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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Pediatr.</journal-id>
<journal-title-group>
<journal-title>Frontiers in Pediatrics</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Pediatr.</abbrev-journal-title>
</journal-title-group>
<issn pub-type="epub">2296-2360</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fped.2025.1663016</article-id>
<article-version article-version-type="Version of Record" vocab="NISO-RP-8-2008"/>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Original Research</subject>
</subj-group>
</article-categories>
<title-group>
<article-title>Toxocariasis in children: seroprevalence after 15 years in a major city in Brazil</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name><surname>Lima</surname><given-names>Viviane dos Santos Vaccaro</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
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<contrib contrib-type="author">
<name><surname>Ferreira</surname><given-names>Isabella Braghin</given-names></name>
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<contrib contrib-type="author">
<name><surname>Rapchan</surname><given-names>Gabriela Geraldi da Silva</given-names></name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author">
<name><surname>Lescano</surname><given-names>Susana Zevallos</given-names></name>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
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<contrib contrib-type="author">
<name><surname>Rodrigues e Fonseca</surname><given-names>Gabriela</given-names></name>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
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<name><surname>Giuffrida</surname><given-names>Rogerio</given-names></name>
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<contrib contrib-type="author">
<name><surname>Kmetiuk</surname><given-names>Louise Bach</given-names></name>
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<contrib contrib-type="author" corresp="yes">
<name><surname>Biondo</surname><given-names>Alexander Welker</given-names></name>
<xref ref-type="aff" rid="aff5"><sup>5</sup></xref>
<xref ref-type="corresp" rid="cor1">&#x002A;</xref>
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<name><surname>Santar&#x00E9;m</surname><given-names>Vamilton Alvares</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<aff id="aff1"><label>1</label><institution>Graduate College in Animal Science, University of Western S&#x00E3;o Paulo (Unoeste)</institution>, <city>Presidente Prudente</city>, <state>S&#x00E3;o Paulo</state>, <country country="br">Brazil</country></aff>
<aff id="aff2"><label>2</label><institution>Graduate College in Health Sciences, University of Western S&#x00E3;o Paulo (Unoeste)</institution>, <city>Presidente Prudente</city>, <state>S&#x00E3;o Paulo</state>, <country country="br">Brazil</country></aff>
<aff id="aff3"><label>3</label><institution>Laboratory of Medical Investigation, Clinical Hospital of the University of S&#x00E3;o Paulo</institution>, <city>FMUSP</city>, <state>S&#x00E3;o Paulo</state>, <country country="br">Brazil</country></aff>
<aff id="aff4"><label>4</label><institution>Center of Environmental Health, Municipal Secretary of Health</institution>, <city>Curitiba</city>, <state>Paran&#x00E1;</state>, <country country="br">Brazil</country></aff>
<aff id="aff5"><label>5</label><institution>Graduate College of Cell and Molecular Biology, Department of Veterinary Medicine, Federal University of Paran&#x00E1; (UFPR)</institution>, <city>Curitiba</city>, <state>Paran&#x00E1;</state>, <country country="br">Brazil</country></aff>
<author-notes>
<corresp id="cor1"><label>&#x002A;</label><bold>Correspondence:</bold> Alexander Welker Biondo <email xlink:href="mailto:abiondo@ufpr.br">abiondo@ufpr.br</email></corresp>
</author-notes>
<pub-date publication-format="electronic" date-type="pub" iso-8601-date="2025-11-19"><day>19</day><month>11</month><year>2025</year></pub-date>
<pub-date publication-format="electronic" date-type="collection"><year>2025</year></pub-date>
<volume>13</volume><elocation-id>1663016</elocation-id>
<history>
<date date-type="received"><day>29</day><month>07</month><year>2025</year></date>
<date date-type="accepted"><day>28</day><month>10</month><year>2025</year></date>
</history>
<permissions>
<copyright-statement>&#x00A9; 2025 Lima, Ferreira, Rapchan, Lescano, Rodrigues e Fonseca, Giuffrida, Kmetiuk, Biondo and Santar&#x00E9;m.</copyright-statement>
<copyright-year>2025</copyright-year><copyright-holder>Lima, Ferreira, Rapchan, Lescano, Rodrigues e Fonseca, Giuffrida, Kmetiuk, Biondo and Santar&#x00E9;m</copyright-holder><license><ali:license_ref start_date="2025-11-19">https://creativecommons.org/licenses/by/4.0/</ali:license_ref>
<license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License (CC BY)</ext-link>. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license>
</permissions>
<abstract><sec><title>Introduction</title>
<p>Children have a potentially greater exposure to <italic>Toxocara</italic> spp. infection via egg ingestion due to playground soil contact. Understanding the associated risk factors may help to prevent infection in children; however, there were no studies that monitored urban seropositivity over time. Therefore, we aimed to assess <italic>Toxocara</italic> seroprevalence and the associated risk factors in children who regularly accessed the Public Health System in a major city area in southeastern Brazil over a period of 15 years (2008&#x2013;2023).</p>
</sec><sec><title>Methods</title>
<p>A cross-sectional study was designed in 2023&#x2013;2024 to screen for anti-<italic>Toxocara</italic> antibodies in children through an enzyme-linked immunosorbent assay alongside an epidemiological questionnaire to assess health status and living conditions. Univariate and multivariate logistic regression analyses were used to investigate the factors associated with seropositivity in children.</p>
</sec><sec><title>Results</title>
