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. 2023 Apr 5;3(4):e0001726. doi: 10.1371/journal.pgph.0001726

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

Kariuki H Njaanake 1,2,*, Job Omondi 1, Irene Mwangi 2, Walter G Jaoko 1,2, Omu Anzala 1,2
Editor: Julia Robinson3
PMCID: PMC10075406  PMID: 37018184

Abstract

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.

Introduction

Schistosoma spp., the causative agents of human schistosomiasis, infect over 250 million individuals worldwide [1, 2]. Currently, morbidity control through mass praziquantel administration is the main strategy for schistosomiasis control which has been adopted by governments in endemic countries with support from WHO and other stakeholders [3]. This is a long-term resource-intensive strategy and monitoring its performance is imperative. However, most of the currently available tools to monitor schistosomiasis morbidity after treatment with praziquantel have inherent weaknesses including low sensitivity, requirement of high performance freezers and electricity in the field which is a major problem in resource poor endemic areas [4, 5]. Despite this, progress in development of more tools that can circumvent these drawbacks has been slow.

Cytokines in urine are increasingly being appreciated as potential biomarkers of urogenital tract morbidity in other infectious and non-infectious inflammatory conditions [6–11]. Urogenital schistosomiasis is associated with considerable inflammation in the urogenital tract [12]. A study in highly endemic area of coastal Kenya assessed different cytokines and suggested that interleukin (IL)-6 and -10 in urine of children infected with S. haematobium are correlated with morbidityand infection intensities [13]. This implied that these cytokines have the potential of being additional tools to monitor S. haematobium-related urogenital morbidity. It is known that control activities, including mass praziquantel administration, tend to reduce infection prevalence and intensities in endemic areas which in turn may reduce the performance of morbidity monitoring tools [4]. It is therefore imperative to investigate the potential of urinary IL-6 and IL-10 in morbidity monitoring under low infection endemicity setting. In addition, it is important to assess how various factors may influence these cytokines. For example, age and sex have been shown to be important factors that influence urinary cytokines in other inflammatory conditions [14]. Such information is also important with regard to urinary schistosomiasis but is currently not available.

Schistosomiasis is endemic in low resource settings and monitoring the performance of morbidity control involves collection of specimens in areas with no electricity or freezers for storage. This means that specimens such as urine may require storage for some time before being assayed. A good tool therefore ought to circumvent the requirement of electricity and high performance freezers for specimen storage. In the present study, we therefore also sought to assess how urinary IL-6 and IL-10 are associated with age and sex in children infected with S. haematobium in coastal Kenya. In addition, we also assessed the effects of storage of urine specimens at –20°C, 4°C and 25°C for two weeks on urinary IL-6 and IL-10.

Methods

Study area and study design

The present cross-sectional study was carried out in the year 2018 in children attending Duncan Ndegwa Primary School. The school is located in Kwale County of coastal Kenya. The County is known to have a low endemicity endemic for S. haematobium compared to Tana Delta of Kenya [13, 15]. The study involved 245 randomly selected children whose parents had given informed consent. Only children, boys and girls, aged between 5 and 15 years who were permanent residents of the area were included in the study. These were children who had had no treatment with praziquantel in the past three months and had no observable clinical illness. Demographic information, including age and sex was collected from the children during recruitment. A 50 ml urine sample was collected from each child on each of three consecutive days [16]. A part (10 ml) of day 1 urine sample from each child was examined for S. haematobium eggs. Another part of the day 1 urine sample from each child was tested for haematuria. A third part was divided into three aliquots which were stored at –20°C, 4°C and 25°C, respectively, for 14 days before being assayed for IL-6 (n = 165) and IL-10 (n = 190). On each of days II and III, a 10 ml urine sample from each child was examined for S. haematobium eggs. The urinary tracts of 165 children were also examined for morbidity. children, boys and girls, aged between 5 and 15 years who were permanent residents of the area. Only children who had had no treatment with praziquantel in the past three months and had no observable clinical illness were included in the study.

Sample size calculation

The minimum sample size for the study was estimated using statistical software (Stata Version 12) as an estimation for a two-sample means comparison using t-test assuming equal standard deviations. The intention was to detect an absolute difference in mean cytokine levels (IL-6 or IL-10) of 0.5 pg/ ml between children with urinary tract morbidity and those without where p-value = 0.05; power = 80% and; standard deviation = 1. This gave a minimum sample size of 168 children. However, since absenteeism in the schools was deemed to be high it was assumed that about 45% of the children may not provide all the specimens required. The minimum sample size was therefore increased by 45% to yield 243.6 children. This was rounded off to 245 children.

