Neuroinvasive Free-Living Amoebae Pathogenesis, Neuroinflammation and Therapeutic Challenges

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Abstact

* The abstract is long and full of extra details. Shorter sentence would improve readability.  Focus on the most critical points.
-  No epidemiological data (case numbers, geographic distribution, incidence) are explored, which could strengthen your idea.  Include a short epidemiological statistic (e.g., “fewer than 300 cases reported worldwide”).
-  Specify gaps more clearly (e.g., limited therapeutic efficacy , lack of clinical trials).  Explicitly state what is missing in the present research (e.g., “no standardized treatment regimens exist”).  
* Several sentences are multi-clausal andlong. shorter statements would enhance clarity.

Introduction
* Many sentences are multi-layered, and long which reduces readability. Breaking them into shorter sentences.
-  Terms like “free-living amoebae” “neuroparasites,” “protozoa,”  are mentioned interchangeably; clarifying distinctions early to avoid confusion.    
-  Some mechanisms (e.g., proteolytic degradation, Trojan horse mechanism) are extra detailed for an introduction.  Keep mechanistic details for later sections in the manuscript; make the introduction set the stage broadly.
* Authors must add brief epidemiological context (e.g., rarity of cases, incidence or mortality rates, geographic distribution) would explore the importance of the topic.  
- The aim is clear but could be more concised and impactful.

Discussion
* The section is full of details, which may overwhelm readers. Consider summarizing some epidemiological and environmental details.  Highlight notable outbreaks only.  
-  Use consistent phrasing for “primary amoebic meningoencephalitis (PAM)” after first mention, rather than alternating between the full name and the abbreviated term.  
-  Some microscopic descriptions (e.g., Virchow–Robin spaces, Schwann cell mesaxons) is very technical for general audience and readers. Simplify this part or move it to a separated pathology section.  Keep only the most critical findings (edema, necrosis, hemorrhage, olfactory bulb involvement) and move highly technical details to supplementary filed if needed.
-  While case numbers are given (~500 worldwide), add incidence rates or comparison to other CNS infections to highlight this topic and severity more effectively.
- Standardize terminology in the manuscript and avoid redundancy (such as repeating “mortality >95%” multiple times).
* Group related virulence factors (e.g., proteases, adhesion proteins, EVs) into concised subparagrahs.  
* Use linking sentences to guide readers from adhesion → invasion → tissue destruction → immune evasion.  
*  Refer to which mechanisms are most promising for drug development (cathepsin B, Nfa1, Nf23, EVs).  
*  Avoid overwhelming readers with excessive molecular data & retain the key findings.
* Explicitly relate high mortality and virulence mechanisms to PAM’s rapid progression and
* Refine treatment part in the manuscript:
   - *Conventional regimens* (CDC recommendations, adjunctive agents)  
   - *Reported survival rates* (Refer to successful combinations and surgical interventions)  
   - *Emerging  new therapies* (nitroxoline, repurposed drugs, auranofin).
* Group findings into categories (metal-based, conjugates, phytochemical-enhanced)  better than listing it separelely.  
* Connect novel therapies to their potential impact on survival rates.  
* Summarize conclusion. Focus on international collaboration, urgency, and translational roadmap in fewer sentences for more powerfull impact.

Author Response

Response to reviewers

We would like to sincerely thank the Reviewers for their careful evaluation of our manuscript entitled “Neuroinvasive free-living amoebae—pathogenesis, neuroinflammation and therapeutic challenges” and for their valuable comments, which helped us improve the quality and clarity of the paper. The manuscript has been thoroughly revised in accordance with the Reviewers’ recommendations.

Reviewer 1

Dear Reviewer 1,

We sincerely thank you for your careful evaluation of our manuscript entitled “Neuroinvasive free-living amoebae—pathogenesis, neuroinflammation and therapeutic challenges” and for your valuable comments. The manuscript has been thoroughly revised in accordance with your recommendations. Below, we provide a point-by-point response to all comments. All changes made to the manuscript have been indicated using Track Changes in the revised version.

