A ribosomal marker-based metataxonomic framework for environmental surveillance of nematodes of public health importance

Juan P. Zuluaga; Katherine Bedoya-Urrego; Juan F. Alzate

doi:10.1371/journal.pone.0348689

Peer Review History

Original SubmissionApril 18, 2026
25 Jun 2026 Decision Letter - Nils Pilotte, Editor Dear Dr. Alzate, 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 Aug 09 2026 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. A 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'. 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Additional Editor Comments: While the reviews of your work were extremely positive, reviewer #1 provided some worthwhile suggestions and raised a number of valid points which merit your consideration. We look forward to receiving your revised manuscript after you have addressed these concerns. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ******** Reviewer #1: Zuluaga et al. have presented novel results for nematode species-specific environmental surveillance using wastewater and agricultural biomass samples in Colombia. The methods and analyses are robust, and the manuscript has a logical flow. The study objectives were outlined and successfully executed. The manuscript would benefit from some minor revisions as outlined below. Namely, a deeper dive into the literature for wastewater and environmental surveillance of nematodes, sequencing target selection can be addressed, and methods can be more detailed by including an in-depth description of the types of samples collected and date of collection. I have suggested a table be included to detail this information. Introduction: Lines 44-47: The nuance between human parasite resistance and animal infective parasite resistance should be better captured. Resistance is established in many animal infective helminths while in humans resistance may be emerging, with limited evidence supporting this claim. Line 54: Animal and zoonotic hookworm eggs cannot be differentiated either. • Some additional literature on other metataxonomic and wastewater/environmental surveillance for intestinal parasitic worms that should be considered: https://doi.org/10.1038/s41467-023-44521-3, https://doi.org/10.1371/journal.pntd.0012416, https://doi.org/10.1371/journal.pwat.0000224, 10.1021/acs.est.1c02168, https://doi.org/10.1371/journal.pwat.0000337, Gilleard Nemabiome papers Material Methods: Were samples collected from WWTPs settled solids, influent (primary?), effluent? What time frame were samples collected? How many samples from each site were collected. A table may be useful here. Line 85: What is the estimated population that uses each individual WWTP? Line 90: More information on type of food samples and how these were processed as well as what the bioinput samples consist of. A breakdown of how many samples from each site and in what years they were collected would be helpful hear to interpret results (lines 233-243). Suggest including a table for this information. 122-123 Incomplete sentence here. Results: Full genus and species should be written out when introducing for the first time in the body of the manuscript followed by abbreviated G. species. The data here is longitudinal which should be highlighted since it makes the results more impactful and relevant for public health monitoring of parasitic spp.. 235: “occasional presence”—can this be more quantitative? (x detects out of y samples or locations) Fig1: legend mentions biofertilizers (bioinput?), should be described in methods or consistently labeled as “bioinput” or “biofertilizer”. Introducing these terms in the methods would be helpful as well. Suggest making circle around each species breakdown in the same colour as the study site. Figure 5 / 6: presence absence data: suggest dropping sample names and rotating 90 degrees such that species names are on the x and the y is sample type and location (or category for figure 6). Remove “_” between genus and species. * / can be added to the right axis. Supplemental: Is the supplemental data the same as in the main figures? If so, there is no need for addition location-stratified supplementary figures. A table in the supplementary data of sample, location, sample type (including sludge, settled solids, influent, effluent, etc.), collection date, and detection +/- would be more fitting. Discussion: Address why the 18s target was chosen over other targets (ie ITS-2) either in the discussion or introduction. Sections of the discussion are repetitive and can be combined to minimize highlighting similar pitfalls (ie limited reference genomes, difficulty with species level resolution). Another pitfall that should be mentioned, however, is low abundance sequencing amplification bias using these approaches. While this is not a quantitative study, the longitudinal data coupled with species-specific detection add novel surveillance data for these Colombian communities and can be emphasized further. 298- 300: If Trichinella spp / Angiostrongylus cantonensis detection is likely from intermediate hosts, what utility does WBE have for these species? Are clinical cases rising in the sampling areas? Additional commentary on this can strengthen the argument for WBE surveillance in these communities. 357-358 More curated sequences can be obtained from WormBase ParaSite databases which is often cross referenced in NCBI and can be mentioned for future studies. 373: Mention how many out of the 6 species are relevant to public health surveillance. 395-397: “Methodological applicability” – this research implements and validates methods for presence absence detection of parasites in diverse environmental samples. Reviewer #2: This is a well-planned study and a well-written manuscript that clearly explains the use of meta taxonomy for environmental surveillance. Rather than assessing the burden of nematodes, the manuscript presents a well-structured methodological framework that can be applied in future research. ****** what does this mean?). If published, this will include your full peer review and any attached files. 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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. Attachments Attachment Submitted filename: PONE-D-26-19237_reviewer.pdf https://doi.org/10.1371/journal.pone.0348689.r001