Test for haematuria

Immediately after collection, day-1 urine samples were examined visually for macrohaematuria and for microhaematuria using URiSCAN dipsticks (YD Diagnostics, Korea) according to the manufacturer’s instructions. Briefly, the dipstick was immersed into the urine sample until all reagent squares were fully immersed. It was then immediately removed. The colour change on the reagent square due to oxidation, catalyzed by hemoglobin in urine, was interpreted qualitatively as haematuria or no haematuria using the urinalysis guide provided by manufacturer.

Urine examination for S. haematobium eggs

On each of three days, a 10 ml urine sample from each of child was filtered through a 12μm pore polycarbonate filter (Sterlitech) using a 13 mm filtration chamber. The filter was examined under a microscope for S. haematobium eggs [17]. A mean egg count was calculated from the three days’ readings and used to classify the infections as negative if no S. haematobium eggs were found, light if 1–49 eggs were found or heavy if ≧50 eggs were found [4].

Urine analysis for IL-6 and IL-10

Day I urine samples from 165 randomly selected children were assayed for IL-6 using Invitrogen Human IL-6 ELISA kit (analytical sensitivity = <0.05 pg/mL; Assay range = 0.064–10000 pg/mL) with 96-well plates pre-coated with human IL-6 antibodies according to manufacturer’s instructions. The assay for IL-10 was done 190 randomly selected samples using DIAsource Human IL-10 96-well plate ELISA kits (Analytical sensitivity = <0.05 pg/mL; Assay range = 0.064–5,000 pg/mL) according to the manufacturer’s instructions. The plates were read at an absorbance of 450 nm using Infinite 200PRO reader.

Urinary tract ultrasound examination

A random sample of 165 children was selected prior to urine examination for S. haematobium eggs and their urinary tracts examined for ultrasound-detectable morbidity by an experienced ultrasonographer using a portable convex sector scanner (SSD-500; Aloka, Tokyo, Japan). Total morbidity was scored according to the Niamey protocol [18]. If any kidney and/ or ureter dilatation was observed the child was asked to empty the bladder and come back for re-examination. Morbidity was based on changes in urinary bladder (shape, wall lesions, wall thickening, masses and pseudopolyps), ureter dilatation and renal pelvis dilatation. Total morbidity scores were arbitrarily graded as 0 = no morbidity, 1–5 = light morbidity, 6–10 = moderate morbidity and ≥ 11 = severe morbidity.

Statistical analysis

Statistical analyses of the data were performed using STATA (Version 12) software. Median ages between boys and girls or between groups were compared using MannWhitney U tests or Kruskal-Wallis test, as appropriate. Medians of S. haematobium eggs, IL6 and IL-10 were compared between groups using Mann-Whitney U test or Kruskal-Wallis test. Prevalences were compared using X2-test or logistic regression. The correlation between cytokine levels and S. haematobium egg counts or ultrasound-detectable pathology levels was analysed using Kendall Tau. Diagnostic performance of haematuria, IL-6 and IL-10 was assessed using Kappa statics. In all tests, p-values less than 0.05 were regarded as statistically significant.

Ethics approval and consent to participate

The study protocol was reviewed by and received approval from the University of Nairobi-Kenyatta National Hospital Ethics and Research Committee (Approval No. P110/03/2017).

Results

Study population

A total of 245 children, 106 (43.3%) boys and 139 (56.7%) were girls, were recruited in the present study. The overall median age of the children was 10 years. The boys were significantly older than girls with median ages 11 and 10 years, respectively (p = 0.003). The children were grouped into 3 age groups; 5–9-year-olds, 10–11-year-olds and 12–15-year olds with 85 (34.7%), 121 (49.4%) and 39 (15.9%) children, respectively.

S. haematobium prevalence in relation to age, sex and ultrasound-detectable urinary tract morbidity

The overall prevalence of S. haematobium infection among the children was 36.3% (89/245). The prevalence of the infection was significantly higher in boys 50.9% (54/106) than in girls 25.2% (35/139) (p<0.001). The infection prevalences were 22.4% (19/85), 42.2% (51/121) and 48.7% (19/39) for the age groups 5–9 years, 10–11 years and 12–15 years, respectively, with a significant difference in prevalences between the groups (X2 = 11.538; p = 0.003).

The overall prevalence of detectable pathology was 35.8% (59/165). The intensity of morbidity ranged from none to severe. Fig 1 shows photographs of urinary bladders of two boys with diffuse bladder wall thickening and bladder masses. The intensity of morbidity in relation to sex, age and S. haematobium infection intensity is shown in Table 1. Overall, a significantly higher proportion of boys had ultrasound-detectable morbidity than girls (X2 = 18.0760; p<0.001). Similarly, a significantly higher proportion of children with infection had pathology (X2 = 14.3969; p<0.002). However, there was no significant difference in proportions of children with pathology in relation to age overall (X2 = 10.2826; p = 0.113).

