Abstract(resumo):

Background: Fecal calprotectin levels can be used to monitor inflammation and vary with age; those of children under 4 years of age should be carefully interpreted. This study aimed to analyze factors contributing to variations in fecal calprotectin levels of apparently healthy public and private schoolchildren aged 4–6 years. Methods: This study was conducted in Rio de Janeiro from 2019 to 2022. Fecal samples were obtained for parasitological examination and fecal calprotectin measurement. School category (public and private from regions with different socioeconomic levels), sex, and weight were compared. The mean and standard deviation for fecal calprotectin levels were determined using the Mann-Whitney U test. Results: A total of 155 fecal samples were evaluated. The mean calprotectin levels in public and private schoolchildren were 57.1 ?g/g and 18.0 ?g/g, respectively (p < 0.03). The mean calprotectin levels were 57.5 ?g/g and 12.4 ?g/g in female public and private schoolchildren, respectively (p < 0.04). Conclusion: Fecal calprotectin levels were significantly different between children attending public and private schools potentially due to varying degrees of intestinal inflammation.

Keywords(palavra-chave):

children, calprotectin, feces, intestinal absorption, inflammatory bowel diseases, gastrointestinal disease.

Content(conteúdo):

Introduction
Calprotectin, a protein found in cell nuclei and cytoplasm across various tissues, functions as an intracellular Ca2+ receptor.1 During inflammatory processes, calprotectin is secreted during cell rupture or by activated monocytes and neutrophils. Elevated calprotectin levels are typically associated with acute inflammation, and these levels can be quantified in either serum or feces.2 Fecal calprotectin (FC) is a useful biomarker for monitoring inflammatory bowel disease and has further been proposed as a biomarker for environmental enteric dysfunction (EED).3 Several studies have previously sought to define the optimal values of FC levels in pediatric populations, recognizing that cut-off values may vary by age group.4,5
Environmental factors related to hygiene conditions and socioeconomic status may be associated with increased FC levels in children. Small intestinal bacterial overgrowth (SIBO) and EED establish a link between environmental influences and a degree of intestinal inflammation, rendering FC a prospective marker for research endeavors in developing countries6 and in malnourished children.7 FC levels can be influenced by various factors and conditions, making it challenging to interpret the results and establish a single cut-off value for different pediatric age groups.
As this is an original study, it aimed to analyze potential causes of variations in FC levels among apparently healthy children attending public and private schools in the municipality of Rio de Janeiro, Brazil. Furthermore, we aimed to elucidate the potential impact of environmental factors on FC levels and their relevance as markers in the pediatric population.

Methods
This observational, cross-sectional, and analytical study was conducted among children in public and private schools in Rio de Janeiro, Brazil, from 2019 to 2022, as part of a larger study that includes other analyses. The neighborhoods of the city were stratified and grouped into five categories according to the municipal Human Development Index (HDI). Neighborhoods were randomly selected from each group, followed by the random selection of public and private schools located in those neighborhoods. Children between the ages of 4 and 6 years were randomly selected for inclusion in the study. Patients with chronic illness, acute diarrhea, abdominal pain, a history of any type of bleeding, or non-steroidal anti-inflammatory drug use within the last 30 days were excluded, as were children with any respiratory illness prior to fecal collection.
After obtaining informed consent, the parents of the children responded to a questionnaire, either in person or remotely, providing information regarding the child’s sex, age, weight, school type (public or private), and neighborhood of residence. Fecal samples were collected from the participants' homes and delivered to the principal researcher, ensuring refrigeration for a maximum of 1 day. Completion of the questionnaire did not precede feces collection by more than 1 day. Upon receipt of the fecal samples, a portion was allocated for parasitological examination, while the remainder was frozen at -20°C. The Central Laboratory of the Instituto de Puericultura e Pediatria Martagão Gesteira (IPPMG) at the Universidade Federal do Rio de Janeiro conducted parasitological examinations of fecal samples using optical microscopy and performed FC measurements.
The Z-score for weight (Zw) was used for nutritional assessment based on guidelines of the Brazilian Ministry of Health and recommendations of the World Health Organization (WHO), with the following cut-off points: Zw < -3, very low weight for age; -3 < Zw < -2, low weight for age; -2 < Zw < +2, adequate weight for age; and Zw > +2, high weight for age.8
The quantitative measurement of FC was performed using the in vitro chemiluminescence immunoassay method with the LIAISON® Calprotectin kit manufactured by DiaSorin S.p.A. (Saluggia, Italy). FC levels were expressed in micrograms per gram (?g/g) of feces, and the assay had a quantitative detection range of 5–800 ?g/g. All stages of the analysis were conducted in accordance with the manufacturer’s instructions.
This study was approved by the Research Ethics Committee of the IPPMG (reference number: CAAE 05132818.4.00005264) in 2019. Written informed consent was obtained from the legal guardians of the children participating in the research. The study was performed in accordance with the Declaration of Helsinki.
Frequency distributions were determined for the following variables: sex and type of school (public or private). The mean and standard deviation of age and weight were calculated along with the mean value of FC. The median value of FC was compared between the groups based on sex, weight, and type of school using the Mann-Whitney U test. The significance level was set at p < 0.05. SPSS software (version 21; IBM Corp., Armonk, NY, USA) was used for statistical analyses.
Os dados desta pesquisa estão disponíveis no SciELO Data (DOI: https://doi.org/10.48331/SCIELODATA.3RWIG5).