1. Abstract Comments
2. A) The abstract is long and full of extra details. Shorter sentence would improve readability.  Focus on the most critical points.
   B) No epidemiological data (case numbers, geographic distribution, incidence) are explored, which could strengthen your idea.  Include a short epidemiological statistic (e.g., “fewer than 300 cases reported worldwide”).
   C) Specify gaps more clearly (e.g., limited therapeutic efficacy , lack of clinical trials).  Explicitly state what is missing in the present research (e.g., “no standardized treatment regimens exist”).
   D) Several sentences are multi-clausal andlong. shorter statements would enhance clarity.

Response: Thank you for this helpful comment. We have substantially revised the Abstract to improve clarity, concision, and readability. Several long, multi-clausal sentences were shortened or divided into shorter statements. We also added concise epidemiological information, including the approximate number of reported PAM and well-documented GAE cases worldwide. In addition, we clarified the major translational and therapeutic gaps, including poor CNS drug penetration, limited treatment efficacy, treatment-related toxicity, the absence of standardized treatment regimens, and the lack of controlled clinical trials.

2.Introduction

Comments
A) Many sentences are multi-layered, and long which reduces readability. Breaking them into shorter sentences. The Introduction was substantially rewritten, and long, multi-clausal sentences were shortened or divided into clearer statements.
B) Terms like “free-living amoebae” “neuroparasites,” “protozoa,”  are mentioned interchangeably; clarifying distinctions early to avoid confusion. The terminology was clarified at the beginning of the Introduction. Free-living amoebae are now defined as environmental protozoa, while amphizoic species and neuroinvasive FLA are introduced separately to avoid confusion.
C) Some mechanisms (e.g., proteolytic degradation, Trojan horse mechanism) are extra detailed for an introduction.  Keep mechanistic details for later sections in the manuscript; make the introduction set the stage broadly. Detailed mechanistic examples, including proteolytic degradation and Trojan horse-related invasion mechanisms, were removed from the Introduction and retained for later mechanistic sections.
D) Authors must add brief epidemiological context (e.g., rarity of cases, incidence or mortality rates, geographic distribution) would explore the importance of the topic.  Epidemiological context was added, including the approximate number of reported PAM and well-documented GAE cases worldwide, the estimated risk after recreational freshwater exposure, underdiagnosis, and high mortality.
E) The aim is clear but could be more concised and impactful. The aim of the review was rewritten to be shorter, more focused, and more impactful, emphasizing CNS invasion, neuroinflammation, neuropathology, diagnostic challenges, therapeutic strategies, and translational priorities.

Response: Thank you for these helpful comments. We have substantially revised the Introduction to improve readability, clarity, and logical flow. Long, multi-layered sentences were shortened or divided into simpler statements. The terminology was clarified at the beginning of the Introduction by defining free-living amoebae as environmental protozoa and explaining the amphizoic nature of clinically relevant species. Detailed mechanistic descriptions, including proteolytic degradation and the Trojan horse mechanism, were removed from the Introduction and are now discussed in the relevant mechanistic sections of the manuscript. We also added concise epidemiological context, including the rarity of reported cases, the estimated risk after recreational freshwater exposure, and the high mortality associated with these infections. Finally, the aim of the review was rewritten to be more concise and focused.

3. Discussion

Comments
A) The section is full of details, which may overwhelm readers. Consider summarizing some epidemiological and environmental details.  Highlight notable outbreaks only.  

Response: Thank you for this helpful comment. We have revised this section to improve readability and reduce excessive epidemiological and environmental detail. The revised version now summarizes the global distribution and key environmental determinants of N. fowleri, while retaining only the most clinically relevant information and selected notable examples, including the Ústí nad Labem outbreak. Highly detailed ecological and environmental survival data were shortened or removed to avoid overwhelming readers and to maintain focus on the clinical relevance of PAM. Analogous revisions were also made in the section on Acanthamoeba spp., where the description was shortened and focused on key information regarding environmental occurrence, human exposure, genotypic diversity, and the clinical burden of GAE. Because mortality data are already presented in the Introduction, they were not repeated in this subsection.

1. 
   B) Use consistent phrasing for “primary amoebic meningoencephalitis (PAM)” after first mention, rather than alternating between the full name and the abbreviated term.  

