The Prevalence of Candida albicans and Malassezia globosa in Preschool Children with Severe Early Childhood Caries: A Case-Control Study

Background: This cross-sectional study aimed to identify the prevalence of Candida albicans and Malassezia globosa in children with severe early childhood caries and caries-free children in Hong Kong. Methods: This study first recruited a total of 80 children aged between 48 and 72 months old, 40 children with severe early childhood caries, and 40 caries-free children. The children were then further divided into four groups, with 20 children in each group: Group 1: Severe early childhood caries—C. albicans, Group 2: Severe early childhood caries—M. globosa, Group 3: Caries-free—C. albicans and Group 4: Caries-free—M. globosa. Saliva, plaque, and caries lesion samples were collected from participants with severe early childhood caries, while only saliva and plaque samples were collected from caries-free participants. Caries status of the primary molars was assessed using WHO’s decayed, missing, and filled tooth index, and the severity of cavitated lesions was determined based on International Caries Diagnosis and Assessment System criteria as caries code 5 or 6. The samples were analyzed using an Internal Transcribed Space and Quantitative Real-Time Polymerase Chain Reaction. Results: C. albicans was more prevalent in saliva and plaque samples of severe early childhood caries than in the caries-free group. Proportion of C. albicans in both saliva and plaque samples differed significantly between severe early childhood caries and caries-free groups (p < 0.05). Within the severe early childhood caries group, the proportion of children with C. albicans varied between 6 and 46%. No significant difference in M. globosa load was found between plaque samples of the severe early childhood caries and caries-free groups (p = 0.159). Conversely, no significant difference in M. globosa load was observed between saliva samples of severe early childhood caries and caries-free groups (p = 0.051). Conclusions: This study demonstrated a strong association between C. albicans and severe early childhood caries. M. globosa was detected in both the caries-free and severe early childhood caries groups, albeit at low levels.


Introduction
Early childhood caries (ECC) poses a significant global public health challenge.According to a recent report by the World Health Organization (WHO), dental caries affects 60-90% of children worldwide [1].Early childhood caries, a specific type of caries that affects preschool children, is characterized by the presence of one or more decayed lesions and missing or filled tooth surfaces in any primary tooth in children younger than 71 months of age [2].Severe early childhood caries (S-ECC) is recognized as a particularly aggressive and difficult-to-treat variant of this disease [1,3].Previous studies have shown that over 90% of decayed teeth in affected children go untreated [4], resulting in extensive damage to the tooth structure, severe pain, tooth loss, and a diminished quality of life [5].It is worth noting that more than 50% of preschool children in Hong Kong suffer from dental caries [4].
Recent in vivo and in vitro studies have suggested that yeasts, mainly Candida, cocolonize with Streptococci to form carious lesions, especially in cases of S-ECC [6][7][8].This cross-kingdom synergism between Streptococci and Candida leads to enhanced biofilm development, facilitated by the simultaneous up-regulation/activation of genes related to biofilm matrix production and carbohydrate metabolism.As a result, this creates a hypervirulent ecological niche that contributes to the pathogenesis of S-ECC [6,9].In a landmark study, Xiao et al. [6] used salivary and plaque analysis to propose that the presence of Candida species, specifically C. albicans, results in the formation of a highly acidogenic and acid-tolerant bacterial community in S-ECC.Therefore, Candida species are hypothesized to be equally significant contributors to the pathological process, particularly in cases of S-ECC [9].Candida species possess critical virulence traits that promote the development of S-ECC.Non-albicans Candida and C. albicans can exist as mono, dual, or mixed infections.They are both acidogenic and aciduric, contributing to enamel demineralization and dentin collagenolysis in deep dentinal lesions [9].However, the precise role of Candida in the pathogenicity of S-ECC has yet to be clarified.While some studies have shown no significant correlation between the presence of C. albicans and caries, others have clearly demonstrated the cariogenicity of C. albicans in rats fed high sucrose/glucose diets [10].A recent systematic review and meta-analysis [11] concluded that children with ECC have significantly greater odds of having a Candida biome compared to caries-free children, with C. albicans being the most predominant species.
Apart from Candida, fungi in the genus Malassezia (previously known as Pityrosporum) are also associated with human skin diseases, such as atopic dermatitis, seborrheic dermatitis, and dandruff [12].Malassezia has been identified to consist of multiple species, namely M. globosa, M. furfur, and M. pachydermatis, based on their characterization of ribosomal DNA and their ability to grow in different media [13].A previous study has examined the supragingival mycobiome in dental caries and found that not only was M. globosa more abundant in caries-free subjects, but it also outnumbered Candida [7].Moreover, it has been suggested that M. globosa has an inhibitory effect against Streptococcus mutans and inhibits biofilm formation by Staphylococcus aureus via a secreted aspartyl protease [7].
The emerging evidence presented above clearly indicates the potential importance of the mycobiome, specifically the Candida biome, in S-ECC.However, there are currently no studies available on the Candida biome of S-ECC from Hong Kong.This is clinically relevant, as it is important to understand the strategic intervention approaches required to manage this widespread childhood disease, which could be considered a silent epidemic in Hong Kong and worldwide.Since Candida is a prevalent colonizer of ECC [6,9], it is necessary to evaluate its role and the mechanisms by which it contributes to the caries process by assessing its cariogenic attributes [8].Therefore, this study aimed to compare the prevalence of Candida, specifically Candida albicans and Malassezia globosa, in S-ECC and caries-free (CF) children in Hong Kong.The null hypothesis of this study was that there is no difference in the prevalence of C. albicans and M. globosa in S-ECC children compared to children without caries.