<p>In total, 30/260 [11.5&#x0025;; 95&#x0025; confidence interval (CI): 8.2&#x0025;&#x2013;16.0&#x0025;] children were found to be seropositive for <italic>Toxocara</italic> spp. Boys were at a higher exposure risk [odds ratio (OR): 4.4] to toxocariasis than girls. Having a mother with a graduate degree (OR: 0.21; <italic>p</italic>&#x2009;&#x003D;&#x2009;0.016), receiving drinkable water supply (OR: 0.29; <italic>p</italic>&#x2009;&#x003D;&#x2009;0.017), and having hygienic habits of washing hands before meals (OR: 0.32; <italic>p</italic>&#x2009;&#x003D;&#x2009;0.033) were protective factors. The findings presented no significant difference (<italic>p</italic>&#x2009;&#x003D;&#x2009;0.879) when compared to those of a previous 2008 serosurvey, with 28/252 (11.1&#x0025;; 95&#x0025; CI: 7.8&#x0025;&#x2013;15.6&#x0025;) seropositive children.</p>
</sec><sec><title>Discussion</title>
<p><italic>Toxocara</italic> seroprevalence in children living in an urban area did not significantly change over the 15 years since the previous serosurvey was conducted. As no mitigatory actions were carried out between the surveys, we speculate that environmental contamination and child exposure to <italic>Toxocara</italic> spp. remained steady. However, future surveys should include soil detection of <italic>Toxocara</italic> spp. to fully establish the role of environmental contamination over time. In conclusion, although toxocariasis in children may not increase over time, such seroprevalence may not decrease either, persisting (even in low levels) for long periods.</p>
</sec>
</abstract>
<kwd-group>
<kwd>epidemiology</kwd>
<kwd>parasite</kwd>
<kwd>pediatric infection</kwd>
<kwd>toxocariasis</kwd>
<kwd>zoonosis</kwd>
</kwd-group>
<funding-group>
<funding-statement>The author(s) declare that no financial support was received for the research and/or publication of this article.</funding-statement>
</funding-group>
<counts>
<fig-count count="2"/>
<table-count count="1"/><equation-count count="0"/><ref-count count="61"/><page-count count="9"/><word-count count="854584"/></counts><custom-meta-group><custom-meta><meta-name>section-at-acceptance</meta-name><meta-value>Pediatric Infectious Diseases</meta-value></custom-meta></custom-meta-group>
</article-meta>
</front>
<body><sec id="s1" sec-type="intro"><title>Introduction</title>
<p>Human toxocariasis is a zoonotic helminth infection primarily impacting lower-socioeconomic populations living in tropical and subtropical countries (<xref ref-type="bibr" rid="B1">1</xref>). Toxocariasis has been classified as a neglected parasitic disease and targeted for public health action by the Centers for Disease Control and Prevention (<xref ref-type="bibr" rid="B2">2</xref>), alongside Chagas disease, cyclosporiasis, cysticercosis, toxoplasmosis, and trichomoniasis. The health burden of common toxocariasis in humans has been estimated to be 23,084 disability-adjusted life years lost annually in 28 selected countries (<xref ref-type="bibr" rid="B3">3</xref>). Based on an average global seroprevalence rate of 19&#x0025;, an estimated 91,714 disability-adjusted life years are lost annually due to toxocariasis across affected countries; of these, 40,912 are related to less severe forms, i.e., common toxocariasis, and 50,731 to cognitive impairment in children. The estimated economic impact in Brazil, adjusted for purchasing power parity, was &#x0024;283,389,884 over a 10-year period, a burden equivalent to that reported in Nigeria, India, the UK, Romania, and Ireland (<xref ref-type="bibr" rid="B3">3</xref>).</p>
<p>Human toxocariasis is mainly acquired through the ingestion of <italic>Toxocara canis</italic> and <italic>Toxocara cati</italic> eggs that have been shed into contaminated soil via the feces of dogs and cats, which act as definitive hosts (<xref ref-type="bibr" rid="B4">4</xref>, <xref ref-type="bibr" rid="B5">5</xref>). <italic>Toxocara</italic> spp. infection may lead to a self-limiting febrile illness (common toxocariasis) in some patients or an asymptomatic (covert toxocariasis) infection. In addition, human toxocariasis may remain subclinical or lead to mild, moderate, or severe symptoms, including larva migrans syndromes (visceral and ocular) and neurotoxocariasis (<xref ref-type="bibr" rid="B4">4</xref>, <xref ref-type="bibr" rid="B6">6</xref>).</p>
<p>Despite mostly affecting vulnerable populations in underdeveloped and developing countries, human toxocariasis remains a devastating zoonotic infection in children living in the USA (<xref ref-type="bibr" rid="B7">7</xref>). The disease is associated with urticaria (<xref ref-type="bibr" rid="B8">8</xref>), bronchial asthma and pneumonia (<xref ref-type="bibr" rid="B9">9</xref>), ocular impairment (<xref ref-type="bibr" rid="B10">10</xref>), and cognitive function decline (<xref ref-type="bibr" rid="B11">11</xref>) in children. Children are considered to be more exposed to infection through the ingestion of soil contaminated with <italic>Toxocara</italic> spp. eggs (<xref ref-type="bibr" rid="B4">4</xref>), along with direct contact with dogs and cats (<xref ref-type="bibr" rid="B12">12</xref>), particularly puppies and kittens (<xref ref-type="bibr" rid="B13">13</xref>).</p>
<p>According to a recent meta-analytic study, the estimated global seroprevalence of anti-<italic>Toxocara</italic> spp. antibodies among children is 25.0&#x0025; [95&#x0025; confidence interval (CI) 22&#x0025;&#x2013;29&#x0025;], particularly in boys (<xref ref-type="bibr" rid="B14">14</xref>). Risk factors for toxocariasis have been reported worldwide, including caregiver education, contact with dogs/cats, and improper handling of pet feces associated to seropositivity in Vietnam (<xref ref-type="bibr" rid="B15">15</xref>) and drinking unpurified water in Iran (<xref ref-type="bibr" rid="B16">16</xref>). Higher risk was reported in asthmatic children with a recorded history of contact with soil and pets as compared to control and pneumonic groups in Egypt (<xref ref-type="bibr" rid="B9">9</xref>). In Brazil, associated risk factors for toxocariasis include family income (up to one minimum wage), the presence of a dog, the habit of playing with soil/sand, and eosinophilia (<xref ref-type="bibr" rid="B17">17</xref>).</p>
