Review Reports - Zoonotic Helminths in the Southern Peruvian Altiplano: A Four-Year Sero-Epidemiological Study and One Health Policy Implications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer Comments

The present study investigated the prevalence of three helminth parasitic, zoonotic infections that are part of the Neglected Tropical Diseases,—echinococcosis, fascioliasis and the taeniasis/cysticercosis complex—among residents of the Chucuito Health Network (Puno Health Region, Peru) over four years (2018–2021). The study detected the presence of antibodies against these parasites using Elisa as the diagnostic tool, from sera (n = 910) collected from health care facility attendees.

The study presents the results in the One-Health context, therefore addresses a very critical public health challenge, particularly the zoonosis by neglected tropical diseases, that often times do not receive priority attention although they still infect large numbers of humans and animals.

Minor comments

Line 75 grammar “to screening”
Line 136 “significance of < 0.05”
The following need to be improved for more clarity Lines 136-137
The spelling error in Figure 4 must be corrected

Clarity on Antibody detection Whether it was both IgM and/or IgGM to differentiate whether acute/recent infection and past exposure

Major comments

The manuscript needs reorganizing for example:

Materials and Methods

1.Table 1 is referenced in Section 2.2 (Sample Collection) lines 96-97 . This section describes sera separation and cold chain maintenance and no relation to the number of samples collected per area, but reflects the text in Section 2.4 Sample Design lines 112-114.

2.In fact the order could be rearranged – Section 2.4- Sample Design could be labelled as Study Design which should follow immediately after Section 2.1; before sera separation and Elisa analysis. Study design should describe the type of study eg cross sectional observational etc. Then Sampling which needs more details on sampling – how the sampling was done in the region and – were there sample size considerations , how the selection was made eg randomization or sequential, or all patients presenting during the recruitment period.

Results

Data in Table 1 should be part of the results .
The prevalence data (Figure 3 ) should come right after the number of samples per area ( Table 1 data), Followed by Figure 2 ( marginal means) before all the statistical data in Figures and Tables

5.Table 2 – a descriptive legend that defines indication the values presented for each year.

6.A better depiction of the statistical differences is needed instead of the generic statement (between the years and diseases) eg fasciolosis and taeniasis in 2018 ; taeniasis in 2018 and 2019 could not be significantly different)

7.In general, the statistical description and presentation / interpretation needs to be improved throughout the manuscript.

8Full description of table/figure headings with legends describing the acronyms/ abbreviations, p values where relevant.

Discussion and conclusions

In the Discussion, construction of sentences with references should follow convention, ie reference the author/s then the reference number

10.A conclusion that the upsurge of infections in 2021 is “plausibly driven by evolving environmental and socio-economic conditions, including intensified human–animal contact and constrained access to health services in remote communities.” is not based on the findings of the current results, since these variables were not analyzed in this study. This need rephrasing.

11.For completeness, the study limitations must be highlighted, including any design vs generalizability of results etc.

Author Response

Comments 1: Line 75 grammar “to screening”

Response 1: We thank the reviewer for pointing this out. We have corrected the sentence to read “This research aims to screen for three zoonotic diseases in the human population …”.

Comments 2: Line 136 “significance of < 0.05”

The following need to be improved for more clarity Lines 136-137

Response 2: We have clarified the description of statistical significance in the Methods and rephrased the Results paragraph, avoiding the ambiguous expression “significance of sig.<0.05”.

The previous sentence:

“From Table 2, with a significance of sig.< 0.05; we can point out evidence that the presence of diseases is significantly different for each year; likewise, statistical differences are shown in each year that was screened.”
has been replaced by:
“At a significance level of p < 0.05, we found evidence that disease prevalence differed across the four survey years; post-hoc comparisons identified year-on-year differences for each condition.”

Comments 3: The spelling error in Figure 4 must be corrected

Response 3: We have corrected the spelling in Figure 4 (axis labels and legend).

Comments 4: Clarity on Antibody detection Whether it was both IgM and/or IgGM to differentiate whether acute/recent infection and past exposure

Response 4: We appreciate this important point. The ELISA kits used detect IgG antibodies (and do not distinguish IgM). Consequently, the results reflect previous exposure (or chronic infection) rather than acute infection. We have clarified this in the Methods and acknowledged it as a limitation.

Changes in manuscript:

Section 2.3. Serological analysis:

“Sera were screened for IgG antibodies against Echinococcus granulosus, Fasciola hepatica and the Taenia solium complex using commercial ELISA kits. These assays detect IgG and therefore reflect past exposure rather than acute infection.”

Discussion – Limitations subsection:

“The ELISA kits detect IgG only and cannot distinguish acute from past infection, so our seroprevalence estimates should be interpreted as indicators of cumulative exposure.”

Comments 5: The manuscript needs reorganizing for example:

Materials and Methods

Response 5: We agree and have reorganised Sections 2.1–2.4 and expanded the study-design description.

Changes in manuscript:

Section titles and order now read:

2.1 Study area (renamed from “Study delimitation” as also suggested by Reviewer 4)
2.2 Study design and sampling (new, expanded)
2.3 Sample collection
2.4 Serological testing
2.5 Statistical analysis

New text in Section 2.2 (Study design and sampling):

“We conducted a cross-sectional, observational study nested within the routine serological screening activities of the Chucuito Health Network. All individuals attending participating primary health centres during the study period and for whom sufficient serum was available were eligible for inclusion. Sampling was therefore by consecutive inclusion rather than probabilistic randomisation. The minimum required sample size (n = 384) to detect a prevalence of 5 % with 95 % confidence and 2 % precision was exceeded, as 910 sera were ultimately analysed. Annual sample numbers and population denominators are summarised in Table 1.”

Table 1 reference moved:
References to Table 1 have been removed from Sample collection (previous 2.2) and are now placed in Results (Section 3.1), where sample numbers are first summarised.

Comments 6: Results

Data in Table 1 should be part of the results .

Response 6: We agree. Table 1 is now clearly introduced and discussed in the Results section.

Change in manuscript:

Results, Section 3.1:

“A total of 910 sera were analysed, with 182 in 2018, 168 in 2019, 376 in 2020 and 184 in 2021 (Table 1).”

Comments 7:

The prevalence data (Figure 3 ) should come right after the number of samples per area ( Table 1 data), Followed by Figure 2 ( marginal means) before all the statistical data in Figures and Tables

Response 7:

We have rearranged the Results section so that:

Table 1 (sample numbers and population)
Figure 3 (prevalence by year and disease)
Figure 2 (marginal means)
Figure 4 and Table 3 (PCA and correlations)

are presented in that logical order.

Change in manuscript:

Reordered the paragraphs and figure references in Section 3.

Comments 8:

5.Table 2 – a descriptive legend that defines indication the values presented for each year.

Response 8: We have expanded the legend to specify that the values are numbers of seropositive individuals and we have improved the description of the significant differences in the text. To avoid clutter, we summarise key findings in the text rather than adding many pairwise p-values in the table, but we now specify which comparisons were significant.

Changes in manuscript:

Table 2 legend now reads:

“Table 2. Number of ELISA-positive sera for each zoonotic disease by year in the Chucuito Health Network, 2018–2021. Pairwise Fisher’s exact tests indicated significant differences between years for echinococcosis and fascioliasis (p < 0.05); taeniasis/cysticercosis differences were not statistically significant.”

Results text updated:

“… Echinococcosis prevalence in 2021 (9.2 %) was higher than in 2018–2020 (4.4–5.4 %), and Fisher’s exact tests confirmed significant between-year differences for echinococcosis and fascioliasis, but not for taeniasis/cysticercosis.”

Comments 9:

7.In general, the statistical description and presentation / interpretation needs to be improved throughout the manuscript.

Response 9: We have clarified the statistical methods (tests, significance level, what was compared) in Section 2.5 and aligned the Results interpretation accordingly.

