Molecular Detection and Genotyping of Enteric Protists in Asymptomatic Schoolchildren and Their Legal Guardians in Madrid, Spain

Round 1

Reviewer 1 Report

This study extends previous results of the group by evaluating the epidemiology of enteric protists in Spain, with a focus on apparently healthy children. Besides, they aimed to investigate risk factors associated with Giardia, Cryptosporidium, and Blastocystis transmission. The authors listed as the main findings: 1) confirmation that the enteroparasites G. duodenalis, Cryptosporidium spp., and Blastocysts sp. are a common finding in the stools of apparently healthy schoolchildren; 2) Evidence for Blastocystis sp within-household transmission. This is an incremental study conducted in an area not previously evaluated in Spain. However, it is not clear which lack of knowledge the present results fulfil  that deserve to be published.

Comments:

• Besides genotypes A and B, genotype E was already detected in humans (Fantinatti et al. 2016, Zahedi et al 2017). Please, update this information.
• The number of legal guardian participants is low (n=6) in comparison to the schoolchildren (n=74). Considering these two groups differ substantially in their demographic characteristics (age, behavior, exposition, etc), the inclusion of adults can potentially bias the study. Then, the data analysis should focus on the 74 schoolchildren, a casuistic well defined in the Material and methods, section 4.1 (lines 86-90). The adults have to be excluded from the global analysis and considered separately. The fact that “all G. duodenalis and most Cryptosporidium isolates were detected in children only”, reinforces this suggestion.
• The authors should include a table contents informing the schoolchildren's demographic and clinical characteristics, according to the age range.
• Figure 1 – The authors should include the number of participants in each one age group in the legends.
• Table 3 should precede tables 1 and 2. As stated before, the substantial data are related to the schoolchildren.
• The aim of the study is mainly to evaluate the infection by enteroparasites in asymptotic schoolchildren. However, table 3 presents the variables “diarrhea in the last 7 days” and “diarrhea in schoolchildren”, as well as, exposed and unexposed categories. What does mean exposed and unexposed? It is not clear whether diarrhea in schoolchildren exposed means symptomatic cases. Please, clarify.
• According to Table 3, the category age was available for 70 out 80 subjects. However, the text informs that “The age of four participants was unknown.” Were the guardians included in this analysis or only the schoolchildren? Please, clarify. This reinforces the importance of the schoolchildren groups in the study.
• The risk factors can vary according to the age range. Then, to better interpret the data, the age groups should be the same applied in Figure 1.
• The sentence “Regarding genotyping data, sequence analyses revealed sub-assemblage BIV as the only G. duodenalis genetic variant circulating in the surveyed paediatric population, although this result should be interpreted with caution due to the LOW number of isolates genotyped in this study.” has to be rephrased. In fact, the SINGLE isolate genotyped belonged to sub-assemblage BIV.

Author Response

Referee #1

This study extends previous results of the group by evaluating the epidemiology of enteric protists in Spain, with a focus on apparently healthy children. Besides, they aimed to investigate risk factors associated with Giardia, Cryptosporidium, and Blastocystis transmission. The authors listed as the main findings: 1) confirmation that the enteroparasites G. duodenalis, Cryptosporidium spp., and Blastocystis sp. are a common finding in the stools of apparently healthy schoolchildren; 2) Evidence for Blastocystis sp within-household transmission. This is an incremental study conducted in an area not previously evaluated in Spain. However, it is not clear which lack of knowledge the present results fulfil that deserve to be published.

We thank Reviewer #1 for his/her constructive criticism. We acknowledge that one of the major limitations of this study was the relatively small number of samples (particularly those from parent/legal guardians) analysed. This issue has been adequately commented in the last paragraph of the Discussion section. Regarding the novelties and contributions of the study, there are two that we would like to highlight: i) please note that there are very few molecular epidemiological surveys investigating household transmission of diarrhoea-causing protist parasites globally. This is one of them; and ii) this study provides molecular evidence favouring the notion that Blastocystis sp. should be regarded as a commensal rather than a pathogen. In this regard, see also our reply to comment #1 by reviewer #2.