<p>Although the prevalence of toxocariasis in Brazil has been estimated in meta-analytic studies to be 28.0&#x0025; (95&#x0025; CI: 22&#x2013;34) in the general population (<xref ref-type="bibr" rid="B18">18</xref>) and 39.3&#x0025; (95&#x0025; CI: 29.8&#x2013;48.7) in children (<xref ref-type="bibr" rid="B14">14</xref>), a higher seroprevalence of 63.6&#x0025; was reported in infants in socioeconomically vulnerable conditions in northeastern Brazil (<xref ref-type="bibr" rid="B19">19</xref>). In a previous study conducted in southeastern Brazil in 2008, 28/252 (11.1&#x0025;) children were seropositive for <italic>Toxocara</italic> spp. seropositivity was observed in 12/126 (9.5&#x0025;) children from a middle-income background and in 16/126 (12.7&#x0025;) children from a lower-income background, indicating an inversely proportional connection to family income (<xref ref-type="bibr" rid="B20">20</xref>). In addition, higher household income and female gender were significant protective factors for both subgroups (<xref ref-type="bibr" rid="B20">20</xref>). Although the knowledge of toxocariasis occurrence and associated risk factors is important for understanding the disease mechanisms of transmission, to our knowledge, no study has been conducted on the dynamics of toxocariasis in children over time. Therefore, in this study, we aimed to assess the seroprevalence and associated risk factors for toxocariasis in children who visited the Public Health System between 2008 and 2023 in the same area of southeastern Brazil that was studied previously.</p>
</sec>
<sec id="s2" sec-type="methods"><title>Methods</title>
<sec id="s2a" sec-type="ethics-statement"><title>Ethics statement</title>
<p>This study was approved by the Ethics Committee of the University of Western S&#x00E3;o Paulo (protocol 8,294), and the protocol has been submitted to the National Commission of Ethics in Research (CAAE: protocol 73476123.2.0000.5515). For data collection, each guardian was informed about the confidentiality of participant identity and the right to refuse participation at any time. The guardian formalized the authorization by signing the Free and Informed Consent Terms form in compliance with Resolution No. 441/2012 of the National Brazilian Health Council. The anonymity of the participants was ensured by assigning a random number that was used to identify each sample and metadata, according to criteria adopted elsewhere (<xref ref-type="bibr" rid="B21">21</xref>). Information produced by the researchers regarding toxocariasis was provided to each child enrolled in the study (<xref ref-type="sec" rid="s11">Supplementary Figure S1</xref>).</p>
</sec>
<sec id="s2b"><title>Study design</title>
<p>This cross-sectional study was designed to detect anti-<italic>Toxocara</italic> spp. antibodies and gather information from a pediatric population accessing the Brazilian Public Health System. The participants were recruited during routine examinations requested by the public health system at the Dr. Jo&#x00E3;o Carlos Grigoli Clinical Analysis Laboratory at the University of Western S&#x00E3;o Paulo between November 2023 and April 2024. The study was conducted in the municipality of Presidente Prudente (22&#x00B0;7&#x2032;16.5540&#x02BA;S and 51&#x00B0;23&#x2032;0.2400&#x02BA;W), S&#x00E3;o Paulo state, southeastern Brazil (<xref ref-type="fig" rid="F1">Figure&#x00A0;1</xref>). Presidente Prudente is a major Brazilian city, currently ranked 25th (top 0.5&#x0025;) in the city human development index (HDI) (0.806, very high), 138th (top 2.5&#x0025;) in population with 225,000 inhabitants, and 159th (top 3.0&#x0025;) in gross domestic product out of 5,565 municipalities in Brazil.</p>
<fig id="F1" position="float"><label>Figure&#x00A0;1</label>
<caption><p>Map of Brazil showing the study location. The municipality of Presidente Prudente is presented, with the city indicated by a red star. The state of S&#x00E3;o Paulo (upper right) and its western region (bottom right) are highlighted in yellow.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="fped-13-1663016-g001.tif"><alt-text content-type="machine-generated">Map displaying a region in Brazil with emphasis on S&#x00E3;o Paulo state. The image highlights a specific area with a star symbol, situated between the Paran&#x00E1; and Paranapanema Rivers. Insets show the location of S&#x00E3;o Paulo within Brazil and the specific focus area within the state. The map includes longitude and latitude markers, and shaded relief indicates major geographical features.</alt-text>
</graphic>
</fig>
<p>The study was conducted at a public prenatal referral health establishment located in the municipality of Presidente Prudente (22&#x00B0;7&#x2032;16.5540&#x02BA;S and 51&#x00B0;23&#x2032;0.2400&#x02BA;W), S&#x00E3;o Paulo state, southeastern Brazil.</p>
</sec>
<sec id="s2c"><title>Sample size calculation</title>
<p>Using the Scalex SP calculator (<xref ref-type="bibr" rid="B22">22</xref>), we calculated the sample size of 259, adjusted to 260 assuming the upper 95&#x0025; confidence limit (19.6&#x0025;) of the prevalence in children in Presidente Prudente as observed in a previous study (<xref ref-type="bibr" rid="B20">20</xref>). We assumed a margin of error of &#x00B1;5&#x0025; in estimating the prevalence with 95&#x0025; confidence and a 5&#x0025; potential loss.</p>
</sec>
<sec id="s2d"><title>Inclusion and exclusion criteria</title>
<p>Children aged 1&#x2013;11 years, regardless of sex, whose legal guardian (&#x003E;18 years) provided written consent were included in the study. All children were randomly invited to participate during routine visits to the Brazilian Public Health System. The age of the children included was established based on the Brazilian Statute of the Child and Adolescent (Law number 8,069, 13 July 1990) (<xref ref-type="bibr" rid="B23">23</xref>), which considers someone to be a child if they are &#x2264;12 years of age. Children living outside Presidente Prudente and/or children who did not accept the invitation to participate in the study were excluded.</p>
</sec>
<sec id="s2e"><title>Sociodemographic data</title>
<p>To gather information on socioeconomic status, residence, contact with pets (dog or cats), and hygienic habits and practices (<xref ref-type="table" rid="T1">Table&#x00A0;1</xref>), a semistructured questionnaire was completed by the guardian. The data were gathered in a spreadsheet (Excel) to evaluate the associated risk factors for toxocariasis.</p>
<table-wrap id="T1" position="float"><label>Table&#x00A0;1</label>