Changes in manuscript:

Section 2.5:

“Proportions were compared using Fisher’s exact tests (2 × 4 contingency tables for each pathogen across years). Where indicated, pairwise post-hoc comparisons were conducted with Bonferroni-adjusted p-values. Principal Component Analysis (PCA) was applied to annual prevalence estimates to explore covariance patterns among the three pathogens.”

Section 3.3:
We removed the incorrect statement about a “negative association between case numbers and calendar year” (see Reviewer 2) and now describe the PCA qualitatively, without over-interpreting correlations.

Comments 10:

8Full description of table/figure headings with legends describing the acronyms/ abbreviations, p values where relevant.

Response 10:
We have revised all table and figure captions to ensure they are self-explanatory and that all abbreviations (CHN, PCA, ELISA) and statistical elements (p < 0.05) are defined in either captions or footnotes.

Comments 11:

Discussion and conclusions

In the Discussion, construction of sentences with references should follow convention, ie reference the author/s then the reference number

A conclusion that the upsurge of infections in 2021 is “plausibly driven by evolving environmental and socio-economic conditions, including intensified human–animal contact and constrained access to health services in remote communities.” is not based on the findings of the current results, since these variables were not analyzed in this study. This need rephrasing.
For completeness, the study limitations must be highlighted, including any design vs generalizability of results etc.

Response 11: We have revised the Discussion to introduce references as “Author(s) [number]” rather than only numbers. We also added a Limitations paragraph.

Changes in manuscript:

Example:

“Guerra and Ramírez [29] reported a prevalence of 5 % …” instead of “as reported by [29].”

New Limitations paragraph at end of Discussion:

This study has several limitations. First, it is based on a cross-sectional convenience sample of health-facility attendees rather than a population-based random sample, which may limit generalisability. Second, we used secondary programme data without unique identifiers and could not exclude repeated testing of some individuals. Third, the ELISA assays detect IgG only and cannot distinguish active from past infection. Fourth, we lacked individual-level covariates such as age, sex, occupation and animal contact, so we cannot model risk factors. These caveats should be considered when interpreting the findings.

Reviewer 2 Report

Comments and Suggestions for Authors

This paper deals with analysis of prevalences of three zoonotic diseases, echinococcocis, fascioliasis and the taeniasis/cysticercosis complex, in human population of a given region (Chucuito) of Peru across annually samples from 2018 to 2021. Authors used ELISA to detect pathogen specific antibodies. The data and analyses are original and interesting, providing important and useful data regarding poorly known zoonotic conditions in this vulnerable area of high Andean communities. The manuscript deserves publication in IJERPH.

However, I would discuss the main conclusion of the authors and also make a couple of minor suggestions, as follows:

First, I agree Pearson correlation analysis supports and qualified the pattern of prevalence observed for echinococcocis in the PCA analysis; however, it is indicating a negative strong correlation r = −0.707, p = 0.010 as currently stated in Ms lines 153-155, meaning a significant decrease in prevalence from 2018 to 2021, echinococcocis was highest in earlier years of sampling and steadily declined over time. Such that conclusions, lines 220 to 224 need some rephrasing please.

Second, the references (bibliography section) must be cited correctly according to the journal IJERPH rules. Please double check this.

Minor points:

Lines 42-43: Helminth-mediated zoonoses include nematodes (trichinosis), cestodes (cysticercosis, echinococcosis) and trematodes (schistosomiasis) and fascioliasis [9]. Incomplete sentence? I do not understand the context of this statment, and reference #9 is for Iran zoonosis. Many more zoonotic helminths are lacking (Anisakiasis, Heterophiids, etc), please clarify the statement.

Lines 123 – 124 PCA allows to explore the tendency of the prevalences across the years of the study, not the temporal relationships.

Line 101, Table 1, please add enough details to understand the data provided in the table. Line 113 avoid repeating data provided in any table/figure. Valid for most tables/ figures: in the legends, provide enough details to understand the values given.

As previously commented, it is not easy to examine the literature as currently stand. However, I suspect several important references are lacking; following I include a brief perusal of references important in the context of this Ms:

Avila, H. G., Santos, G. B., Cucher, M. A., Macchiaroli, N., Pérez, M. G., Baldi, G., ... & Kamenetzky, L. (2017). Implementation of new tools in molecular epidemiology studies of Echinococcus granulosus sensu lato in South America. Parasitology international, 66(3), 250-257.

Bargues, M. D., Gayo, V., Sanchis, J., Artigas, P., Khoubbane, M., Birriel, S., & Mas-Coma, S. (2017). DNA multigene characterization of Fasciola hepatica and Lymnaea neotropica and its fascioliasis transmission capacity in Uruguay, with historical correlation, human report review and infection risk analysis. PLoS Neglected Tropical Diseases, 11(2), e0005352.

Carmona, C., & Tort, J. F. (2017). Fasciolosis in South America: epidemiology and control challenges. Journal of helminthology, 91(2), 99-109.

Cucher, M. A., Macchiaroli, N., Baldi, G., Camicia, F., Prada, L., Maldonado, L., ... & Kamenetzky, L. (2016). Cystic echinococcosis in South America: systematic review of species and genotypes of Echinococcus granulosus sensu lato in humans and natural domestic hosts. Tropical Medicine & International Health, 21(2), 166-175.

Cuervo, P. F., Mera y Sierra, R., Artigas, P., Fantozzi, M. C., Bargues, M. D., & Mas-Coma, S. (2025). Impact of climate change on the spread of fascioliasis into the extreme south of South America. PLOS Neglected Tropical Diseases, 19(8), e0013433.

Laurimäe, T., Kinkar, L., Andresiuk, V., Haag, K. L., Ponce-Gordo, F., Acosta-Jamett, G., ... & Saarma, U. (2016). Genetic diversity and phylogeography of highly zoonotic Echinococcus granulosus genotype G1 in the Americas (Argentina, Brazil, Chile and Mexico) based on 8279 bp of mtDNA. Infection, Genetics and Evolution, 45, 290-296.

Manterola, C., Totomoch-Serra, A., Rojas, C., Riffo-Campos, A. L., & Garcia-Mendez, N. (2022). Echinococcus granulosus sensu lato genotypes in different hosts worldwide: a systematic review. Acta parasitologica, 67(1), 161-185.

Martins, I. V., & Verocai, G. G. (2024). Fasciola hepatica. Trends in Parasitology.

Pavletic, C. F., Larrieu, E., Guarnera, E. A., Casas, N., Irabedra, P., Ferreira, C., ... & Vilas, V. J. (2017). Cystic echinococcosis in South America: a call for action. Revista panamericana de salud publica, 41, e42.

Poggio, T. V., Jensen, O., Chacon Saravia, T., Pino Nuñez, A., Boado, L. A., Gomez, J. M., & Heath, D. (2022). Past and future methods for controlling Echinococcus granulosus in South America.

Rojas-Pincheira, C., Manterola, C., García-Méndez, N., & Totomoch-Serra, A. (2022). Distribución espacial de los Genotipos de Echinococcus granulosus sensu lato por hospedero en el continente americano. Revisión sistemática. Revista médica de Chile, 150(7), 944-957.

Author Response

Comments 1:

Response 1: We are grateful to the reviewer for catching this inconsistency. We re-examined the correlation analysis and confirmed that the previously reported negative correlation was misinterpreted and not correctly specified in the text. In the revised manuscript we remove the erroneous statement about a significant negative correlation between case numbers and calendar year. Instead, we rely on the descriptive prevalence values and Fisher’s exact tests only.

We have also softened the language in the Conclusions: we now state that echinococcosis peaked in 2021 and that the apparent increase should be interpreted cautiously, given the small number of time points and sample sizes.

Changes in manuscript:

Section 3.3 (PCA and correlations):
The sentence:

“Pearson correlation analysis corroborated this pattern, revealing a significant negative association between case numbers and calendar year (r = −0.707, p = 0.010)….”
has been deleted.

