Characterization of Lophomonas spp. Infection in a Population of Critical Care Patients

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Brief report demonstrated the presence of Lophomonas spp infection in hospitalized patients.

 In my opinion, the report is well written.

When analysing, I did not see any points to be revised. In my opinion, the manuscript can be accepted for publication.

Author Response

Dear reviewer,

We kindly thank you for work in reading and reviewing our manuscript, as well as your validation of its quality.

Kind regards,

Francisco das Neves Coelho

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors report 11 cases of Lophomonas spp. infection found in ICU patients. Briefly, the main finding of the present study demonstrated the infection of Lophomonas in ICU patients which may cause or exacerbate the outcome of respiratory diseases. Below are some points that need to be clarified.

1. As the diagnosis of Lophomonas infection is difficult based on morphological evidence alone, it would be better to include the result of Wheatley's trichrome staining in Figure 1 to show cytoplasmic vacuoles if still possible.

2. Yosra et al. have reported that the nucleus of Lophomonas is not
apparent in Giemsa stained sample (DOI: 10.1007/s00436-015-4554-4), whereas in this study the nucleu is clearly observed and has similar appearance with bronchial epithelial ciliated cells. Is there any explaination about this difference?

Author Response

Dear reviewer,

I am grateful for your comments regarding the different staining techniques, which we consider relevant and contributes to improving our performance.

We currently establish the identification of Lophomonas using fresh and extemporaneous observation of respiratory samples. Our experience reveals that, in suspected cases (patients with comorbidities which are associated with immunosuppression, or lung infection that does not respond to the established therapy), an extemporaneous observation of the samples is essential so that we can identify the characteristic movement of Lophomonas. As demonstrated in the videos we included with the present manuscript, the movements produced by the Lophomonas protozoa are very characteristic and are completely different from the synchronized movements of bronchial epithelial cells. Flagellates have different shapes with cytoplasmic plasticity, from ovoid to round (15-40 microns), completely different from ciliated columnar cells. In wet mounts, the ciliary movement from columnar cells was coordinate and synchronous, while the flagellar movement produced by Lophomonas was wavy and leaded to active swimming of the protozoa.

We consider that staining is not essential to perform an accurate identification of Lophomonas. However, we use trichrome and Giemsa stains daily to stain vegetative forms of protozoa, namely amoebae and flagellates. In this case we only used Giemsa staining because we considered that we obtained satisfactory results regarding the shape, size, position of the nuclei, Lophomonas flagella and implantation of the cilia of the bronchial epithelium cells.

In the future we will compare the performance of the two staining techniques. As stated by Yosra et al, Trichrome was the best stain in demonstration of cellular details of Lophomonas, but H & E, PAP, and Giemsa stains showed good quality of stains as well.

I would like to highlight that in the literature (e.g. Zhang et al, 2011) we can repeatedly read that this is a rare pathogen mainly diagnosed in geographical areas with more disadvantaged socio-economic conditions. We consider that Lophomonas infection is underdiagnosed, not only due to lack of awareness and clinical suspicion, but also because of inadequate laboratory management. Bacteriological, mycological, and viral tests require samples to be stored at 4ºC, but the characteristic Lophomonas movements cannot be properly identified in a sample that has been subject to these conditions. Before collecting BAL in the ICU, the laboratory must be informed that the samples will be collected, so that they will not be stored at 4ºC or left for observation the following morning, which will not allow the characterization of Lophomonas movements. The reason for this is still unknown, but it's likely that active protozoa specimen cannot survive for long outside of the host and in such adverse circumstances. We updated the manuscript, and this information is mentioned in the Discussion section.

We've made minor corrections to the manuscript to include the use of Wheatley's Trichome. Unfortunately, we currently do not have any respiratory samples with positive Lophomonas identification to color with Wheatley's Trichrome. If, by chance, we come to identify a respiratory sample positive for Lophomonas before publication, we'll apply Wheatley's Trichome to it and contact the journal so that the respective pictures may be added.

Kind regards,

Teresa Baptista Fernandes

Francisco Neves Coelho

Reviewer 3 Report

Comments and Suggestions for Authors

I have read with interest the manuscript “Characterization of Lophomonas spp. infection in a population of critical care patients.”

It is important to make known this potential human pathogen, Lophomonas, which is why I consider that the paper is of relevant interest given that it seems to especially affect immunosuppressed patients.

However, several topics should be answered by the authors.

In Materials and methods section

-Although, the approval of the ethics committee of the hospital where the study was carried out is included, please include the name and location of the hospital in this section.

-It would be interesting to know if all the patients came from the same geographic area. Could that information be added to the table?

Minor comments

Spp should not be italicized.

Author Response

Dear reviewer,

I gladly thank you for work in reading and reviewing our manuscript, as well as your suggestions on how to further improve it.

I've corrected the italicized Spp. in the manuscript. I've also introduced a short sentence regarding the approval from our ethics committee in the Materials and Methods section, according to your recommendation.

Regarding the introduction of the patient's geographic area, while it's indeed a relevant information, we're hesitant to add this information to the manuscript. Our ethics committee requires us to disclose what categories of information we will consult from the patient's personal file and, since information regarding place of residency has not been a disclosed item of research, it would breach the original research protocol which the committee had agreed upon.

Our hospital complex provides healthcare to roughly a third of Lisbon's population (about 500.000 habitants), and it serves as a referral center to five other hospitals within the Greater Lisbon district. At the moment we can only disclose that all patients had a residence located within this geographic area.

