing lesions (4.7% vs 9.7%) at 6 months. An increase in the effectors of cellular immunity that could be responsible for their effect was also observed in supplemented patients. We conclude that nutritional supplementation with this casein hydrolysate-based formula could improve the outcomes of observational management of HPV infection.

Subject Areas

hpv; papilloma; lsil; hsil; hpv clearance; immunomodulation; nk cells; casein hydrolysate; ditriamino; hupavir

Comments (2)

Comment 1
Received: 27 March 2020
The commenter has declared there is no conflict of interests.
Comment: == '''EVALUATION OF ORAL SUPPLEMENTATION WITH A CASEIN HYDROLYSATE-BASED FORMULA TO FAVOR THE CLEARANCE OF HR-HPV INFECTIONS AND THEIR DERIVED LESIONS ''' == We are a group of biomedical students (3rd year) from Universidad Francisco de Vitoria (Madrid, Spain). As part of our assignments, we were asked to review this paper published as preprint. We would like to kindly share our thoughts and positive criticism in case that it would be of any help for the authors as well as the scientific community. Please see below our comments: Pingarrón et al. evaluated the benefits of a nutritional supplementation with a casein hydrolysate-based formula in patients with HR-HPV infection. The study compared two groups, one with the casein supplementation and one without and concluded that the supplementation can improve the outcomes of HR-HPV infection. We believe that the article should not be published in its current form because, after an exhaustive review of it, we have found major issues that require revision. === '''1. ABSTRACT AND INTRODUCTION ''' === List of comments: 1. The casein-hydrolysate formula administered to patients contains vitamins and minerals. Importantly, the fact that these supplements have already shown a positive effect on infection clearance and on the persistence of the lesions is problematic. The effect of the casein alone should be examined (i.e., on one hand the casein and on the other hand) because by combining the formula with the vitamins, we cannot know if the patients are having an improvement due to the casein or to the vitamins. This could be accomplished conducting a clinical trial organized in three groups: intake of vitamin and mineral supplements, casein supplements and the control (daily diet). 2. The latency period between the infection and the derived lesions is 9.3 months (for CIN-1 lesions) and 11.8 months (for CIN-2 lesions) (doi: 10.1186/1750-9378-2-15). Therefore, we consider that the duration of the study should have been longer to support the recommendation of this treatment. 3. In the abstract is mentioned that life habits such as diet and exercise have an impact on the virus infection progression. Therefore, these parameters should all have been taken into account to control for the impact of different life habits amid groups. 4. In the introduction is noted that HPV infections are associated to cervical cancer in 100% of the cases. We have contrasted this information and could not find evidence supporting this statement. There is an association between HPV and cervical cancer, but it is not a 100% reciprocity [1] [2]. 5. Line 2. Currently, more than 200 different HPV types have been identified, not just 100 types (https://www.cancer.gov/about-cancer/causes-prevention/risk/infectious-agents/hpv-and-cancer). 6. Line 5-7. The HPV classification used is incomplete: • High risk: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 62, 66, 67, 68, 69, 70, 71, 73, 82, 83, 84, 85 y 89. • Low risk: 6, 11, 32, 40, 42, 43, 44, 54, 55, 57, 61, 64, 72, 74, 81, 87 y 91. ''Oncology: Breakthroughs in Research and Practice, June 29, 2016. Management Association, Information Resources, IGI Global''. 7. Line 14-15 and 42-44. There is a contradiction between both affirmations. The first sentence mentions that a hundred percent of HPV infections are associated to cancer. The second sentence indicates that the oncogenic HPV is not enough for malignant progression, which has been corroborated. (https://www.cancer.gov/about-cancer/causes-prevention/risk/infectious-agents/hpv-and-cancer). === '''2. MATERIALS, METHODS AND RESULTS ''' === The use of PCR for HR-HPV detection has several disadvantages: - This method may not identify all HVP genotypes present in the sample. - In many cases, multiple HPV infection occur. Some HPV sequences might be amplified more than others, which complicates the detection of the genotype, and in consequence, the diagnosis (Abreu, A.L.P., Souza, R.P., Gimenes, F. et al. A review of methods for detect human Papillomavirusinfection. Virol J 9, 262 (2012). (https://doi.org/10.1186/1743-422X-9-262). List of comments: 1. One to six patients who had not eliminated the virus were excluded from the result because the type of HPV that they were infected with could not be identified. These patients were part of the casein-treated groups (groups one and three). There are currently multiple tests for the different types of HR-HPV including tests that detect high-risk types such as PapilloCheck HPV screening, so there were additional methods available for determining high-risk virus types in case of doubt. If these six patients had not been excluded, the results may not have been significantly different due to the small sample size of the study. We cannot determine if this difference is statistically significant due to lack of data. Also, according to this result, the number of patients participating in each group of study has been modified to make the results more remarkable. In Figure 1, the first group has 30 patients and if we compared it to line 6 of 3.1 of the results, the group has 26, we do not understand why the number of patients change between each group. As we can observe, in the first graph the results are modified because on table 1 the number of patients is different. __Figure 1a__. The HR-HPV clearance is classified in three groups, the third one is “Total or partial HR-HPV clearance”. In the investigation, a third group is created in which is added a total clearance and partial clearance. This is the only group in which the differences reached statistical significance. 2. __Figure 2__."Control 6 meses" is incorrect. It should be written “6 month control”. 3. __Table 2__. It shows parameters of casein hydrolysate-based formula treated patients. It is also important to include data from control patients to assess the impact of the casein hydrolysate-based formula between infected and healthy individuals. 4. Related to the statistical analysis we found three basics errors. First, Chi-squared test and T-Student are normally used when the sample is less than 30 people, in this paper the n number is >100. Moreover, these methods are used when the null hypothesis is true, and this is related with random odds between variables. In this case, to solve the problem our class suggested using the PICO method [4]., which may be more appropriate to analyze clinical trials. In some figures of the paper (e.g. Figure 2) it is considered statistical significance when p < 0.1. When the p level is higher than the confidence level, the results obtained are not statistically significant; making the differences between the results not significant. [5]. '''=== 3. BIBLIOGRAPHY ===''' [1] ''Human papillomavirus and cervical cáncer''. DOI: 10.1128/cmr.16.1.1-17.2003. [2] ''Epidemiology and burden of HPV-related disease''. DOI: ''10.1016/j.bpobgyn.2017.08.006''. [3] ''Human papillomavirus persistence and nutrients involved in the methylation pathway among a cohort of young women''. PMID: 11927495. [4] ''Thomas D.Gauthier, Mark E.Hawley., (2007), ScienceDirect, Statistical Methods-Chapter 5''. DOI:10.1016/B978-012369522-2/50006-3 [5] ''Greenland, S., Senn, S., Rothman, K., Carlin, J., Poole, C., Goodman, S. and Altman, D., 2020. Statistical Tests, P Values, Confidence Intervals, And Power: A Guide To Misinterpretations''.
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Comment 2
Received: 27 March 2020
Commenter: 3ºBiomedicine
The commenter has declared there is no conflict of interests.
Comment:

EVALUATION OF ORAL SUPPLEMENTATION WITH A CASEIN HYDROLYSATE-BASED FORMULA TO FAVOR THE CLEARANCE OF HR-HPV INFECTIONS AND THEIR DERIVED LESIONS



We are a group of biomedical students (3rd year) from Universidad Francisco de Vitoria (Madrid, Spain). As part of our assignments, we were asked to review this paper published as preprint. We would like to kindly share our thoughts and positive criticism in case that it would be of any help for the authors as well as the scientific community. Please see below our comments:

Pingarrón et al. evaluated the benefits of a nutritional supplementation with a casein hydrolysate-based formula in patients with HR-HPV infection. The study compared two groups, one with the casein supplementation and one without and concluded that the supplementation can improve the outcomes of HR-HPV infection.

We believe that the article should not be published in its current form because, after an exhaustive review of it, we have found major issues that require revision.

1. ABSTRACT AND INTRODUCTION



List of comments: 1. The casein-hydrolysate formula administered to patients contains vitamins and minerals. Importantly, the fact that these supplements have already shown a positive effect on infection clearance and on the persistence of the lesions is problematic. The effect of the casein alone should be examined (i.e., on one hand the casein and on the other hand) because by combining the formula with the vitamins, we cannot know if the patients are having an improvement due to the casein or to the vitamins. This could be accomplished conducting a clinical trial organized in three groups: intake of vitamin and mineral supplements, casein supplements and the control (daily diet).

2. The latency period between the infection and the derived lesions is 9.3 months (for CIN-1 lesions) and 11.8 months (for CIN-2 lesions) (doi: 10.1186/1750-9378-2-15). Therefore, we consider that the duration of the study should have been longer to support the recommendation of this treatment.

3. In the abstract is mentioned that life habits such as diet and exercise have an impact on the virus infection progression. Therefore, these parameters should all have been taken into account to control for the impact of different life habits amid groups.