Response: Thank you for this comment. We have applied this change throughout the manuscript and now use the abbreviations “PAM” and “GAE” consistently after their first definition.

1. C) Some microscopic descriptions (e.g., Virchow–Robin spaces, Schwann cell mesaxons) is very technical for general audience and readers. Simplify this part or move it to a separated pathology section.  Keep only the most critical findings (edema, necrosis, hemorrhage, olfactory bulb involvement) and move highly technical details to supplementary filed if needed.

Response: Thank you for this helpful comment. We have revised this section to improve readability and reduce excessive technical detail. Highly specialized microscopic descriptions, including references to Schwann cell mesaxons and Virchow–Robin spaces, were removed or simplified. The revised text now focuses on the most clinically and pathologically relevant features of PAM, including olfactory bulb involvement, cerebral edema, hemorrhage, necrosis, increased intracranial pressure, and the presence of trophozoites without cyst formation in brain tissue.

1. D) While case numbers are given (~500 worldwide), add incidence rates or comparison to other CNS infections to highlight this topic and severity more effectively.

Response: We thank the Reviewer for this important suggestion. We agree that case numbers alone do not fully convey the epidemiological rarity and clinical severity of PAM. Therefore, we added information on the very low reported annual occurrence of PAM and included an exposure-related risk estimate for recreational freshwater contact. We also clarified that, although PAM is far less common than bacterial or viral CNS infections, its abrupt onset, rapid neurological progression, and very high case-fatality rate make it one of the most devastating infectious emergencies of the CNS

1. E) Standardize terminology in the manuscript and avoid redundancy (such as repeating “mortality >95%” multiple times).

Response: Thank you for this helpful suggestion. The manuscript has been revised to ensure consistent terminology throughout, and redundant statements, including repeated references to mortality exceeding 95%, have been removed or consolidated.

1. 
   F) Group related virulence factors (e.g., proteases, adhesion proteins, EVs) into concise subparagrahs.  Use linking sentences to guide readers from adhesion → invasion → tissue destruction → immune evasion.   Refer to which mechanisms are most promising for drug development (cathepsin B, Nfa1, Nf23, EVs).

Response: Thank you for this helpful suggestion. We have reorganized the section on Naegleria fowleri virulence mechanisms into concise subsections focused on adhesion and phagocytosis, invasion and tissue penetration, and extracellular vesicles with cytotoxicity and stress-adaptation mechanisms. We added linking sentences to guide the reader through the pathogenic sequence from adhesion to invasion, tissue destruction, and adaptation to host-derived stress signals. Excessive molecular detail was shortened, while the key virulence factors with translational relevance, including cathepsin B, Nfa1, Nf23, and extracellular vesicle-associated pathways, were retained and highlighted as promising preclinical targets for future therapeutic or immunological intervention.

Comment G:
Standardize terminology in the manuscript and avoid redundancy, such as repeated statements that mortality exceeds 95%.

Response: Thank you for this helpful suggestion. The manuscript has been revised to ensure consistent terminology throughout. Redundant statements, including repeated references to mortality exceeding 95%, were removed or consolidated. Mortality is now discussed only where it directly supports the clinical or translational relevance of the section..

Comment H: The section on Acanthamoeba spp. virulence mechanisms has been reorganized into concise mechanistic subsections covering adhesion, phagocytosis and host cell invasion, secreted enzyme-mediated barrier disruption, and apoptosis. Excessive molecular detail was reduced, while key findings related to MBP-mediated adhesion, protease-dependent tight junction disruption, phospholipase activity, and apoptosis were retained. Linking sentences were added to guide the reader through the sequence from initial attachment to tissue invasion, blood–brain barrier damage, host cell death, and progression of GAE.

Response: Thank you for this helpful suggestion. The section on Acanthamoeba spp. virulence mechanisms has been reorganized into concise mechanistic subsections covering adhesion, host-cell invasion and phagocytosis, enzyme-mediated barrier disruption, and host-cell death with dissemination. Excessive molecular detail was reduced, while key findings related to MBP-mediated adhesion, protease-dependent tight-junction disruption, phospholipase activity, apoptosis, and ADP-associated calcium-dependent host-cell death were retained. We also added linking sentences to guide the reader through the sequence from initial attachment to tissue invasion, blood–brain barrier damage, host-cell death, dissemination, and progression of GAE.