The paragraph now reads:

“Using PCA, we identified a strong loading of echinococcosis on the first component in the final three years of sampling (Figure 4). This pattern is consistent with the descriptive observation that echinococcosis prevalence was higher in 2021 than in previous years, although formal trend testing with only four time points has limited power and should be interpreted with caution.”

Conclusions, point 2 (Recent upsurge):
Previously:

“… showed a pronounced increase in 2021, plausibly driven by evolving environmental and socio-economic conditions …”
Now:
“All three diseases—most conspicuously echinococcosis—showed their highest observed prevalence in 2021. With only four annual surveys and modest case counts, this apparent increase should be regarded as a signal for further investigation rather than definitive evidence of a sustained upward trend.”

Comments 2:

Second, the references (bibliography section) must be cited correctly according to the journal IJERPH rules. Please double check this.

Response 2:
We have carefully revised all references to conform to MDPI/IJERPH style (abbreviated journal names, year after journal, author initials, no “and”/“et al.” in the author list, complete volume and page numbers). Incorrect or incomplete references have been corrected, and duplicated entries removed.

Comments 3:

Minor points:

Response 3: We have rephrased the sentence to clarify that we are providing examples, not an exhaustive list, and removed the ambiguous link to a single reference.

Change in manuscript:

Introduction, helminth zoonoses paragraph:

“Key helminth-mediated zoonoses include nematodes (e.g., trichinellosis), cestodes (e.g., cysticercosis, echinococcosis) and trematodes (e.g., schistosomiasis, fascioliasis), among others [8].”

Comments 4:

Lines 123 – 124 PCA allows to explore the tendency of the prevalences across the years of the study, not the temporal relationships.

Response 4: We agree and have rephrased the description to avoid implying causal “temporal relationships”.

Change in manuscript:

Section 2.5:

“PCA was used to explore patterns in annual prevalence across the three pathogens” instead of “temporal relationships”.

Comments 5:

Response 5:
We have expanded the legend to specify that Table 1 shows provincial population and number of sera analysed per year in the CHN, and we removed redundant repetition of the same numbers from the text.

Comments 6:

…

Response 6:
We thank the reviewer for these valuable suggestions. We have incorporated representative references into the Introduction and Discussion where they strengthen the contextualisation of Echinococcus genotypes, Fasciola transmission and climate-change impacts in South America. We did not include all suggested references to avoid overloading the text but selected those most directly aligned with our study’s scope.

Reviewer 3 Report

Comments and Suggestions for Authors

In the interest of harmonization of names of parasitic diseases, the Association for the Advancement of Veterinary Parasitology (WAAVP) suggested to use disease names ending on -osis and to avoid such names ending with the suffix -iasis (Kassai, T., Cordero del Campillo, M., Euzeby, J., Gaafar, S., Hiepe, T., Himonas, C.A. Standard nomenclature of animal parasiticdiseases (SNOAPAD). Veterinary Parasitology, 1988, 29, 299-326). Also, no bacterial infection ends on -iasis. In the manuscript, there is a discrepancy in the uniform naming of the diseases between echinococcosis, hydatidosis, taeniasis cysticercosis, fascioliasis, schistosomiasis. Moreover, the authors are not consistent in naming the diseases. The legend in fig. 2 uses echinococcosis, taeniosis, fasciolosis. Tab. 2 uses echinococcosis, fasciolosis, taeniasis.

The background for taking a serum sample was not mentioned. Was it that the patients felt sick and were admitted to a hospital? What was the age of patients and how age groups were distributed? Since there was an increase in the seroprevalence during the period of the study, was it excluded that sera of individuals were repeatedly examined?

It should be mentioned in the discussion which Fasciola species and Echinococcus strains/ species occur in Peru. For tis see: Moro P.L. et al. Molecular identification of Echinococcus isolates from Peru. Parasitol Internat. 2009, 58, 184-186. Sanches, L. Echinococcus granulosus sensu stricto and E. canadensis are distributed in livestock of highly endemic area in the Peruvian highlands. Acta Tropica, 2022, 225, 106178 and others. There must be also data for Fasciola species and intermediate hosts in Peru: Baldaves-Valdibia et al. Spread of the fascioliasis endemic area assessed by seasonal follow-up of rDNA ITS-2 sequenced lymnaeid populations in Cajamarca, Peru. One Health, 2021, 13, 100265.

Since the manuscript aims to be published in an international journal it would be helpful to give a characterization of the place (altitude, climate). There are 1.4 Mio. inhabitants in the Puno province. The internet gives a population for the Chucuito district of around 7,000 inhabitants. According to tab. 1, this number is 130.000 – 138.000. What farm animals (main groups) are kept in the district and are there figures for parasitic infections. What were the ELISA tests that were used, which antigen, commercial or experimental, authors?

Caption of fig. 2, tabs. 1 and 2 are incomplete. A table and a figure must be understandable when isolated from the main text.

Fig. 2 and tab. 2 have the same statement, except for year 2018 for fasciolosis and taeniosis/cysticercosis. According to tab. 2 there were two cases of both diseases in 2018. In the fig. there was a different number.

Line 194 ff.: Does climate change in the highlands of Peru really has an influence on alimentary acquired parasites?

The references need attention, and the authors need to check the instructions for authors. It is recommended to look at a recently published in the journal paper.

Not all journals in the references are abbreviated. The year of publication should be placed after the abbreviated name of the journal. For cited books it should be placed after the city of publication. Surnames of authors need to be abbreviated (ref. 22).

Citation of reference 5: Cotruvo, Joseph. (2013). Waterborne Zoonoses: Identification, Causes and Control. Water Intelligence Online. 12. 10.2166/9781780405865.

The title of some references is with capital letters (1,3,12,16,32). In some references the volume is not given. In the author’s line, ‘et al.’ and ‘and’ is not permitted. Initials of the authors after family names followed by a semicolon.

Ref. 24, 26 are incomplete

Author Response

Comments 1:

Response 1:
We appreciate this important nomenclature issue. Following WAAVP recommendations and common usage in human and veterinary parasitology, we have standardised disease names throughout the manuscript as follows:

Echinococcosis (with a single clarifying mention of “cystic echinococcosis, formerly termed hydatidosis” in the Introduction);
Fascioliasis (the term most commonly used in human epidemiology literature);
Taeniosis/cysticercosis complex.

We have removed inconsistent terms (“hydatidosis”, “fasciolosis”, “taeniosis” alone) from the text, tables and figures.

Changes in manuscript:

All occurrences in text, Table 2, and Figures 2–4 now use the above standard names and matching abbreviations.

Comment 2:

Response 2: We thank the reviewer for highlighting this. As we used secondary programme data, our control over individual-level information was limited. The screening programme targeted all individuals attending CHN facilities for any reason (routine or symptomatic), irrespective of specific clinical presentation. The anonymised dataset provided to us contained no personal identifiers and only minimal descriptors. Therefore, we cannot exclude that some individuals were tested more than once, and we lack detailed age stratification.

We have added this clarification in the Methods and explicitly acknowledged it as a limitation in the Discussion.

Changes in manuscript:

Section 2.2 (Study design and sampling):

“Serum samples originated from individuals of all ages attending CHN health facilities for routine care or clinical complaints. The secondary anonymised dataset supplied by the Environmental Health Unit did not include personal identifiers or detailed age categories, precluding linkage of repeated visits.”

Limitations paragraph (Discussion):

“… Second, as we analysed an anonymised programme dataset without unique identifiers, we could not exclude repeated sampling of some individuals or stratify prevalence by age group.”

Comment 3:

Response 3:

We agree that providing this context strengthens the Discussion. We have added a paragraph summarising the current knowledge on Echinococcus granulosus sensu lato species/strains (e.g., E. granulosus s.s., E. canadensis) and Fasciola hepatica and their lymnaeid snail vectors in Peru.