4. In the introduction is noted that HPV infections are associated to cervical cancer in 100% of the cases. We have contrasted this information and could not find evidence supporting this statement. There is an association between HPV and cervical cancer, but it is not a 100% reciprocity [1] [2].

5. Line 2. Currently, more than 200 different HPV types have been identified, not just 100 types (https://www.cancer.gov/about-cancer/causes-prevention/risk/infectious-agents/hpv-and-cancer).

6. Line 5-7. The HPV classification used is incomplete:

• High risk: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 62, 66, 67, 68, 69, 70, 71, 73, 82, 83, 84, 85 y 89.
• Low risk: 6, 11, 32, 40, 42, 43, 44, 54, 55, 57, 61, 64, 72, 74, 81, 87 y 91.

Oncology: Breakthroughs in Research and Practice, June 29, 2016. Management Association, Information Resources, IGI Global.

7. Line 14-15 and 42-44. There is a contradiction between both affirmations. The first sentence mentions that a hundred percent of HPV infections are associated to cancer. The second sentence indicates that the oncogenic HPV is not enough for malignant progression, which has been corroborated.
(https://www.cancer.gov3/about-cancer/causes-prevention/risk/infectious-agents/hpv-and-cancer).

2. MATERIALS, METHODS AND RESULTS



The use of PCR for HR-HPV detection has several disadvantages:

- This method may not identify all HVP genotypes present in the sample.
- In many cases, multiple HPV infection occur. Some HPV sequences might be amplified more than others, which complicates the detection of the genotype, and in consequence, the diagnosis (Abreu, A.L.P., Souza, R.P., Gimenes, F. et al. A review of methods for detect human Papillomavirusinfection. Virol J 9, 262 (2012).
(https://doi.org/10.1186/1743-422X-9-262).

List of comments: 1. One to six patients who had not eliminated the virus were excluded from the result because the type of HPV that they were infected with could not be identified. These patients were part of the casein-treated groups (groups one and three). There are currently multiple tests for the different types of HR-HPV including tests that detect high-risk types such as PapilloCheck HPV screening, so there were additional methods available for determining high-risk virus types in case of doubt.
If these six patients had not been excluded, the results may not have been significantly different due to the small sample size of the study. We cannot determine if this difference is statistically significant due to lack of data.
Also, according to this result, the number of patients participating in each group of study has been modified to make the results more remarkable. In Figure 1, the first group has 30 patients and if we compared it to line 6 of 3.1 of the results, the group has 26, we do not understand why the number of patients change between each group. As we can observe, in the first graph the results are modified because on table 1 the number of patients is different.

Figure 1a. The HR-HPV clearance is classified in three groups, the third one is “Total or partial HR-HPV clearance”. In the investigation, a third group is created in which is added a total clearance and partial clearance. This is the only group in which the differences reached statistical significance
2. Figure 2."Control 6 meses" is incorrect. It should be written “6 month control”.
3. Table 2. It shows parameters of casein hydrolysate-based formula treated patients. It is also important to include data from control patients to assess the impact of the casein hydrolysate-based formula between infected and healthy individuals.
4. Related to the statistical analysis we found three basics errors. First, Chi-squared test and T-Student are normally used when the sample is less than 30 people, in this paper the n number is >100. Moreover, these methods are used when the null hypothesis is true, and this is related with random odds between variables. In this case, to solve the problem our class suggested using the PICO method [4]., which may be more appropriate to analyze clinical trials.
In some figures of the paper (e.g. Figure 2) it is considered statistical significance when p < 0.1. When the p level is higher than the confidence level, the results obtained are not statistically significant; making the differences between the results not significant. [5].

3. BIBLIOGRAPHY



[1] Human papillomavirus and cervical cáncer. DOI: 10.1128/cmr.16.1.1-17.2003.

[2] Epidemiology and burden of HPV-related disease. DOI: 10.1016/j.bpobgyn.2017.08.006.

[3] Human papillomavirus persistence and nutrients involved in the methylation pathway among a cohort of young women. PMID: 11927495.

[4] Thomas D.Gauthier, Mark E.Hawley., (2007), ScienceDirect, Statistical Methods-Chapter 5. DOI:10.1016/B978-012369522-2/50006-3

[5] Greenland, S., Senn, S., Rothman, K., Carlin, J., Poole, C., Goodman, S. and Altman, D., 2020. Statistical Tests, P Values, Confidence Intervals, And Power: A Guide To Misinterpretations.
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