International Journal of
Environmental Research
and Public Health
Article
Candida Species in Children Undergoing Orthodontic
Treatment with Removable Appliances: A Pilot Study
˛ 1 , Magdalena Sycińska-Dziarnowska 1 , Gianrico Spagnuolo 2,3 ,
Aleksandra Brzezińska-Zajac
Liliana Szyszka-Sommerfeld 1, *,† and Krzysztof Woźniak 1,†
Citation: Brzezińska-Zajac,
˛ A.;
Sycińska-Dziarnowska, M.;
Spagnuolo, G.; Szyszka-Sommerfeld,
L.; Woźniak, K. Candida Species in
Children Undergoing Orthodontic
Treatment with Removable
Appliances: A Pilot Study. Int. J.
Environ. Res. Public Health 2023, 20,
4824. https://doi.org/10.3390/
ijerph20064824
Academic Editor: Paul B. Tchounwou
Received: 28 January 2023
Revised: 1 March 2023
Accepted: 7 March 2023
Published: 9 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Environ. Res. Public Health 2023, 20, 4824. https://doi.org/10.3390/ijerph20064824
1 Department of Orthodontics, Pomeranian Medical University in Szczecin, Al. Powst. Wlkp. 72,
70-111 Szczecin, Poland
2 Department of Neurosciences, Reproductive and Odontostomatological Sciences,
University of Naples “Federico II”, 80131 Napoli, Italy
3 School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
* Correspondence: liliana.szyszka@gmail.com
† These authors contributed equally to this paper.
Abstract: The purpose of this study was to analyze the effect of orthodontic treatment with removable
appliances on the growth of Candida spp. in children undergoing orthodontic treatment. The
study included 60 patients of equal numbers as to gender from the orthodontics department of the
Pomeranian Medical University in Szczecin, Poland. All patients were aged 6–12 years and were
qualified for orthodontic treatment with removable appliances. The following examinations were
performed on the day of treatment initiation (T1) and 6 months after the start of treatment (T2); a
collection of oral swabs for culture on Sabouraud’s medium and the identification of fungal colonies
using the VITEK® 2 YST. At T1, 42 (70%) subjects, were free of Candida, while after 6 months of
treatment, the number decreased to 25 (41.67%). Two types of fungi, C. albicans and C. parapsilosis,
predominated in the test performed at T1. The study at T2 showed that C. albicans most frequently
colonized the oral cavity in 23 children (38.33%). Three new strains C. dubliniensis, C. kefyr, and
C. krusei were identified at T2. Statistical analysis showed a significant correlation between the culture
results and the age of the patient at T2. Patients older than 9 years had significantly more positive
tests. Orthodontic treatment with removable appliances contributes to increased oral colonization by
Candida spp.
Keywords: Candida species; Candida albicans; orthodontic treatment in children; removable orthodontic
appliance; oral health during orthodontic treatment
1. Introduction
Removable orthodontic appliance treatment is a popular method of orthodontic ther-
apy among young, growing patients. The undoubted advantages of removable appliances
include the ease of maintaining hygiene, both of the appliance and of the entire oral cavity.
There are no restrictions on access to the tooth surface during cleaning. In adolescent
patients, who quite often struggle to maintain proper oral hygiene, this is an important
factor in favor of treating malocclusion with removable devices [1,2]. On the other hand,
orthodontic treatment with removable devices is often a rather uncomfortable process. For
the youngest patients, appliances are perceived as “foreign bodies”, placed in a highly
sensitive area. This can result in difficulties in accepting the plate, both for physiological
reasons such as discomfort while wearing it or difficulties in activities previously per-
formed subconsciously such as swallowing saliva, as well as for psychological reasons
such as questioning of the appearance and the effect on the child’s pronunciation during
treatment [3].
Removable orthodontic appliances are made up of several components, among which
the acrylic plate that makes up the majority of the surface of the appliance is the part
https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2023, 20, 4824 2 of 14

