Ocular Toxoplasmosis: Mechanisms of Retinal Infection and Experimental Models
Round 1
Reviewer 1 Report
This is a review on Toxoplasma gondii infection, with an emphasis on the Retina. Various aspects are addressed such as; generalities of the infection, description of the retinal cellular organization, diffusion routes of the parasite through the retinal epithelium, cellular transport, and cellular route. Then the infection of endothelial cells by Toxoplasma gondii and other routes of infection is explained. Furthermore, the movement of T gondii within the human retina and its importance in this infection are introduced. Then the infection of retinal epithelial cells is described. The behavior of T. gondii strains in relation to eye damage is described. Finally, two in vivo and ex vivo study models are described for the study of Toxoplasma infection.
From my point of view, I believe that the damage caused by the parasite in both immunocompromised and immunocompetent patients should have been addressed. However, the review is approached from the Immunobiological point of view between the parasite Toxoplasma gondii and its host.
This is a review of interest for the understanding of ocular toxoplasmosis. Which is limited to the points previously described.
Observations.
There are some exceptions where Toxoplasma is not italicized and must be italicized.
Figure 1. Organization of the human retina. Created with BioRender.com. It has different font sizes that must be corrected.
Figure 3. The in vivo and ex vivo models can be described around the objects that form the figure for a better understanding of it. For example, they put a slide that is supposed to have the retinal cells infected with T. gondii to be seen in fixed and stained samples or intraocular fluid. In the ex vivo model, the green figure represents the cells of the retina that are extracted to be infected in culture. They can put explanatory titles.
Author Response
See the attached file.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The idea of the manuscript entitled «Looking in the eye: in search for Toxoplasma» by V. Rodriguez Fernandez is a good one summarizing the landmark papers by Montoya and Liesenfeld 2004 as well as that from J. Smith and colleagues 2020 and enrich the paper with additional information describing how Toxoplasma enters the retina and how it makes use of biological signaling pathways to achieve this aim (chapters 2-5), and how the host and parasite genetic disposition modify its effectiveness (chapters 6-7). This is not, wht the title reflects, the that the title has to be replaced accordingly.
The linguistic situation needs a professional work up to facilitate the understanding of the manuscript, and the abstract needs re-wording since it does currently not contain any information inviting to further read the article.
There are mind gaps at several points that hopefully will be removed with re-writing, such as in chapter 1 the linkage of humidity (not moisture!) to social factors linked to the prevalence of Toxoplasma in a population.
Figure 1 is textbook knowledge and probably is not helpful for the reader of this article.
Lines 73-75: repetition, remove
Figure 2c: this route has in vivo not been shown to be of any relevance, no more than speculation should clearly be stated: I personally would stick to facts and consider to remove this and the corresponding in the absence of clinical relevance.
Line 178 and line 187: check/replace references, which do not support the corresponding statements.
Line 196: remove the whole 1st sentence, does not contain any information.
Line 197: gene polimorphisms
Line 234: unless not supported by a recent evidence, I would assume 85% to be the wrong number .
Lines 238-42: No meaningful content. Remove.
Chapter 8: Completely detached from the rest, nothing in common with the rest of the manuscript, not helpful. Would completely remove or link to the aforementioned data.
Based on the above-mentioned, The conclusions (chapter 9) require adaptation.
Author Response
See the attached file.
Author Response File: 
Author Response.pdf
Round 2
Reviewer 2 Report
The revised manuscript has tried to cope with the reviewer comments, but still is linguistically not supportable, and major points have not been answered. Moreover, several of the changes require further attention. In concrete:
The title is not representative of the content of this manuscript which is how Toxoplasma reaches into the eye.
The aim of the study seems to be to summarize how the retina gets infected, but not to “summarize the main features of ocular toxoplasmosis and indicate possible models for the study of the disease” is not what the manuscript presents. This is also what is contained in the summary which has received major improvement. Since the host immune response is not in the focus of this study, I would already in the summary clearly state that this manuscript focusses on the parasite and not the host.
P3: “Transmission of infection may occur by …” is a repetition, pls remove
P3: “… but more research is needed in order to better understand the main route” This postulate is far from being understandable based on the aforementioned. Clinically, damage starts except in rare exceptions in the inner retina (nerve fiber layer) from where the parasite extends towards RPE and choroid. So why the call for more research for outer retinal toxoplasmosis ?
P4: During in utero, the posterior pole is not normally involved, it is the most frequent location, but this is far from being normal. Tachyzoite diffusion is again a completely wrong term, there is no diffusion, but active movement as described by the authors later!
P6: Recent studies show … (25) in vitro. Please specifiy that this has not been reported in vivo.
P6: pls argue why “more research is needed to elucidate the mechanisms underlying free tachyzoite movement into the retina”. I do not see the impact of the way to reach the retina for the pathophysiology and evolution of disease.
P8: “Müller glial cells are supporting…” is correct, but not meaningful. Please be more concise of their effect.
P9: reference 48 wrong
P10: The statement that “Most of these cases in Europe are congenital” does not represent the current opinion, this was believed until the 90ies.
P10: “A recent study where mice were infected with type I, type II and two types from haplogroup 6 strains showed that all of them induce OT 43,44.” If the authors see no differ3nce between infection with different strains, why do the postulate that “more studies are needed in order to shed light on the pathogenesis of the different strains”
P11: please elaborate as clearly as the advantages also the limitations of organotypic tissue cultures which are time-limited and independent of the systemic host immune response.
P12: Mouse models mimic some aspects of human disease, while human ocular disease typically starts several weeks to months after systemic infection, and there exists no animal model for spontaneously recurrent disease after oral infection and in congenital disease.
P4, fig 1 AND P13, fig 3: educational slides, not contributing to the understanding of this review à remove
P14, conclusions: “… migrate among the different layers, mostly infecting Müller glial cells and RPE”: In contrast to Müller cells, RPE is NOT a cell population of primary interest with the inner retina being primarily affected in the vast majority of instances (i.e. >99%). If the authors disagree, they will have to provide water-tight published evidence including OCT and histopathology images from human OT.
P14, conclusions: “… there is still work ahead to identify the cells most affected and to gain more knowledge about the immune response” This is no conclusion of this paper, neither local nor systemic immune response having been tackled.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
The current manuscript is the result of several author-reviewer discussion rounds and seems to have reched its maximum now