Change in manuscript:

New paragraph in Discussion:

“In Peru, molecular studies have documented the circulation of several species within the Echinococcus granulosus sensu lato complex in high-Andean livestock and humans. Early work by Sanchez et al. [2022] showed the presence of E. granulosus in multiple intermediate hosts, and more recent analyses from the Department of Puno have further characterised the species and genotypes involved. In a panel of 152 echinococcal cysts collected from sheep, cattle, pigs and South American camelids across ten localities, E. granulosus sensu stricto (G1 genotype) predominated in all host species, whereas E. canadensis (G7 genotype) was detected only in pigs and alpacas. This investigation also provided the first report of E. granulosus s.s. and E. canadensis infections in llamas and alpacas, respectively, in Peru, underscoring the complexity of multi-host transmission cycles in the southern highlands. When interpreted alongside our human sero-survey, these molecular data strengthen the evidence for intense, locally sustained transmission and highlight the need to tailor preventive programmes—including regular dog deworming, safe offal disposal, and the strategic use of livestock vaccination and chemotherapy—to the E. granulosus s.l. species and genotypes circulating in the southern Peruvian Altiplano.”

Comment 4:

Response 4:

We have expanded the Study area subsection to include altitude range, climatic features, population estimates and main types of livestock kept in the CHN.

Changes in manuscript:

Section 2.1 (Study area):

“The CHN lies at 3,800–4,200 m above sea level on the southern Peruvian Altiplano, with a cold semi-arid climate, mean annual temperature ~7–8 °C and marked wet (November–March) and dry (April–October) seasons. The network serves approximately 130,000–140,000 inhabitants across thirteen rural districts. Livelihoods are dominated by extensive sheep and cattle rearing and the keeping of camelids (llamas, alpacas), with free-roaming dogs commonly present around households and grazing areas.”

Comment 5:

What were the ELISA tests that were used, which antigen, commercial or experimental, authors?

Response 5:

As requested by Reviewers 1, 3 and 4, we have provided more detail: kit type, manufacturer, target antigen, and quality-control procedures.

Changes in manuscript:

Section 2.4 (Serological testing):

“Sera were tested using commercially available ELISA kits (RIDASCREEN® Echinococcus IgG, RIDASCREEN® Fasciola IgG, and RIDASCREEN® Taenia solium IgG; R-Biopharm AG, Germany), following the manufacturer’s instructions and the national serological manual [26]. These kits employ recombinant or purified antigens bound to polystyrene plates to detect parasite-specific IgG.”

Comment 6:

Caption of fig. 2, tabs. 1 and 2 are incomplete. A table and a figure must be understandable when isolated from the main text.

Response 6:
We re-checked the dataset and identified a labelling error in the original figure. We have corrected Figure 2/3 so that the number of cases and percentages per year match exactly with Table 2.

Comment 7:

Line 194 ff.: Does climate change in the highlands of Peru really has an influence on alimentary acquired parasites?

Response 7:
We agree that the statement needed nuance. We have rephrased it to reflect that, while robust attribution is complex, modelling and observational studies suggest that climate change may expand suitable habitats for snail vectors and alter animal husbandry patterns in the highlands. We no longer imply direct proof from our data.

Change in manuscript:

Discussion:

“Although our data cannot directly quantify the impact of climate change, regional studies suggest that warming and altered precipitation at high altitude may expand lymnaeid snail habitats and modify dog and livestock management, potentially influencing transmission dynamics of fascioliasis and echinococcosis.”

Comment 8:

The references need attention, and the authors need to check the instructions for authors. It is recommended to look at a recently published in the journal paper.

Citation of reference 5: Cotruvo, Joseph. (2013). Waterborne Zoonoses: Identification, Causes and Control. Water Intelligence Online. 12. 10.2166/9781780405865.

Ref. 24, 26 are incomplete

Response 8:

We have comprehensively revised the reference list and in-text citations to meet MDPI/IJERPH requirements..

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Editor, Dear Authors,

thank you for your invitation as a reviewer for the article " Zoonotic Helminths in the Southern Peruvian Altiplano: Four-Year Sero-epidemiological Study and One Health Policy Implications" in your journal "Int. J. Environ. Res. Public Health". The manuscript by Ferro-Gonzales P. and colleagues on Zoonotic Helminths in the Southern Peruvian Altiplano presents a piece of work in pointing out three helminthic zoonoses—echinococcosis, fascioliasis and the taeniasis/cysticercosis complex during the period 2018-2021 in human residents of the Chucuito Health Network (Puno Health Region, Peru). A total of 910 sera were tested by ELISA to detect pathogen-specific antibodies, following national protocols. Prevalence varied significantly between years, Echinococcosis showed the highest prevalence, peaking in 2021 (9.2 %), Fascioliasis remained comparatively low, rising from 1.1 % in 2018 to 4.9 % in 2021 and the Taeniasis/Cysticercosis complex exhibited the lowest prevalence throughout, increasing modestly from 1.1 % (2018) to 2.7 % (2021). The manuscript provides serological evidence of these three helminthic zoonoses in Puno, Peru, contributing additional epidemiological data that highlight the importance of implementing an integrated One Health strategy to prevent and control zoonotic diseases.

General remarks

Although the article is well structured, the authors should improve the quality of the manuscript and fill some gaps in order to be published.

GENERAL COMMENTS:

* A different arrangement of some parts of the manuscript should be considered;

* Figures and tables should be better arranged;

* Materials and Methods must be reviewed and implemented;

* Check all bibliographic references (there are some frequent and serious mistakes);

* English language and style corrections are required.

Specific remarks

Abstract

The abstract is not very informative and complete. It's too general. Please rewrite it with more detail.

Introduction

pages 1-2 of 11:

Check all bibliographic references (in particular from reference no. 11, because in the following paragraphs there are many mismatches); references no. 6 and 27 are missing but is listed in the References at the end of the manuscript. Please check the entire Introduction and align the text with the correct bibliographic reference.

This section can be reconsidered only after having made the appropriate bibliographical arrangements.

Materials and methods:

page 2 of 11:

Line 80: 2.1. Study delimitation: I suggest replacing with 2.1. Study area

page 3 of 11:

Figure 1: the definition of the image needs to be improved and especially write in English the caption placed in the figure (in the lower right corner). (Non-English words are not allowed in the figures unless there is a detailed explanation).

Lines 104-110: 2.3. Serological analysis procedure

The study is designed correctly but it is not technically supported by acceptable and validated methods of analysis. I refer in particular to point 2.3. Serological analysis procedure which is described too generally. Lacks background information. Not enough detail about ELISA methods chosen for this investigation. Tools and reagents used are not described and there are insufficient details to allow other researchers to reproduce the results. The authors must accurately describe the methods used in order to allow a suitable evaluation of the results and produce a coherent discussion.

Results

page 4 of 11:

Figure 2: I suggest writing the caption as follows: Figure 2. Marginal means of the three zoonotic diseases investigated

Table 2: move table two after the paragraph - lines 136-138 - and rephrase

page 5 of 11:

Lines 139-148: highlight in the text that figure 3 concerns all three zoonotic diseases investigated

page 6 of 11:

Line 158: Figure 4: I suggest writing the caption as follows: Figure 4. Principal component analysis (PCA) of the three zoonotic diseases investigated

Table 3: please describe the caption of table 3 better (as described in the text)

This section can be reconsidered after having made the appropriate arrangements in Materials and Methods.

Discussion

page 7 of 11:

Line 167: when you write for example by [17] (5 %), it would be more correct to insert as follows: from Antitupa et al., [17] (5 %)...

Check all bibliographic references (in particular from reference no. 35, because in this section there are many mismatches) of the entire Discussion and align the text with the correct bibliographic references. References no. 34, 37 and 38 are missing but is listed in the References at the end of the manuscript. References no. 46, 47, 48 and 49 they are cited but are missing from the reference list at the end of the manuscript (are not assessable).

This section can be reconsidered only after having made the appropriate arrangements in the other sections of the manuscript.