Participant Recruitment and Examination
Prior approval from the HKU/HA HKW Institutional Review Board (UW 20-270) was obtained for this study.Eighty children between the ages of 48 and 72 months, who were attending the Multi-Specialty Clinic of the Institute for Advanced Dentistry, Faculty of Dentistry, the University of Hong Kong, were recruited as participants.Informed consent was obtained from the parents of each child participant.A full dental examination was conducted for all the healthy and co-operative participants by a postgraduate student in Paediatric Dentistry, under the close supervision of two experienced clinicians (Cynthia Kar Yung Yiu and Prasanna Neelakantan).

Experimental Design
The experimental study design for the present study is shown in Figure 1.This crosssectional study was conducted on 80 healthy children aged between 48 and 72 months old, 40 children with severe early childhood caries, and 40 caries-free children.The children were then further divided into four groups, with 20 children in each group: Paediatric Dentistry, under the close supervision of two experienced clinicians (Cynthia Kar Yung Yiu and Prasanna Neelakantan).

Experimental Design
The experimental study design for the present study is shown in Figure 1.This crosssectional study was conducted on 80 healthy children aged between 48 and 72 months old, 40 children with severe early childhood caries, and 40 caries-free children.The children were then further divided into four groups, with 20 children in each group:   The anticipated presence proportion of Candida in the S-ECC lesions of children was estimated as 31.9%from a previous pilot study [9], and the sample size was calculated based on precision.To achieve a 95% confidence interval with a width of 20% (i.e., ±10%), a total of 84 children were required.However, due to the COVID-19 outbreak and suspension of dental services, only samples from 80 children could be obtained.

Inclusion Criteria
The American Academy of Paediatric Dentistry classification was used to identify children with S-ECC.The children selected for sample collection had at least two decayed (occlusal or proximal lesions) asymptomatic primary molars.The severity of the cavitated lesions was assessed by the same examiner using the well-established International Caries Diagnosis and Assessment System (ICDAS) classification.According to ICDAS, S-ECC was further sub-categorized as code 5 (a distinct cavity with visible dentine involving less than half the tooth surface) and code 6 (an extensive and distinct cavity with visible dentine affecting more than half of the surface).For this study, samples were only collected from decayed lesions coded as 5 and 6, as these are the most severe cavitated dentine caries lesions according to the ICDAS classification system [14].

Exclusion Criteria
The exclusion criteria for this study included children who had been on antibiotics or antifungals within the past 4 weeks, those who were wearing orthodontic appliances, children with any congenital tooth anomaly, and children whose teeth had a likelihood of pulp exposure during the caries removal process or were uncooperative during dental examination.