Fig 1.

Fig 1

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Urinary bladder pathology among the children; a) 10-year-old boy with diffuse thickening of the bladder wall (red arrows); b) 11-year-old boy with diffuse thickening of bladder wall (red arrows) and bladder masses (yellow arrows).

Table 1. Urinary tract morbidity in relation to sex, age and S. haematobium infection intensity.

Group n No morbidity Light morbidity Moderate morbidity Severe morbidity p-value
Boys 72 47.2% 25.0% 18.1% 9.7%
Girls 93 77.4% 15.1% 5.4% 2.2% <0.001
5–9 years old 40 77.5% 15.0% 7.5% 0.0%
10–11 years old 89 62.9% 16.9% 11.2% 9.0%
12–15 years old 36 52.8% 30.5% 13.9% 2.8% 0.114
Not infected 105 74.2% 16.2% 6.7% 2.9%
Light Infection 36 66.7% 22.2% 11.1% 0.0%
Heavy infection 24 16.6% 29.2% 29.2% 0.0% <0.001
Overall 165 64.2% 19.4% 10.9% 5.5%
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Haematuria

Overall, the prevalence of haematuria among the children was 14.8% (n = 245). Among the boys, 23 (21.9%) had haematuria whereas 13 (9.4%) of the girls had haematuria. Girls had lower odds of having haematuria than boys (OR = 0.37, 95% CI: 0.18–0.77). This was statistically significant (p<0.008). Age was significantly associated with prevalence of haematuria among the children where 6 (7.1%), 18 (15.0%) and 12 (30.1%) of those in age groups 5–9 years, 10–11 years and 12–15 years, respectively, had haematuria (X2 = 11.9610; p = 0.003). In relation to infection intensity, 21 (60.0%) of children with heavy S. haematobium infection had haematuria whereas 7 (13.0%) of those with light infection and 8 (5.2%) of those with no infection had haematuria. This was statistically significant (X2 = 68.4476; p<0.001). The sensitivity and specificity of IL-6 and IL-10 ELISA and haematuria using urine microscopy as the reference are shown in Table 2. The performances of IL-6, IL-10 and haematuria were also plotted against that of urine microscopy. The area under the curve was largest for haematuria followed by IL-6 and IL-10 had the smallest area as shown in Fig 2.

Table 2. Sensitivity and specificity of urinary IL-6, IL-10 and haematuria using microscopy as a reference.

Sensitivity Specificity PPV NPV EA Kappa
IL-6 72.1% 48.1% 57.0% 37.0% 47.6% 0.1796
IL-10 86.1% 22.9% 46.8% 37.9% 42.6% 0.0738
Haematuria 31.5% 94.8% 71.7% 36.5% 59.5% 0.3012
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PPV = Positive Predictive Value

NPV = Negative Predictive Value

EA = Expected agreement.

Fig 2. ROC curve showing the areas under the curve for IL-6, IL-10 and haematuria using urine microscopy as the reference for the diagnosis of infection with S. haematobium.

Fig 2

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il6 = IL-6, il10 = IL-10.

IL-6 in relation to sex and age

Urine samples from randomly selected 165 children were assayed for IL-6 levels. The prevalence of detectable levels of IL-6 in urine in relation to sex and age is shown in Table 3. Of the children tested, 98 (59.4%) had detectable levels of IL-6 in urine. There was no significant difference in the proportion of children with detectable levels of IL-6 in urine between boys and girls (n = 93) (p = 0.301, OR = 0.72, 95%CI: 0.38–1.35).

Table 3. Association between IL-6 and gender, age, S. haematobium infection status, and haematuria.

Factor Category No. of children (n) % with detectable IL-6 levels in urine X2 p-value Median (pg/ml) p-value
Gender Females 72 55.9% 0.301 112.7
Males 93 63.9% 1.0702 129.0 0.550
Age (years) 5–9 54 72.2% 0.045 242.9
10–11 88 51.1% 101.0
12–15 23 60.9% 6.1935 108.7 0.019
S. haematobium infection status Positive 60 72.1% 0.011 157.2
Negative 105 51.9% 6.5102 107.2 0.086
Haematuria Present 29 54.4% 0.005 106.7
Absent 136 82.8% 7.9640 108.9 0.021
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There was no statistically significant difference in the levels of IL-6 in urine between boys and girls (p = 0.535). The comparisons of urinary IL-6 levels between boys and girls, children with and without infection, children with and without haematuria and, children with and without urinary bladder are shown in Fig 3. In relation to age, there was a borderline statistical difference in proportions with detectable IL-6 among the age groups and a statistically significant difference in the levels of IL-6 in urine among the age groups (p = 0.015).

Fig 3.