Results
A total of 200 questionnaires were completed, and 155 fecal samples were examined in the study (Figure 1).
Among the 24 children excluded, one had an FC level >800 µg/g, which was much higher than the average for the other children in the study. This child was referred for further investigation.
Among the 177 samples that underwent parasitological examination, 18 showed positive findings (10.2%), all for Giardia lamblia, 14 (77.8%) of which were collected from public school children. Four individuals who had previously tested positive were reevaluated after undergoing treatment with metronidazole, administered at a dosage of 15 mg/kg, twice daily for 5 days.9 These reevaluations included new parasitological examinations conducted 30 days or more after completing the medication regimen, along with the completion of a new questionnaire. No significant difference was noted between FC levels before and after treatment (p=0.4). Unfortunately, the limited number of samples available for reevaluation prevents the assessment of statistical significance between the dosage results.
The study included a total of 155 children aged 4–6 years. For 11 samples, the measurement was repeated as suggested by the software for consistency assessment. Table 1 summarizes the participants’ data according to sex.
A comparison of children from public and private schools revealed no significant difference in weight between girls (p=0.37) and boys (p=0.92). The mode was 20 kg (14.2% of the children) in public schools and 18 kg (17.1%) in private schools. The mode was 18.3 kg (14.3%) for girls, 20 kg (18.3%) for boys, and the total mode was 20 kg for all participants (14.2%).

Tab.1

The median FC level is listed in Table 2. The mode was 5.0 ?g/g in both the school types, corresponding to 10% in public schools and 17.1% in private schools. A smaller number of samples from private schools was analyzed, which may have resulted in wider confidence intervals for this group. However, the proportionality observed in official enrollment data for public (78%) and private (22%) schools in the municipality of Rio de Janeiro was maintained, and this was considered an attenuating factor in the comparisons between groups.10

Tab.2

Only one sample from each school category (public and private; 0.6% and 2.9%, respectively)
presented maximum FC levels according to sex and age (Table 3).

Tab.3
No significant differences in FC levels were noted between sex (p=0.40) and boys and girls in public and private schools (p=0.33 and 0.18, respectively). When comparing FC levels in boys by school type, we found no significant difference (p=0.14). In contrast, among girls, FC levels were higher in public schools than in private schools (p=0.04).
The mean ages and weights of the boys and girls were similar. Among girls, the lowest weight was 12 kg (Z < -2), whereas the highest was 39 kg (Z > +3). For boys, the lowest weight was 13.9 kg (Z < -2), whereas the highest was 32 kg (Z > +3). No significant difference was noted in FC levels between samples of children with a Zw below -2 and samples with Zw between -2 and +2 (p=0.26). Five children (3.2%) had Zw values > +3, and the highest FC level was 37.9 ?g/g. None of the children in this study had Zw values < -3.

