Influence of oral biofilm index, caries experience, and laboratory markers of disease progression on the oral carriage of Candida in HIV-infected and non-infected children: a cross-sectional study

Abstract

The present study aimed to compare the oral Candida rate between infected and uninfected children with the human immunodeficiency virus (HIV), as well as analyze the association between Candida spp. and predisposing factors of colonization, like oral biofilm index, caries experience, and laboratory markers of AIDS progression. A cross-sectional study was employed. Candida species were identified and quantified from saliva samples of 50 HIV-infected and 50 uninfected children. Biofilm index and decayed, missing, and filled teeth (dmft/DMFT) indices were assessed by oral clinical examinations. Additionally, CD4⁺ T lymphocyte count and viral load were obtained from medical records of the HIV-infected children. Candida species were cultured from 74% of the HIV-infected children and 46% of uninfected ones (p = 0.0076). Candida albicans and Candida parapsilosis were the most frequently isolated species in both studied groups. The isolation of Candida species was significantly higher in HIV-infected children with CD4 ≤ 15% (p = 0.0146); it had influence of mature oral biofilm and the caries index (dmft + DMFT ≥ 8) (p < 0.05) and was associated with the plasma viral load. The present data show that the HIV infection, oral biofilm index, caries experience, and laboratory markers of AIDS progression exert an influence on the prevalence of oral Candida in children.

Introduction

The prevalence of HIV-related oral manifestations ranges from 30 to 80%, especially when CD4⁺ T-cell count is low (< 200 cell/mm³) and the viral load is high (> 20,000 copies/mL) [1–3]. Pediatric patients often present oral mucosal lesions among the first manifestations of HIV infection due to the dysregulation of the oral microbiota and the consequent development of opportunistic infections [4]. The immature immune system in children exposes those with HIV to faster disease development [5], with clinical manifestations of symptoms in the first year of life or even before 1 year of age in approximately 50% of this specific population [6].

The consensus in the literature is that candidiasis is one of the most frequent oral manifestations in children with HIV [4]. Oral candidiasis is an opportunistic fungal infection caused mainly by Candida albicans [7]. However, non-albicans Candida species (NACs) such as C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and more recently, C. auris have also been associated with it [8–11]. Most candidiasis manifestations are associated with biofilm formation, one of the well-known virulence attributes produced by Candida species [12, 13]. Biofilms are defined as organized microbial communities that can attach to biological or artificial surfaces, and they are typically surrounded by a self-produced matrix. From a clinical perspective, the biofilm formation can increase the resistance of cells against antifungals and help them evade host immune defense, impacting patient management [14–16].

Children with HIV often have a higher occurrence of fungi than non-infected children, and the high prevalence of fungal species in saliva implies mucosal colonization and an increased risk for the development of clinically significant oral candidiasis [17, 18]. Studies also associate high oral colonization by Candida with early carious lesions in tooth enamel, as Candida has been identified in the supragingival dental plaque of children with HIV and a high frequency of caries [19]. This is an important aspect, as children with HIV have a greater caries experience (prevalence of 12 to 54%), and dental caries can also exert an influence on the dissemination of Candida [5, 20, 21].

The number of pediatric patients affected by HIV remains high, especially in developing countries [4], and HIV mother-to-child transmission is an important cause of child mortality worldwide [22]. According to the Joint United Nations Program on HIV/AIDS (UNAIDS) [23], about 4500 new cases of HIV infection (adults and children) were recorded per day in 2019, among which children aged 0 to 14 years accounted for 9%. In the same year, 1.8 million children/adolescents were living with HIV, and 9500 deaths occurred in this group, representing an important cause of child mortality worldwide [4, 22, 23]. In this context, dentists can play a role in monitoring the evolution of HIV infection by diagnosing associated oral manifestations, thereby assisting in the early diagnosis of HIV infection, which could contribute to improving the quality of life of the patients [24].