Fig 3

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Box plot showing urinary IL-6 levels in relation to (a) sex, (b) S. haematobium infection, (c) haematuria and, (d) urinary bladder pathology. Solid line represents the median; X represents the mean and error bars represent the ranges.

IL-10 in relation to sex and age

Urine samples from 190 children, of whom 83 were boys and 107 were girls, were assayed for IL-10 levels (Table 4). Of these children, 153 (80.5%) had detectable levels of IL10 in urine. There was no significant difference in the proportion of children with detectable levels of IL-10 in urine between boys (n = 83) and girls (n = 107) (p = 0.243, OR = 0.64, 95%CI: 0.30–1.35).

Table 4. Association between the prevalence of IL-10 and gender, age, S. haematobium infection status, and haematuria.

Factor Category No. of children (n) % with detectable IL-10 in urine X2 p-value Median (pg/ml) p-value
Sex Females 107 77.3% 59.6
Males 83 84.3% 1.3650 0.243 59.6 0.995
Age (years) 5–9 66 75.8% 80.0
10–11 94 80.9% 59.6
12–15 30 90.0% 2.6805 0.262 59.6 <0.001
S. haematobium infection status Positive 72 86.1% 69.2
Negative 118 72.1% 2.3059 0.129 59.6 0.301
Haematuria Present 33 87.9% 59.6
Absent 157 79.0% 1.3767 0.241 59.6 0.216
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There was no statistically significant difference in the levels of IL-10 in urine between boys and girls (p = 0.996). The comparisons of urinary IL-10 levels between boys and girls, children with and without S. haematobium infection, children with and without haematuria and, children with and without urinary balder pathology are shown in Fig 4. In relation to age, there was no statistical difference among the age groups but there was a statistically significant difference in the levels of IL-10 in urine among the age groups (p<0.001). There was no significant correlation between IL-10 and S. haematobium eggs (Tau = 0.047; p = 0.266).

Fig 4.

Fig 4

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Box plot showing urinary IL-10 levels in relation to (a) sex, (b) S. haematobium infection, (c) haematuria and, (d) urinary balder pathology. Solid lines represent the median; X represents the mean and error bars represent the ranges.

IL-6 and IL-10 in relation to morbidity

Of the 165 children examined for ultrasound-detectable urinary tract pathology, 112 also had their urine samples analysed for IL-6. Of these children, 37 (33.0%) had ultrasound-detectable pathology and 60 (53.6%) had detectable levels of IL-6 in urine. IL-6 levels were detected in urine samples from 39 (52.0%) of children without detectable pathology. The relationship between IL-6 levels and gender, age, infection status, haematuria and morbidity are shown in Fig 3. There was no significant correlation between IL-6 levels and ultrasound detectable pathology (Tau = 0.0794, p = 0.322). There was no significant difference between those with and those without pathology (X2 = 1.8916; p = 0.635). Similarly, there was no significant statistical difference in IL-6 levels between children with and without ultrasound detectable pathology (p = 0.384).

A random sample of 127 had both their urinary tracts examined for pathology and their urine samples analysed for IL10. Of these children, 43 (33.9%) had ultrasound-detectable pathology and 101 (79.5%) had detectable levels of IL-10 in urine. The relationship between IL-6 levels and gender, age, infection status, haematuria and morbidity are shown in Fig 4. Of those without detectable pathology 68 (81.0%) had detectable IL-10 levels in their urine whereas of those with detectable pathology 33 (76.7%) had detectable levels of IL-10. There was a negative but not significant correlation between IL-10 and ultrasound-detectable pathology levels (Tau = -0.0211; p = 0.768). There was no significant difference in proportion between those with and those without pathology (X2 = 1.3806; p = 0.701). Similarly, there was no significant statistical difference in IL-10 levels between children with and without ultrasound detectable pathology (p = 0.333).

Effects of temperature on IL-6

The proportions of urine specimens with detectable levels of IL-6 after a 14-day storage at different temperatures were compared (Fig 5). A total 97 (59.4%) urine specimens had detectable levels of IL-6 after 14 days of storage at –20°C. Of these, 61 (62.9%) also had detectable levels of IL-6 after storage at 4°C for 14 days. Only 31 (31.6%) of urine specimens with detectable levels of IL-6 after storage at –20°C also had detectable levels of IL-6 after storage at 25°C. A total of 36.4% of urine specimens with detectable levels of IL-6 after storage at 4°C also had detectable levels of IL-6 after storage at 25°C. There were significant differences in IL-6 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).

Fig 5. Proportions of specimens with detectable levels of IL-6 or IL-10 after a 14-day storage at –20°C, 4°C or 25°C.