that most affects the oral microbiota. The acrylic plate is considered a major risk factor
for allergic lesions and causing fungal infections in the oral cavity. The substance in-
cluded in the acrylic plastic used in orthodontics is methacrylic acid methyl ester (methyl
methacrylate monomer, MMA-C5H8O2). According to the classification of the substance
in accordance with Annex VI of the Regulation of the European Parliament and of the
Council of 16 December 2008, MMA is classified as highly flammable, irritant to the skin
and respiratory tract, and sensitizing upon skin contact. However, it is not considered
a human carcinogen [4]. At the end of the polymerization process, depending on the
conditions under which it is carried out, between 0.2% and 7% of free methyl methacrylate,
the so-called residual monomer, remains in the appliance. It is in methacrylate that most of
the causes of sensitization reactions are attributed which can lead to changes in the oral
microflora. In recent years, opportunities have been sought to reduce the number of Candida
species in removable appliances. In a study by Shahabi et al., the addition of chitosan
nanoparticles (NPs), known for their antimicrobial properties, to cold-cured acrylic resin
had no significant negative effect on its flexural and compressive strength [5].
A healthy oral cavity provides a natural habitat for a diverse group of microorganisms.
The ecological niches are primarily bacteria, fungi, protozoa, or mycoplasmas, and in some
cases, viruses are also identified. All of them together mainly inhabit the gastrointestinal
tract, including just the oral cavity, which is its first section and forms the endogenous
microflora, also known as the microbiota [6]. The discovery of the ability of host cells
and bacterial cells to communicate with each other proves the existence of a network of
relationships forming one large ecosystem that can determine the health and disease of the
human body [7,8]. Dworecka-Kaszak [9] noted the occurrence of 74 species of fungi that
could be grown under laboratory conditions. Among them, Candida (75%), Cladosporium
(65%), Aureobasidium (50%), Saccharomyces (50%), Aspergillus (35%), Fusarium (30%), and
Cryptococcus (20%) predominated. The microbiome located in the oral cavity is characterized
by a fairly high stability in the abundance of individual groups of microorganisms so that
the individual microbial species present there coexist with each other under conditions
of homeostasis. The qualitative composition of the human microbiome is more complex
and individually diverse than initially thought. It is subject to development and constant
change. The microbiome is influenced not only by diet or the type and quality of food
consumed but also by age, cultural and social conditions, and genetic and environmental
factors [10].
Yeast-derived fungi, which include all of the Candida species, have not been clearly
classified as strictly pathogenic. Their importance in oral biocenosis has not yet been
clearly explained. Some authors believe that they are present in the healthy oral cavity
as commensal microorganisms, and thus are part of its physiological microflora. On the
other hand, according to other researchers, Candida fungi are pathogens, being the cause
of systemic mycoses of many organs, including the oral cavity [11]. Depending on the
literature, the prevalence of fungal infections in healthy study volunteers ranges from
20% to 60%. In addition, it has been proven that fungi in the oral cavity in children
and adolescents are detected twice as often as in healthy adults [12]. The most common
oral yeast fungus is Candida albicans. It is also considered the main causative agent of
oral candidiasis. The recently conducted systematic review showed Candida colonies
increase, especially C. albicans species, during the first month of therapy with removable
orthodontic appliances, followed by a decrease after a few months [13]. Less commonly
described are C. crusei, C. glabrata, C. tropicalis, C. parapsilosis, C. famata, C. pseudotropickalis,
and C. dubliniensis. These fungi can reside in the oral cavity for a long time without
producing any disease symptoms. However, when the body’s homeostasis is disturbed
and unfavorable conditions exist, they can cause risk to the health and sometimes even
to the life of their host. Most of the described cases of oral candidiasis involve patients
using prosthetic restorations and orthodontic appliances—mainly removable—as well as
patients burdened with immune-compromised diseases, including AIDS or autoimmune
diseases [14,15]. The pathogenic properties of C. albicans are manifested by its antigenic and
Int. J. Environ. Res. Public Health 2023, 20, 4824 3 of 14

morphological variability, ability to adhere to vascular endothelium, immunomodulatory

activities, and production of lytic enzymes.
A healthy body benefits from a multitude of defense mechanisms that protect it from
Candida expansion. These include a continuous process of exfoliation of epithelium, the
flow of saliva and the organic and inorganic components it contains, the pH of the mucous
membranes and skin, the coexistence of saprophytic bacteria competing for a place in the
host cells or, finally, an efficient immune system. Superficial colonization of the oral cavity
by the fungus is conditioned by its overcoming local environmental conditions, such as
the salivary barrier or the continuity of the mucosa. Once the microorganisms penetrate
inside the tissues, the colonization transforms into an infection. Local factors that increase
the risk of oral candidiasis include changes in the composition of the microflora, a decrease
in salivary flow, a diet rich in carbohydrates, and thinning and/or ulcerated epithelium
resulting from the use of not-fitting dental restorations or orthodontic appliances. Moreover,
regarding systemic factors, immunosuppression, prolonged antibiotics use, endocrine
disruption, and folic acid and iron deficiencies should be mentioned [12,16].
In addition, the patient should be instructed on the necessity and importance of
proper oral hygiene. Local irritants should be eliminated and properly selected vitamin
supplementation should be implemented to correct deficiencies [17]. There are numerous
publications in the literature on the effect of acrylic restorations on the adult population.
Kozak et al. studied the effect of fixed orthodontic appliances on the distribution of dental
biofilm [18]. However, few studies in this area concern removable orthodontic appliances
used by the youngest patients [19–21].
Given the paucity of research on Candida species in children undergoing orthodontic
treatment with removable appliances, the aim of this study was to evaluate the impact of
orthodontic treatment with removable appliances on the growth of Candida spp.

2. Materials and Methods

This study was conducted in accordance with the guidelines of the Helsinki Declara-
tion and the protocol was approved by the Local Bioethics Committee of the Pomeranian
Medical University in Szczecin, Poland (protocol number: KB-0012/55/17). Parents of
participating patients gave written consent for their children to participate in the research
project. At the same time, they were informed in detail about the type of research, the
technique used to perform it, and the possibility of opting out of participation in the study
at any stage.