The current literature reports the association between the prevalence of oral Candida and systemic conditions in HIV-infected children [17, 19, 25, 26]. However, it is also important to evaluate possible factors that may exert influence on the Candida rate in those patients; hence, controlling those factors may improve the patient’s quality of life. In Brazil, there is relatively little information on the association of oral Candida carriage in children, such as oral biofilm index and caries experience. Therefore, the present study consists in assessing quantitatively and qualitatively the colonization of oral Candida in Brazilian children, considering dental biofilm accumulation, levels of decayed, missing, and filled teeth indices (dmft/DMFT), as well as laboratory markers of disease progression (CD4⁺ T lymphocytes and viral load) on the prevalence of oral Candida in HIV-infected and uninfected children.

Materials and methods

Study design

The present study conforms to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines [27]. For the study, one hundred children were divided in two groups. The power of the sample was subsequently calculated using the OpenEpi software.

Subjects and eligibility criteria

The first group consisted of 50 children who tested positive for anti-HIV antibodies by both enzyme-linked immunosorbent assay (ELISA) and Western blotting assays. The children were routinely monitored as part of the outpatient population at the Instituto de Puericultura e Pediatria Martagão Gesteira of the Federal University of Rio de Janeiro (UFRJ), Brazil. This institute was chosen because it is a reference center for the treatment of children with HIV in the State of Rio de Janeiro. All children with HIV were receiving antiretroviral therapy at the time of sampling. The second group consisted of 50 children with no symptoms or previous history of HIV infection recruited from the pediatric dentistry clinic of the UFRJ School of Dentistry. For ethical and financial reasons, these children were not tested for the presence of HIV antibodies. A thorough medical history was taken of each child. Those with a history of diabetes mellitus, any form of immunosuppression, and those taking immunosuppressive medication were excluded from the study. To be included in the study, the children could not have any clinical signs of oral candidiasis or mucosal lesions at the time of screening or have received antifungal therapy in the previous 3 months.

Sample collection

A sterile swab was rubbed against the dorsal surface of the tongue of each subject and then transferred to a sterilized test tube containing 0.3 ml of a 0.9% sodium chloride solution [28]. The swab method was selected rather than the oral rinses method because there were children under 2 years old who participated in the study and would not be able to collaborate in the rinses method, so we wanted to standardize the collection of the sample. The samples were processed within 2 h of collection and were mixed with a vortex mixer for 30 s before culturing. Aliquots (0.1 ml) of undiluted samples were plated onto CHROMagar Candida medium (BD Diagnostic Systems, Paris, France). After 72 h of incubation at 37 °C under aerobic conditions, the number of colony-forming units (CFUs) per milliliter of saliva was determined. All colonies with different morphologies and/or colors were identified by conventional methods (morphological, physiological, and biochemical characteristics), such as the germ tube test in serum, chlamydospore formation, growth at different temperatures (37, 42, and 45 °C), assimilation of carbon and nitrogen compounds, fermentation of carbohydrates, hydrolysis of urea, cycloheximide resistance, tolerance to acetic acid, and the diazonium blue B color reaction [29].

Oral biofilm index, caries experience, and laboratory markers of disease progression

All children were submitted to a meticulous oral examination conducted by two expert clinicians. Oral biofilm was evaluated (presence and maturation) using the modified index described by Ribeiro et al. [30], as follows: (0) no visible biofilm, (1) thin, scattered visible biofilm, and (2) thick, firmly attached visible biofilm (mature). Caries experience was determined using the decayed, missing, and filled teeth (dmft [primary teeth] and DMFT [permanent teeth]) indices recommended by the World Health Organization (WHO) [31], as follows: (i) sound (no evidence of caries, whether treated or not), (ii) decayed (lesion or decay on the surface of a tooth or a temporary restoration), (iii) missing (tooth extracted due to caries and not for other reasons), and (iv) filled (one or more restorations and no primary or recurrent caries). Moreover, the medical records of each child with HIV were used to collect laboratory markers of disease progression (CD4⁺ T lymphocyte count and viral load).

Statistical analysis

Associations were tested considering the group (HIV-infected and non-infected children) and its relation to Candida isolation, the frequency and density of Candida, Candida species isolated from the oral cavity, the isolation of multiple Candida species, the density of Candida isolation according to the visible biofilm index, and the density of Candida isolation according to caries experience. Associations were also tested considering the occurrence of Candida according to the CD4⁺ T lymphocyte count and viral load in the HIV-infected children. For such, a correlation coefficient test, Fisher’s exact test, and the chi-square test were used, when appropriate. A p value < 0.05 was considered indicative of statistical significance.