Caries Diagnosis
The caries status was evaluated using the WHO criterion of decayed, missing, and filled (dmft) tooth index, while the severity of cavitated lesions was determined as either caries code 5 or 6 according to ICDAS criteria [13], as described previously.Intra-examiner reproducibility at the tooth surface level was assessed using Cohen's kappa coefficient (0.82).A single, trained, and calibrated operator collected infected dentine samples from 40 S-ECC children under a strict aseptic protocol.

Site-Specific Sample Collection
Three samples, saliva, plaque, and caries lesions, were collected from each participant with S-ECC.From each CF participant, two samples, saliva and plaque samples, were collected.The samples from caries lesions were obtained from symptom-free, caries-active, deep dentin lesions categorized as ICDAS codes 5 and 6.The teeth were cleaned using a prophy brush without the use of prophy paste and then dried.A sterile spoon excavator was used to collect the infected dentine samples from the occlusal and proximal lesions [9].If gingival bleeding occurred during sample collection or if gingival bleeding contaminated the cavity, the samples were excluded.Immediately after collection, the samples from caries lesions were placed in a sterilized container containing PBS solution.
For the plaque samples, a supragingival dental plaque sample was collected from the buccal surfaces of maxillary incisors [15][16][17] using a sterilized cotton swab [17].Samples were collected from clinically sound gingival areas for the CF group, and around the most cavitated enamel for the cervical and proximal caries group.Immediately after collection, the cotton swab was placed in a sterilized container containing PBS solution.Regarding the saliva samples, 2 mL of unstimulated whole saliva was collected from each child by having them spit directly into a sterile container.All samples were then transferred immediately to the laboratory for analysis.

Internal Transcribed Space (ITS) and Quantitative Real Time Polymerase Chain Reaction (PCR)
The samples were initially centrifuged at 9880 rpm, and the resulting pellet was resuspended in 80 µL of 20 mM Tris-HCl, pH 8.0; 2 mM EDTA; 1.2% Triton with 20 µL of 20 mg/mL lysozyme and incubated overnight at 37 • C. The DNA was then extracted from the samples using the manufacturer's protocol (QIAamp DNA Mini Kit, QIAGEN, Hilden, Germany).PCR analysis was conducted using the universal yeast primer sequences ITS4 and ITS86, as well as the universal primers for M. globosa.The load of the organisms was measured in real time using a Taqman TAMRA probe.Table 1 shows the sequence of primers for C. albicans and M. globosa.

Organism Sequence
C. albicans

Statistical Analysis
The baseline data, including Ct values of C. albicans and M. globosa for the samples, were entered into a Microsoft Office Excel (Microsoft, Redmond, WA, USA) spreadsheet.For the analysis of C. albicans data, statistical tests such as Fisher's exact test and binomial exact test were used.Fisher's exact test was employed to compare the prevalence of C. albicans in saliva and plaque samples between the S-ECC and CF groups.A binomial exact 95% confidence interval was also provided to estimate the population prevalence (proportion of children having C. albicans) within the S-ECC group.For the analysis of M. globosa data, a two-sample t-test was performed when the results followed a normal distribution.However, if the results did not follow a normal distribution, the Mann-Whitney U test was performed.The statistical analysis was performed using SPSS Version 28.0 (SPSS Inc., Chicago, IL, USA).The results were considered statistically significant at p ≤ 0.05.

Demographic Characteristics of the Participants
The study included a total of 80 children, consisting of 48 boys and 32 girls.Among them, 40 children had S-ECC, and 40 children had CF.All participants were asked about their history of antibiotics or antifungal treatment for any condition.The participants were healthy children aged between 48 and 72 months, with a mean age of 52 months.A total of 200 samples were collected, including saliva, plaque, and caries samples for each S-ECC participant (120 samples from the S-ECC group), and saliva and plaque samples for each CF child (80 samples from the CF group).Two samples (1 from the S-ECC group and 1 from the CF group for processing C. albicans) were excluded due to blood contamination.