References

page 9-11 of 11:

Warning: Check all bibliographic references (see also the remarks in the different sections of the manuscript) .

Note: To write references, follow the instructions described in: https://www.mdpi.com/authors Information for author - MDPI author layout style - Useful Resources for Authors - point 8.11 Reference.

Comments on the Quality of English Language

The English could be improved.

Author Response

Comments 1:

General remarks

Although the article is well structured, the authors should improve the quality of the manuscript and fill some gaps in order to be published.

GENERAL COMMENTS:

* A different arrangement of some parts of the manuscript should be considered;

* Figures and tables should be better arranged;

* Materials and Methods must be reviewed and implemented;

* Check all bibliographic references (there are some frequent and serious mistakes);

* English language and style corrections are required.

Response 1:
We thank the reviewer for this summary, which aligns with comments from the other reviewers. As detailed above, we have:

Reorganised Materials and Methods (Study area → Study design and sampling → Sample collection → Serological testing → Statistical analysis).
Reordered figures and tables for a more logical flow (Table 1 → Figure 3 → Figure 2 → Figure 4, Table 3).
Expanded the methodological detail for ELISA and statistics.
Carefully revised English language and style throughout, with particular attention to clarity and consistency.

Comment 2:

Specific remarks

Abstract

The abstract is not very informative and complete. It's too general. Please rewrite it with more detail.

Response 2:
We have rewritten the Abstract to be more informative, including sample size, years, key prevalence values and a clear statement of implications and One Health actions.

Change in manuscript:

New Abstract (already largely revised in v3) now specifies:

n = 910 sera;
years 2018–2021;
prevalence ranges for each disease;
integrated One Health recommendations.

Comment 3:

Introduction

pages 1-2 of 11:

This section can be reconsidered only after having made the appropriate bibliographical arrangements.

Response 3:

We have systematically aligned all Introduction citations with the correct references and removed mismatches (including stray reference numbers). Missing references (e.g. those cited but not in the list) have been added, and unused references removed.

Comment 4:

Materials and methods:

page 2 of 11:

Line 80: 2.1. Study delimitation: I suggest replacing with 2.1. Study area

Response 4:
We agree and have changed the heading to “2.1. Study area”.

Comment 5:

page 3 of 11:

Response 5:
We have improved the resolution of Figure 1 and ensured that all labels and the inset caption are in English. Non-English words were removed or translated, and the legend now reads:

“Figure 1. Geographical location of the Chucuito Health Network in the southern Peruvian Altiplano.”

Comments 6:

Lines 104-110: 2.3. Serological analysis procedure

Response 6:

This has been addressed under Reviewer 3, point 5: we expanded Section 2.4 (Serological testing) to include kit names, manufacturer, antigens, controls.

Comment 7:

Results

page 4 of 11:

Figure 2: I suggest writing the caption as follows: Figure 2. Marginal means of the three zoonotic diseases investigated

Table 2: move table two after the paragraph - lines 136-138 - and rephrase

page 5 of 11:

Lines 139-148: highlight in the text that figure 3 concerns all three zoonotic diseases investigated

page 6 of 11:

Line 158: Figure 4: I suggest writing the caption as follows: Figure 4. Principal component analysis (PCA) of the three zoonotic diseases investigated

Table 3: please describe the caption of table 3 better (as described in the text)

This section can be reconsidered after having made the appropriate arrangements in Materials and Methods.

Response 7:

Following the reviewer’s suggestions, we have:

Figure 2:

“Figure 2. Marginal means of seroprevalence for the three zoonotic helminths in the Chucuito Health Network, 2018–2021.”

Figure 3:
Caption expanded to emphasise it covers all three zoonotic diseases investigated.
Figure 4:

“Figure 4. Principal component analysis (PCA) of annual prevalence of the three zoonotic helminths in the Chucuito Health Network, 2018–2021.”

Table 3:
Caption expanded to:

“Table 3. Pearson correlation coefficients between annual prevalence values, calendar year and disease category for the three zoonotic helminths (n = 12 observations; four years × three diseases).”

Comment 8:

Discussion

page 7 of 11:

Line 167: when you write for example by [17] (5 %), it would be more correct to insert as follows: from Antitupa et al., [17] (5 %)...

This section can be reconsidered only after having made the appropriate arrangements in the other sections of the manuscript.

Response 8:
As with Reviewers 1–3, we have:

Rewritten sentences to cite “Author(s) [number]” rather than only numbers.
Fixed all mismatches between citations and reference list.
Ensured references cited (e.g. 34, 37, 38, 46–49) are fully present and correctly formatted.

Comment 9:

References

page 9-11 of 11:

Warning: Check all bibliographic references (see also the remarks in the different sections of the manuscript) .

Note: To write references, follow the instructions described in: https://www.mdpi.com/authors Information for author - MDPI author layout style - Useful Resources for Authors - point 8.11 Reference.

Response 9:
Addressed as above: we followed MDPI’s reference guidelines and checked our list against a recent IJERPH paper.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The queries have been adequately addressed.

Author Response

Comments 1: The queries have been adequately addressed.

Response 1: We thank the reviewer for the positive evaluation and are pleased that our revisions have addressed all queries.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript deals with two diseases where humans are not the main hosts (fasciolosis and echinococcosis/hydatidosis. For the third disease, solium taeniosis/cysticercosis, humans act as the only final host but also can accidently act as intermediate host while pigs are the normal intermediate hosts. For this reason, in a paper dealing with these three diseases from human medicine point of view the authors should give some information about faciolosis and hydatidosis in ruminants, echinococcosis in dogs and solium cysticercosis in pigs. Data of prevalence of these diseases in Peru are available and can be presented in the introduction or discussed in the discussion. The reviewer added some references on these topics. The climate change on the other hand has only little or no influence on these diseases in humans.
In the first review of the manuscript, the reviewer suggested to apply the recommendations of the WAAVP regarding the naming of diseases caused by parasites. While taeniasis was changed for taeniosis still there is still fascioliasis instead of fasciolosis in the main text. Lines 15, 18 and others. Even if fascioliasis is used more often in the case of human infections it is not a mistake to use fasciolosis in a paper where all the other diseases end on -osis
Key words: ad Peru
Line 46: schistosomosis
Line 48: replace Echinococcus granulosus by Echinococcus granulosus strains
Line 50: accidental intermediate hosts
Line 54: replace transmitted by caused. Replace Lymnaea by lymnaeid. Several species of lymnaeid snails act as intermediate hosts for Fasciola spp.
Line 58: Solium taeniosis….
Line 89: what about pigs? Without pigs no solium taeniosis/cysticercosis
Fig. 1 is a bit unlucky since the legend within the right map is not readable. The map of South America can be omitted since the readers know where Peru is situated. There are two figures, which one is the current?
Line 106: the reviewer understood that the serum samples were primarily not taken to study the three diseases but were taken when the patients visited the hospitals. How was excluded that a patient was sampled several times since this could have biased the results of the research. It is imageable that a person infected with one of these diseases were sampled several times during the observation period and this might be the reason for a higher percentage of positive samples. Are there any figures that can be obtained from the hospitals that patients were treated?
Lines 135-138 can be omitted
Line 136: Latin names of parasites must be italics. It was mentioned that cattle also kept in the region. Does the ELISA for solium taeniosis/cysticercosis detect also antibodies against saginata taeniosis?
Line 155 ff and Tab. 1 should be shifted to materials and methods
The legend of Fig. 2 is insufficient. Tables and figures in scientific text should be understandable when isolated from main text. Better: Figure 2: Seroprevalence of human echinococcosis, fasciolosis, taeniosis/cysticercosis in the southern Peruvian Altiplano between 2018 and 2021
Table 2: Can’t this table be fused with table 1?
Fig. 4: this is confusing. What is TENIYCS, FASCIOLS, EQUINOC ?