Discussion
This study analyzed potential factors for variations in FC levels in apparently healthy children aged between 4 and 6 years attending public and private schools.
Since 2009, school enrollment in Brazil has been mandatory from the age of 4,11 under the responsibility of the municipalities.12 Since then, there has been an undeniable increase in the attendance rate of children between 4 and 5 years old, reaching the national mark of 90.5% in 201513 and 97.36% in the Municipality of Rio de Janeiro between 2016 and 2019.14 However, social inequalities in childhood and adolescence within the municipality remain.15 Thus, two groups of children were chosen, one from public schools and the other from private schools, aiming to ensure greater potential for social, economic, and cultural heterogeneity. In Brazil, 78% of children enrolled in preschool attend public schools with 15.8% of private school spots being occupied through agreements with government entities.16 This research aimed to maintain this distribution percentage.
No statistically significant difference was observed between sexes, which is consistent with previously published findings.17 A difference was found between the FC values of these children, especially among girls. This finding led to an assessment of the socio-economic and socio-environmental interference on FC values, considering that these children live in the same city and many of them in the same neighborhood. This may indicate the presence of a greater degree of intestinal inflammation18 among less privileged children, even in children without clinical manifestations.
In Brazil, the HDI,19 a general measure of human development, is high20 as well as the Municipal HDI of the city of Rio de Janeiro.21 This index is composed of three dimensions – longevity, education and income – and stratified according from very low to very high.22 Thus, it can be speculated that within the same neighborhood, residents should exhibit similarities in life expectancy index (longevity), education index which reflect adult literacy rate and gross schooling rate) and gross domestic product rates (income).23 The HDI of the neighborhoods in this research ranged from high to very high24 with 6% being high and 94% very high, regardless of whether they were enrolled in public or private schools. Information on the household income of the children participating in the study was not obtained, but data on their place of residence were available. It is important to note that the high percentage of children in this age group enrolled in municipal preschools may have attenuated differences in the study outcomes.
Rio de Janeiro has neighborhoods with family incomes ranging from very high to low.25 As in any developing country, extremes involve family income, housing conditions, basic sanitation, and education levels, all of which affect the environment in which children are raised. A Brazilian study in a single city found that children from socioeconomically disadvantaged areas have markedly different gut microbiota compared to those with higher socioeconomic status and better sanitary conditions.26 Another Brazilian study, using questionnaires, identified low household income, shared soap, improper storage of toys and toothbrushes, and poor sanitation as risk factors for intestinal infestations.27 In this city, some areas have inadequate sanitation, which therefore resulting in an environment that is highly detrimental to the growth and development of children.28-30 However, the results we obtained regarding nutritional status (with no child having a weight below the Zw -2 range) indicate that our study did not encompass this underprivileged population. This is perhaps because these children did not have access to school or the scope of the sample did not reach them. The enrolment rate of children aged 4–5 years in preschool is influenced by family income; that is, a low family income poses a challenge for the family to send these children to school.31,32 Nevertheless, the statistical relevance we observed may have been related to the influence of the environment, even if that influence was not extreme. Interestingly, one study did not demonstrate a statistically significant association between calprotectin levels and growth indicators among low-income, apparently healthy children who moved freely between home and daycare environments.33 Research targeting populations that may have been excluded should be conducted in order to strengthen the association of the aspects identified in this study.
We found a significant difference in the FC levels of girls from public and private schools but not for boys. Although consistent with differences observed between public and private school children, no epidemiological data or evidence beyond speculation were found to explain this. Occasionally, we identified some mentions of sex subgroups; however, these were inconsistent, not related to primary findings, nor associated with socioeconomic factors. As the main focus of most studies has been directed toward age or variables influencing fecal calprotectin measurements, as well as differences between sexes, no consistent evidence supporting this discussion was considered. Further targeted studies are warranted to assess these findings.
Interestingly, Giardia lamblia, the most frequently found protozoan, was the only causative organism detected in all positive fecal parasitological examinations.34 Among the 18 positive exams, only 1 came from a private school located in a neighborhood with the lowest HDI in this category; all 4 participants who re-enrolled in the study went to public schools. This may be related to high environmental and personal hygiene conditions, as the prevalence of soil-transmitted helminthiases is declining in Brazil, and it is no longer considered an endemic region by the WHO.35 This infection can be self-limiting, and an expected negative stool test in approximately 3 weeks36 justifies caution in repeating the test 30 days after treatment. In general, in the city of Rio de Janeiro, public schools are attended by children with lower family income than private schools.37 This observation may help elucidate our finding of a higher prevalence of this protozoan, particularly in circumstances marked by less favorable sanitary conditions, poorer hygiene practices, or lower levels of education.38 Hence, this outcome may reflect the broader pattern of this infection within the localized context of the city. A study conducted in an African city with a high incidence of giardiasis, involving children aged 4 to 12 years, did not demonstrate a statistically significant difference between those with positive and negative tests for Giardia, despite a small difference in values in agreement with this observation.39 Studies with an adequate number of samples evaluating fecal calprotectin after two to four weeks of antiparasitic treatment should be conducted to provide statistically significant data.
There was no statistically significant difference in Zw between either girls or boys from public and private schools, and indeed the Zw of most children was normal, between -2 and +2 (Table 2). As there is generally a difference in family income between public and private schools, this lack of statistical significance may reflect better access to food or improved family income, which has been occurring in Brazil for many years, but may have been negatively influenced by the COVID-19 pandemic40 The data for this study were collected both before and after the COVID-19 pandemic, and this time period may reflect some influence from the rapid impoverishment that affected a vulnerable population. If we consider that weight loss is a marker that occurs more rapidly in the absence of proper nutrition, the impact of impoverishment may have been greater in lower social classes that, in some way, may not have been included in this survey.
