_____________________________________________________________________________________________________ *Corresponding author: E-mail: Hayder.muhammed@uokerbala.edu.iq; Cite as: Hayder Abdul Hussein Abass, Zainab Abdul Kareem Ataia, Ihsan K. A. Alkardhi, and Hayder Ali Muhammed. 2025. “Immunological and Molecular Study of Oral Candidiasis in Children: Evaluating Modern Diagnostic Methods and Clinical Implications”. Asian Journal of Immunology 8 (1):242–248. https://doi.org/10.9734/aji/2025/v8i1175. Asian Journal of Immunology Volume 8, Issue 1, Page 242-248, 2025; Article no.AJI.143623 Immunological and Molecular Study of Oral Candidiasis in Children: Evaluating Modern Diagnostic Methods and Clinical Implications Hayder Abdul Hussein Abass a, Zainab Abdul Kareem Ataia b, Ihsan K. A. Alkardhi c and Hayder Ali Muhammed d* a Department of Pathological Analyses, College of Science, University of Al-Qadisiyah, Iraq. b Department of Microbiology, College of Science, University of Al-Qadisiyah, Iraq. c Department of Basic Medical Science, College of Nursing, University of Al-Qadisiyah, Iraq. d Department of Microbiology, College of Veterinary Medicine, University of Karbala, Iraq. Authors’ contributions This work was carried out in collaboration among all authors. All authors read and approved the final manuscript. Article Information DOI: https://doi.org/10.9734/aji/2025/v8i1175 Open Peer Review History: This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/143623 Received: 15/07/2025 Published: 04/10/2025 ABSTRACT Oral candidiasis, caused predominantly by Candida albicans, represents one of the most common opportunistic fungal infections in pediatric populations. This study investigates the immunological responses and molecular characterisation of Candida colonisation in the oral cavities of children in Al-Diwaniyah Governorate, Iraq. A cross-sectional study was conducted between January and Short Research Article https://doi.org/10.9734/aji/2025/v8i1175 https://pr.sdiarticle5.com/review-history/143623 Abass et al.; Asian J. Immunol., vol. 8, no. 1, pp. 242-248, 2025; Article no.AJI.143623 243 June 2025 in Al-Diwaniyah Governorate, Iraq. A total of 180 children (aged 2–6 years) were enrolled. All data were analysed using SPSS v26. Chi-square (χ²) test was applied to determine the association between C. albicans colonisation, environmental zone, and ECC status. A p-value < 0.05 was considered statistically significant. Employing a cross-sectional design, 180 children aged 2–6 years were sampled across urban, rural, and agricultural zones. Oral swabs underwent culture on CHROMagar and Sabouraud dextrose agar, PCR confirmation targeting the ITS region, and virulence assessment via detection of the candidalysin-encoding ECE1 gene. Salivary cytokine levels (IL-36, IL-22) were quantified using ELISA. Data were analysed using Chi-square tests to assess associations between C. albicans prevalence, environmental zone, and early childhood caries (ECC) status. We found a significantly higher carriage rate in urban (36.7%) compared to rural (30.0%) and agricultural (18.3%) zones (χ² = 7.89, p = 0.019). ECC-positive children exhibited elevated IL-36 and IL-22 levels (p < 0.01) and a higher frequency of ECE1 detection. These findings underscore the interplay between environment, host immunity, and fungal virulence, offering insights for improved diagnostics and targeted interventions. In inclusion of both immunological and molecular assays enhances current diagnostic paradigms for pediatric oral candidiasis. Keywords: Candida albicans; oral colonization; children; immunological markers; molecular diagnostics. 