<caption><p>Data for assessing the potential children&#x0027;s exposure to toxocariasis.</p></caption>
<table>
<thead>
<tr>
<th valign="top" align="left">Topics</th>
<th valign="top" align="center">Gathered information</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left">Socioeconomic and demographic characteristics</td>
<td valign="top" align="left">Sex, age, mother&#x0027;s educational level, monthly household income, household location</td>
</tr>
<tr>
<td valign="top" align="left">Residence</td>
<td valign="top" align="left">Drinkable water, unpaved yard</td>
</tr>
<tr>
<td valign="top" align="left">Domestic animals</td>
<td valign="top" align="left">Owning dog, dog contact, owning cat, cat contact</td>
</tr>
<tr>
<td valign="top" align="left">Hygienic habits and practices</td>
<td valign="top" align="left">Washing hands before meals, contact with soil, playing on public parks, ingestion of raw meat, biting nails (onychophagy), and pica behavior</td>
</tr>
</tbody>
</table>
</table-wrap>
</sec>
<sec id="s2f"><title>Collection of blood samples</title>
<p>Blood samples (3.0&#x2005;mL) were collected by an authorized health professional belonging to the health establishment staff once per child via peripheral venipuncture using commercial vacuum tubes (Vacutainer, BD Co., Curitiba, Brazil). Blood samples were then centrifuged at 1,295&#x2005;<italic>g</italic> for 5&#x2005;min; serum was collected and frozen (&#x2212;20&#x00B0;C) until the test.</p>
</sec>
<sec id="s2g"><title>Screening anti-<italic>Toxocara</italic> immunoglobulin (Ig)G antibodies</title>
<sec id="s2g1"><title><italic>Toxocara</italic> antigen for enzyme-linked immunosorbent assay test</title>
<p>The antigen used for detecting anti-<italic>Toxocara</italic> IgG antibodies was obtained from female <italic>T. canis</italic> adult specimens spontaneously shed by naturally infected puppies. The roundworms were rinsed in 1&#x0025; sodium hypochlorite (5&#x2005;min) and the washed with 0.9&#x0025; saline (3&#x2005;min). After removing debris, the anterior third of the female worm was dissected to obtain eggs. Protocols previously described were employed to produce <italic>T. canis</italic> excretory-secretory (TES) antigen <italic>in vitro</italic> (<xref ref-type="bibr" rid="B24">24</xref>) and concentrate the protein (<xref ref-type="bibr" rid="B25">25</xref>).</p>
<p>Owing to the possibility of cross-reactivity with other ascarids, serum samples were preincubated (30&#x2005;min at 37&#x00B0;C) with <italic>Ascaris suum</italic> adult worm extract solution (25.0&#x2005;&#x03BC;g/&#x03BC;L) in 0.01&#x2005;M phosphate-buffered saline (PBS, pH 7.2) containing 0.05&#x0025; Tween 20 (PBS-T) (Sigma, St. Louis, MO, USA), following a published protocol (<xref ref-type="bibr" rid="B24">24</xref>).</p>
</sec>
<sec id="s2g2"><title>Serological testing</title>
<p>IgG antibodies against TES were detected through indirect enzyme-linked immunosorbent assay (ELISA) at a dilution of 1:200. Polystyrene 96-well microtiter plates (Corning, Costar, NY, USA) were coated (1&#x2005;h at 37&#x00B0;C) and then incubated for 18&#x2005;h at 4&#x00B0;C, with 2.0&#x2005;&#x03BC;g/&#x00B5;L per 100&#x2005;&#x00B5;L/well of TES antigens in 0.06&#x2005;M carbonate-bicarbonate buffer (<xref ref-type="bibr" rid="B22">22</xref>, <xref ref-type="bibr" rid="B23">23</xref>), at pH 9.6. Then, they were blocked (1&#x2005;h at 37&#x00B0;C) using 3&#x0025; Molico&#x00AE; skimmed milk 5&#x0025; PBS-T. After blocking, the wells were washed three times with PBS-T (5&#x2005;min each). Subsequently, 100&#x2005;&#x00B5;L of the sera preadsorbed with <italic>A. suum</italic> somatic antigen was added to the wells in duplicate. The plates were incubated for 1&#x2005;h at 37&#x00B0;C, washed three times with PBS-T (5&#x2005;min each), and antihuman IgG (Fc-specific) peroxidase antibody&#x2014;produced in goat (Sigma A6029)&#x2014;was added at a 1:10,000 dilution (45&#x2005;min at 37&#x00B0;C). The plates were then washed three times (5&#x2005;min each).</p>
<p>The reaction was initiated using the substrate o-phenylenediamine (0.4&#x2005;mg/mL, Sigma), and it was stopped by adding 2 N sulfuric acid. Absorbance was read at 492&#x2005;nm. A serum previously shown to be non-reactive (negative control) and a known reactive serum (positive control) were tested in each plate. Antibody levels are expressed as reactivity indexes, which were calculated as the ratio between the absorbance values of each sample, and the cutoff value was 0.285.</p>
</sec>
</sec>
<sec id="s2h"><title>Data analysis</title>
<p>All statistical analyses were performed using R v.4.1.1 (R Core Team, 2022). To assess risk factors related to seropositivity, the outcome data were categorized (variables shown in <xref ref-type="table" rid="T1">Table&#x00A0;1</xref>). Then, univariate analysis (Pearson chi-square or Fisher&#x0027;s exact test) was performed. Variables presenting a significance of &#x003C;0.20 in the univariate analysis were considered suitable for logistic regression analysis (multivariate analyses) to assess the risk/protective factors associated with the likelihood of <italic>Toxocara</italic> spp. seropositivity. To improve the final model, the predictor variables were tested for collinearity and the presence of influential values. Variables with a high degree of collinearity were excluded from the logistic model (variance inflation factor &#x003E;4.0). From the regression coefficients for each predictor variable, odds ratios (ORs) were estimated per point with 95&#x0025; CIs. The best-fitting model was considered the one that included significantly associated variables (<italic>p</italic>-value&#x2009;&#x003C;&#x2009;0.05) and minimized the Akaike information criterion.</p>
<p>A chi-square test for equality of proportions without Yates&#x0027; continuity correction was performed to compare the seroprevalence for anti-<italic>Toxocara</italic> spp. antibodies at two distinct time points (2008 vs. 2023) within the target population. A 5&#x0025; significance level was adopted for all statistical tests.</p>
</sec>
</sec>
<sec id="s3" sec-type="results"><title>Results</title>
<sec id="s3a"><title>Demographic characteristics of participants</title>