2.1. Characteristics of the Study Group

The study included 60 patients (30 girls and 30 boys) of the Department of Orthodon-
tics at the Pomeranian Medical University in Szczecin, Poland, aged 6 to 12 years, who were
qualified for orthodontic treatment with removable appliances; Schwarz active plate (upper
and/or lower). All the subjects had to meet all of the inclusion criteria, and individuals who
did not meet all of the listed inclusion criteria were excluded from the study. The following
inclusion criteria were applied: malocclusion, no syndrome diagnosis, female and male
gender, no coexistence of systemic diseases, no drugs taken that could affect the results, also
without taking antibiotics for 6 months before the start of the study, no history of trauma or
surgical treatment in the orofacial region, no previous orthodontic treatment, and voluntary
consent to participate in the research. Patients with good/optimal oral hygiene (<40%)
as assessed by the approximal plaque index (API) were included in the study. Subjects
with systemic diseases, taking medications that could affect the research outcomes, taking
antibiotics 6 months or less before the start of the study or during the research, a history of
trauma or surgery in the orofacial area, previous orthodontic treatment, as well as subjects
with abnormal lip seal, mouth breathing, and/or individuals who were mentally unable
to participate in the study procedures were excluded from the group. Decayed, missing,
and filled teeth (DMFT) in permanent dentition and decayed, extraction-needed, and filled
teeth (dmft) in the primary dentition index value > 6 and/or average/poor oral hygiene
Int. J. Environ. Res. Public Health 2023, 20, 4824 4 of 14

according to the API were criteria for excluding a patient from participating in the study.
Both patients and their parents were instructed about the requirement to use the devices
for 14–16 h a day, in order to achieve the best therapeutic results. Patients were advised to
attend follow-up appointments every 4–5 weeks.
The results of the subject and physical examination of the participating patients were
recorded in the orthodontic examination card. In addition, due to the need to include addi-
tional data obtained for the purpose of this research project, a special patient examination
card was created. The card recorded the results of microbiological cultures—the appearance
of Petri dishes with Sabouraud’s medium after an appropriate incubation period and the
results of fungal identification tests.

2.2. Collection of Samples and Preparation and Isolation of Candida colonies, Identification of
Candida Species by VITEK® 2 YST
The following tests were performed on each patient on the day of handing out the
devices (T1) and 6 months after the onset of treatment (T2):
1. Taking swabs from the oral cavity including the hard palate, buccal mucosa, and
tongue using sterile swab sticks with a transport medium;
2. Performing cultures on Sabouraud’s medium;
3. Identification of fungal colonies was performed using the VITEK® 2 YST automated
card system (bioMérieux, Warszawa, Poland).
During the intraoral examination, a dental diagram was completed. The degree of
loosening of deciduous teeth, the presence of carious cavities, and the degree of attrition of
both deciduous teeth (physiological attrition) and permanent teeth (pathological attrition)
were also noted. The dental status index for permanent teeth (DMFT) and deciduous teeth
(dmft) was calculated. Oral hygiene was assessed during the examination using the API.
The canine classes and first angle classes were initially assessed, as well as the amount of
overbite and overjet. The final verification of the obtained results was carried out on the
diagnostic cast of each patient. Each patient’s records consisted of an analysis of casts; a
panoramic radiograph; and extraoral and intraoral photographs documenting the condition
of the teeth, periodontium, and surrounding tissues, which also served as study material.
When handing over the removable orthodontic appliance, detailed instructions on oral
hygiene and appliance care were given. A brushing technique based on the principle of
sweeping (roll) was recommended. All patients were advised to brush their teeth twice
a day with fluoride toothpaste (1450 ppm) for at least 4 min. In younger children and
those with less manual dexterity, it was recommended that an adult monitor the brushing
process each time and repeat the brushing activity if necessary. Each child’s toothbrush
was individually selected in terms of bristle hardness and length of the brush’s working
part, taking into account, among other things, the child’s age, size of the mouth, and teeth.
Flossing was suggested to ensure proper hygiene in the interdental spaces, especially in
young patients with crowded teeth. In addition to verbal instruction, phantom models were
used during the instruction to better illustrate a proper brushing and flossing technique.
Oral swabs were taken with a 15-cm-long stick tipped with a sterilized swab. Opening
of the package containing the tube with the swab stick was performed directly at the patient.
The swab was taken from the base of the tongue, buccal mucosa, and hard palate by gently
rubbing the test site for 3 s. All swabs were taken early in the morning, after a 12 h fast.
After collection, the swab stick was immediately placed in a tube of AMIES transport
medium, signed with the code number assigned to each patient and placed in the transport
refrigerator. Immediately after swab collection, the tube was transported to the Department
of Microbiology at the Pomeranian Medical University in Szczecin, Poland. Sabouraud’s
solid agar medium with gentamicin and chloramphenicol (bioMérieux, Warszawa, Poland)
in the form of round amber-colored Petri dishes was used for the isolation and culture of
fungi. Sabouraud’s medium was prepared in accordance with the recommendations for
the composition of medium C in the European Pharmacopoeia. The main components
included in the medium were peptones, glucose, and agar. The medium on the dishes
Int. J. Environ. Res. Public Health 2023, 20, 4824 5 of 14

was stored at a temperature of 2 to 8 degrees Celsius. Using a sterile inoculation loop,

material was taken from the swab stick. Then, a small amount of the test material was
spread with a zigzag over the entire surface of the agar medium located in the Petri dish
and placed at 34 degrees Celsius. Incubation of the material lasted 48 h, and the culture
was viewed daily. After incubation, further observation of microbial colony growth was
conducted. In order to correctly identify yeast species, it was decided to use the VITEK® 2
YST automated card system (bioMérieux, Warszawa, Poland). Each card contains 64 special
wells and 47 fluorescent biochemical tests and is designed for single use. It detects more
than 50 species of pathogenic yeast fungi.