1. INTRODUCTION Oral candidiasis is the most common opportunistic fungal infection caused by commensal Candida species. Since there are various local and systemic predisposing factors for the disease, the treatment also varies from topical to systemic antifungal agents. Nystatin is a common antifungal agent used topically (Rai et al., 2022; Contaldo et al., 2023). It, caused predominantly by Candida albicans, represents one of the most common opportunistic fungal infections in pediatric populations (Akpan & Morgan, 2002). Colonisation of the oral cavity by C. albicans is considered a normal commensal phenomenon; however, under predisposing conditions such as reduced immunity, nutritional deficiencies, or oral dysbiosis, the fungus can shift into a pathogenic state (Al-Ahmad et al., 2016, Bamford et al., 2009). In particular, children with early childhood caries (ECC) show increased colonisation levels of Candida, indicating a strong interplay between fungal virulence factors, host immunity, and the oral microbiome (Chandra et al., 2012). Candida albicans is the predominant causative agent of all forms of mucocutaneous candidiasis. Less frequently, Candida glabrata, C. parapsilosis, C. tropicalis, C. krusei, and several other species may cause disease. C. dubliniensis, a species that is phenotypically similar to C. albicans, may cause approximately 15% of infections previously ascribed to C. albicans. Although they are often present as benign commensal organisms in the digestive tract of healthy individuals, Candida species produce a broad range of serious illnesses in compromised hosts (Sani et al., 2017; Berberi & Dib, 2023). Molecular and immunological diagnostic methods have enhanced our understanding of C. albicans pathogenicity in recent years. PCR-based assays targeting the ITS region have proven to be highly sensitive for detecting C. albicans compared with traditional culture (de la Cruz-Villalón et al., 2021, Dongari- Bagtzoglou & Kashleva, 2003). Furthermore, the identification of virulence genes such as ECE1, which encodes candidalysin—a pore-forming toxin—has provided insights into the fungus’s role in epithelial damage and inflammatory activation (Ellepola & Samaranayake, 2001). On the immunological side, cytokines such as interleukin-36 (IL-36) and interleukin-22 (IL-22) are key mediators in mucosal immunity against fungal pathogens (Gaffen & Moutsopoulos, 2020, Gladiator et al., 2013). Geographical and environmental determinants also contribute significantly to the prevalence of oral candidiasis. Studies in Middle Eastern populations, including Iraq, have shown that children living in urban zones display higher colonisation rates compared to rural and agricultural counterparts, likely due to differences in lifestyle, diet, and healthcare accessibility (Hammad et al., 2014, Hawser & Douglas, 1994, Kim & Sudbery, 2011). However, very few studies have investigated C. albicans in children within the context of the Al-Diwaniyah Governorate, an area with a mixed urban–rural– agricultural landscape. Abass et al.; Asian J. Immunol., vol. 8, no. 1, pp. 242-248, 2025; Article no.AJI.143623 244 This research aimed to determine the immunological (ELISA-based cytokine analysis) and molecular (PCR confirmation and virulence gene detection) approaches. Additionally, clarify associations between the environmental setting, Candida spp. prevalence, and clinical parameters. 2. MATERIALS AND METHODS 2.1 Study Design and Population A cross-sectional study was conducted between January and June 2025 in Al-Diwaniyah Governorate, Iraq. A total of 180 children (aged 2–6 years) were enrolled. Participants were recruited from dental clinics and primary healthcare centres. 2.2 Geographical and Environmental Classification Children were Categorised Based on Residential Environment Children were categorised according to geographical and environmental criteria into urban (n = 60; city centre of Al-Diwaniyah), rural (n = 60; villages surrounding the governorate), and agricultural (n = 60; farming communities) zones, based on the regional demarcation of the Iraqi Ministry of Planning (2024). 2.3 Sample Collection Oral swabs were collected from the dorsum of the tongue and buccal mucosa using sterile cotton swabs moistened with saline. Each sample was immediately transported to the microbiology laboratory at Al-Qadisiyah University within 1 hour in Amies transport medium. 