Fig 5

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Effects of temperature on IL- 10

The proportions of urine specimens with detectable IL-10 after a 14-day storage at different temperatures were compared. A total of 38 (27.9%) of urine specimens with detectable levels of IL-10 after storage at –20°C also had detectable levels of IL-10 after storage at 4°C for 14 days. A total of 41 (31.5%) of urine specimens with detectable levels of IL-10 after storage at -20°C also had detectable levels of IL-10 after storage at 25°C. A total of 17 (39.5%) of urine specimens with detectable levels of IL-10 after storage at 4°C also had detectable levels of IL-10 after storage at 25°C. There were significant differences in IL-10 levels between urine specimens stored at –20°C and those stored at 4°C (p<0.001) but not between those stored at 4°C and those stored at 25°C (p = 0.871).

Discussion

Urinary tract inflammation is associated with cytokines in urine which are increasingly being appreciated as biomarkers of urogenital pathology [6, 8–10, 19, 20]. A previous study in an area highly endemic for S. haematobium suggested that urinary IL-6 and IL-10 have this potential but not much is known in relation to urinary cytokines and the influential factors in schistosomiasis [13]. The present study therefore assessed how urinary IL-6 and IL-10 are associated with age, sex and urinary tract pathology in children from an area with low S. haematobium prevalence.

The overall prevalence of S. haematobium infections (36.3%) was low compared to what was reported elsewhere in a similar study [13]. Boys had higher prevalence of infections and parasite egg counts than girls. However, both IL-6 and IL-10 in urine were not associated with sex of the children. No immediate explanation was available for this as it is expected that the sex with higher infections would have higher pro-inflammatory cytokines such as IL-6 and lower anti-inflammatory cytokines such as IL-10. It is however probable both boys and girls had the inflammation threshold to cause secretion of detectable cytokines in urine despite differences in shedding of parasite eggs in urine.

Urinary tract pathology was significantly higher in boys than in girls which corroborates the findings of a study in Angola [21]. However, in line with the association between sex and the cytokines, there were no significant differences in IL-6 and IL-10 between children with pathology and those without. Urinary cytokines are most likely secreted by activated immune cells in the urinary tract whereas ultrasound usually detects old pathology in which there could be minimal cellular activation and thus little cytokine secretion [22]. This could, at least partially, explain the lack of correlation between ultrasound-detectable pathology and urinary cytokines but requires further investigations. In addition, depending on the condition, cytokines such as IL-10 could have been produced by other non-inflammatory cells so that a huge proportion of the children had detectable levels regardless of infection status [23].

The prevalence and intensity of infections and increased with age. The prevalence of ultrasound detectable pathology also increased with age, although this was not significant, which corroborates the findings of a study in Sudan [24]. An increase in prevalence and levels of urinary IL-6 with age was also observed among the children. Taken together with the association between infection status and IL-6 levels, these suggest that inflammation processes continue with persistent infections as older children may have been exposed to infections for longer [25]. Eventually, immunomodulation kicks in and resulting in less pathology [26]. This could be the reason why children in the 10–11 years age group had higher morbidity and cytokine levels than those below or above them.

Our findings corroborate the findings of other studies that haematuria is a useful marker of S. haematobium infections in children [27–29]. Using urine microscopy as the reference, all three had poor agreement but kappa statistics showed that haematuria had higher agreement followed by IL-6 ELISA whereas IL-10 ELISA had the lowest. Low sensitivity of haematuria in low infections intensities have been reported elsewhere [28, 30]. Low infection prevalence and intensities could also be the reason for low sensitivity of urinary IL-6 and IL-10 but further studies should shed more light on this. In general these findings suggest that, similar to other urogenital schistosomiasis morbidity markers, the sensitivity of urinary cytokines may be reduced in areas with low infection prevalence and intensities [4].

Urine contains proteolytic enzymes that may denature proteins [31]. Urine storage temperatures, may therefore influence proteolytic degradation of urinary cytokines thus affecting their levels [32, 33]. There was observable reduction of proportion of specimens with detectable levels of IL-6 and IL-10 following a 14-day storage at 4°C and 25°C although above 4°C there was less reduction for IL-10. These findings show that urine samples for cytokine assays should be frozen immediately after collection [34].

The present study only assayed a one-day urine specimen for cytokines which could be a potential limitation. Whereas previous studies have shown that schistosome egg shedding by infected individuals vary from day to day, no studies have been carried out on daily variation of urinary cytokine levels in relation to S. haematobium infections. Another limitation in the study is that after collection and exposure of the urine specimens to different temperatures, the specimens were stored at –80°C for more than 8 weeks. During this period, due to the low temperatures, some of the vials broke or the lids came off and therefore such samples were not included in cytokine assays as there could have been possible contamination. However, this was deemed to be random and could not introduce any systemic bias in the results.