Meanwhile, this finding may be related to food insecurity, as it relates to the quantity and quality of food consumed.41 Families facing economic hardship may tend to consume higher quantities of energy-dense foods as a means to alleviate hunger, with dietary variety and quality often improving as family income increases. However, habits, beliefs, and eagerness to consume, which can be signs of opulence, can indicate the difference between consumed and recommended foods, through Zw.42 Additionally, families facing food insecurity may prioritize protecting children, with less impact on income decline.43 Public schools provide meals to children44 to reduce food deficiencies, a practice maintained during the pandemic even in the suspension of classes.45
Environmental conditions, basic sanitation, and lack of hygiene education can all influence intestinal contamination in children, resulting in an inflammatory response independent of dietary patterns. One example is the impact of handwashing on the reduction of diarrheal mortality but not on the improvement of linear growth.46 FC has drawn attention as a possible biomarker of EED, a subclinical disease related to intestinal inflammation and growth impairment due to chronic exposure to pathogens.47 The hypothesis that EED is an entity in which the biomarkers currently used for its study are not specific enough to avoid confounding factors, including asymptomatic carriers of enteric pathogens, may lead to the consideration of intestinal contamination as a possible factor contributing to the statistical difference between public and private schools. Indeed, EED has been associated with a certain degree of inflammation and increased FC observed in preschool children raised in low-income environments.33 Therefore, children from lower-income families, who generally attend public schools, could have a greater degree of gut inflammation and, thus, higher FC levels. Low family income is generally associated with low education and hygiene education.46 The absence of personal, food, and environmental hygiene can increase the risk of pathogen exposure in the gastrointestinal system, potentially leading to a state of inflammation that would justify the increased FC levels in these children.47 In this study, seven (4.5%) children had a weight below Zw -2, with only one being a private school student. The maximum FC values were 81.6 µg/g. Considering that the study was conducted on apparently healthy children and that the European Society for Pediatric Gastroenterology, Hepatology and Nutrition recommends interpreting the cut-off point of 50 µg/g with caution,48 it is reasonable to consider that level may be normal or could indicate some degree of intestinal inflammation in an asymptomatic child,17 regardless of their Zw. There was no statistical significance in the FC level results between children with Zw below -2 and those with Zw between +2 and -2. None of the children in this study had a Zw below -3.
This study did not assess the height of the children involved; however, information regarding the absence of disease, such as answers to the questionnaire, negative parasitological examination, and the weight of most children being in the normal range based on the Z -2 score (WHO-Anthro for personal computers) supports the hypothesis that frequent contact with pathogens in populations with low hygiene education may generate some degree of intestinal inflammation and result in an increase in FC levels compared to that in populations with better conditions. Thus, the presence of inflammation in the intestine could lead to an increase in CF without immediately affecting the height of the children.
Another condition that could explain the difference in FC between public and private schools through hygiene education and socioeconomic conditions is SIBO,49 which has a higher frequency in developing countries and is associated with an increase in symbiotic bacteria in the small intestine.50 Children with SIBO and the healthy controls did not show a statistically significant difference in FC values,51 suggesting no inflammation.52 However, FC appears to increase in children with concomitant SIBO and EED,53 and it is not recommended to use FC to diagnose SIBO in previously healthy children.47
This study has some limitations, primarily due to the lack of data on the height of children, which is an essential component for diagnosing EED. Although the initial study design did not include this evaluation, the results were striking to the extent that, despite the presence of incomplete parameters, we decided to publish the findings. More comprehensive studies should be conducted to strengthen these data. The criterion used to consider children as apparently healthy was similar to that used in other studies but may represent an inaccurate methodology in that it does not serve as a fully reliable marker. Another limitation is the small sample size, which, as a prospective study, faced the impact of the lockdown imposed by the COVID-19 pandemic. However, the available literature on this subject is scarce, and the inferences relating to the findings showed that there is scope for future research to better understand inflammatory bowel activity in healthy children.
Our primary points of discussion were the statistically significant differences in FC levels between students from public and private schools, and between girls in these schools. Despite the well-established link between EED and malnutrition, no relationship was observed between low weight and FC levels in this study. Further studies are therefore required to evaluate the usefulness of this tool as a predictor of subclinical EED. Additionally, the statistically significant differences between FC values involving girls from public and private schools generated hypotheses that extend beyond environmental factors. These hypotheses warrant exploration into educational, social, and organic factors as potential contributors to the observed differences.
In addition, other factors that may directly or indirectly influence the reliable measurement of FC in children should be evaluated in future studies. Many studies have investigated increased fecal calprotectin in gastrointestinal diseases—from infant colic and cow’s milk allergy to functional abdominal pain, celiac disease, and cystic fibrosis—showing some variability toward higher levels, but consistency supporting its use as a diagnostic or monitoring marker still requires further research..17 In research involving apparently healthy children, ethical constraints limit invasive methods needed to exclude subclinical diseases, inherently creating the possibility that such limitations may affect the results.
Limitations in established knowledge mean we still lack definitive explanations for the differences observed between girls attending public and private schools. Although some hypotheses—such as possible involvement of SIBO and functional disorders—can be proposed, they remain highly speculative and lack solid scientific support.54-56 No statistically significant sex difference was observed, consistent with previous studies. However, a small difference was noted that apparently did not influence cutoff values for this age group and was beyond our study’s scope. This aspect may be investigated in future studies. Our study focused on children aged 4 to under 6 years, with a sample size considered small but comparable to other studies with slightly different numbers and much broader age ranges.16,33,39,57
In summary, in this study, we compared FC values in asymptomatic children attending public and private schools. Although no significant differences were found regarding Zw, there was a statistically significant difference between individuals from public and private schools, especially among girls. This potential increase in intestinal inflammation could contribute to changes in the growth and development of less privileged children in the same city, even when they are asymptomatic. In the future, the use of this tool may contribute to improving the assessment of government strategies aimed at enhancing hygiene education programs as well as the environment in which low-income families live.