<p>In total, 260 individuals were sampled including 137/260 (52.7&#x0025;) boys and 123/260 (47.3&#x0025;) girls, aged 1&#x2013;11 years (median: 6 years). In terms of the monthly family wage, 246 individuals responded to the questionnaire, while 14 individuals did not provide the information and were not included in familial income statistics. Thus, 112/246 (45.5&#x0025;) guardians declared an income of less than one minimum wage (USD 312.00), 116 (47.2&#x0025;) declared an income of up to USD 928.00, and 18 (7.3&#x0025;) declared an income of more than USD 1,000.00. All participants had an indoor bathroom, and the majority (235/260; 90.4&#x0025;) had a domestic sewage system. Approximately 50&#x0025; (122/260; 46.7&#x0025;) lived in a residence with a dirty yard.</p>
</sec>
<sec id="s3b"><title>Seropositivity and risk factors for toxocariasis</title>
<p>Anti-<italic>Toxocara</italic> spp. antibodies were detected in 30/260 children (11.5&#x0025;; CI 95&#x0025;: 8.2&#x2013;16.0&#x0025;). The final logistic regression model revealed that boys were 4.4-fold more likely to be seropositive than girls. Having a mother with a graduate degree (OR: 0.2; <italic>p</italic>&#x2009;&#x003D;&#x2009;0.016), access to a drinkable water supply (OR: 0.3; <italic>p</italic>&#x2009;&#x003D;&#x2009;0.033), and washing hands before meals (OR: 0.3; <italic>p</italic>&#x2009;&#x003D;&#x2009;0.008) were protective factors for toxocariasis (<xref ref-type="fig" rid="F2">Figure&#x00A0;2</xref>).</p>
<fig id="F2" position="float"><label>Figure&#x00A0;2</label>
<caption><p>Risk/protective factors for <italic>Toxocara</italic> spp. seropositivity in a pediatric population (<italic>n</italic>&#x2009;&#x003D;&#x2009;260) assisted by the Public Health System, in southeastern Brazil, assessed using univariate and multivariate analyses. The vertical line with a value of 1 represents the line of null effect. OR is significant (&#x002A;) if the mean with 95&#x0025; confidence intervals (95&#x0025; CI) does not touch the vertical line (value of 1). Risk factor: male sex (OR: 4.4; 95&#x0025; CI: 1.8&#x2013;12.2; <italic>p</italic>&#x2009;&#x003D;&#x2009;0.003). Protective factors: maternal education level (OR: 0.2; 95&#x0025; CI: 0.1&#x2013;0.7; <italic>p</italic>&#x2009;&#x003D;&#x2009;0.016), washing hands before meals (OR: 0.3; 95&#x0025; CI: 0.1&#x2013;0.7; <italic>p</italic>&#x2009;&#x003D;&#x2009;0.008), and drinkable water supply (OR: 0.3; 95&#x0025; CI: 0.1&#x2013;0.9; <italic>p</italic>&#x2009;&#x003D;&#x2009;0.033).</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="fped-13-1663016-g002.tif"><alt-text content-type="machine-generated">Forest plot illustrating odds ratios (OR) for various factors across four groups: Individual, Hygiene, Environmental, and Animal. Each factor is analyzed through univariate and multivariate analyses. Variables include household income, education, gender, age, hygiene behaviors, environmental aspects, and animal interactions. Dots represent ORs, with lines indicating confidence intervals. Asterisks (*) highlight significant results. Factors like having a dog or contact with a cat show higher ORs in multivariate analysis.</alt-text>
</graphic>
</fig>
<p>The AI service Recraft V3 (<xref ref-type="bibr" rid="B26">26</xref>) text-to-image model (October 2024) was used to produce the four illustrations in <xref ref-type="fig" rid="F2">Figure&#x00A0;2</xref>. The specific prompts used for generating the images were as follows: <xref ref-type="fig" rid="F2">Figure&#x00A0;2</xref>, column 1, row 1&#x2014;&#x201C;Mother and children in a park, vector, realistic, white background&#x201D;; <xref ref-type="fig" rid="F2">Figure&#x00A0;2</xref>, column 1, row 2&#x2014;&#x201C;Beef on a plate with a cup of water, vector, realistic&#x201D;; <xref ref-type="fig" rid="F2">Figure&#x00A0;2</xref>, column 1, row 3&#x2014;&#x201C;A child playing in a park with a sandcastle, realistic drawing, white background&#x201D;; and <xref ref-type="fig" rid="F2">Figure&#x00A0;2</xref>, column 1, row 4&#x2014;&#x201C;Dog and cat, realistic vector, white background.&#x201D;</p>
<p>Although the univariate analysis included having dog in the logistic regression, this variable was not considered significant in the final model (<italic>p</italic>&#x2009;&#x003D;&#x2009;0.162). Contact with a dog or a cat was considered to be suitable for inclusion in the multivariate analysis but was not retained in the final logistic regression model. The other evaluated variables were not significant in the univariate analysis (<xref ref-type="sec" rid="s11">Supplementary Table S1</xref>): monthly income (<italic>p</italic>&#x2009;&#x003D;&#x2009;0.267), living in a house with an unpaved yard (<italic>p</italic>&#x2009;&#x003D;&#x2009;0.346), playing on public playground (<italic>p</italic>&#x2009;&#x003D;&#x2009;0.893), having a cat (<italic>p</italic>&#x2009;&#x003D;&#x2009;0.654), onychophagy (<italic>p</italic>&#x2009;&#x003D;&#x2009;0.27), pica (<italic>p</italic>&#x2009;&#x003D;&#x2009;0.764), raw/undercooked meat consumption (<italic>p</italic>&#x2009;&#x003D;&#x2009;0.268), and cat ownership (<italic>p</italic>&#x2009;&#x003D;&#x2009;0.654). The performance of the final regression model was considered to have a good to very good discriminative power (AUC: 79.8&#x0025;; 95&#x0025; CI: 71.2&#x2013;88.4) (<xref ref-type="sec" rid="s11">Supplementary Figure S2</xref>).</p>
</sec>
</sec>
<sec id="s4" sec-type="discussion"><title>Discussion</title>
<p>The present study reassessed the seroprevalence and risk factors of toxocariasis in a pediatric population in southeastern Brazil who accessed services through the Public Health System. The seropositivity and 95&#x0025; confidence interval (28/252; 11.1&#x0025;; 95&#x0025; CI: 7.8&#x2013;15.6) were highly similar to those observed previously in the same study area (<xref ref-type="bibr" rid="B20">20</xref>), suggesting the stability of <italic>Toxocara</italic> spp. seroprevalence. Furthermore, the male gender was considered to be a risk factor for <italic>Toxocara</italic> seropositivity in both the previous (<xref ref-type="bibr" rid="B20">20</xref>) and present studies.</p>