While geographic distribution may be of relevance, we suspect that social and economic factors may play a greater role in exposure to Lophomonas cysts. Cockroaches are the commonest carrier of Lophomonas and are a very common pest which may be found throughout most urban centers in Portugal, especially in older buildings where sanitary installations and sewage draining may be suboptimal. As such, geographic distribution may play a lesser role in exposure to these protozoa in comparison to other factors such as economic capacity, household sanitary conditions and age, history of recent travel to endemic areas, and other factors which are commonly studied within the domain of public health.

I hope you may understand our restrictions to comply to your suggestions.

 

Kind regards

Francisco das Neves Coelho

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors

Your research and dates on Lophomonas infections in humans is of high scientific interest and very important for infectious diseases clinicians.

However it is necessary improve some things.

First of all you have to specify, in Materials, the sample size relating to all the patients you have analyzed and from which you identified the eleven patients positive for the protozoa.

Secondly, you have to demonstrate that patients with only Lophomonas presence as etiological agent was screened for all bacterial and virus infections.

As you know, it is necessary to consider also that respiratory symptoms due to SARS-COV-2 infection may to persist long after the infection resolution. Therefore, although the Lophomonas presence is correctly demonstrated, respiratory symptoms coud be of non-specific origin.

In Discussion you have at least talk about the opportunistic feature of this protozoan infection.

Lastly, bibliography must be improved with all the bibliographic items on this matter, as, e.g., Tyagi R.,2016; Zhang X., 2011; Pinos Vélez N., 2021; etc.

Author Response

Dear reviewer

I thank you for your work and patience in reading our manuscript, as well as your suggestions on how to improve it.

An updated version of the manuscript has been uploaded. Regarding your suggestions for improvement of this manuscript:

1) The total number of ICU patients admitted from January 2021 to June 2023, which amounts to 1859 patients. The number of patients where Lophomonas spp. was identified was 11, which represents 0,6% of the total population of ICU patients. We've added this information to the beginning of the Results section.

2) As specified in Materials and Methods section, lines 62 to 67, it is our policy to conduct a bundle of examinations to screen for unusual airway pathogens in patients with diagnosed lower airway infection who have no identifiable pathogens in microbiological samples and fail to improve under standard care and empirical antibiotic regimen. Among these, we include screening for several atypical bacteria, direct observation and PCR testing for Mycobacterium tuberculosis, Pneumocystis jirovecii, measurement of galactomannan, mycologic cultures, PCR testing for main respiratory viruses and parasitological evaluation. Other tests may be performed to exclude other relevant diagnoses, but they are usually not performed to all patients and are considered only in specific patients.

Because we perform this bundle of tests, we were able to identify a number of co-infecting organisms, such as Parainfluenza, CMV, Aspergillus spp., etc. in addition to Lophomonas in our patients. These pathogens were specified in Appendix B, Table 1, in the “Other organisms” section, when the tests were positive for any agent likely involved in lung infection. A “No” means that, to our best ability, we were not able to identify other present pathogens other than Lophomonas as the causative agent for lung infection.

The patients where we failed to identify other respiratory pathogens aside from Lophomonas underwent the same extensive respiratory screening we usually perform in our patients. This includes patients who were originally admitted to our care due to SARS-CoV-2 pneumonia, displayed clinical improvement under standard care and therapy, and later developed a new exacerbation of respiratory symptoms where standard tracheal aspirates failed to identify other pathogens such as bacteria, mold or reactivation of viral agents.

As a curiosity, in the past we did perform parasitological screening mostly to exclude Strongyloides stercoralis, as we've also had a significant number of patients undergoing steroid therapy presenting with pneumonia and multiorgan failure due to Strongyloides hyperinfection, although in our experience this diagnosis appears to be far more common in patients who were born or spent most of their lives in former Portuguese colonies in Africa (DOI: 10.1155/2023/4412935). The identification of a number of flagellated protozoa appears to be relatively recent, and it has further reinforced the necessity to exclude the presence of lower-airway parasites in patients who are not improving their respiratory symptoms under our care.

3) You are right in pointing out that patients initially admitted under our care due to SARS-CoV-2 pneumonia could present with persistent and sometimes long-term respiratory symptoms. As we mentioned above, we only conducted a thorough screening for opportunistic agents in patients who failed to clinically improve under standard care, or presented with a relapse of respiratory symptoms. We recognize that the conjoint presence of an ongoing severe viral infection and opportunistic parasitic infection may account for an exacerbation of symptoms, such as a lower PaO2/FiO2, a higher likelihood of hemodynamically instability, and introduces entropy to the interpretation of blood results due to a number of reasons. We've included this factor as a limitation to our study (Discussion section).

4) You are right in pointing out that the opportunistic character of this pathogen has not been emphasized in the Discussion section. We've expanded the section regarding immunosuppression to better address this point. We also included a short reference to co-infection between Lophomonas spp. and Mycobacterium tuberculosis, as well as the possible role of structural lung disease in the development of this infection.  

5) Thank you for your suggestions regarding additional literature. We've found and removed a duplicate citation from our list and added four other citations related to point nr 4, using some of the examples you provided. This manuscript was intended to be a brief report mainly concerning our own findings and as such we had refrained from using an excessive number of sources.

If you have any other suggestions for improvement of this manuscript or any questions you feel it is still unanswered, please feel free to contact us.

Kind regards

Francisco das Neves Coelho