Results and discussion

The present study showed that the isolation of oral Candida species was higher in children with HIV than in non-infected children, and its relation with demographic parameters was shown in Table 1. In the HIV-infected group, 87.5% of the boys and 61.5% of girls had positive cultures for Candida compared to 42.9% of the boys and 48.3% of the girls in the non-infected group. Thus, no positive correlation was found regarding sex within the same group. However, Candida isolation was significantly more frequent among the boys in the HIV-infected group compared to boys in the non-infected group (p = 0.0035).

Table 1.

Relationship between Candida isolation in HIV-infected and non-infected children and demographic parameters

Groups	Age ranges (year)			Gender
	2–5	6–9	10–13	Male	Female
HIV +	16/17 (94.1%)	14/21 (66.7%)	7/12 (58.3%)	21/24 (87.5%)	16/26 (61.5%)
HIV −	6/10 (60%)	11/25 (44%)	6/15 (40%)	9/21 (42.9%)	14/29 (48.3%)
p value Fisher’s exact test	p 0.0473	p 0.1485	p 0.4495	p 0.0035	p 0.4185

HIV + (HIV-infected children); HIV − (non-infected children).

The group formed by children with HIV consisted of 24 boys and 26 girls (mean age: 7 years; range: 2 to 13 years). The group of non-infected children consisted of 21 boys and 29 girls (mean age: 8 years; range: 2 to 13 years). Based on the comparison of Candida species cultures between the two groups of children, the statistical power was 82.6%, confirming that the sample was sufficient to generate statistically reliable results. As age in the overall sample ranged from 2 to 13 years, the participants were allocated to three categories to facilitate the comparisons between the two groups: (A) 2 to 5 years, (B) 6 to 9 years, and (C) 10 to 13 years. Interestingly, we found statistically significant differences in the frequency of Candida isolation in the youngest age group compared to the other age groups in both HIV-infected and non-infected children (Table 1).

The frequency of Candida isolation was significantly higher in HIV-infected children, reaching a percentage of 74% (37/50) when compared to non-infected children that had a percentage of 46% (23/50) (Fisher’s exact test, p = 0.0076). Likewise, the density of Candida isolation was greater in the HIV-infected group ranging from 10 to 17,030 CFU/mL (mean 1219.5, median 110, and geometric mean 124.7 CFU/mL) when compared to the non-infected group that ranged from 10 to 1040 CFU/mL (mean 171.3, median 40, and geometric mean 51.8 CFU/mL). This finding is in agreement with data reported by Cerqueira et al. [17], Rosa Oliveira et al. [19], Portela et al. [25], and Alves et al. [26], in which in all studies the carrier rate of Candida in HIV-infected children was significantly higher when compared to HIV non-infected children.

The higher density of Candida in HIV-infected children may be associated with changes in the oral environment and local immunologic reactivity, enabling a larger number of Candida species to colonize the oral cavity in children with HIV [18, 26]. This may partially explain the high incidence of oral candidiasis in children with HIV [7, 32]. It has been suggested that HIV proviral DNA may be present in oral epithelial cells [33], which could, in turn, influence Candida colonization. Moreover, 32% of cases of HIV infection are associated with xerostomia [34], which is a well-known cause of oral candidiasis in children [35]. The increase in salivary lactoferrin is another factor associated with greater colonization by Candida species in children with HIV [26].

In the present study, a total of 102 isolates belonging to the Candida genus were retrieved from the 100 participants. The amount of Candida isolates was twofold higher in the HIV-infected group compared to the non-infected group (p = 0.0221) (Table 2). Candida albicans was the most frequent species found in both groups, with a remarkably high frequency in HIV-infected children (Table 2). C. parapsilosis was the most prevalent species belonging to the NACs in both HIV-infected and non-infected children. C. famata, C. dubliniensis, C. guilliermondii, and C. pelliculosa were only recovered from HIV-infected children (Table 2).

Table 2.