<p>The seroprevalence of <italic>Toxocara</italic> spp. infection observed in the present study (11.5&#x0025;; 95&#x0025; CI: 8.2&#x2013;16.0) was lower than the estimated seroprevalence among children worldwide (25&#x0025;; 95&#x0025; CI: 22&#x2013;29) and in Brazil (39.3&#x0025;; 95&#x0025; CI: 29.8&#x2013;48.7) (<xref ref-type="bibr" rid="B14">14</xref>). In Brazil, the seroprevalence ranges between 4.2&#x0025; (7/167) in the south (<xref ref-type="bibr" rid="B27">27</xref>) and 63.6&#x0025; (503/791) in the northeast (<xref ref-type="bibr" rid="B19">19</xref>). Our results contradict those of a meta-analysis which revealed a decrease in <italic>Toxocara</italic> spp. seroprevalence in children over time, estimated at 33.6&#x0025; (23.3&#x2013;44.0) in 2000&#x2013;2005, 23.3&#x0025; (15.5&#x2013;31.1) in 2006&#x2013;2011, 29.1&#x0025; (22.0&#x2013;36.2) in 2012&#x2013;2017, and 10.6&#x0025; (7.08&#x2013;13.4) in 2018&#x2013;2021 (<xref ref-type="bibr" rid="B14">14</xref>). One of the reasons for such a decrease&#x2014;according to the latest authors&#x2014;is the level of health knowledge of the population.</p>
<p>Some factors may influence the rates of seropositivity in the population, including a low HDI, income level, latitude, and high environmental temperature (<xref ref-type="bibr" rid="B5">5</xref>). Thus, regional socioeconomic and climatic differences may explain the wide range of seropositivity in Brazil. In the present study, approximately 50&#x0025; of the population lived with more than one monthly salary, but monthly household income was not associated with seropositivity. This result contrasts with the results of a serosurvey on schoolchildren in S&#x00E3;o Paulo, the largest city of Brazil (<xref ref-type="bibr" rid="B28">28</xref>), and the previous study conducted in Presidente Prudente (<xref ref-type="bibr" rid="B20">20</xref>), in which the frequency of <italic>Toxocara</italic> spp. infection was higher in children whose families had the lowest monthly income. In contrast with previous findings, household income in the present study was not associated with toxocariasis seropositivity. This outcome could be a consequence of the city&#x0027;s high HDI, which may have evenly impacted the overall population (<xref ref-type="bibr" rid="B5">5</xref>). Thus, the steady low toxocariasis seropositivity over time in the children observed in our study could be the result of adequate population hygiene habits and water supply.</p>
<p>Sanitation conditions have been linked to toxocariasis, with conflicting results in pediatric populations, with poor sanitation associated (<xref ref-type="bibr" rid="B29">29</xref>) or not (<xref ref-type="bibr" rid="B30">30</xref>) with seropositivity. In Argentina, highly unsanitary conditions and lack of water supply and sewer networks led to a high toxocariasis seroprevalence in abandoned, institutionalized children (<xref ref-type="bibr" rid="B31">31</xref>). In Brazil, adequate sanitary facilities were considered to be a protective factor, leading to the relatively low seroprevalence previously observed in children from Presidente Prudente (<xref ref-type="bibr" rid="B32">32</xref>). In addition, in the present study, access to drinkable water supply was revealed to be a protective factor for seropositivity, corroborating the results of a population-based study of children in Amazonia, northern Brazil, in which multivariate analysis revealed piped water inside the household to be a protective factor (<xref ref-type="bibr" rid="B33">33</xref>). Furthermore, in Iran (<xref ref-type="bibr" rid="B16">16</xref>) and Thailand (<xref ref-type="bibr" rid="B34">34</xref>), consumption of untreated water was considered a risk factor for toxocariasis in children.</p>
<p>Hygienic habits and residence conditions contribute to <italic>Toxocara</italic> spp. infection (<xref ref-type="bibr" rid="B35">35</xref>). The habit of washing hands before meals was a protective factor for toxocariasis in the present study. This finding is in line with those of similar serosurveys involving children in China (<xref ref-type="bibr" rid="B36">36</xref>) and Brazil (<xref ref-type="bibr" rid="B30">30</xref>). Although poor hygiene habits, such onychophagy and pica, may increase the risk of <italic>Toxocara</italic> spp. infection via egg ingestion (<xref ref-type="bibr" rid="B35">35</xref>, <xref ref-type="bibr" rid="B37">37</xref>, <xref ref-type="bibr" rid="B38">38</xref>), these habits were not associated with seropositivity in the present study. Poor hygienic habits are considered to be more common in socioeconomically disadvantaged children (<xref ref-type="bibr" rid="B30">30</xref>). A study conducted in Saudi Arabia to evaluate the level of handwashing knowledge, attitudes, and practices among school students showed a positive correlation between the education level of the mother and hand hygiene practices (<xref ref-type="bibr" rid="B39">39</xref>). Thus, the influence of maternal education on hygienic practices observed in the present study should be considered another reason for the seropositivity that we observed. In a previous study conducted in northeastern Brazil, low maternal education was associated with a higher seroprevalence to <italic>Toxocara</italic> spp. and reflected the family hygiene habits (<xref ref-type="bibr" rid="B40">40</xref>). Likewise, a higher educational level was previously reported to be a protective factor for toxocariasis among adults in vulnerable populations in Brazil, including people experiencing homelessness (<xref ref-type="bibr" rid="B41">41</xref>) and inmates (<xref ref-type="bibr" rid="B32">32</xref>).</p>
<p>Food-borne transmission may also play a role in human toxocariasis. There is a lower risk of consuming larvated eggs in unwashed spinach than in undercooked meat (<xref ref-type="bibr" rid="B42">42</xref>). Such research highlights the importance of reducing environmental <italic>Toxocara</italic> spp. contamination in farms and correctly cooking meat to prevent toxocariasis<sans-serif>.</sans-serif></p>