2.3. Statistical Analysis

The results of the study were statistically analyzed using STATISTICA 10 (StatSoft,
Kraków, Poland) computer programs. The Shapiro–Wilk W test was used to verify that the
results of the study conformed to a normal distribution. In order to verify the hypothesis
regarding the existence and nonexistence of differences between the mean values for the
independent variables, the Mann–Whitney U test and Student’s t-test of the independent
variables were used. Spearman’s r-rank correlation coefficient and Pearson’s r-linear
correlation coefficient were used to assess correlations between variables. To assess the
relationship between qualitative or step variables, the chi2 test of independence with Yates’s
correction or Fisher’s exact test was used. All results whose significance level p was less
than 0.05 (p < 0.05) were considered statistically significant.

3. Results
The average follow-up time for treatment of children in the study group was
6.118 ± 0.212 months, with no statistical significance between girls and boys. The mean
age of all patients participating in the study at the start was 9.02 ± 1.38 years. Given
that the subsequent study was to be conducted approximately 6 months after the first
study, the mean age of the subjects increased to 9.53 ± 1.39 years. The mean age of the
girls at T1 was 8.83 ± 1.29 years, while that of the boys was 9.21 ± 1.47 years. The age
of the patients at T2 changed—this was due to the requirements of the experiment being
conducted. The mean age for girls was 9.34 ± 1.29 years, and for boys 9.72 ± 1.47 years.
The difference in the mean age of the subjects at T1 and T2 between girls and boys was not
statistically significant.
Swabs taken from the oral cavity allowed an overall assessment of the presence or
absence of fungal colonization in the study group of patients. At the first examination (T1),
42 subjects, representing 70% of the study group, were free of Candida, while after 6 months
of treatment, the number decreased to 25 (41.67%) (Table 1). In 17 subjects (28.33%), who
initially showed no evidence of fungi in the mouth, the appearance of Candida strains was
observed (Table 1).
Of the 18 subjects who were found to be colonized with fungi in their mouths during
the T1 examination, the most commonly identified strain was Candida albicans—13 subjects
(72.22% of all identified fungi). The study at T2 also showed that C. albicans was the
fungus most frequently colonizing the oral cavity of 23 persons (38.33%). Three new strains
C. dubliniensis, C. kefyr, and C. krusei were identified at T2 (Table 1).
Table 1 shows the percentage results of the occurrence of specific fungal species
according to the time at which the study was conducted. Based on the analysis of the
results obtained, there was a correlation between the microorganisms present in the second
examination and orthodontic treatment with removable appliances (Rs = 0.29, p = 0.001).
There were significant differences in the percentage distribution of specific fungal species
depending on the time of examination. In addition, the prevalence of each type of Candida
was analyzed. At T2, a statistically significantly less frequent negative culture result was
obtained (p = 0.015), while Candida albicans (Rs = 0.18, p = 0.046) and Candida dubliniensis
(Rs = 0.21, p = 0.022) were statistically significantly more frequently identified.
Int. J. Environ. Res. Public Health 2023, 20, 4824 6 of 14

Table 1. Relationship between the prevalence of each type of Candida to the time at which the study
was conducted.

T1 T2 Chi2 Fisher’s Spearman’s ρ

Type of Candida Total No. R Rank
No. % No. % p-Value p-Value p-Value

Absence 42 70.00 25 41.67 67 0.002 0.003 −0.29 0.002

C. albicans 13 21.67 23 38.33 36 0.046 0.072 0.18 0.046
C. parapsilosis 3 5.00 4 6.67 7 0.696 1.000 0.04 0.699
C. tropicalis 1 1.67 0 0 1 0.315 1.000 −0.09 0.319
C. lusitaniae 1 1.67 0 0 1 0.315 1.000 −0.09 0.319
C. dubliniensis 0 0 5 8.33 5 0.022 0.057 0.21 0.022
C. kefyr 0 0 2 3.33 2 0.153 0.495 0.13 0.156
C. krusei 0 0 1 1.67 1 0.315 1.000 0.09 0.319
Total 60 100.00 60 100.00 120
Pearson’s chi2 test 17.23 df = 7 p = 0.015
Spearman’s ρ 0.29 t = 3.344 p = 0.001

In the first examination (T1), no patient showed more than 50 colonies in the prepara-
tion, 20% showed single colony growth, while 10% showed moderately abundant colony
growth. The examination performed at T2 showed abundant fungal colony growth
(>50 colonies in the culture) in 12 patients (20%) (Table 2).

Table 2. Analysis of the relationship between the number of isolated Candida colonies and the time at
which the study was conducted.

T1 T2 Total Chi2 Fisher’s Spearman’s ρ

No. of Candida p-Value R Rank p-Value
No. % No. % No. p-Value
Absence 42 70.00 25 41.67 67 0.001 0.003 −0.29 0.002
1–9 colonies 12 20.00 7 11.67 19 0.211 0.317 −0.11 0.214
10–50 colonies 6 10.00 16 26.67 22 0.018 0.032 0.22 0.018
>50 colonies 0 0 12 20.00 12 0.0002 0.0002 0.33 0.0002
Total 60 100.00 60 100.00 120
Pearson’s chi2 test 22.17 df = 3 p = 0.0001
Spearman’s ρ 0.37 t = 4.3177 p = 0.0001

An analysis of the relationship between the number of fungal colonies grown in

the preparation and the time at which the study was conducted showed a tendency for
the number of fungal colonies to increase over time (Table 2). A statistically significant
correlation was observed between fungal colony growth and orthodontic treatment with
removable appliances. Analysis of the relationship between the number of isolated fungal
colonies and the use of removable plates showed a statistically significant percentage
increase in the number of these colonies at T2 (Rs = 0.37, p = 0.0003).