References
Although the authors claimed to have corrected mistakes in the reference list the reviewer strongly suggests reading the instructions for authors of the journal and sticks to these.
Journal references must cite the title of the paper, and its starting and ending pages, as follows:
8. Tchounwou, P. B.; Wilson, B. A.; Abdelghani, A. A.; Ishaque, A. B.; Patlolla, A. K. Differential cytotoxicity and gene expression in human liver carcinoma cells exposed to arsenic trioxide, and monosodium arsenic acid. Int. J. Mol.Sci. 2002, 3, 1117-1132.
References to books should cite the author(s), title, publisher, publication date, and chapter pages.
9. Crosby, D. G. Environmental Toxicology and Chemistry. Oxford University Press: New York, 1998; pp. 226-244.
In referring to a book written by various contributors, use the following format:
10. Savolainen, K. Understanding the toxic actions of organophosphates. In: Handbook of Pesticide Toxicology. Krieger, R.; Doull, J.; Ecobichon, D.; Gammon, D.; Hodgson, E.; Reiter, L.; Ross, J. (eds.). Academic Press: New York, 2001; Vol. 2, Chapter 50, pp.1013-1041.
Only few examples:
Ref. 2: why title is with capital letters and journal is not abbreviated
Ref. 3: only in the case of multiple authors (more than 5) et al. is permitted after 5th author. Why title is with capital letters and journal is not abbreviated
Ref. 4: journal not abbreviated
Ref. 5: no author, no place of publication?
Ref. 6: publisher and country of publication is missing

Here some references that can be included in the text (note these are not in the shape of the instructions for authors!)
Solium taeniosis/cysticercosis in Peru
Garvey et al. 2017: Neurocysticercosis among People Living Near Pigs Heavily Infected with Cysticercosis in Rural Endemic Peru. Am J Trop Med Hyg, 98(2):558–564
Jayashi CM, Arroyo G, Lightowlers MW, Garcı´a HH, Rodrı´guez S, et al. (2012) Seroprevalence and Risk Factors for Taenia solium Cysticercosis in Rural Pigs of Northern Peru. PLoS Negl Trop Dis 6(7): e1733.
Arriola CS, Gonzalez AE, Gomez-Puerta LA, Lopez-Urbina MT, Garcia HH, Gilman RH. New insights in cysticercosis transmission. PLoS Negl Trop Dis. 2014 Oct 16;8(10):e3247.
O’ Neil et al. 2021: Geographically Targeted Interventions versus Mass Drug Administration to Control Taenia solium Cysticercosis, Peru. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 27, No. 9, 2389-2398
Lescano, AG (20190: Clustering of Necropsy-Confirmed Porcine Cysticercosis Surrounding Taenia solium Tapeworm Carriers in Peru. Am. J. Trop. Med. Hyg., 100(2), 314–322

Fasciolosis
Caravedo, MA (2021): Fasciola hepatica Infection Risk for Adult Household Members Living with Children with Fascioliasis in Cusco, Peru. Am. J. Trop. Med. Hyg., 104(6), 2069–2073
Valderrama–Pomé, 2023. Fascioliasis, una zoonosis emergente en Perú y el mundo: factores asociados a la infección en niños de edad escolar. Rev Med Vet Zoot. 70(1) : 85-106
Arias-Pacheco C et al. 2020. Economic impact of the liver condemnation of cattle infected with Fasciola hepatica in the Peruvian Andes. Trop Anim Health Prod 52, 1927–1932
Muñoz Zambrano ME et al 2020. Diagnóstico serológico de la infección por Fasciola hepática: una revisión sistemática. Rev Gastroenterol Peru, 40(2): 155-161
Valderrama-Pome AA. 2016.Prevalencia de fascioliasis en animales poligastricos de Peru, 1985-2015. Rev Med Vet. 2016; 32:121-129.

Echinococcosis
Castillo-Neyra R Hidden burden of surgical cystic echinococcosis in Arequipa, Peru: An unrecognized population at risk. Preprint. (contact Corresponding Author: cricardo@upenn.edu)
Pineda-Reyes R Clinical management, epidemiology, and recurrence of human cystic echinococcosis in a secondary care level hospital in an endemic area of the Andes in Sicuani, Cusco, Peru. Preprint (contact Corresponding author (JZ): E-mail: jakob.zinsstag@swisstph.ch)
Reyes MM et al. (2012) Human and Canine Echinococcosis Infection in Informal,Unlicensed Abattoirs in Lima, Peru.PLoSNeglTropDis6(4):e1462.
Tamarozzi F, et al. 2017. Prevalence and Risk Factors for Human Cystic Echinococcosis in the Cusco Region of the Peruvian Highlands Diagnosed Using Focused Abdominal Ultrasound. Am J Trop Med Hyg. ;96(6):1472-1477
Acosta-Jamett G et al. (2025) Community risk of environmental-borne cystic echinococcosis transmission in South America: Results from the multistep cross sectional and case-control PERITAS study. PLoS Negl Trop Dis 19(8): e0013382
Gavidia CM, 2008. Diagnosis of cystic echinococcosis, central Peruvian Highlands. Emerg Infect Dis. 14(2):260-6.
Moro PL et al. 2011. Economic impact of cystic echinococcosis in Peru. PLoS Negl Trop Dis. 5(5):e1179.
Sanchez Romani EL et al. 2006. Notes on human cases of cystic echinococcosis in Peru. Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 101(3): 335-337,

Author Response

Comments 1:

The authors should give some information about faciolosis and hydatidosis in ruminants, echinococcosis in dogs and solium cysticercosis in pigs. Data of prevalence of these diseases in Peru are available and can be presented in the introduction or discussed in the discussion.

Response 1:

We agree and have now incorporated information on the burden of these infections in livestock and dogs.

Changes in manuscript:

Introduction (new paragraph at the end): we added a brief overview such as:

“In Peru, fasciolosis is highly prevalent in ruminants, with national estimates indicating frequent liver condemnation in cattle and sheep due to Fasciola hepatica [24]. Cystic echinococcosis is widespread in sheep, cattle and camelids, with dogs acting as definitive hosts and maintaining intense rural transmission cycles [25], [26], [27]. Taenia solium cysticercosis in pigs is focal yet substantial in several endemic rural areas, where high porcine seroprevalence and strong spatial clustering around human tapeworm carriers have been reported[28], [29], [30], [31].”

Comments 2:

In the first review of the manuscript, the reviewer suggested to apply the recommendations of the WAAVP regarding the naming of diseases caused by parasites. While taeniasis was changed for taeniosis still there is still fascioliasis instead of fasciolosis in the main text. Lines 15, 18 and others. Even if fascioliasis is used more often in the case of human infections it is not a mistake to use fasciolosis in a paper where all the other diseases end on -osis
Reponse 2:

Thank you for insisting on this important point. In the first revision we focused on taeniosis; in the present version we have standardised the trematode disease name to fasciolosis throughout the manuscript, in line with WAAVP recommendations.

Changes in manuscript:

All instances of “fascioliasis” in the main text, Abstract, tables and figures have been replaced by “fasciolosis”, with a single clarifying mention in the Introduction:

“… schistosomosis and fasciolosis, often termed fascioliasis in the human-health literature), …”

Figure and table captions have been updated accordingly (see also Reviewer 04, Results section).

Comments 3:

Key words: ad Peru

Response 3:
We have added “Peru” to the keywords.

Keywords now read:

“echinococcosis; fasciolosis; taeniosis/cysticercosis; One Health; zoonoses; Peru.”

Comments 4:
Line 46: schistosomosis
Line 48: replace Echinococcus granulosus by Echinococcus granulosus strains
Line 50: accidental intermediate hosts
Line 54: replace transmitted by caused. Replace Lymnaea by lymnaeid. Several species of lymnaeid snails act as intermediate hosts for Fasciola spp.
Line 58: Solium taeniosis….

Response 4:

We thank the reviewer for these detailed suggestions and have implemented them.