Revision 1
14 Jul 2026 Author Response Response to Reviewers Manuscript PONE-D-26-19237 A Ribosomal Marker-Based Metataxonomic Framework for Environmental Surveillance of Nematodes of Public Health Importance We thank the Academic Editor and both reviewers for their careful and encouraging evaluation. We were glad that all technical criteria (soundness, statistical rigour, data availability, and language) were assessed positively by both reviewers, and we are grateful to Reviewer #1 for a set of suggestions that have measurably improved the manuscript. Every point has been addressed. Below, comments are grouped by manuscript section; each is quoted, followed by our response and, where useful, the exact wording introduced in the revised text. Note on line numbers. The line numbers below are those of the original submission, as cited by the reviewer. The precise location of each change is marked in the “Revised Manuscript with Track Changes” file. Throughout, we also made minor grammatical and stylistic corrections (punctuation, italicization of species names, and consistent use of the term “bioinput”) that are not itemized individually. A. Response to Journal Requirements 1. Style and file naming Response. The manuscript has been formatted to the PLOS ONE style templates, and all files have been named according to the journal's conventions (e.g., Fig1.tiff, Fig5.tiff, S1_Table, S2_Table, S1_File). 2. Code sharing Response. All author-generated code underpinning the findings is provided, without restriction, as Supporting Information (S1 File), and will be freely available under the article's CC BY 4.0 license upon publication. S1 File contains the automated metataxonomic pipeline and the Python and R analysis/plotting scripts, making the complete analytical workflow openly reproducible. 3. Data availability Response. We have revised our data-availability plan to comply fully with the open-data policy (updated statement in Section E). The raw amplicon sequencing reads are publicly available in the NCBI Sequence Read Archive under BioProject accession PRJNA976754; the per-sample detection data underlying every figure are provided in the new S2 Table; the sampling design is provided in the new S1 Table; and the complete analysis code is provided in S1 File. 4. Figure 1 (map/satellite imagery) Response. Figure 1 contains no copyrighted map or satellite imagery from proprietary sources such as Google Maps, Street View, or Google Earth. The map was produced entirely by the authors in QGIS v3.40.11 from openly licensed data. The terrain relief is based on the digital elevation model distributed with the ALOS PALSAR High-Resolution Radiometrically Terrain-Corrected product of the Alaska Satellite Facility DAAC — a resampled copy of the public-domain SRTM elevation model, shared without restriction under the NASA EOSDIS Data Use Policy (ref 38) — and the administrative boundaries were obtained from the open-data repository of the Instituto Geografico Agustin Codazzi (IGAC; ref 39), released as public information under Colombian Law 1712 of 2014. These sources are equivalent to the public-domain repositories recommended in the journal's figure-replacement guidance. Source attribution has been added to the Methods and to the legend of Figure 1. 5. Suggested references Response. We reviewed all works suggested by Reviewer #1 and incorporated the two most directly relevant to our study: Siko et al. 2025 (PLOS Water; environmental surveillance of soil-transmitted helminths in settings without networked sewerage; now ref 23, Introduction) and Avramenko et al. 2015 (the foundational strongylid “nemabiome” ITS-2 approach; now ref 47, Discussion). The remaining suggested works were evaluated but not cited, as these two best support the specific points on environmental surveillance and marker selection that the reviewer raised. 6. Reference list Response. The reference list was reviewed for completeness and accuracy. Five references were added (Section D), of which three correspond to reviewer/editor suggestions (refs 23, 47, 48); the other two (refs 38, 39) document the open data sources of Figure 1. No retracted articles are cited. B. Response to Reviewer #1 We thank Reviewer #1 for recognizing that “the methods and analyses are robust” and that “the study objectives were outlined and successfully executed,” and for the constructive suggestions detailed below. Introduction Lines 44–47 — anthelmintic resistance nuance Reviewer comment. The nuance between human parasite resistance and animal infective parasite resistance should be better captured. Resistance is established in many animal infective helminths while in humans resistance may be emerging, with limited evidence supporting this claim. Response. We rewrote the passage to separate the two contexts, distinguishing established resistance in animal-infective helminths from the still-limited evidence in human helminths. Text in the revised manuscript: “Anthelmintic resistance is well established in many animal-infective helminths, where intensive drug use has selected for resistant populations and compromised veterinary control programs (8,9). In human helminths, by contrast, resistance is not yet firmly established: reduced or suboptimal treatment responses have been reported, but the supporting evidence remains limited (10).” Line 