<p>As observed both in this study and the previous study conducted in the same study area (<xref ref-type="bibr" rid="B20">20</xref>), boys were more likely to be seropositive for <italic>Toxocara</italic> spp. than girls. This finding corroborates the conclusions of a global meta-analysis on seroprevalence for toxocariasis in pediatric populations, which reported a higher prevalence in boys, potentially linked to social behaviors and higher exposure to <italic>Toxocara</italic> spp. eggs (<xref ref-type="bibr" rid="B14">14</xref>), previously related to pica, geophagia, and poor hygiene practices (<xref ref-type="bibr" rid="B43">43</xref>, <xref ref-type="bibr" rid="B44">44</xref>). A higher risk of contact with the soil and easy contamination&#x2014;due to the engagement of boys in outdoor activities and games&#x2014;have been reported as contributing factors for infection by other pathogens (<xref ref-type="bibr" rid="B45">45</xref>). These authors observed significantly more <italic>Escherichia coli</italic> on the hands of boys than on the hands of girls because boys were less likely to wash their hands with soap after playing outside. Studies have shown a positive association between seropositivity and children playing in public squares (<xref ref-type="bibr" rid="B46">46</xref>), as well as living in homes with a dirty backyard (<xref ref-type="bibr" rid="B28">28</xref>). In the present study, neither of these variables (playing in public parks or the presence of a dirty backyard) was associated with seropositivity. A study conducted in Presidente Prudente verified a high number of contaminated public parks (24/25; 96.0&#x0025;) and reported that the climatic conditions and soil characteristics in its parks were conducive to the year-round maintenance of <italic>Toxocara</italic> spp. eggs (<xref ref-type="bibr" rid="B47">47</xref>).</p>
<p>People living in urban areas are reportedly more likely to be seropositive due to higher exposure to toxocariasis agents (<xref ref-type="bibr" rid="B16">16</xref>, <xref ref-type="bibr" rid="B48">48</xref>). However, in the present study, no differences were noted when comparing children living in urban and rural locations, corroborating the findings of a nationwide serosurvey in Israel (<xref ref-type="bibr" rid="B49">49</xref>). According to these authors, the contamination of soil with <italic>Toxocara</italic> spp. eggs might be greater in urban areas and domiciled dogs, instead of stray dogs, may contribute as the primary source of human infection. In the present study, most of the studied population lived in urban areas; however, it is difficult to infer a reason justifying our findings.</p>
<p>Contact with cats or dogs was not associated with seropositivity for <italic>Toxocara</italic> spp. antibodies in children in this study, contrasting with findings from a meta-analysis of a moderate positive correlation between dogs and human prevalence and a significant positive correlation between cats and human prevalence (<xref ref-type="bibr" rid="B3">3</xref>). In addition, contact with dogs and cats was significantly associated with seropositivity, particularly in younger individuals and in regions such as the Americas, the Middle East, and the Western Pacific, according to another meta-analysis (<xref ref-type="bibr" rid="B12">12</xref>). A previous study conducted at the Veterinary Teaching Hospital in Presidente Prudente reported that only 11/165 (6.7&#x0025;) dogs were contaminated with non-embryonated <italic>T. canis</italic> eggs, suggesting low risk of toxocariasis transmission by direct contact in well-maintained dogs (<xref ref-type="bibr" rid="B50">50</xref>).</p>
<p>In this study, no information regarding the hygienic or health status of dogs or cats was collected. Nevertheless, independent of the significance of the presence of <italic>Toxocara</italic> spp. eggs on the hair, regular deworming and hygiene of pets are important considerations for minimizing environmental contamination. As a general guideline, dogs and cats older than six months should be dewormed four times a year, particularly in endemic areas, to reduce the toxocariasis risk due to pet contact and environmental contamination (<xref ref-type="bibr" rid="B12">12</xref>, <xref ref-type="bibr" rid="B51">51</xref>). Although pet neutering may not directly reflect on toxocariasis transmission, a decrease in stray pet populations can indirectly reduce environmental contamination, primarily in public parks (<xref ref-type="bibr" rid="B52">52</xref>). In addition to pet neutering and deworming, general hygiene measures may decrease exposed populations to egg ingestion from contaminated environments, particularly children (<xref ref-type="bibr" rid="B53">53</xref>).</p>
<p>The ingestion of raw/undercooked meat from paratenic hosts has been reported to be responsible for toxocariasis transmission in Brazil, with sources including sheep (<xref ref-type="bibr" rid="B54">54</xref>, <xref ref-type="bibr" rid="B55">55</xref>), chickens (<xref ref-type="bibr" rid="B56">56</xref>, <xref ref-type="bibr" rid="B57">57</xref>), and cattle (<xref ref-type="bibr" rid="B58">58</xref>). However, no association with seropositivity was observed in the present study. Although the consumption of raw/undercooked meat in children could influence seropositivity, supporting data are lacking. Thus, future investigations should assess the consumption of raw/undercooked meat as an associated risk factor for toxocariasis in children.</p>
<p>Regarding age, the frequency of seropositivity was higher in older (6&#x2013;11 years) rather than younger (1&#x2013;5 years) children, but no association to seropositivity was revealed in the logistic regression analysis. It has been stated that the behavioral habits of younger individuals increase exposure to contaminated soil. However, anti-<italic>Toxocara</italic> seropositivity increases with the age of children age due to repeated infection, leading to the persistence of antibodies (<xref ref-type="bibr" rid="B59">59</xref>).</p>