3.1. Analysis of the Relationship between the Results of Fungal Colonies and Gender of Patients
Statistical analysis showed significant correlations between the number of isolated
fungal colonies in both girls (Rs = 0.41, p = 0.001) and boys (Rs = 0.33, p = 0.0105) (Table 3).
We also analyzed how specific categories of study variables differed from each other.
Int. J. Environ. Res. Public Health 2023, 20, 4824 7 of 14

Table 3. Analysis of the relationship between fungal colony results and patient gender.

No. of T1 T2 Total Chi2 Fisher’s Spearman’s ρ

R Rank
Candida No. % No. % No. p-Value p-Value p-Value

Absence 21 70.00 11 36.67 32 0.009 0.019 −0.33 0.009

1–9 colonies 6 20.00 5 16.67 11 0.738 1.000 −0.04 0.743
10–50 colonies 3 10.00 7 23.33 10 0.165 0.298 0.18 0.171
Girls >50 colonies 0 0 7 23.33 7 0.004 0.010 0.36 0.004
Total 30 100.00 30 100.00 60
Pearson’s chi2 test 11.82 df = 3 p = 0.008
Spearman’s ρ 0.41 t = 3.421 p = 0.001
Absence 21 70.00 14 46.67 35 0.066 0.115 −0.24 0.068
1–9 colonies 6 20.00 2 6.67 8 0.128 0.254 −0.20 0.133
10–50 colonies 3 10.00 9 30.00 12 0.052 0.104 0.25 0.054
Boys >50 colonies 0 0 5 16.67 5 0.019 0.052 0.30 0.019
Total 30 100.00 30 100.00 60
Pearson’s chi2 test 11.40 df = 3 p = 0.009
Spearman’s ρ 0.33 t = 2.643 p = 0.0105

The subgroups of girls and boys had fairly similar results for the type of fungi isolated.
In the study at T1, 21 girls and 21 boys had negative culture results. In a follow-up study,
conducted after six months, there were more positive results in both subgroups, with girls
having slightly more positive results than boys. Candida albicans was the most common
fungus isolated in both subgroups (Table 4).

Table 4. The results of the prevalence of specific fungal species according to the time at which the
study was conducted and the gender of the patients.

Type of T1 T2 Chi2 Fisher’s Spearman’s ρ

Total No. R Rank
Candida No. % No. % p-Value p-Value p-Value

Absence 21 70.00 11 36.67 32 0.009 0.019 −0.33 0.009

C. albicans 6 20.00 11 36.67 17 0.152 0.252 0.18 0.157
C. parapsilosis 2 6.67 2 6.67 4 1.000 1.000 0.00 1.000
C. tropicalis 1 3.33 0 0 1 0.313 1.000 −0.13 0.321
C. lusitaniae 0 0 0 0 0 1.000 1.000 - -
Girls C. dubliniensis 0 0 3 10.00 3 0.075 0.237 0.23 0.077
C. kefyr 0 0 2 6.67 2 0.150 0.491 0.19 0.155
C. krusei 0 0 1 3.33 1 0.313 1.000 0.13 0.321
Total 30 100.00 30 100.00 60
Pearson’s chi2
11.60 df = 7 p = 0.115
test
Spearman’s ρ 0.35 t = 2.852 p = 0.006
Absence 21 70.00 14 46.67 35 0.067 0.115 −0.24 0.069
C. albicans 7 23.33 12 40.00 19 0.165 0.266 0.18 0.171
C. parapsilosis 1 3.33 2 6.67 3 0.553 1.000 0.08 0.561
C. tropicalis 0 0 0 0 0 1.000 1.000 - -
C. lusitaniae 1 3.33 0 0 1 0.313 1.000 −0.13 0.321
C.dubliniensis 0 0 2 6.67 2 0.150 0.491 0.19 0.155
Boys
C. kefyr 0 0 0 0 0 1.000 1.000 - -
C. krusei 0 0 0 0 0 1.000 1.000 - -
Total 30 100.00 30 100.00 60
Pearson’s chi2
6.05 df = 7 p = 0.534
test
Spearman’s ρ 0.24 t = 1.857 p = 0.068
Int. J. Environ. Res. Public Health 2023, 20, 4824 8 of 14

3.2. Analysis of the Relationship between the Results of Fungal Cultures and the Age of
the Patients
There was an overall increase in the number of Candida fungal colonies in both the
younger and older patient groups. In the group of patients aged 9 years and older, there
was a statistically significant increase in the number of fungal colonies with a p = 0.005
(Table 5).

Table 5. Analysis of the relationship between fungal colony results and patient age.