Comment I: Avoid overwhelming readers with excessive molecular data & retain the key findings

Response:We thank the Reviewer for this helpful suggestion. We revised the section on the immune response to Naegleria fowleri to reduce excessive molecular detail while retaining the key findings. The revised text now focuses on the main immunological stages, including mucosal barrier disruption, humoral responses, neutrophil recruitment, glial activation, neuroinflammation, and immunopathology. Detailed lists of individual mediators and signaling pathways were shortened and integrated into broader mechanistic categories to improve readability and emphasize their biological relevance.

Comment J: Explicitly relate high mortality and virulence mechanisms to PAM’s rapid progression
Response: Thank you for this helpful suggestion. We have revised this section to explicitly link the high mortality of PAM with the rapid progression driven by N. fowleri virulence mechanisms, including efficient adhesion, olfactory-route migration, protease-mediated tissue degradation, extracellular vesicle-associated cytotoxicity, and resistance to host-derived stress signals.

Comment K: Refine treatment part in the manuscript:
 Conventional regimens* (CDC recommendations, adjunctive agents); Reported survival rates* (Refer to successful combinations and surgical interventions); Emerging  new therapies* (nitroxoline, repurposed drugs, auranofin). Group findings into categories (metal-based, conjugates, phytochemical-enhanced)  better than listing it separelely.  Connect novel therapies to their potential impact on survival rates.  

Response: Thank you for this valuable comment. We have extensively revised and reorganized the Treatment section in accordance with the Reviewer’s recommendations. The revised version now clearly separates conventional CDC-based regimens, adjunctive therapeutic measures, reported survival-associated combinations, surgical interventions, and emerging experimental therapies. We also expanded the discussion of nitroxoline, repurposed drugs, auranofin, and other novel approaches, grouping them into clearer mechanistic categories rather than listing them separately. Finally, we emphasized that these strategies remain experimental or preclinical, while highlighting their potential future relevance for improving survival through enhanced CNS penetration, reduced toxicity, and improved anti-amoebic activity.

Comment L:  Summarize conclusion. Focus on international collaboration, urgency, and translational roadmap in fewer sentences for more powerfull impact.

Response: Thank you for this helpful suggestion. We have revised and shortened the Conclusions and future directions section to provide a more focused and impactful final message. The revised conclusion now emphasizes the urgency of improving outcomes in PAM and GAE, the need for coordinated international collaboration, and a translational roadmap linking early diagnosis, therapeutic innovation, environmental surveillance, One Health strategies, and collaborative research. Figure 4 has been retained as a graphical summary of these key milestones, current challenges, and future priorities.

Reviewer 2 Report

Comments and Suggestions for Authors

Oliwia et al. summarize (current) knowledge about the neurological manifestations induced by the free-living amoebae Naegleria fowleri and Acanthamoeba spp. They summarize causative agents, virulence factors, and therapeutic options for these manifestations. This is interesting review. While the review is well-structured, modifications are still required for the clarity and novelty of the presentation. I went through this review and found the following points:

1. The authors insisted that they “synthesize” the data shown by other people (as indicated in lines 9 and 58). This is not clear for me. Did the author perform a systematic review, or meta-analysis? For me, it looks like a normal narrative review. The authors need to clarify this.
2. Some of the terms mentioned in this manuscript are not clear. The authors need to provide explanations of terms like neuroinflammation or granulomatous inflammation. The explanation should be readable and understandable by the normal reader.
3. The authors insisted that Naegleria fowleri induces acute infection. This is misleading. In some studies, the infections started 5 days following daily exposure to contaminated water. These 5 days exposure is absolutely not acute. Did that author mean that the symptoms acutely developed? If so, then this will be explained.
4. An interesting point which is sightly mentioned in this review is the effect of species on the symptoms or manifestations (line 48). I think the authors need to cover this part in detail.
5. An extremely important point for the treatment is the differentiation between trophozoites and cysts. The authors need to clearly indicate the difference in the treatment options available for these life stages.
6. The point of investigating the environmental effects on the pathogenicity of these protozoa is very nice. These effects appeared in some communities (PMID: 29401275). Since the authors mentioned some information about the geographical distribution of these infestations, they should include these effects in this work.
7. Certain species of Acanthamoeba, especially Acanthamoeba castellanii induces keratitis. Could this represent a way of transmission to the brain? Even if there is no literature available, the authors could still provide few sentences about this possible/non-possible way of transmission.
8. One of the misunderstood points in the review is the assumption that these infections are rare (line 306), making them as one of the neglected infections. However, the high fatality rate (95%; line 291) opposes this assumption. Could the authors explain this and indicate whether these infections are categorized under this art of neglected infections.
9. The review must include novel literatures indicating potential treatment options. This include using various compounds such as PHMB, PD, or even plant extracts (PMID: 31833381, and 33262903, and 37229821). Although these options are still experimental so far; however, they provide a possible application in the future.
10. More should be written about the dissemination of these infections. One of the possible mechanism of dissemination of these infections is the release of ADP which induces cellular death in a calcium dependent-manner (PMID: 11349088).
11. The review is supported by nice figures. The authors need to state which program was used for the creation of these figures.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Dear Reviewer 2,

We sincerely thank you for your careful evaluation of our manuscript entitled “Neuroinvasive free-living amoebae—pathogenesis, neuroinflammation and therapeutic challenges” and for your valuable comments. The manuscript has been thoroughly revised in accordance with your recommendations. Below, we provide a point-by-point response to all comments. All changes made to the manuscript have been indicated using Track Changes in the revised version.

Oliwia et al. summarize (current) knowledge about the neurological manifestations induced by the free-living amoebae Naegleria fowleri and Acanthamoeba spp. They summarize causative agents, virulence factors, and therapeutic options for these manifestations. This is interesting review. While the review is well-structured, modifications are still required for the clarity and novelty of the presentation. I went through this review and found the following points:

Comment 1.The authors insisted that they “synthesize” the data shown by other people (as indicated in lines 9 and 58). This is not clear for me. Did the author perform a systematic review, or meta-analysis? For me, it looks like a normal narrative review. The authors need to clarify this.

Response:
We thank the Reviewer for this valuable comment. We have clarified this issue in the revised manuscript. Accordingly, the wording in the Abstract and Introduction has been revised to avoid suggesting a formal systematic evidence synthesis. We have also added a dedicated section entitled “Literature Search Strategy”, in which we describe the databases searched, the scope of the literature considered, and the types of studies included. This addition is intended to make the methodological approach transparent and to clearly define the manuscript as a narrative review integrating clinical, experimental, and translational evidence.

Comment 2. Some of the terms mentioned in this manuscript are not clear. The authors need to provide explanations of terms like neuroinflammation or granulomatous inflammation. The explanation should be readable and understandable by the normal reader.

Response: We thank the Reviewer for this valuable comment. In response, we revised the Introduction to improve conceptual clarity and make the text more accessible to readers. Specifically, we added brief explanations of key terms used throughout the manuscript, including “neuroinflammation” and “granulomatous inflammation”, and incorporated them into the introductory section in a concise and reader-friendly manner. In addition, we modified the surrounding text to improve terminological consistency and provide a clearer clinical and neuropathological context for the discussed concepts.

Comment 3. The authors insisted that Naegleria fowleri induces acute infection. This is misleading. In some studies, the infections started 5 days following daily exposure to contaminated water. These 5 days exposure is absolutely not acute. Did that author mean that the symptoms acutely developed? If so, then this will be explained.

Response: We thank the Reviewer for this important clarification. We agree that the term “acute infection” may have been imprecise, as it could be interpreted as referring to the exposure period or incubation phase. Our intention was to describe the acute onset of symptoms and the rapidly progressive clinical course of PAM after a short incubation period. We have therefore revised the relevant sentences to clarify that N. fowleri infection is characterized by a short incubation period followed by acute symptom onset and rapid neurological deterioration.