We would also like to inform Referee #1 about two issues detected during the review process of the manuscript:

1. First, please note that we have opted to consider as parasite-positive samples only those confirmed by Sanger sequencing. Consequently, we have lost few samples previously considered positive to Cryptosporidium and Blastocystis. We have now modified the results section (including Figure 1 and Table 3) to adapt them to the introduced changes. Please note that these changes are minor and have not affected the essence of the manuscript, the results obtained and the conclusions reached.
2. We detected an unnoticed row shift in the previous version of Table 3, produced during copy-and-paste. This issue has been now solved and the correct version of the Table has been provided ion the revised version of the manuscript.
3. Besides genotypes A and B, genotype E was already detected in humans (Fantinatti et al. 2016, Zahedi et al 2017). Please, update this information.

Reply: please note that human infections by G. duodenalis assemblages C-F have been sporadically reported globally, primarily in children and immunocompromised individuals. This issue has been now stated in the introduction section.

2. The number of legal guardian participants is low (n=6) in comparison to the schoolchildren (n=74). Considering these two groups differ substantially in their demographic characteristics (age, behavior, exposition, etc), the inclusion of adults can potentially bias the study. Then, the data analysis should focus on the 74 schoolchildren, a casuistic well defined in the Material and methods, section 4.1 (lines 86-90). The adults have to be excluded from the global analysis and considered separately. The fact that “all duodenalis and most Cryptosporidium isolates were detected in children only”, reinforces this suggestion.

Reply: we strongly disagree with Referee #1. First, please note that one of the goals of the present study was to investigate within-household transmission of diarrhoea-causing protist pathogens, as clearly stated in the last paragraph of the Introduction section. As commented above, this is precisely one of the novel approaches followed in the present survey. This goal would have not been accomplished if the adult population was removed from the study. Second, please note that we have devoted three previous research papers (see Mateo et al. Medicine 2014, 93, e75; Reh et al. Euro Surveill. 2019, 24, 1900241 and Muadica et al. Microorganisms 2020, 8, 466) to the description of the frequency, molecular diversity and factors associated to a higher risk of infection by protist enteric parasites in paediatric populations of the Madrid area. None of these tackled the presence of these pathogens in adult populations in close contact with infected children. Third, we provide evidence that asymptomatic, unnoticed infections (at least for Blastocystis sp.) in members of the same household (including children and adults) may be due to person-to-person transmission. Fourth, differences in prevalence rates for Giardia and Cryptosporidium in children and adult populations were highly expected, as it is well-known that children are particularly exposed to these infections due to poor personal hygiene and immature immune system. However, please note that we have shown a marked age-related pattern in the case of Blastocysts carriage, with higher rates in adult individuals. As commented before, this finding may support the non-pathogenic nature of Blastocystis sp.

3. The authors should include a table contents informing the schoolchildren's demographic and clinical characteristics, according to the age range.

Reply: Please note that this information is already shown (although broken down) in current Table 3. In an attempt to make available to the interested reader the data used to determine the prevalence rates and molecular diversity of G. duodenalis, Cryptosporidium spp., Blastocystis sp., and E. bieneusi and the sociodemographic and clinical variables used to estimate risk infections in the present study we have now included the full dataset as Supplementary Table S1.

4. Figure 1 – The authors should include the number of participants in each one age group in the legends.

Reply: Following Reviewer #1 advice, the number of participants by age group has been stated in the first paragraph of sub-section 2.1.

5. Table 3 should precede tables 1 and 2. As stated before, the substantial data are related to the schoolchildren

Reply: We disagree with Referee #1. Please note that we have followed the same order of the Material and method section: first detection and genotyping of enteric protist parasites, second risk analyses. For consistency purposes the same order has been kept in the Results section.

6. The aim of the study is mainly to evaluate the infection by enteroparasites in asymptomatic schoolchildren. However, table 3 presents the variables “diarrhea in the last 7 days” and “diarrhea in schoolchildren”, as well as, exposed and unexposed categories. What does mean exposed and unexposed? It is not clear whether diarrhea in schoolchildren exposed means symptomatic cases. Please, clarify.