No. of T1 T2 Total Chi2 Fisher’s Spearman’s ρ

R Rank
Candida No. % No. % No. p-Value p-Value p-Value

Absence 24 80.00 17 56.67 41 0.052 0.009 −0.25 0.053

1–9 colonies 5 16.67 2 6.67 7 0.227 0.423 −0.16 0.234
<9 10–50 colonies 1 3.33 5 20.00 7 0.044 0.103 0.26 0.045
years >50 colonies 0 0 5 16.67 5 0.019 0.05 0.30 0.019
old Total 30 100.00 30 100.00 60
Pearson’s chi2 test 11.05 df = 3 p = 0.011
Spearman’s ρ 0.31 t = 2.513 p = 0.014
Absence 18 60.00 8 26.67 26 0.009 0.018 −0.34 0.008
1–9 colonies 7 23.33 5 16.67 12 0.517 0.748 −0.08 0.526
≥9 10–50 colonies 5 16.67 10 33.33 15 0.136 0.233 0.19 0.141
years >50 colonies 0 0 7 23.33 7 0.005 0.011 0.36 0.005
old Total 30 100.00 30 100.00 60
Pearson’s chi2 test 12.85 df = 3 p = 0.005
Spearman’s ρ 0.44 t = 3.697 p = 0.0005

Table 6 shows the results of the prevalence of specific fungal species according to
the time at which the study was conducted in children in two age groups—the first for
younger children and the second for older children. In both groups, a statistically significant
relationship was observed between the detection of more fungal species and the use of
removable appliances in the group of younger children (Rs = 0.28, p = 0.031) and in the
group of older children (Rs = 0.31, p = 0.017).

Table 6. Results of the incidence of each fungal species according to the time at which the study was
conducted in children in two age groups.

Type of T1 T2 Total Chi2 Fisher’s Spearman’s ρ

R Rank
Candida No. % No. % No p-Value p-Value p-Value

Absence 24 80.00 17 56.6 41 0.052 0.094 −0.25 0.053

C. albicans 5 16.67 7 23.33 12 0.518 0.748 0.08 0.526
C. parapsilosis 0 0 1 3.33 1 0.312 1.000 0.13 0.321
C. tropicalis 1 3.33 0 0 1 0.313 1.000 −0.13 0.321
<9 C. lusitaniae 0 0 0 0 0 1.000 1.000 - -
years C. dubliniensis 0 0 3 10.00 3 0.075 0.237 0.23 0.077
old C. kefyr 0 0 2 6.67 2 0.150 0.491 0.19 0.155
C. krusei 0 0 0 0 0 1.000 1.000 - -
Total 30 100.00 30 100.00 60
Pearson’s chi2
8.53 df = 7 p = 0.288
test
Spearman’s ρ 0.28 t = 2.204 p = 0.031
Int. J. Environ. Res. Public Health 2023, 20, 4824 9 of 14

Table 6. Cont.

Type of T1 T2 Total Chi2 Fisher’s Spearman’s ρ

R Rank
Candida No. % No. % No p-Value p-Value p-Value

Absence 18 60.00 8 26.67 26 0.009 0.018 −0.34 0.009

C. albicans 8 26.67 16 53.33 24 0.035 0.064 0.27 0.035
C. parapsilosis 3 10.00 3 10.00 6 1.000 1.000 0.00 1.000
C. tropicalis 0 0 0 0 0 1.000 1.000 - -
≥9 C. lusitaniae 1 3.33 0 0 1 0.313 1.000 −0.13 0.321
years C. dubliniensis 0 0 2 6.67 2 0.150 0.491 0.19 0.155
old C. kefyr 0 0 0 0 0 1.000 1.000 - -
C. krusei 0 0 1 3.33 1 0.313 1.000 0.13 0.321
Total 30 100.00 30 100.00 60
Pearson’s chi2
10.51 df = 7 p = 0.161
test
Spearman’s ρ 0.31 t = 2.441 p = 0.017

In addition, individual types of Candida were analyzed quantitatively (Table 6). At T2,
older children were statistically significantly less likely to have a negative culture result
(Rs = −0.34, p = 0.009). Older patients had significantly more positive fungal cultures.
Additionally, in the subgroup of children aged 9 years and older, statistically significantly
more often Candida albicans fungi were isolated from collected samples (Rs = 0.27, p = 0.035).

4. Discussion
The oral cavity is a place where many types of microorganisms—from bacteria and
viruses to fungi—coexist, as confirmed by the work of numerous researchers [22,23]. Some
of these microorganisms are part of the normal oral microbiome and do not cause any
negative effects in the host body. Among many researchers, there is no consensus on which
group the fungi inhabiting the oral cavity should be classified in. Some scientists believe
that they are part of the natural microbiome and do not have a destructive effect on the
host body. From this point of view, the relationship between humans and fungus is typical
commensalism, so the existence of the fungus in the oral cavity brings neither loss nor
benefit to the host. On the other side of the scale are scientists for whom any number, even
of single fungal colonies in the human body is a typical example of parasitism, i.e., an inter-
action on the basis of which the host suffers losses associated with the presence of a foreign
genome [24]. Therefore, in the presented research, it was decided to check how often fungi
colonize the oral cavity of children in whom there is no clinical manifestation of infection.
According to our own research, the oral cavity of the majority of children presenting to the
Orthodontics Clinic in Szczecin for orthodontic treatment was not colonized by any type of
fungi. Of the 60 patients participating in the study, only 18 patients had positive cultures
for fungi, accounting for 30% of all subjects. Among the positive results, more than 70% of
the fungi were Candida albicans. In most of the children tested, the Candida spp. detected
were single colonies of no more than nine colonies in the specimen.
However, analyzing the results obtained six months after the beginning of treatment,
there is a reversal trend of the predominance of negative results over positive ones. In
our study, positive results were obtained in 58.33% of the participants, indicating the
detection of fungal colonies in the analyzed material, which shows an almost twofold
increase in positive results compared to the study before the start of treatment. The most
frequently detected fungal species was Candida albicans. This was the result of almost
66% of positive cultures. According to our own observations, there was an increase in
the number of colonies in the preparation after six months of treatment—most of the
positive patients had a moderately abundant colony count, which meant the presence of
10 to 50 fungal colonies in the analyzed preparation. The first references in the scientific
literature relating to changes in Candida count date back to 1979. Arendorf and Walker
studied the relationships regarding the appearance of fungi in the oral cavity in patients
Int. J. Environ. Res. Public Health 2023, 20, 4824 10 of 14