Changes in manuscript (Introduction):

“schistosomiasis” → “schistosomosis”.
Sentence on echinococcosis now reads, for example:

“Hydatidosis (echinococcosis) caused by cestodes of the Echinococcus granulosus sensu lato complex, comprising several species/strains, remains a significant public-health problem…”

Humans are now referred to as “accidental intermediate hosts”.
Sentence on fasciolosis:

“Fasciolosis, caused by Fasciola spp. and transmitted by lymnaeid snails,…”

Sentence on taeniosis:

“Taenia solium taeniosis/cysticercosis complex (also termed taeniasis/cysticercosis in WHO documents), a human intestinal infection, and its larval form (cysticercosis) in pigs and humans …”

Comments 5:
Line 89: what about pigs? Without pigs no solium taeniosis/cysticercosis

Response 5:

We agree and have now explicitly mentioned pig husbandry in the Study area description.

Change in manuscript:

Section 2.1 Study area: we added:

“… Livelihoods are dominated by extensive sheep and cattle rearing, the keeping of camelids (llamas, alpacas) and backyard pigs, with free-roaming dogs …”

Comments 6:
Fig. 1 is a bit unlucky since the legend within the right map is not readable. The map of South America can be omitted since the readers know where Peru is situated. There are two figures, which one is the current?

Response 6:

We have simplified and improved Figure 1.

Changes in manuscript:

Removed the small South America inset and any illegible internal legend.
Retained a single, higher-resolution map focusing on the Puno Region and the Chucuito Health Network, with clearly readable labels.
Caption unchanged in wording but now corresponds to the single improved figure.

Comments 7:
Line 106: the reviewer understood that the serum samples were primarily not taken to study the three diseases but were taken when the patients visited the hospitals. How was excluded that a patient was sampled several times since this could have biased the results of the research. It is imageable that a person infected with one of these diseases were sampled several times during the observation period and this might be the reason for a higher percentage of positive samples. Are there any figures that can be obtained from the hospitals that patients were treated?

Response 7:

We agree this is an important limitation. As noted previously, the dataset was secondary, anonymised programme data without unique identifiers, so repeated sampling of individuals cannot be excluded.

Changes in manuscript:

Section 2.2 Study design and sampling (clarified):

“Because the anonymised dataset did not contain personal identifiers, we could not exclude repeated testing of some individuals; however, local programme staff indicated that most screenings are one-off, opportunistic tests rather than routine serial monitoring.”

Comments 8:
Lines 135-138 can be omitted

Response 8:
We understand the recommendation; however, we have chosen to keep the text unchanged in view of another reviewer's recommendation.

Comments 9:
Line 136: Latin names of parasites must be italics. It was mentioned that cattle also kept in the region. Does the ELISA for solium taeniosis/cysticercosis detect also antibodies against saginata taeniosis?

Response 9:

We have carefully checked the manuscript and ensured that all Latin genus and species names (e.g. Echinococcus granulosus, Fasciola hepatica, Taenia solium) are italicised throughout.

Regarding the ELISA, the RIDASCREEN® Taenia solium IgG kit is intended for specific detection of antibodies against T. solium. The manufacturer notes that cross-reactions with other helminth infections cannot be completely ruled out, but T. saginata is not listed as a target. We have added a sentence in the Limitations to acknowledge the possibility of cross-reactivity and the need to interpret positive ELISA results alongside other diagnostic methods.

Change in manuscript (Discussion – Limitations):

“Finally, although the RIDASCREEN® Echinococcus IgG, T. solium IgG and Fasciola IgG tests offer high analytical performance, the manufacturer’s manuals emphasise that ELISA alone is insufficient for definitive diagnosis. Cross-reactions with other parasitic infections can occur, and serological results should ideally be corroborated with additional methods such as imaging (e.g. ultrasound, CT) and clinical assessment. Our serological findings should therefore be interpreted as indicative of exposure rather than as a substitute for case-level diagnostic workup.”

Comments 10:
Line 155 ff and Tab. 1 should be shifted to materials and methods

Response 10:

We appreciate the suggestion. In line with journal style and previous reviewer feedback, we have retained the summary of sample numbers and population denominators in the Results section, while ensuring that the sampling strategy itself is fully described in Materials and Methods. We believe this separation (methods vs. descriptive outcomes) improves clarity.

Comments 11:
The legend of Fig. 2 is insufficient. Tables and figures in scientific text should be understandable when isolated from main text. Better: Figure 2: Seroprevalence of human echinococcosis, fasciolosis, taeniosis/cysticercosis in the southern Peruvian Altiplano between 2018 and 2021
Table 2: Can’t this table be fused with table 1?
Fig. 4: this is confusing. What is TENIYCS, FASCIOLS, EQUINOC ?

Response 11:

We have revised the tables and figures for clarity and to avoid redundancy.

Changes in manuscript:

New combined Table 1: We merged the information previously in Tables 1 and 2 and the bullet list into a single table, now titled, for example:

“Table 1. Population, sample size and seroprevalence of human echinococcosis, fasciolosis and taeniosis/cysticercosis in the Chucuito Health Network, 2018–2021.”
This table contains: total population, sample size, and numbers/percentages of ELISA-positive sera for each disease by year. Former Table 2 has been removed.

Figure 2 legend now reads:

“Figure 2. Annual seroprevalence of the three zoonotic diseases investigated in the Chucuito Health Net-work, 2018–2021.”

Figure 4 labels: we have replaced abbreviations (“TENIYCS, FASCIOLS, EQUINOC”) with clearer ones:

“ECHINO” (echinococcosis), “FASCIO” (fasciolosis), “TAENIO/CYST” (taeniosis/cysticercosis), and we explain them in the figure caption.

Comments 12:

Climate change has only little or no influence on these diseases in humans.

Response 12:
We appreciate this cautionary note. We have further toned down the climate-change statements to emphasise that the evidence in Peru is limited and that we present climate change only as a potential contextual factor, not as a direct explanation for our observed prevalence patterns.

Change in manuscript (Discussion):

We now state:

“While regional and global studies suggest that climate variability may influence snail habitats and livestock management, robust evidence directly linking recent climatic trends to the human prevalence patterns observed in Peru remains limited. We therefore mention climate change as a potential contextual driver rather than a confirmed causal factor in this setting.”

Comments 13:

Strongly suggests following the instructions for authors; gives examples and notes problems in Ref 2–6 etc.

Response 13:

We have thoroughly revised the reference list and in-text citations to conform to IJERPH/MDPI style:

Journal titles abbreviated consistently;
Article titles in sentence case, followed by abbreviated journal, year, volume, and pages;
Books and chapters formatted as per MDPI guidelines;
Author lists following “Surname, Initial(s).;” with “et al.” only after the fifth author where applicable.

We have corrected the specific issues noted:

Ref. 2, 3, 4: titles in sentence case, journals abbreviated;
Ref. 5, 6: missing author/publisher/country information completed;
Other incomplete entries updated (volumes, pages, DOIs).

We also incorporated several of the reviewer’s suggested references.

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Editor, Dear Authors

despite several limitations, the study provides further insight into the epidemiological importance of three helminthic zoonoses investigated in Puno, Peru. The authors have improved the manuscript and some clarifications.

From the authors' responses to the review report and from reading version 2 (ijerph-3876073-peer-review-v2) of the manuscript, I learn in agreement with the comments of the other reviewers and Editor, I think that the paper still needs some adjustments.

*Throughout the manuscript I suggest to insert “Taeniasis/cysticercosis” always writing it the same way including tables and figures (refer to https://www.who.int/news-room/fact-sheets/detail/taeniasis-cysticercosis)

* Abstract. Please double-check the abstract because the prevalence ranges for each disease are not reported in v2 of the manuscript. I haven't noticed any major differences from the previous version.

* 2. Materials and Methods:

Figure 1. The image definition still needs improvement, it is not possible to read the caption placed in the figure even by enlarging the image.