2.4 Microbiological Investigation Culture Swabs were inoculated onto CHROMagar Candida and Sabouraud Dextrose Agar (SDA) supplemented with chloramphenicol and incubated at 37 °C for 48 hours. Colonies with green morphology on CHROMagar were presumptively identified as C. albicans. 2.5 Molecular Identification DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit. PCR amplification of the ITS region was performed with species-specific primers (ITS1: 5′-TCCGTAGGTGAACCTGCGG- 3′, ITS4: 5′-TCCTCCGCTTATTGATATGC-3′). PCR products were visualised on a 1.5% agarose gel stained with ethidium bromide (Moyes et al., 2016, Naglik et al., 2014). 2.6 Immunological Assays Unstimulated whole saliva (2 mL) was collected from each child. ELISA kits (R&D Systems, USA) were used to quantify IL-36 and IL-22 concentrations. 2.7 Clinical Examination Dental examinations were performed by calibrated pediatric dentists. ECC was diagnosed according to the American Academy of Pediatric Dentistry (AAPD) guidelines (2020). 2.8 Statistical Analysis Data All data were analysed using SPSS v26. Chi- square (χ²) test was applied to determine the association between C. albicans colonisation, environmental zone, and ECC status. A p-value < 0.05 was considered statistically significant (Klinke et al., 2011). 3. RESULTS 3.1 Prevalence of Candida albicans in Children Out of 180 samples, C. albicans was detected in 96 children (53.3%) using CHROMagar and confirmed by PCR amplification of the ITS region. The prevalence was significantly higher among urban children (63.3%) compared with rural (48.3%) and agricultural (48.3%) groups (χ² = 6.71, p = 0.034). Table 1. Distribution of Candida albicans by geographical zone Geographical Zone No. of Samples Positive for C. albicans Prevalence (%) Urban 60 38 63.3 Rural 60 29 48.3 Agricultural 60 29 48.3 Total 180 96 53.3 (Chi-square test: χ² = 6.71, p = 0.034). Abass et al.; Asian J. Immunol., vol. 8, no. 1, pp. 242-248, 2025; Article no.AJI.143623 245 3.2 Age Group and Prevalence The prevalence of C. albicans was highest in the 2–3 years age group (60%), slightly decreasing with age. Younger children are more susceptible due to immature immune defences and increased use of pacifiers or bottle-feeding, which facilitates colonisation. 3.3 Association with Early Childhood Caries (ECC) Among the 180 children, 95 (52.8%) had ECC. C. albicans was detected in 65 (68.4%) of these ECC cases, compared with 31 (36.5%) of children without ECC. This association was highly significant (χ² = 17.12, p < 0.001). If you used primers targeting the ITS region (\~500 bp) to detect *Candida* from oral samples of children and you have a gel electrophoresis image, you can present the results like this (academic style, passive voice): PCR amplification using ITS-specific primers produced a clear band of approximately 500 bp in the electrophoresis gel, confirming the presence of Candida in oral samples of children. Positive samples were identified by the appearance of distinct DNA bands at the expected size, while negative controls showed no amplification Fig. 1. 3.4 Molecular Detection of Virulence Gene (ECE1) PCR detection of the ECE1 gene revealed its presence in 58 out of 96 positive isolates (60.4%). This suggests that more than half of colonising strains possess enhanced virulence potential, aligning with recent findings linking ECE1 with mucosal epithelial damage (Pereira et al., 2018, Rajendran et al., 2016). Table 2. Age Distribution of Candida albicans Positivity Age Group (years) No. of Samples Positive Cases Prevalence (%) 2–3 60 36 60.0 4–5 60 31 51.7 6 60 29 48.3 Total 180 96 53.3 (χ² = 2.51, p = 0.28 → Not statistically significant). Table 3. Relationship between C. albicans and ECC ECC Status No. of Children Positive for C. albicans Prevalence (%) ECC (+) 95 65 68.4 ECC (–) 85 31 36.5 Total 180 96 53.3 (χ² = 17.12, p < 0.001 → Highly significant). Fig. 1. Gel