using dental restorations and orthodontic appliances. The authors observed a significant
increase in the number of fungi after six months of treatment with removable appliances,
both on the surface of the teeth and on the oral mucosa [25]. In contrast, in 1982, a study
by Addy et al. showed no statistically significant differences between groups of healthy
adolescents without orthodontic treatment, with fixed orthodontic appliances, and treated
with removable appliances. Candida carriage was demonstrated in 46–52% of subjects
regardless of the group. However, the researchers noted a significant increase in fungal
counts in those who had previously been diagnosed with Candida carriage and were under
orthodontic treatment compared to untreated subjects. The authors of the study suggested
that fixed and removable appliances may stimulate Candida proliferation in those who are
Candida carriers; however, they did not determine whether fungal-free patients may develop
an infection initiated by orthodontic treatment [20]. The first long-term study on the effects
of removable appliances on fungal development was conducted in 1985 by Arendorf and
Addy [21]. The authors observed 33 British youths between the ages of 8 and 17, varying in
gender and age. They proved that before starting orthodontic treatment, 39% of the patients
were Candida carriers, while after nine months, the number increased to 79%. Thirteen of the
non-carrier patients showed the appearance of Candida during the course of treatment, of
which only three patients still had fungal colonies in their mouths after orthodontic therapy.
Two of the patients who were carriers of C. albicans before the start of treatment had negative
culture results after the end of treatment, indicating that even single fungal colonies had
not been isolated. Arendorf and Addy proved that there is a close dependence between
acrylic removable appliances and the emergence of Candida. According to the authors,
these appliances temporarily change the microbial status in the oral cavity [21]. The results
of our study confirm the findings regarding the increase in the number of patients with
oral fungal infection during orthodontic treatment with removable appliances. Moreover,
similar results were obtained by Khanpayeh et al. [26]. In the studied group of 40 patients
(21 female and 19 male) with an average age of 11.7 years and treated with removable
appliances for about 9 months, 77.5% of positive cultures for Candida were obtained. In
9 patients, not even a single fungal colony was detected, which accounted for 22.5% of
all the subjects. The authors published the results of microbiological cultures including
the type of fungi isolated. The vast majority of the detected fungi were Candida albicans
(62.5%). In addition, C. tropicalis (7.5%), C. parapsilosis (5%), and C. krusei (2.5%) were also
isolated. Consistent with the results of our research, a similarly dominant group of fungi
was C. albicans, which was detected in 38.33% of the subjects. The second most prevalent
was C. dublienensis, isolated in 8.33% of individuals, followed by C. parapsilosis (6.67%),
C. kefyr (3.33%), and C. krusei (1.67%). Comparing the results of Khanpayeh et al. and
our study, we can risk the thesis that removable appliances predispose the patient to the
development of Candida, with C. albicans being the most common species. Moreover, in a
study conducted in Mexico, an increase in Candida spp. occurred after 4 weeks of treatment
with removable appliances and was found in oral mucosa material in 30.9% of patients [19].
Additionally, in the study by Arika et al., both fixed and removable space maintainers
caused an increase in the number of microorganisms in the oral cavity [27]. As confirmed
by various analyses, the slight differences between studies regarding the prevalence of
other types of fungi may be due to microbiological variability in the patient groups studied,
which may be influenced by the latitude where the study was carried out [28–30].
In the conducted study, it was shown that positive Candida cultures before the start
of treatment were at the same level regardless of gender. Six months after the start of
treatment, there was a greater increase in positive Candida cultures in girls than in boys.
When analyzing the culture results obtained in relation to the age of the patients, it can be
seen that negative results predominated in children under nine years of age, both before
and after the start of treatment. The general population trend in our study was maintained.
Although the number of negative T2 tests decreased in this age group, it was still higher
than the number of positive tests. In the group of older patients, 9 years and over, there
were more negative results at T1, while there were more positive results at T2. From the
Int. J. Environ. Res. Public Health 2023, 20, 4824 11 of 14

