Urinary interleukins (IL)-6 and IL-10 in schoolchildren from an area with low prevalence of Schistosoma haematobium infections in coastal Kenya

Urinary cytokines are gaining traction as tools for assessing morbidity in infectious and non-infectious inflammatory diseases of the urogenital tract. However, little is known about the potential of these cytokines in assessing morbidity due to S. haematobium infections. Factors that may influence the urinary cytokine levels as morbidity markers also remain unknown. Therefore the objective of the present study was to assess how urinary interleukins (IL-) 6 and 10 are associated with gender, age, S. haematobium infections, haematuria and urinary tract pathology and; 2) to assess the effects of urine storage temperatures on the cytokines. This was a cross-sectional study in 2018 involving 245 children aged 5–12 years from a S. haematobium endemic area of coastal Kenya. The children were examined for S. haematobium infections, urinary tract morbidity, haematuria and urinary cytokines (IL-6 and IL-10). Urine specimens were also stored at –20°C, 4°C or 25°C for 14 days before being assayed for IL6 and IL-10 using ELISA. The overall prevalence of S. haematobium infections, urinary tract pathology, haematuria, urinary IL-6 and urinary IL-10 were 36.3%, 35.8%, 14.8%, 59.4% and 80.5%, respectively. There were significant associations between prevalence of urinary IL-6, but not IL-10, and age, S. haematobium infection and haematuria (p = 0.045, 0.011 and 0.005, respectively) but not sex or ultrasound-detectable pathology. There were significant differences in IL-6 and IL-10 levels between urine specimens stored at –20°C and those stored at 4°C (p<0.001) and, between those stored at 4°C and those stored at 25°C (p<0.001). Urinary IL-6, but not IL-10, was associated with children’s age, S. haematobium infections and haematuria. However, both urinary IL-6 and IL-10 were not associated with urinary tract morbidity. Both IL-6 and IL-10 were sensitive to urine storage temperatures.


Long sentences in all sections including abstract should be split into short and clearer sentences.
Response: Long sentences have shortened. 3.2. Some grammatical and typographic errors were noticed. Response: Where these errors have been noticed, the same have been corrected in the revised version.
3.3. Some incomplete sentences; e.g. last 2 nd line to end in page 18 "pathology in the urinary tracts of the was comparable to", and some other sentences in different sections.
Response: The sentence on page 18 had a typographical omission and has since been corrected. Others, where noticed, have also been corrected. 3.4. Text, particularly results section can be largely shortened.
Response: This has now been extensively done. 3.5. Methods, Text of haematuria: "Briefly, Avoiding touching the ………. side of the testing strip container." Remove all these unnecessary details Response: These have been removed.
3.6. Discussion section should also be rewritten. It should be more focused and concise.
Response: This has now been. The Discussion have been rewritten and focused. It has been made more concise. 3. 7. Avoid over repetition of results in discussion section.
Response: This has been corrected. 4. What is the study design? It is stated in abstract that it was a cross-sectional study but not mentioned in "Study area and study design". This should be stated with more information in methods section. Moreover, it seems to be a cross-sectional controlled study, taking in account the 165 children only, 60 (cases) were infected and 105 were not infected (controls). However, description on how those 165 were selected was not described. What was the rationale of selecting the school? How children were selected at the school? Similarly, the area was described as low prevalence area in the title but this was not mentioned or described in methods.

Response:
The correction has been made in page 5. However, the no controlling was consciously done in the study design but, rather, only comparisons of groups were done. In addition, not all children availed themselves for all the tests. Those who did not avail some specimens or data were not systematically different from the others and therefore it is no appropriate to only consider 165 children. That the area, compared to "Njaanake et al., 2014", was a low endemic area has been mentioned in the methodology.

5.
In general, the manuscript can be entirely rewritten based on 165 children selected for assessing the association between ILs and infection. Describe the main survey first with 425 children then 165 were selected (explain), and focus on the 165 children. Minimize text about demographic factors associated with infection. For example, Discussion, 3 rd paragraph "Boys had higher prevalence …": This paragraph can be shortened or ignored. The core of this study was the 165 children, and the epidemiological results might be incomplete as some important factors were not assessed. When stating "suggest possible gender-based differences in exposure with boys having a higher exposure." This needs other factors to be investigated and confounders to be controlled. Refer to major comments. Response: There were 245 children and not 425 children as stated by the reviewer. Whereas 165 children had their urinary tracts examined for morbidity and their urine specimens assayed for IL-6, 190 children had their urine specimens assayed for IL-10. It therefore follows that restricting the study to 165 children then data from 25 children will be lost. It was not possible for all the children to undergo all the tests as these were not done at one point or in one day. It is noteworthy that the statement that "suggest possible gender-based differences in exposure with boys having a higher exposure" is a suggestion, based on well known facts in schistosomiasis, and not a conclusion. This statement has been removed.

MINOR REVISION (arranged according to text flow)
6. Abstract, 1 st sentence: remove "due to infections with Schistosoma haematobium". Response: This was removed. 7. Abstract, 2 nd sentence: The sentence can be removed. "However, there is no universally recommended tool for assessing the inflammation and resultant morbidity especially in early stages." Response: This was removed.
8. Abstract, methods: split the 1 st sentence into short sentences. Response: The first sentence in the Abstract is already short and shortening it further will make it lose its meaning.
9. Abstract, results: last sentence is unclear; split this long sentence into short sentences.
Response: This was corrected. 10. Abstract, Conclusion: positively! How this was concluded?
Response: This has now been rephrased.
11. Abstract, Conclusion: "prevalence of urinary IL-6 is positively associated with S. haematobium infection and morbidity". Morbidity!. But it was stated that "S. haematobium-related urinary tract morbidity using ultrasonography" & "There was no significant association between prevalence or levels of IL-6 and ultrasound detectable".

Response: This has now been corrected.
Response: This has now been done. 26. "had significantly higher egg counts". Please state the values (mean or medians or geometric mean) for each group. Response: Based on the distribution of eggs, this statement is not very informative and has been removed. 27. "A total of 165 children had their urinary tracts examined for morbidity using ultrasound." How was these 165 children selected? When (before or after urine examination)? How many of them infected? 60 infected and 105 not as shown in table 1; this should also be stated and explained in the text.
Response: This has been corrected. 28. "The intensity of morbidity ranged from none to severe (Fig 1)." Fig. 1 does not show this range of morbidity! Response: This has been corrected.
29. "A significantly higher proportion of boys had ultrasound-detectable morbidity than girls (X2= 18.0760; p<0.001)." This should be revised and rephrased. Higher proportion of girls had light morbidity than males, although the overall proportion of the boys was higher.
Response: This has been corrected.
30. "Significantly higher proportion of children with infection had pathology (X2= 14.3969; p<0.002)" This should be revised and rephrased. Which group had higher proportions at morbidity groups? 29.2% of heavily infected children had moderate morbidity, this was over double the proportion of light infected and over 4 times of not infected, etc.