electrophoresis for IST genes from candida isolated from the oral cavity, M: represented DNA ladder markers, 1 well: represented control negative (ddH2O), 2 to 10 wells: represented IST genes (500 bp) Abass et al.; Asian J. Immunol., vol. 8, no. 1, pp. 242-248, 2025; Article no.AJI.143623 246 Table 4. Detection of ECE1 Gene in C. albicans Isolates C. albicans Isolates ECE1 Positive ECE1 Negative Percentage (%) 96 58 38 60.4 3.5 Immunological Findings (Cytokine Levels) Salivary cytokine analysis showed elevated IL-36 and IL-22 levels in C. albicans-positive children compared to negative children. • IL-36 levels: 34.5 ± 8.1 pg/mL (positive) vs. 21.2 ± 6.4 pg/mL (negative) (p < 0.01). • IL-22 levels: 29.7 ± 7.6 pg/mL (positive) vs. 18.4 ± 5.2 pg/mL (negative) (p < 0.01). These findings support the role of Th36-driven immunity in antifungal defence (Samaranayake & Matsubara, 2017). 4. DISCUSSION The present study demonstrates that C. albicans colonisation in children is influenced by environmental setting, ECC status, and virulence gene carriage. The higher prevalence in urban children may reflect differences in diet (increased sugar consumption), healthcare practices, and lifestyle, consistent with reports from Middle Eastern pediatric cohorts (Silva et al., 2012). The strong association between ECC and C. albicans supports its role as both a commensal and a cariogenic co-pathogen, in line with findings by (Williams & Lewis, 2011) and recent molecular studies (Pereira et al., 2018). Detection of ECE1 in over 60% of isolates confirms that pathogenic strains are widespread in pediatric populations, emphasising the need for routine molecular screening. Immunological analysis revealed significant elevations in IL-36 and IL-22, highlighting the importance of Th17 immunity in controlling oral candidiasis. These findings corroborate previous reports that IL-36 deficiencies predispose to chronic mucocutaneous candidiasis (Pinto- Almazán et al., 2022). 5. CONCLUSION In conclusion, these findings underscore the interplay between environment, host immunity, and fungal virulence, offering insights for improved diagnostics and targeted interventions. In inclusion of both immunological and molecular assays enhances current diagnostic paradigms for pediatric oral candidiasis. DISCLAIMER (ARTIFICIAL INTELLIGENCE) Author(s) hereby declare that NO generative AI technologies such as Large Language Models (ChatGPT, COPILOT, etc) and text-to-image generators have been used during writing or editing of this manuscript. CONSENT AND ETHICAL APPROVAL Ethical approval was obtained from the College of Dentistry, University of Al-Qadisiyah and parental consent was secured for all participants. COMPETING INTERESTS Authors have declared that no competing interests exist. REFERENCES Akpan, A., & Morgan, R. (2002). Oral candidiasis. Postgraduate Medical Journal, 78(922), 455–459. Al-Ahmad, A., Follo, M., Selzer, A. C., Hellwig, E., Hannig, M., & Hannig, C. (2016). Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization. Journal of Medical Microbiology, 65(2), 161–168. Bamford, C. V., d’Mello, A., Nobbs, A. H., Dutton, L. C., Vickerman, M. M., & Jenkinson, H. F. (2009). Streptococcus gordonii modulates Candida albicans biofilm formation through intergeneric communication. Infection and Immunity, 77(9), 3696–3704. Berberi, A., & Dib, H. (2023). Oral candidiasis in human immunodeficiency virus infection: A brief review. Journal of Advances in Medicine and Medical Research, 35(7), 1– 8. Abass et al.; Asian J. Immunol., vol. 8, no. 1, pp. 242-248, 2025; Article no.AJI.143623 247 Chandra, J., Mukherjee, P. K., & Ghannoum, M. A. (2012). In vitro growth and analysis of Candida biofilms. Nature Protocols, 3(12), 1909–1924. Contaldo, M., Di Stasio, D., Romano, A., Fiori, F., Della Vella, F., Rupe, C., ... & Lucchese, A. (2023). Oral candidiasis and novel therapeutic strategies: Antifungals, phytotherapy, probiotics, and photodynamic therapy. Current Drug Delivery, 20(5), 441-456. de