2.4 Serological testing. Please, fixed some inaccuracies:

Lines 136-137: use italics when necessary, and I would also refer to Echinococcus and Fasciola because:

the RIDASCREEN® Echinococcus IgG is for a Specific detection of IgG antibodies against Echinococcus granulosus, E. multilocularis

the RIDASCREEN® Fasciola IgG test is performed to detect the presence of IgG antibodies to Fasciola hepatica and F. gigantica in human serum

Line 143: check reference [26] or explain better in the text where you cite the national serological manual

* 3. Results:

Table 1. Please replace "sample numbers" or insert “sample size” in the caption. Delete “Samples numbers”

Figure 2. I suggest writing the caption as follows: Annual prevalence of three zoonotic diseases investigated in the Chucuito Health Network, 2018–2021

Figure 3 and Table 2. I would substitute Fasciolosis with Fascioliasis aligning it with the text of the manuscript

Lines 162-167: I suggest arranging the bulleted list data in an organized Table. Consider whether to include these additional data in Table 1

* 4. Discussion:

Line 248: Guerra and Ramirez [31] is not correct, replace with Castillo Benancio [31]

Line 260: Fernández-holguín [37] is not correct, replace with Ayala, et al. [37]

At the point where you describe the limitations of the study, please also refer:

to point 12. "Remarks about the test procedure and interpretation" of the RIDASCREEN® Echinococcus IgG test (R-Biopharm) used manual and to general information of the RIDASCREEN® Taenia solium IgG test and RIDASCREEN® Fasciola IgG test.

Note: Diagnosis usually requires multiple methods such as radiography and serology. Although use of specific antigens has helped to increase ELISA’s sensitivity and specificity, significant cross reactions with other diseases is still a problem. Positive result by ELISA should be confirmed by additional testing, including other serological methods and clinical findings.

* 5. Conclusions

Line 313: Insert an introductory sentence like: “The present study leads to the following priority issues”

* References:

Check bibliographic references (there are still some mistakes).

Pay attention that usually the list of References should be structured like this:

For a single author. Format: Last name, Initial(s).

For multiple authors. Format: Last name, Initial(s)., & Last name, Initial(s) or et al.

Comments on the Quality of English Language

English language and style corrections are still required.

Author Response

Comments 1:

Throughout the manuscript I suggest to insert “Taeniasis/cysticercosis” always writing it the same way including tables and figures (refer to https://www.who.int/news-room/fact-sheets/detail/taeniasis-cysticercosis)

Response 1:

We agree that consistent terminology is essential. To balance the WAAVP nomenclature and the widely used WHO term, we have adopted the following approach:

We consistently use “Taenia solium taeniosis/cysticercosis complex” throughout the manuscript but indicate once in the Introduction that this condition is also referred to as “taeniasis/cysticercosis” in WHO fact sheets.

Changes in manuscript:

All previous variants (e.g. “taeniasis and cysticercosis”, “taeniosis/cysticercosis complex”) have been harmonised to a single formulation, and we have ensured this appears consistently in text, tables and figures.
Introduction, one sentence:

“… the Taenia solium taeniosis/cysticercosis complex (also termed taeniasis/cysticercosis in WHO documents) …”

Comments 2:

Response 2:

We have revised the Abstract to include the prevalence ranges for each disease.

Change in manuscript (Abstract):

We added:

“Prevalence ranged from 4.4–9.2 % for echinococcosis, 1.1–4.9 % for fasciolosis, and 1.1–2.7 % for taeniosis/cysticercosis across the four years.”

Comments 3:

* 2. Materials and Methods:

Figure 1. The image definition still needs improvement, it is not possible to read the caption placed in the figure even by enlarging the image.

Response 3:

We have replaced Figure 1 with a higher-resolution map. Labels are now clearly legible when zoomed to 100 %, and the figure focuses solely on the Puno Region and CHN.

Comments 4:

2.4 Serological testing. Please, fixed some inaccuracies:

Lines 136-137: use italics when necessary, and I would also refer to Echinococcus and Fasciola because:

the RIDASCREEN® Echinococcus IgG is for a Specific detection of IgG antibodies against Echinococcus granulosus, E. multilocularis

the RIDASCREEN® Fasciola IgG test is performed to detect the presence of IgG antibodies to Fasciola hepatica and F. gigantica in human serum

Response 4:

We have corrected formatting and clarified species coverage.

Changes in manuscript:

All genus/species names are in italics.

Comments 5:

Line 143: check reference [26] or explain better in the text where you cite the national serological manual

Response 5:

We have clarified the reference in the text and completed the bibliographic details.

Changes in manuscript:

Section 2.3 and 2.4:

“… following the manufacturer’s instructions and the Peruvian National Institute of Health procedures manual for the Serological Diagnosis of Parasitic Zoonoses [34].”

Reference [34] now includes full title, institution, year and city of publication.

Comments 6:

* 3. Results:

Table 1. Please replace "sample numbers" or insert “sample size” in the caption. Delete “Samples numbers”

Figure 2. I suggest writing the caption as follows: Annual prevalence of three zoonotic diseases investigated in the Chucuito Health Network, 2018–2021

Figure 3 and Table 2. I would substitute Fasciolosis with Fascioliasis aligning it with the text of the manuscript

Response 6:

We have restructured the tables and captions in line with this and Reviewer 03’s suggestions.

Changes in manuscript:

Combined Table 1 now includes population, sample size and seroprevalence by disease and year; the caption uses “sample size (n)” and no longer uses “Samples numbers”.
Figure 2 caption has been adjusted to:

“Figure 2. Annual seroprevalence of the three zoonotic diseases investigated in the Chucuito Health Network, 2018–2021.”

Fasciolosis/fascioliasis: as explained to Reviewer 03, we have standardised on “fasciolosis” throughout, including in Figure 3 and the new Table 2, with a brief note in the Introduction that fascioliasis is the more common term in human medicine.

Comments 7:

Lines 162-167: I suggest arranging the bulleted list data in an organized Table. Consider whether to include these additional data in Table 1.

Response 7:

We agree and have implemented this, as described above. The prevalence percentages that were previously listed in bullets have been moved into the new combined Table 1, which now provides a concise, self-contained summary.

Comments 8:

* 4. Discussion:

Line 248: Guerra and Ramirez [31] is not correct, replace with Castillo Benancio [31]

Line 260: Fernández-holguín [37] is not correct, replace with Ayala, et al. [37]

Response 8:

Thank you for these corrections. We have reviewed the entire Discussion and corrected the mapping between in-text citations and the reference list.

Changes in manuscript:

The sentence referring to a 4.9 % prevalence now cites Castillo Benancio [40].
The sentence referring to Ayacucho data now correctly cites Ayala et al. [46].

Comments 9:

At the point where you describe the limitations of the study, please also refer:

Response 9:

We agree this is an important nuance and have strengthened the Limitations accordingly.

Change in manuscript (Discussion – Limitations paragraph):

We added:

This complements the existing statement on IgG-only detection and cumulative exposure.

Comments 10:

* 5. Conclusions

Line 313: Insert an introductory sentence like: “The present study leads to the following priority issues”

Response 10:

We have added the suggested introductory phrase to lead into the numbered items.

Change in manuscript (Conclusions):

New opening line of Section 5:

“The present study leads to the following priority issues:”

Comments 11:

* References:

Check bibliographic references (there are still some mistakes).

Pay attention that usually the list of References should be structured like this:

For a single author. Format: Last name, Initial(s).

For multiple authors. Format: Last name, Initial(s)., & Last name, Initial(s) or et al.

Response 11:

In response to this and Reviewer 03’s comments, we have once again reviewed and standardised the entire reference list:

For a single author: “Surname, Initial(s).”
For multiple authors: “Surname, Initial(s).; Surname, Initial(s).; … et al.” where appropriate, following MDPI guidelines.
Journal references include full article title, abbreviated journal name, year, volume and page range.
Book and chapter references include publisher, city, year and page numbers.

We have verified that all references cited in the text appear in the list and that all listed references are cited.