review of the literature, it appears that there are no scientific reports that take into account
differences between the age and gender of the subjects. It would be advisable to continue
research in this area, because of the resulting possibility of learning more about groups at
increased risk of contracting fungal infections during orthodontic therapy. In addition, our
study showed that there were no statistically significant correlations between the type of
fungi isolated and the gender and age of the patients studied. In our own material, the
group of patients consisted of 30 female and 30 male patients. These relationships were
quite similar with respect to the work of other researchers on orthodontic treatment in
children, with girls more often being the predominant patient group [31–33]. In contrast,
in analyses carried out in Brazil and Italy [34,35], both the groups of patients treated with
fixed and removable appliances were predominantly boys. The significant predominance
of female patients in most studies may be due to the fact that in girls, from an early age,
more importance is placed on appearance, facial aesthetics, and smiling [35]. In order to
allow multiple comparisons of the results obtained, depending on the gender and age
differences of the study group, in this study it was decided to take both of these factors into
account when analyzing the impact of removable appliances on the parameters studied.
The results presented by other researchers largely coincide with those of our own
study. Various authors have analyzed not only removable appliances as hypothesized
to affect the oral microbiome, but also other removable prosthetic restorations [36,37].
The consideration of removable appliances and removable prosthetic restorations at the
same time is justified by the fact that, in both cases, the acrylic plate can obstruct the flow
of saliva between the plate and the oral mucosa in a significant way, which definitely
impedes the self-cleaning process. In addition, an increase in temperature is observed
beneath the mucosa-adherent surface of the denture plate or orthodontic appliance. This
factor is considered to be conducive to the growth and proliferation of microorganisms,
especially various types of fungi. Another issue often raised by researchers is the presence
of micro-leaks and pores in the acrylic device, which combined with a warm and moist
oral environment, may promote the growth of pathogens. In addition, the long hours of
use of appliances and dentures promote micro-trauma to the oral mucosa, making it more
vulnerable to fungal penetration and colonization of its deeper layers. The eradication of
Candida spp. from the infected oral cavity is significantly hindered [37].
In our study, no clinical manifestations of fungal infections were observed. None of
the patients developed full-blown oral candidiasis and did not show the typical symptoms
that can accompany this disease, such as pain, itching, burning or differences in taste
perception [38,39]. In spite of this, the results regarding candida colony number in the
swabs taken show a significant increase over time. The increase in the number of oral
fungal infections in the absence of clinical symptoms of candidiasis can be explained by
the hypothesis of a high resistance of young, healthy organisms to the development of
pathogenic microorganisms. The theory of commensal interdependence between fungus
and human seems to be closest to the truth. As long as the host organism is characterized
by intrinsic homeostasis and its well-being is not disturbed, the oral co-occurring pathogen
brings neither loss nor benefit to the host [40–43]. Our results are consistent with a study by
Hibino et al. where no healthy patient developed Candida infection as a result of orthodontic
treatment. On the other hand, some non-Candida carriers appear to have converted to
Candida carriers after the use of the appliance as a result of an unknown mechanism [15].
However, according to other researchers, when immunity declines and the organism’s
internal balance is disturbed, commensalism can be replaced by parasitism. There is an
intensive proliferation of microorganisms, manifested by the appearance of local clinical
signs of infection. When the organism’s immunity further declines, the fungi can occupy
further tissues and organs, resulting in the development of systemic mycosis [41,44,45].
In conclusion, the results of this study demonstrate that removable orthodontic appli-
ances may adversely affect the oral cavity environment and disrupt its stable ecosystem. In
many patients, this manifests itself as an increase in fungal colonies. The undesirable effects
of removable appliances must therefore always be taken into account in the planning of
Int. J. Environ. Res. Public Health 2023, 20, 4824 12 of 14

orthodontic treatment and, as far as preventive measures are available, be efficiently coun-
teracted. During orthodontic treatment, special attention should be paid to oral hygiene.
It is important to keep in mind the limitations of the study, such as the small number of
participants taking part in the study, and the limited age range of the participants while
remembering that the problem has not been widely studied before. We are currently study-
ing several of these topics. Further research is required on a larger group of subjects to
confirm these study results.

5. Conclusions
1. Orthodontic treatment with removable appliances contributes to an increase in the
colonization of the oral cavity by Candida spp. Fungi;
2. Candida albicans is the most commonly found in patients undergoing orthodontic
treatment with removable appliances;
3. Patients in the older age group over 9 years old are more vulnerable to oral coloniza-
tion by Candida spp. fungi in comparison to patients younger than 9 years old.

Author Contributions: Conceptualization, A.B.-Z., L.S.-S. and K.W.; methodology, L.S.-S. and K.W.;
software, A.B.-Z. and L.S.-S.; validation, L.S.-S. and K.W.; formal analysis, A.B.-Z., L.S.-S. and K.W.;
investigation, A.B.-Z.; resources, A.B.-Z., L.S.-S. and K.W.; data curation, A.B.-Z.; writing—original
draft preparation, A.B.-Z., M.S.-D. and L.S.-S.; writing—review and editing, A.B.-Z., M.S.-D., L.S.-S.,
K.W. and G.S.; visualization, A.B.-Z. and L.S.-S.; supervision, L.S.-S., G.S. and K.W.; project adminis-
tration, L.S.-S. and K.W.; funding acquisition, K.W. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Local Bioethics Committee of the Pomeranian Medical
University (protocol number: KB-0012/55/17, accessed on 27 March 2017).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The datasets used to support the conclusions of this article are included
within the article. Access to other data will be considered by the corresponding author upon request.
Conflicts of Interest: The authors declare no conflict of interest.

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