Comment 4. An interesting point which is sightly mentioned in this review is the effect of species on the symptoms or manifestations (line 48). I think the authors need to cover this part in detail.

Response: We thank the Reviewer for this valuable suggestion. Because PAM and GAE are discussed in detail in separate sections, we added a concise comparative table summarizing species-dependent differences in clinical manifestations. The table highlights how the causative amoebic species influences the clinical phenotype, neurological presentation, neuropathological pattern, diagnostic challenges, and therapeutic implications of neuroinvasive FLA infections.

Comment 5. An extremely important point for the treatment is the differentiation between trophozoites and cysts. The authors need to clearly indicate the difference in the treatment options available for these life stages.

Response: We thank the Reviewer for this valuable comment. We have added a new paragraph at the beginning of the Treatment section to clarify the therapeutic relevance of trophozoite and cyst stages. We now explain that PAM therapy is directed mainly against trophozoites because N. fowleri does not form cysts in human tissue, whereas GAE treatment should ideally target both trophozoites and cysts due to the ability of Acanthamoeba spp. to persist in both forms.

Comment 6. The point of investigating the environmental effects on the pathogenicity of these protozoa is very nice. These effects appeared in some communities (PMID: 29401275). Since the authors mentioned some information about the geographical distribution of these infestations, they should include these effects in this work.

Response:
We thank the Reviewer for this valuable suggestion. We agree that environmental factors are important determinants of the persistence, proliferation, and exposure risk of pathogenic free-living amoebae. In response, we added a new paragraph to the section discussing the geographic distribution and emergence of N. fowleri. This paragraph now addresses the influence of environmental and water-system conditions, including elevated water temperature, turbidity, low disinfectant residuals, biofilm formation, and algal or microbial overgrowth. We also incorporated the example highlighted by the Reviewer, namely the PAM case associated with an artificial whitewater river, to illustrate how suboptimal water-quality conditions in engineered aquatic systems may favor N. fowleri persistence.

Comment 7. Certain species of Acanthamoeba, especially Acanthamoeba castellanii induces keratitis. Could this represent a way of transmission to the brain? Even if there is no literature available, the authors could still provide few sentences about this possible/non-possible way of transmission.

Response: Thank you for this valuable suggestion. We agree that Acanthamoeba keratitis raises an interesting question as to whether ocular infection could, at least theoretically, contribute to CNS dissemination. We have added a short paragraph addressing this possibility. In the revised manuscript, we emphasize that this route remains hypothetical and should be interpreted with considerable caution.

Comment 8. One of the misunderstood points in the review is the assumption that these infections are rare (line 306), making them as one of the neglected infections. However, the high fatality rate (95%; line 291) opposes this assumption. Could the authors explain this and indicate whether these infections are categorized under this art of neglected infections.

Response: We thank the Reviewer for this important clarification. We would like to clarify that our intention was not to classify neuroinvasive free-living amoebae infections as formally recognized neglected tropical diseases. In the revised manuscript, we clarified that the term “rare” refers to the relatively low number of reported and laboratory-confirmed cases, whereas the high fatality rate reflects the severe clinical course, frequent diagnostic delay, and limited therapeutic efficacy of these infections. Thus, rarity and high mortality are not contradictory; rather, neuroinvasive FLA infections are rare but clinically devastating. To avoid ambiguity, we revised the relevant text to emphasize that these infections remain underdiagnosed and insufficiently studied from diagnostic, therapeutic, and translational perspectives, without implying formal classification as neglected tropical diseases.

Comment 9. The review must include novel literatures indicating potential treatment options. This include using various compounds such as PHMB, PD, or even plant extracts (PMID: 31833381, and 33262903, and 37229821). Although these options are still experimental so far; however, they provide a possible application in the future.

Response: Thank you for this valuable suggestion. We agree that recent experimental studies evaluating potential anti-Acanthamoeba compounds should be included. Accordingly, we have incorporated the suggested publications into the treatment section and expanded the discussion of emerging therapeutic approaches, including PHMB, propamidine isethionate, photodynamic therapy, and selected plant-derived compounds.

Comment 10.  More should be written about the dissemination of these infections. One of the possible mechanism of dissemination of these infections is the release of ADP which induces cellular death in a calcium dependent-manner (PMID: 11349088).