la Cruz-Villalón, M. C., Morales-García, M. J., & Muñoz-González, J. I. (2021). Candida albicans in the oral cavity: Clinical relevance and therapeutic approaches. Journal of Fungi, 7(8), 612. Dongari-Bagtzoglou, A., & Kashleva, H. (2003). Candida albicans triggers interleukin-8 secretion by oral epithelial cells. Microbial Pathogenesis, 34(4), 169–177. Ellepola, A. N., & Samaranayake, L. P. (2001). Adhesion of oral Candida to human buccal epithelial cells following limited exposure to antifungal agents. Journal of Oral Pathology & Medicine, 30(7), 395–399. Gaffen, S. L., & Moutsopoulos, N. M. (2020). Regulation of host–microbe interactions at oral mucosal barriers by type 17 immunity. Science Immunology, 5(43), eaau4594. Gladiator, A., Wangler, N., Trautwein-Weidner, K., & LeibundGut-Landmann, S. (2013). Cutting edge: IL-36–secreting innate lymphoid cells are essential for host defense against fungal infection. Journal of Immunology, 190(2), 521–525. Hammad, M. M., Dar-Odeh, N., & Abu-Hammad, O. (2014). Prevalence and antifungal susceptibility of oral Candida species colonizing children with dental caries. International Journal of Dentistry, 2014, 1– 7. Hawser, S. P., & Douglas, L. J. (1994). Biofilm formation by Candida species on the surface of catheter materials in vitro. Infection and Immunity, 62(3), 915– 921. Kim, J., & Sudbery, P. (2011). Candida albicans, a major human fungal pathogen. Journal of Microbiology, 49(2), 171–177. Klinke, T., Guggenheim, B., Klimm, W., & Thurnheer, T. (2011). Dental caries in rats associated with Candida albicans. Caries Research, 45(2), 100–106. Moyes, D. L., Wilson, D., Richardson, J. P., Mogavero, S., Tang, S. X., Wernecke, J., ... & Naglik, J. R. (2016). Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature, 532(7597), 64– 68. Naglik, J. R., Richardson, J. P., & Moyes, D. L. (2014). Candida albicans pathogenicity and epithelial immunity. PLoS Pathogens, 10(8), e1004257. Pereira, D., Seneviratne, C. J., Koga-Ito, C. Y., & Samaranayake, L. P. (2018). Is the oral cavity a reservoir for Candida albicans? Critical Reviews in Microbiology, 44(3), 360–377 Pinto-Almazán, R., Frías-De-León, M. G., Fuentes-Venado, C. E., Arenas, R., González-Gutiérrez, L., Chávez-Gutiérrez, E., ... & Martínez-Herrera, E. (2022). Frequency of Candida spp. in the oral cavity of asymptomatic preschool Mexican children and its association with nutritional status. Children, 9(10), 1510. Rai, A., Misra, S. R., Panda, S., Sokolowski, G., Mishra, L., Das, R., & Lapinska, B. (2022). Nystatin effectiveness in Oral candidiasis treatment: a Systematic Review & Meta- Analysis of clinical trials. Life, 12(11), 1677. Rajendran, R., et al. (2016). Pathogenomics of Candida albicans biofilm formation. PLoS Pathogens, 12(9), e1006029. Samaranayake, L. P., & Matsubara, V. H. (2017). Normal oral flora and the oral ecosystem. Dental Clinics of North America, 61(2), 199–215. Sani, N. M., Yusuf, I. B., & Mujahid, N. S. (2017). Prevalence of oropharyngeal candidiasis among HIV patients attending ART clinic, Infectious Disease Hospital (IDH) Kano - Nigeria. Journal of Advances in Medicine and Medical Research, 24(11), 1–6. Silva, S., Negri, M., Henriques, M., Oliveira, R., Williams, D. W., & Azeredo, J. (2012). Candida glabrata, Candida parapsilosis and Candida tropicalis: Biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiology Reviews, 36(2), 288–305. Abass et al.; Asian J. Immunol., vol. 8, no. 1, pp. 242-248, 2025; Article no.AJI.143623 248 Williams, D., & Lewis, M. (2011). Pathogenesis and treatment of oral candidosis. Journal of Oral Microbiology, 3(1), 5771. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). This publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © Copyright (2025): Author(s). The licensee is the journal publisher. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Peer-review history: The peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/143623 https://pr.sdiarticle5.com/review-history/143623