54 — hookworm eggs Reviewer comment. Animal and zoonotic hookworm eggs cannot be differentiated either. Response. We extended the example so that it now also covers animal and zoonotic hookworms, which are morphologically indistinguishable from the human species at the egg stage — species that are, moreover, represented in our reference tree (Fig 4). Text in the revised manuscript: “…microscopic examination of hookworm eggs cannot differentiate Necator americanus from Ancylostoma duodenale, nor can it distinguish these human hookworms from the morphologically indistinguishable eggs of animal and zoonotic hookworm species such as Ancylostoma ceylanicum and Ancylostoma caninum (14).” Additional literature on environmental surveillance Reviewer comment. Some additional literature on other metataxonomic and wastewater/environmental surveillance for intestinal parasitic worms that should be considered: [https://doi.org/10.1038/s41467-023-44521-3; https://doi.org/10.1371/journal.pntd.0012416; https://doi.org/10.1371/journal.pwat.0000224; 10.1021/acs.est.1c02168; https://doi.org/10.1371/journal.pwat.0000337], Gilleard Nemabiome papers. Response. We incorporated the two most pertinent works. Siko et al. 2025 (ref 23) was added to the Introduction to situate our study within the growing body of environmental surveillance of soil-transmitted helminths; the Gilleard-group nemabiome reference (Avramenko et al. 2015, ref 47) was added to the Discussion to support the 18S-versus-ITS-2 comparison. Text in the revised manuscript: “In parallel, wastewater- and soil-based environmental surveillance is increasingly being explored as a complementary strategy for monitoring soil-transmitted helminths in endemic settings (23).” Materials and Methods Sample matrices, timeframe, and number of samples per site (and Line 85, population per WWTP) Reviewer comment. Were samples collected from WWTPs settled solids, influent (primary?), effluent? What time frame were samples collected? How many samples from each site were collected. A table may be useful here. [Line 85] What is the estimated population that uses each individual WWTP? Response. We substantially expanded the Sample Collection subsection. Each sample is now attributable to a defined matrix (influent water, biosolid, effluent water, or residual sludge); the treatment train of each plant is specified (Cañaveralejo applies advanced primary treatment, the other three use activated sludge); the population served, influent flow, elevation, and daily biosolids output are given per plant; and the sampling years are stated. As suggested, we added S1 Table summarizing the full sampling design for every site. Text in the revised manuscript: “San Fernando treats an influent of ~1.3 m³/s of municipal wastewater by activated sludge, serving 700,000 inhabitants … Aguas Claras treats ~5 m³/s by activated sludge, serving 2,200,000 inhabitants … Cañaveralejo, in the city of Cali, applies advanced primary treatment to ~4 m³/s … serves 2,600,000 inhabitants … El Retiro is a smaller rural plant … serves 20,000 inhabitants … Sampling design, sample types, and collection years for all sites are summarized in S1 Table.” Line 90 — food and bioinput description Reviewer comment. More information on type of food samples and how these were processed as well as what the bioinput samples consist of. Response. We added a generic description of both matrix types. As these were commercial products, their specific brands and formulations are proprietary and cannot be disclosed; this is now stated explicitly. All samples were processed with the same extraction protocol. Text in the revised manuscript: “Five commercial bioinput samples, solid and liquid organic fertilizers marketed for agricultural use (three from Medellín and two from Cali) and three food samples from the Aburrá Valley, comprising fresh produce intended for raw consumption and a processed cereal-based product, were analysed. As these were commercially available products, their specific formulations and brands are proprietary and are not disclosed.” Lines 122–123 — incomplete sentence Reviewer comment. Incomplete sentence here. Response. The sentence was completed. Text in the revised manuscript: “For this analysis, nematodes of public health importance were selected, including human-parasitic and zoonotic species (27,28).” Results Full genus/species at first mention; longitudinal emphasis Reviewer comment. Full genus and species should be written out when introducing for the first time in the body of the manuscript followed by abbreviated G. species. The data here is longitudinal which should be highlighted since it makes the results more impactful and relevant for public health monitoring. Response. All taxa are now written in full at first mention in the Results and abbreviated thereafter. We also made the longitudinal design explicit: the Aguas Claras plant, sampled in 2021 and 2023, is now framed as a two-year series, and a dedicated Discussion paragraph underscores the value of this longitudinal signal for public-health monitoring. Text in the revised manuscript: “Aguas Claras WWTP, sampled in both 2021 and 2023, showed a consistent longitudinal pattern of detection … Overall, Trichinella spp. was recovered from every Aguas Claras sample, and geohelminth DNA (N. americanus and Ascaris spp.) was detected in both sampling years, indicating sustained circulation rather than sporadic contamination.” Line 235 — “occasional presence” Reviewer comment. “occasional presence”—can this be more quantitative? (x detects out of y samples or locations) Response. We replaced the qualitative phrasing with an explicit detection frequency, and the per-species frequencies across all 20 samples are now available in S2 Table (Trichinella spp., 14/20; N. americanus, 7/20; Ascaris spp., 