







































_____________________________________________________________________________________________________ 
 
*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