Reply: Referee #1 is right. This is direct consequence of the row shift that we have mentioned above. The variable “Diarrhoea in the last seven days” has been removed as only asymptomatic children and adults were considered in the study. Exposed individuals to a given factor/variable were those at risk (e.g. contact with family members known to have diarrhoea). Unexposed individuals were those that had no contact with the risk factor (e.g., none of the family members had diarrhoea).

7. According to Table 3, the category age was available for 70 out 80 subjects. However, the text informs that “The age of four participants was unknown.” Were the guardians included in this analysis or only the schoolchildren? Please, clarify. This reinforces the importance of the schoolchildren groups in the study.

Reply: Please see our comments above about the issues with Table 3 and how this problem was sorted out.

8. The risk factors can vary according to the age range. Then, to better interpret the data, the age groups should be the same applied in Figure 1.

Reply: Referee #1 is right. In Table 3 we grouped participating individuals only in two age groups (≤ 7 years and >years) to gain statistical power. By doing so please note that the age-related patter shown for Blastocystis carriage was close (0.1) to statistical significance. When we repeated the same estimation considering the original four age groups (see subsection 2.5.), this value raised to 0.6. This was due to the low sample sized available for each age group. This limitation of the study has been already acknowledged in the discussion section of the manuscript.

9. The sentence “Regarding genotyping data, sequence analyses revealed sub-assemblage BIV as the only duodenalis genetic variant circulating in the surveyed paediatric population, although this result should be interpreted with caution due to the LOW number of isolates genotyped in this study.” has to be rephrased. In fact, the SINGLE isolate genotyped belonged to sub-assemblage BIV.

Reply: following Reviewer #1 advice, this sentence has been now rephrased as “Regarding genotyping data, sequence analyses revealed sub-assemblage BIV in the only G. duodenalis isolate successfully genotyped in the present study”.

Reviewer 2 Report

The study described in this manuscript concerns a relatively small number of samples however the paper is interesting since it provided the latest results of Madrid schoolchildren examination for enteric protozoa.

I have a few issues which should be corrected/considered:

1. Line 3: Please remove the ‘legal guardian’ from the title since only 6 samples of this group were tested.
2. Line 74-85: This part of the manuscript is more of a conclusion than an introduction. Please consider removing/shortening this part of the introduction section.
3. Line 90: Please correct the percentage in the sentence ‘The sex of two participants (2%) was unknown’ (it should be 2.5% or 3%).
4. Please italicize species genus and species names in the whole manuscript (see for example lines 113, 124, 131, 103, 104, 134, 140, 149, 184, 188, 189, 204-207)
5. Please explain in the materials and methods section how the 95% CI was calculated.
6. Table 3: Please write in the caption of the table which statistical test was used for calculation p-values.
7. Table S1 is missing.
8. Discussion: Because only a few samples of legal guardians were examined please consider rebuilding this sentence: ‘The main findings of the study are the confirmation that the protist enteroparasites G. duodenalis, Cryptosporidium spp., and Blastocysts sp. are a common finding in the stools of apparently healthy schoolchildren, and to a lesser extent,  in their legal guardians.’ Please correct also in this sentence spelling of Blastocystis sp.
9. I wonder if the authors considered using the internal amplification control to avoid possible false-negative results.
10. I wonder if the authors considered performing a copromicroscopic examination of faeces samples.

Author Response

Referee #2

The study described in this manuscript concerns a relatively small number of samples however the paper is interesting since it provided the latest results of Madrid schoolchildren examination for enteric protozoa. I have a few issues which should be corrected/considered.

Reply: We thank Reviewer #2 for his/her initial positive appraisal. We have considered and addressed all the issues raised by the Reviewer. Changes introduced in the text have been highlighted in red in the revised version of the manuscript for better identification.