4/20; A. cantonensis, 3/20; T. trichiura, 2/20; E. vermicularis, 1/20). Text in the revised manuscript: “Nematode DNA was detected in 10 of the 12 WWTP samples, with defined spatial distribution patterns at each study site (Fig 5).” Figures Figure 1 — terminology and site-coloured circles Reviewer comment. Fig1: legend mentions biofertilizers (bioinput?), should be described in methods or consistently labeled as “bioinput” or “biofertilizer”. Introducing these terms in the methods would be helpful as well. Suggest making circle around each species breakdown in the same colour as the study site. Response. We standardized the terminology to “bioinput” throughout the manuscript, figures, and legends, and introduced the term in the Methods. Figure 1 was redrawn so that each species-breakdown pie is now outlined in the colour assigned to its study site, visually linking every pie to its location. Figures 5 and 6 — layout Reviewer comment. Presence absence data: suggest dropping sample names and rotating 90 degrees such that species names are on the x and the y is sample type and location (or category for figure 6). Remove “_” between genus and species. * / ** can be added to the right axis. Response. Both figures were redrawn exactly as suggested: rotated 90° so that species names are on the x-axis and sample type/location (Fig 5) or category (Fig 6) on the y-axis; the sample codes were removed; the underscores between genus and species were removed and species names set in italics. In place of the asterisk notation, we display the sample collection dates explicitly along the right axis, which we felt made the temporal information clearer. Supporting information Redundant location-stratified figures Reviewer comment. Is the supplemental data the same as in the main figures? If so, there is no need for additional location-stratified supplementary figures. A table … of sample, location, sample type … collection date, and detection +/- would be more fitting. Response. We agree, and we followed this recommendation. The location-stratified supplementary figures, which duplicated Figure 5, were removed. In their place we provide S2 Table (sample, category, site, region, sample type, collection year, and detection expressed as read counts per taxon) and S1 Table (sampling design). S1 Fig (Clade IV phylogeny) is retained because it conveys information not shown elsewhere. Discussion 18S versus ITS-2 target selection Reviewer comment. Address why the 18s target was chosen over other targets (ie ITS-2) either in the discussion or introduction. Response. We added a paragraph justifying the marker choice. Because the taxa present in an environmental matrix are not known a priori, a broad-range marker with conserved priming sites, informative hypervariable regions, and extensive well-aligned reference coverage is required; the 18S rDNA V4 region meets these criteria. We contrast it with ITS-2, which yields higher intraspecific resolution within defined groups (the nemabiome approach) but is difficult to align across the phylum and has uneven reference coverage for zoonotic taxa. Text in the revised manuscript: “…internal transcribed spacer markers such as ITS-2 provide higher intraspecific resolution and have proven powerful for quantifying species composition within defined groups, most notably the strongylid “nemabiome” approach (47), but their high length and sequence variability hampers alignment across the phylum, and their reference coverage remains uneven for many environmentally relevant and zoonotic nematodes.” Repetitive sections Reviewer comment. Sections of the discussion are repetitive and can be combined to minimize highlighting similar pitfalls (ie limited reference genomes, difficulty with species level resolution). Response. We consolidated these points. The intraspecific-resolution and limited-reference-genome limitations are now discussed once, and the “Constraints and prospects” section opens by referring back to them rather than restating them. Text in the revised manuscript: “Beyond the taxonomic-resolution limitations discussed above, several additional constraints should be considered when interpreting these results.” Low-abundance amplification bias Reviewer comment. Another pitfall that should be mentioned, however, is low abundance sequencing amplification bias using these approaches. Response. We added this limitation and, in the spirit of transparency, tied it to our own data: several detections — including all three positive samples for Angiostrongylus cantonensis — rested on single-read mOTUs and therefore require orthogonal confirmation. Text in the revised manuscript: “A further limitation intrinsic to amplicon-based approaches is amplification bias against low-abundance templates … several detections including all three positive samples for Angiostrongylus cantonensis were supported by single-read mOTUs, underscoring that low-abundance signals warrant confirmatory approaches (e.g., targeted qPCR or deeper sequencing) before epidemiological interpretation.” Lines 298–300 — WBE utility for zoonotic taxa Reviewer comment Attachments Attachment Submitted filename: Rebuttal_letter.docx https://doi.org/10.1371/journal.pone.0348689.r002
11 Aug 2026 Decision Letter - Nils Pilotte, Editor A Ribosomal Marker-Based Metataxonomic Framework for Environmental Surveillance of Nematodes of Public Health Importance PONE-D-26-19237R1 Dear Dr. Alzate, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support. 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, Nils Pilotte, Ph.D. Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: (No Response) ****** what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No ******** https://doi.org/10.1371/journal.pone.0348689.r003
Formally Accepted
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