<p>Despite our promising results, the study has some limitations. First of all, as a cross-sectional study involving pediatric populations spread out over a period of time and relying on epidemiological questionnaires, inaccuracy or falseness of responses by guardians could have partially biased the results and statistical analysis. Furthermore, the findings reflect associations rather than causative factors. Second, it was not possible to verify the association between <italic>Toxocara</italic> spp. infection and different levels of exposure to pets, soil, and raw/undercooked meat. In addition, the ELISA assay (sensitivity 78.3&#x0025;; specificity 92.3&#x0025;) for antibody detection does not distinguish between recent and past toxocariasis infection due to IgG persistence. Although the absence of confirmatory tests, such as western blotting, may raise concerns about false positives, <italic>A. suum</italic> adsorption was used in previous studies conducted by our research group to prevent cross-reactivity (<xref ref-type="bibr" rid="B20">20</xref>, <xref ref-type="bibr" rid="B32">32</xref>, <xref ref-type="bibr" rid="B41">41</xref>, <xref ref-type="bibr" rid="B56">56</xref>, <xref ref-type="bibr" rid="B60">60</xref>). All children sampled were randomly invited to participate while attending routine examinations at the Brazilian Public Health Service. Unlike previous studies conducted by our research group in other vulnerable populations, this study did not include further access to pets of the participants for veterinary intervention, including deworming and vaccination. Furthermore, we could not assess environmental samples and contamination. Finally, no assessment of hematological results was possible, limiting the clinical interpretation of seropositive cases, as eosinophilia is an important laboratorial finding associated with toxocariasis in children (<xref ref-type="bibr" rid="B7">7</xref>, <xref ref-type="bibr" rid="B13">13</xref>, <xref ref-type="bibr" rid="B61">61</xref>). Future studies should include hematological markers (eosinophilia) and possibly clinical symptoms to strengthen the epidemiological interpretation, as well as environmental sampling (soil and playgrounds) to clarify the transmission routes.</p>
<p>In conclusion, the present study suggests that although toxocariasis in children may not increase over time, such seroprevalence may not decrease either, persisting (even in low levels) for long periods, particularly if no intervention has been made. Further studies should be conducted to fully establish the environmental stabilization of <italic>Toxocara</italic> spp. in such a scenario, when no mitigatory actions were taken over time. Future studies should also aim to screen toxocariasis in children in a One Health approach, with concomitant evaluation of household conditions, human and pet health status, dog/cat raising practices, and soil contamination.</p>
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</body>
<back>
<sec id="s5" sec-type="data-availability"><title>Data availability statement</title>
<p>The original contributions presented in the study are included in the article/<xref ref-type="sec" rid="s11">Supplementary Material</xref>; further inquiries can be directed to the corresponding author.</p>
</sec>
<sec id="s6" sec-type="ethics-statement"><title>Ethics statement</title>
<p>The studies involving humans were approved by the Ethics Committee of University of Western S&#x00E3;o Paulo (protocol 8,294) and protocoled in National Commission of Ethics in Research (CAAE: protocol 73476123.2.0000.5515). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants&#x0027; legal guardians/next of kin.</p>
</sec>
<sec id="s7" sec-type="author-contributions"><title>Author contributions</title>
<p>VL: Formal analysis, Investigation, Methodology, Writing &#x2013; original draft, Writing &#x2013; review &#x0026; editing. IF: Formal analysis, Investigation, Methodology, Writing &#x2013; original draft, Writing &#x2013; review &#x0026; editing. GRa: Formal analysis, Investigation, Methodology, Writing &#x2013; review &#x0026; editing. SL: Formal analysis, Investigation, Methodology, Validation, Writing &#x2013; review &#x0026; editing. GRo: Formal analysis, Investigation, Methodology, Writing &#x2013; review &#x0026; editing. RG: Formal analysis, Methodology, Software, Writing &#x2013; original draft, Writing &#x2013; review &#x0026; editing. LK: Investigation, Writing &#x2013; original draft, Writing &#x2013; review &#x0026; editing. AB: Investigation, Resources, Writing &#x2013; original draft, Writing &#x2013; review &#x0026; editing. VS: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing &#x2013; original draft, Writing &#x2013; review &#x0026; editing.</p>
</sec>
<ack><title>Acknowledgments</title>
<p>Authors are particularly grateful to all the parents and guardians who authorized the participation of their children in the present study. A special thanks to Lygia Maria Delfino and the staff of the Laboratory Jo&#x00E3;o Grigoli (Unoeste). Authors also kindly thank the Brazilian Higher Education Improvement Coordination (CAPES) for sponsoring the PhD fellowship (code 001) of VSVL and IBF.</p>
</ack>
<sec id="s9" sec-type="COI-statement"><title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="s10" sec-type="ai-statement"><title>Generative AI statement</title>
<p>The author(s) declare that no Generative AI was used in the creation of this manuscript.</p>
<p>Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.</p>
</sec>
<sec id="s12" sec-type="disclaimer"><title>Publisher&#x0027;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
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<sec id="s11" sec-type="supplementary-material"><title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fped.2025.1663016/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fped.2025.1663016/full&#x0023;supplementary-material</ext-link></p>
<supplementary-material xlink:href="Datasheet1.pdf" id="SM1" mimetype="application/pdf"/>
<supplementary-material xlink:href="Datasheet2.docx" id="SM2" mimetype="application/vnd.openxmlformats-officedocument.wordprocessingml.document"/>
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<fn-group>
<fn id="n1" fn-type="custom" custom-type="edited-by"><p>Edited by: <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/1088278/overview">Mourad Ben Said</ext-link>, University of Manouba, Tunisia</p></fn>
<fn id="n2" fn-type="custom" custom-type="reviewed-by"><p>Reviewed by: <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/2626687/overview">Wadee Abdullah Al-shehari</ext-link>, Harbin Medical University, China</p>
<p><ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/2775303/overview">Alistair Antonopoulos</ext-link>, Kreavet, Belgium</p></fn>
</fn-group>
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