Prevalence of Candida albicans
Real-time PCR (RT-PCR) analysis was performed to determine Candida albicans load, and the results are presented in Table 2, showing the log number of C. albicans in each sample.The PCR analysis clearly indicates that the load of C. albicans in saliva, plaque samples, and the samples from the caries lesions obtained from the S-ECC children was higher compared to the samples isolated from CF children.Intriguingly, the load of C. albicans was not detected in either the plaque or saliva samples from all CF children.This could be due to the C. albicans load in these samples being much lower than the detection threshold level.Table 3 represents the results of Fisher's exact test, comparing the proportion of C. albicans in saliva and plaque samples between children with S-ECC and CF children.In the S-ECC group, the C. albicans proportion was 10.5% in saliva samples and 15.8% in plaque samples, whereas in the CF group, it was 0% in both samples.The differences in the C. albicans proportion between the S-ECC and CF groups for both saliva and plaque samples were found to be statistically significant (p < 0.05).Table 4 shows the proportion of C. albicans in saliva and plaque samples in the S-ECC group.The proportion of C. albicans varied between 6 and 46% based on the binomial exact 95% confidence interval (Table 4).Table 6 compares the average cycle of threshold value of M. globosa in plaque and saliva samples between the S-ECC and CF groups.No significant difference was found in the mean load of M. globosa between the plaque samples of the S-ECC (39.5 ± 1.3) and CF groups (40.0 ± 0.9) (p = 0.159).Conversely, no significant differences were observed in the mean load of M. globosa between the saliva samples of the S-ECC group (38.3 ± 1.4) and CF group (40.0 ± 0.9) (p = 0.051).