Conclusions

The findings of the present study suggest that, unlike urinary IL-10, urinary IL-6 is associated with age, S. haematobium infection intensity and haematuria in children from low endemicity areas. However, it is not associated with ultrasound-detectable urinary tract morbidity. The results also suggest that both urinary IL-6 and IL-10 are significantly sensitive to storage temperatures above –20°C. These findings call for further detailed studies to evaluate the potential of urinary IL-6 as S. haematobium-related morbidity marker.

Supporting information

S1 Database

(XLSX)

Click here for additional data file. (37.6KB, xlsx)

Acknowledgments

We are grateful to the children from Duncan Ndegwa Primary School who agreed to participate in the study, and to the parents and teachers for allowing us to carry out the study in the school. Special thanks are extended to Charles Ng’ang’a and Jackson Muinde (Msambweni sub-District Hospital) for their unconditional support and participation during field sample collection. We are greatly indebted to Farah Bashir (KAVI-ICR) for his technical support and material contribution to this work. This study was funded by the European and Developing Countries Clinical Trials Partnership (EDCTP) for which we are greatly indebted.

Data Availability

The data set analyzed for this manuscript has been provided as supporting information.

Funding Statement

This work was solely funded by European and Developing Countries Clinical Trials Partnership (EDCTP) grant number 100440 UCE TMA 2015 CDF - 995 (http://www.edctp.org/projects-2/edctp2-projects/career-development-fellowships/) as post-doctoral support for KHN. No other author received funding from any award, commercial salary, or other funding source. The funder played no role in Study design, Data collection and analysis, Decision to publish, Preparation of the manuscript.

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PLOS Glob Public Health. doi: 10.1371/journal.pgph.0001726.r001

Decision Letter 0

Raquel Muñiz-Salazar

28 Mar 2022

PGPH-D-22-00101

Urinary Interleukins (IL)-6 and IL-10 in Schoolchildren from an Area with low Prevalence of Urogenital Schistosomiasis in coastal Kenya

PLOS Global Public Health

Dear Dr. Njaanake,

Thank you for submitting your manuscript to PLOS Global Public Health. After careful consideration, we feel that it has merit but does not fully meet PLOS Global Public Health’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 12 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at globalpubhealth@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgph/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Raquel Muñiz-Salazar, Ph.D.

Academic Editor

PLOS Global Public Health

Journal Requirements:

1. We do not publish any copyright or trademark symbols that usually accompany proprietary names, eg (R), (C), or TM  (e.g. next to drug or reagent names). Therefore please remove all instances of trademark/copyright symbols throughout the text, including (R) on pages 6-7.

2. Your manuscript is missing the following sections: Introduction. Please ensure these are present, and in the correct order, and that any references to subheadings in your main text are correct. An outline of the required sections can be consulted in our submission guidelines here:

https://journals.plos.org/globalpublichealth/s/submission-guidelines#loc-parts-of-a-submission

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This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons by return email and your exemption request will be escalated to the editor for approval. Your exemption request will be handled independently and will not hold up the peer review process, but will need to be resolved should your manuscript be accepted for publication. One of the Editorial team will then be in touch if there are any issues.

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ii). State the initials, alongside each funding source, of each author to receive each grant.

iii). State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

iv). If any authors received a salary from any of your funders, please state which authors and which funders.

Additional Editor Comments (if provided):

Your manuscript has now been reviewed by experts in the field.

One of the significant issues is reinforcing the discussion and rewriting the conclusion.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Aim of the study: The author designed the study to assay for levels of urinary cytokine Il-6 and Il-10 to be used used as diagnostic biomarker for urogenital schistosomiasis.

The title is not concise and clear.

The draft is composed of long sentences.

The references used in the study to justify the use of levels of cytokines and chemokine is far fetched since bacterial colonization in the urogenital organs and resultant pathology is different from parasitic infections. In S. hematobium infection, the cytokines released are as result of adult worms migrating into the venules surrounding the organs of the pelvis. Where they lay eggs and subsequently penetrate the vessel wall, move towards the lumen of the bladder. Some of the eggs are sequestered in the pelvic organs like the urinary bladder, lower ureters, vagina, prostate glands, and seminal vessicles where they cause chronic inflammation. Therefore, the assay of IL-6 and IL-10 is an understatement. In this parasitic phenomenon we should assay for a panel of urinary cytokines and chemokines to weed out the most prominent ones.

The author mixed up issues by further going looking at the effect of storage temperature on cytokine titers. This should have been an optimization protocol in the lab.

The author did not show any cytokine graphs or images of ultrasound detected pathologies.

Reviewer #2: Kariuki H Njaanake et.al. investigated the urine parameters and assays for Urogenital Schistosomiasis in schoolchildren in Kenya. They investigate the association of cytokines/protein ( IL6, IL10, ECP) with the Schistosoma haematobium egg detection from urine. Also, the clinical association analysis was also investigated to show the correlation of IL-6 and morbidity levels. Overall, the quality of the study is sound and the writing is structurally prepared. To improve the quality of the work, these questions and suggestions might help.