We would also like to inform Referee #1 about two issues detected during the review process of the manuscript:

1. First, please note that we have opted to consider as parasite-positive samples only those confirmed by Sanger sequencing. Consequently, we have lost few samples previously considered positive to Cryptosporidium and Blastocystis. We have now modified the results section (including Figure 1 and Table 3) to adapt them to the introduced changes. Please note that these changes are minor and have not affected the essence of the manuscript, the results obtained and the conclusions reached.
2. We detected an unnoticed row shift in the previous version of Table 3, produced during copy-and-paste. This issue has been now solved and the correct version of the Table has been provided ion the revised version of the manuscript.

1. Line 3: Please remove the ‘legal guardian’ from the title since only 6 samples of this group were tested.

Reply: We disagree with Referee #2. Even considering the relatively low samples size used in the present study, the inclusion of stool samples from parents/legal guardians is precisely one of the main contributions of the survey. Please also note that there are very few molecular studies that simultaneously investigate the presence and potential household transmission of diarrhoea-causing protist enteric parasites between children and their parents. This is mainly due to the usual reluctancy of adult populations to volunteer participating in this kind of surveys. In addition, please also note that the Blastocystis age-related pattern (including adult subjects) observed in this study supports previous data suggesting that this microorganism should be considered a commensal rather than a pathogen. Considering all the above we wish to keep the title of the manuscript as it stands now.

2. Line 74-85: This part of the manuscript is more of a conclusion than an introduction. Please consider removing/shortening this part of the introduction section.

Reply: We again disagree with Reviewer #2. In our opinion, the above-mentioned paragraph summarized the main findings of the previous molecular studies conducted in the Madrid area by our research group. The idea here was to show previous knowledge in this particular epidemiological scenario, and to establish the link with the present study and its justification. Despite this, and following Reviewer #2 advice, we have shortened the paragraph by removing one of our statements (that devoted to the asymptomatic carriage).

3. Line 90: Please correct the percentage in the sentence ‘The sex of two participants (2%) was unknown’ (it should be 2.5% or 3%).

Reply: Corrected as per requested.

4. Please italicize species genus and species names in the whole manuscript (see for example lines 113, 124, 131, 103, 104, 134, 140, 149, 184, 188, 189, 204-207).

Reply: Corrected as per requested.

5. Please explain in the materials and methods section how the 95% CI was calculated.

Reply: Statistical analyses including estimation of 95% confidence intervals were conducted using R software. Please note that this information is already provided in sub-section 4.8.

6. Table 3: Please write in the caption of the table which statistical test was used for calculation p-values.

Reply: we used the chi-squared and/or Fisher's exact test to compare G. duodenalis, Cryptosporidium spp. and Blastocystis sp. infection rates by the variables potentially considered as risk factors. Please note that this information is already provided in sub-section 4.8., so there is no need to duplicate it in Table 3.

7. Table S1 is missing

Reply: Table S1 is now added in the resubmission.

8. Discussion: Because only a few samples of legal guardians were examined please consider rebuilding this sentence: ‘The main findings of the study are the confirmation that the protist enteroparasites duodenalis, Cryptosporidium spp., and Blastocysts sp. are a common finding in the stools of apparently healthy schoolchildren, and to a lesser extent, in their legal guardians.’ Please correct also in this sentence spelling of Blastocystis sp.

Reply: Please see our answer to your comment #1. The spelling mistake has been corrected.

9. I wonder if the authors considered using the internal amplification control to avoid possible false-negative results.

Reply: The Reviewer raised an important point. Our qPCR protocol does not include internal amplification control to detect the occurrence of inhibited amplification reactions. This point has been now commented in the paragraph devoted to the limitations of the study in the Discussion section.

10. I wonder if the authors considered performing a copromicroscopic examination of faeces samples.

Reply: No. The study was initially conceived and designed as a molecular-based epidemiological study. The reason for selecting PCR methods as diagnostic tools for the detection of diarrhoea-causing protist species is because these assays are far more sensitive than conventional microscopy examination. This feature is key when investigating asymptomatic populations in medium- to high-income countries (such as Spain) characterised by relatively low infection rates and low parasitic burdens.