Discussion
Fungi, specifically Candida, are commensal microorganisms that can colonize dental surfaces [18].C. albicans is the most frequently found Candida species in the oral cavity [9].Our PCR analysis revealed a significantly higher load of C. albicans in saliva, plaque, and caries lesion samples from S-ECC children compared to samples from CF children.This finding is consistent with previous studies.Sziegoleit et al. [19] reported a higher frequency of Candida species in saliva samples from individuals with active caries (67%) compared to CF individuals (2%).
Similarly, Akdeniz et al. [20] found a statistically significant difference in Candida prevalence between caries and CF children.Our study further supports the association of C. albicans with dental caries, as it was exclusively identified in children with S-ECC.These findings are in line with the growing evidence suggesting the potential role of C. albicans in caries development in preschool children.Our results also showed a higher proportion of C. albicans in the S-ECC group compared to the CF group in both saliva and plaque samples.However, no significant difference was observed in the prevalence of M. globosa between the plaque and saliva samples of the S-ECC and CF groups.Therefore, the null hypothesis that there is no difference in the prevalence of C. albicans and M. globosa in S-ECC children compared to CF children has to be partially rejected.Reducing C. albicans colonization can be an effective approach in reducing caries risk.Further investigations are warranted.
A series of seminal studies [8,[21][22][23][24] have demonstrated that Candida species possess biofilm-forming abilities, acidogenic and aciduric potential, and other enzyme-mediated virulence attributes, all of which could contribute to cariogenicity.These fungi can survive in extremely low pH environments and produce enamel-dissolving short-chain carboxylic acids, as well as several protein-degrading hydrolases.The production of these acids leads to demineralization of enamel and dentine, while hydrolases (such as hemolysins, phospholipases, DNAses, acidic hydrolases) have the potential to degrade the organic components of dentine.
Within the S-ECC group, we observed a wide range in the proportion of children with C. albicans, varying between 6 and 46%.This high variability can be attributed to differences in the dmft scores of the children.The presence of C. albicans may contribute to a lower maintenance of salivary pH, which can lead to the initiation of dental caries [25].This finding is consistent with previous studies that reported a correlation between the isolation of yeast and an increased number of caries lesions [15,26,27].The severity of caries may be attributed to the higher acidogenic potential of C. albicans [17].A decrease in pH levels in the oral cavity creates a favorable environment for the activity of extracellular enzymes, which serve as important virulence factors for Candida [28].Ten Cate et al. [29] further reported that C. albicans not only has the ability to adhere to hydroxyapatite (HAP), but they also dissolve HAP to a greater extent (approximately 20-fold) compared to S. mutans.In addition to adhering to tooth surfaces, C. albicans also exhibits high collagenolytic activity, denaturing collagen in exposed dentine [16].
Intriguingly, the prevalence of C. albicans was not detected in any of the saliva or plaque samples from CF patients.It is possible that the load of C. albicans in caries-free children was too low to be detected by RT-PCR analysis.This could be due to factors such as good oral hygiene practices.Thomas et al. [17] reported that Candida carriage ranges from 7 to 21% in CF children.Our finding contradicts the study by Thomas et al. [16], which demonstrated 100% Candida prevalence in saliva samples of caries-free children.However, our findings are consistent with the study by Thaweboon et al. [30], which reported an absence of Candida in the CF group.A future longitudinal study that follows a cohort of children over time to investigate the development of the oral microbiome and the prevalence of C. albicans in children with and without early childhood caries would provide a more comprehensive understanding of the relationship between C. albicans and early childhood caries.
Apart from C. albicans, other fungi are also commonly found in dental plaque in the oral cavities of children suffering from S-ECC [15].These fungi are believed to contribute to caries progression by facilitating carbohydrate fermentation and producing acids.One such fungus is Malassezia, which is a lipid-dependent yeast known to be highly prevalent and abundant [31].Interestingly, this study revealed that M. globosa was present in both the S-ECC and CF groups, suggesting that its presence alone may not be a definitive indicator of caries development.Baraniya et al. [7] reported a significant association between M. globosa and CF children.This could be attributed to the inhibitory effect of M. globosa against S. mutans through the secretion of antibacterial substances.Moreover, M. globosa can also inhibit biofilm formation by Staphylococcus aureus through a secreted aspartyl protease [32].This suggests that M. globosa may play a role in preventing caries development by inhibiting the growth of cariogenic bacteria and preventing the formation of biofilms.Further research is needed to fully understand the role of M. globosa and other fungi in dental plaque and caries progression.In future studies, increasing the sample size and incorporating different investigation methods, such as metagenomic analysis and next-generation sequencing, can offer a more comprehensive understanding of the microbial dynamics involved in the relationship between the Candida biome and S-ECC.
There is compelling evidence to suggest that S-ECC has a detrimental impact on the quality of life of affected children, making it a significant public health concern [3,33].These prevalence findings highlight the necessity for the development of more effective evidencebased strategies for preventing S-ECC [4].However, without a clear understanding of the underlying causes of the disease, it is challenging to provide appropriate care to alleviate the condition and to design suitable care programs.
This study has a few notable limitations.First, the small sample size and recruitment of preschool children only from the Multi-Specialty Clinic limit the representativeness of the study to the wider population of children in Hong Kong.Additionally, the study's focus on children aged 48-72 months may not capture the full spectrum of severe early childhood caries.To address these biases, future studies could recruit participants from multiple clinics or regions in Hong Kong, increase the sample size, and include a wider age range of children with severe early childhood caries.This would enhance the study's generalizability.Second, the exact quantity of M. globosa could not be determined due to the absence of standard DNA samples.Thirdly, the study did not investigate the proportion of C. albicans and M. globosa or their association with the severity of the caries condition.Lastly, in vivo research was conducted on young children, which posed challenges related to compliance and obtaining parental consent.These factors hinder proper clinical examination and sample collection.

Figure 1 .
Figure 1.Flowchart of the experimental design.

Figure 1 .
Figure 1.Flowchart of the experimental design.

Table 1 .
Sequence of primers for C. albicans and M. globosa.

Table 2 .
The load of C. albicans from samples isolated from patients.

Table 3 .
Comparison of C. albicans in saliva and plaque samples between severe early childhood caries and caries-free groups.

Table 4 .
The proportion of children with C. albicans in the severe early childhood caries group.

Table 5
The Ct values in all samples, both in the S-ECC and CF groups, were above 30, indicating a very low M. globosa load.
represents the cycle threshold values (Ct mean values) of M. globosa.

Table 5 .
The average cycle of threshold value of M. globosa from samples isolated from patients.

Table 6 .
Comparison of the average cycle of threshold value of M. globosa in saliva and plaque samples between severe early childhood caries and caries-free groups.