Major comments

-Title

” Urinary Interleukins (IL)-6 and IL-10 in schoolchildren from an area

with Low Prevalence of Urogenital Schistosomiasis in coastal Kenya”.

Why the low prevalence region in coastal Kenya is the appropriate site of study?, please clarify or add this clarified info somewhere in the paper.

-Abstract

“However, it is highly sensitive to urine storage temperatures”, this sentence interrupts the flow of reading.

Please exclude this, or add the result of temperature and IL-6 in the result section.

Method

- What is the inclusion /exclusion criteria of the participants?

- Authors analyzed the effect of temperature on IL-6in urine. How about the 2 consecutive urine samples were analyzed and interpreted. This could provide more insight into the IL-6 variation.

- How is the level of confidence to assess the level of hematuria from Urisxan Dipsticks. Is this the limitation?

- How the author confirms the result of S. haematobium eggs microscopic detection from the urine samples. Is this the limitation?

- -Why not all samples were tested for ECP (n=164), IL-6 (n=165) and IL-10 (n=190).

- Redundant info of ethical approval.

Result

Figure 1, how many times the author investigate the temperature effect to IL-6/IL10. I should be the error bar and statistical analysis.

Discussion

-Why aged group 10-11 has higher severe morbidity

-please add the rationale to focus only IL-10 and IL-6, why not other cytokines.

-Why specificity of ECP, IL6 IL10 and haematuria are so low.

-The part of cytokines IL6 IL10 seems not relevant to the main objective. Two options 1. Keep it, but need to add some info to link this into the main text, this can make the study more beneficial to the readers 2. Exclude to make the study concise and focus.

- Conclusion can be rewritten. Some info should be in the discussion rather than the conclusion.

Minor comment

-Title of table 2 should be revised, it is not only the sensitivity.

Reviewer #3: The following are the correction:

1. The title is not appropriate with the objectiveof the study. The study has shown the relationship with the IL-6 and IL-10 with the detection of S. haematobium egg. In that circumstances, the study will be design as diagnostic validity of IL-6 ad IL-10. Please explain.

2. The methodology section of abstract is missing with the study design, study place, study period. It should be mentioned in this section.

3. The introdction is nicely written. but the rationale of the study is missing.

4. The methodology section is nicely written. The details of the ELISA procedure can be avoided. No need to elaborate the procedure. Please mention "Th ELISA was performed accoridng to the manufacturer's instruction."

5. The results section should be written according to the objective of the study. The relevant table should be mentioned.

6. Rewrite the conclusion related with the findings of the study.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Do you want your identity to be public for this peer review? If you choose “no”, your identity will remain anonymous but your review may still be made public.

For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Nyamongo Onkoba, PhD

Reviewer #2: No

Reviewer #3: Yes: Dr. Md. Abdullah Yusuf

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLOS Glob Public Health. doi: 10.1371/journal.pgph.0001726.r003

Decision Letter 1

Raquel Muñiz-Salazar

14 Sep 2022

PGPH-D-22-00101R1

Urinary Interleukins (IL)-6 and IL-10 in Schoolchildren from an Area with low Prevalence of Schistosoma haematobium infections in coastal Kenya

PLOS Global Public Health

Dear Dr. Njaanake,

Thank you for submitting your manuscript to PLOS Global Public Health. After careful consideration, we feel that it has merit but does not fully meet PLOS Global Public Health’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 14 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at globalpubhealth@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgph/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Raquel Muñiz-Salazar, Ph.D.

Academic Editor

PLOS Global Public Health

Journal Requirements:

Additional Editor Comments (if provided):

The authors have improved the manuscript after addressing all reviewers’ comments. However, the manuscript still has significant drawbacks in terms of quality of writing and data and results from the presentation.

Major and minor comments are provided below. They should be addressed before this manuscript can be accepted for publication.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #4: (No Response)

Reviewer #5: All comments have been addressed

Reviewer #6: (No Response)

**********

2. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #4: No

Reviewer #5: Yes

Reviewer #6: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #4: I was not one of the initial reviewers for this so will have different comments than those that were previously addressed.

The stated objective of the paper was to evaluate the relationship of IL-6 and IL-10 to pathology associated with urinary schistosomiasis. While there was an association of IL-6 with hematuria, neither cytokine showed any association with ultrasound-detectable pathology.

--on page 12 and 13, the authors mention that boys have a higher prevalence of infection and hematuria, respectively, yet the OR given was < 1.

--the quality of the figures is poor, especially figures 1 and 4. This may be an issue with the submission system but as seen in the reviewed material, they are not suitable for publication.