Response: Thank you for this valuable suggestion. Accordingly, we have expanded the relevant section to include the release of ADP and other soluble metabolites by Acanthamoeba, which may induce host-cell apoptosis through calcium-dependent signaling and thereby contribute to tissue damage, immune-cell impairment, and dissemination of infection.

Comment 11.  The review is supported by nice figures. The authors need to state which program was used for the creation of these figures.

Response: We thank the Reviewer for this positive comment and helpful suggestion. We have added information to the figure legends stating that the figures were created by the authors using Microsoft PowerPoint.

Reviewer 3 Report

Comments and Suggestions for Authors

I consider the work is well-written and attempts to provide an overview of amphizoic amoebae and the pathogenic processes they carry out.

It is important that, as this is a review, the authors consider literature that can complement the descriptions of invasion processes. Regarding adherence, the authors only refer to phagocytosis and do not mention studies that describe paracellular invasion processes, the importance of migration and invasion by the mechanisms and/or enzymatic processes that facilitate it.

Besides, authors do not describe neuroinflammatory processes in more detail, even though the manuscript title specifies otherwise. There are studies that describe, through experimental models of GAE in the early stages of invasion, that inflammatory processes are not observed.

The authors could certainly enrich the work to provide a more complete view of the processes these amoebae carry out during their invasion of target tissue.

Author Response

Dear Reviewer 3,

We sincerely thank you for your careful evaluation of our manuscript entitled “Neuroinvasive free-living amoebae—pathogenesis, neuroinflammation and therapeutic challenges” and for your valuable comments. The manuscript has been thoroughly revised in accordance with your recommendations. Below, we provide a point-by-point response to all comments. All changes made to the manuscript have been indicated using Track Changes in the revised version.

Comment 1.  I consider the work is well-written and attempts to provide an overview of amphizoic amoebae and the pathogenic processes they carry out. It is important that, as this is a review, the authors consider literature that can complement the descriptions of invasion processes.

Response: We thank the Reviewer for this positive comment. In response to the Reviewer’s recommendation, we have expanded the discussion of invasion processes by incorporating additional literature on paracellular migration, barrier traversal, enzymatic tissue remodeling, and contact-dependent invasion mechanisms.

Comment 2.   Regarding adherence, the authors only refer to phagocytosis and do not mention studies that describe paracellular invasion processes, the importance of migration and invasion by the mechanisms and/or enzymatic processes that facilitate it.

Response: Thank you for this important comment. We have revised both the PAM and GAE sections to expand the discussion beyond adhesion and phagocytosis. The revised text now includes paracellular invasion, amoeboid migration, tight-junction disruption, extracellular matrix degradation, protease-mediated tissue remodeling, and epithelial/endothelial barrier traversal as key mechanisms supporting tissue invasion and CNS dissemination.

Comment 3. Besides, authors do not describe neuroinflammatory processes in more detail, even though the manuscript title specifies otherwise. There are studies that describe, through experimental models of GAE in the early stages of invasion, that inflammatory processes are not observed. The authors could certainly enrich the work to provide a more complete view of the processes these amoebae carry out during their invasion of target tissue.

Response: Thank you for this important comment. We have revised and expanded the sections on neuroinflammation in both PAM and GAE. In the PAM section, we added a more detailed description of fulminant innate neuroinflammation, including neutrophil infiltration, microglial and astrocytic activation, cytokine and chemokine release, oxidative stress, inflammasome activation, blood–brain barrier dysfunction, edema, and hemorrhagic–necrotic tissue injury. In the GAE section, we incorporated the study by Omaña-Molina et al. (2017) to clarify that early Acanthamoeba neuroinvasion may proceed through mechanically mediated, contact-dependent migration before a fully developed inflammatory response becomes detectable. These revisions provide a more balanced and temporally nuanced view of neuroinflammatory processes during CNS invasion

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I would like to thank the authors for addressing my comments. The review is fine and i recommend accepting it.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors enriched the manuscript, which allowed for a more objective and complete description of the research carried out to date on these protozoa and their clinical importance.