Declarations
Ethics approval and consent to participate: This study was approved by the IPPMG Research Ethics Committee (reference number: CAAE 05132818.4.00005264) in 2019.
Consent for publication: Not applicable.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Competing interests: The authors declare that they have no competing interests.
Funding: Not applicable.
Authors' contributions: SRC formulated the project, collected the data, analyzed the samples, and described the results. CCS, MTA, and JCFJ formulated the project, contributed to data analysis, and reviewed the final manuscript. RRJ contributed to the statistical analysis of the data. All authors contributed to written sections of the manuscript.
Acknowledgments: We would like to express our thanks to Eduardo de Almeida Pernambuco for generously dedicating his time and providing the Laboratory of Clinical Analysis at IPPMG's resources, greatly contributing to the completion of the laboratory tests. Likewise, we extend our gratitude to Manoel Antonio Cardoso, Professor of Pediatrics at the Federal University of the State of Rio de Janeiro, for his collaboration in the sample collection and laboratory testing. We would also like to thank Professor Malvina Tania Tuttman, Full Professor at the School of Education and former Rector of the Federal University of the State of Rio de Janeiro, for endorsing relevant articles. Clemax Couto Sant'Anna thanks CNPq from where he received a grant (301984/2022-0).

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Figure Legend
Figure 1: Flowchart showing the process of obtaining samples