--the figure legends are inadequate. It should be possible to understand what is being shown and the significant findings without referring to the text. In figures 2 and 3, what does the bar represent? what does the "x" represent? what do the error bars represent? Do any of the panels represent statistical differences?

--figure 1 should include a panel from someone with no pathology to better show the contrast.

--figure 4 would be better as the overall concentration of IL-6 and IL-10 at the different time points although this experiment is somewhat unnecessary and it is well expected that storage at the wrong temperature would result in loss. It is true that transporting samples from the field cannot guarantee -20 during transport but any location that has an ELISA reader can reasonably be expected to have a freezer. At most, the experiment on getting the urines to a more appropriate temperature should be ~24 hours rather than 2 weeks.

--what are the normal ranges of IL-6 and IL-10 and what is considered abnormal? The authors seemed to have used "detectable" cytokine as the cutoff for tables 2, 3, and 4. This is not informative without knowing the limit of detection of the assays.

--references should be numbered in the text and bibliography

Reviewer #5: see attached

Reviewer #6: This manuscript investigated the association between urinary cytokine levels (IL6 & IL10) with the prevalence, intensity and morbidity of S. haematobium infection among schoolchildren in Kenya.

I've been given this manuscript as a revised (R1) version and I noticed that the manuscript has been improved after addressing reviewers’ comments given during previous reviewing stage. However, the manuscript still has major drawbacks in quality of writing and data & results presentation. Major and minor comments are provided below. They should be addressed before this manuscript can be acceptable for publication.

Note: A list of other major and minor comments is provided in the attached file.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Do you want your identity to be public for this peer review? If you choose “no”, your identity will remain anonymous but your review may still be made public.

For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #4: No

Reviewer #5: No

Reviewer #6: Yes: Hesham M. Al-Mekhlafi

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLOS Glob Public Health. doi: 10.1371/journal.pgph.0001726.r005

Decision Letter 2

Julia Robinson

27 Feb 2023

Urinary Interleukins (IL)-6 and IL-10 in Schoolchildren from an Area with low Prevalence of Schistosoma haematobium infections in coastal Kenya

PGPH-D-22-00101R2

Dear Dr. Njaanake,

We are pleased to inform you that your manuscript 'Urinary Interleukins (IL)-6 and IL-10 in Schoolchildren from an Area with low Prevalence of Schistosoma haematobium infections in coastal Kenya' has been provisionally accepted for publication in PLOS Global Public Health.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they'll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact globalpubhealth@plos.org.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Global Public Health.

Best regards,

Julia Robinson

Executive Editor, PLOS Global Public Health

***********************************************************

Please review the final comments from the two reviewers.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #7: All comments have been addressed

Reviewer #8: All comments have been addressed

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2. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #7: Yes

Reviewer #8: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #7: Yes

Reviewer #8: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

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Reviewer #7: Yes

Reviewer #8: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #7: Yes

Reviewer #8: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #7: This is an interesting article covering an important area of clinical symptoms / morbidity related to urogenital schistosomiasis. As national programs make progress in reducing prevalence and intensity of infection through chemotherapeutic control (and other measures) in line with the WHO’s 2030 roadmap, increasingly focus will turn to control and elimination of related morbidity.

An early stage test for schistosomiasis-related morbidity would be especially useful.

I found this article to be well-written, clearly laid out and methodologically sound. I was invited to review at the second revision stage and I note from earlier versions that the authors have made substantial changes to the manuscript. I only have some minor questions and assuming those are answered am happy to recommend it for publication.

Comments

• This is a low prevalence area as the authors state. Is it low infection at baseline (endemic equilibrium) or following treatment? Would this have an impact on the study?

• Would it be useful to also test in adults? Why was it restricted to children?

• Why is there a drop off in sample size from 245 to 165 children for ultrasound, and then to 112 for IL6

• The authors use gender and sex interchangeably. I’d suggest keeping consistent to sex.

Reviewer #8: The statememnt on page 5: The study was carried out in 2018 at Duncan Ndegwa primary school in Kwale County.

Page 6: The word is oxidation?

Throughout the discription in the manuscript it is rather appropriate to replace sex with gender.

Thank you for addressing all the comments as raised in the previous comments by the reviewers.

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Reviewer #7: Yes: Michael French

Reviewer #8: Yes: Prof. Takafira Mduluza

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Database

    (XLSX)

    Click here for additional data file. (37.6KB, xlsx)
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    Submitted filename: Response to Reviewers.pdf

    Click here for additional data file. (143.5KB, pdf)
    Attachment

    Submitted filename: Rebuttal Letter II.pdf

    Click here for additional data file. (272.1KB, pdf)

    Data Availability Statement

    The data set analyzed for this manuscript has been provided as supporting information.

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