Candida species isolated from the oral cavity of the HIV-infected and non-infected children

Candida species	Total number of Candida isolates (%)	Number of Candida isolates (%)a		Relative frequency of each Candida species (%)		p value (Fisher’s exact test)
		HIV +	HIV −	HIV +	HIV −
C. albicans	44 (43.1)	29 (42.7)	15 (44.1)	65.9	34.1	0.0085
C. parapsilosis	23 (22.6)	11 (16.2)	12 (35.3)	47.8	52.2	1.0
C. famata	11 (10.8)	11 (16.2)	0	100	0	0.0005
C. tropicalis	8 (7.8)	6 (8.8)	2 (5.9)	75	25	0.2687
C. glabrata	6 (5.9)	2 (2.9)	4 (11.8)	33.33	66.67	0.6777
C. dubliniensis	3 (2.9)	3 (4.4)	0	100	0	0.2424
C. guilliermondii	3 (2.9)	3 (4.4)	0	100	0	0.2424
C. pelliculosa	2 (2.0)	2 (2.9)	0	100	0	0.4949
C. lusitaniae	2 (2.0)	1 (1.5)	1 (2.9)	50	50	1.0
Total	102 (100)	68 (100)	34 (100)			0.0221

^aFor the total 50 HIV-infected children and 50 healthy children. Eighteen HIV-infected children and seven healthy children had two or more species isolated simultaneously. HIV + (HIV-infected children); HIV − (non-infected children).

More than one Candida species was recovered from 25 of the 100 children analyzed. Mixed cultures were predominantly found in the HIV-infected group (n = 18/25) compared to the non-infected group (n = 7/25) (Fisher’s exact test, p = 0.0714). The various combinations of species detected and the quantity of cases are listed ahead. Candida isolates found in both HIV-infected and non-infected children: C. albicans + C. parapsilosis (HIV+ = 4; HIV− = 4), and C. albicans + C. tropicalis (HIV+ = 2, HIV− = 1). The combination of C. albicans + C. glabrata (HIV− = 2) was only found in non-infected children. The following Candida isolates were only identified in HIV-infected children: C. albicans + C. famata (n = 3), C. albicans + C. guilliermondii (n = 2), C. tropicalis + C. dubliniensis (n = 1), C. famata + C. parapsilosis (n = 1), C. albicans + C. famata + C. guilliermondii (n = 1), C. albicans + C. famata + C. parapsilosis (n = 1), C. parapsilosis + C. pelliculosa + C. lusitaniae (n = 1), C. parapsilosis + C. tropicalis + C. dubliniensis (n = 1), and C. tropicalis + C. dubliniensis + C. famata + C. glabrata (n = 1). The isolation of three or four yeasts only occurred in the HIV-infected children. Approximately 58% of the mixed cultures contained C. albicans and at least one NAC.

Considering all Candida species identified in the present study, C. albicans was the most frequently isolated in both HIV-infected and non-infected children. The same pattern has been reported in previous studies [17, 19, 25]. Moreover, most of the Candida species identified are related to cases of candidemia. While C. albicans is the most common, a growing number of cases are attributed to NACs, such as C. parapsilosis [36, 37]. Indeed, C. parapsilosis was the most prevalent NAC recovered from the children in the present investigation. However, the present results differ from those reported by Portela et al. [25], who found C. dubliniensis, C. glabrata, and C. tropicalis to be the most common NACs isolated from the subgingival plaque of children with HIV. Aspects related to the sampling and processing methods employed may cause variability in the quantitative microbial analysis of the oral cavity [38].

C. famata, C. dubliniensis, C. guilliermondii, and C. pelliculosa were only identified in the children with HIV, which suggests that changes in host immune defenses make individuals more susceptible to colonization by more resistant species of Candida [39, 40]. These findings may also be attributed to pathobiological effects of HIV infection, including changes in the salivary flow rate and composition, changes on the surface of epithelial cells, and the use of broad-spectrum antibiotics [41], which may alter the microenvironment and contribute to the selection of Candida species [25]. Moreover, it has been demonstrated that Candida species isolated from individuals with HIV have a greater adherence capacity compared to those isolated from non-infected individuals [39]. However, further studies are needed to clarify the causes of preferential strain selection in HIV-infected patients, which may lead to novel therapeutic interventions [39].

The oral biofilm index (Table 3) and its association with multiple Candida isolation (Table 4) were evaluated, as well as caries experience (Table 5).

Table 3.

Density of Candida isolation in different subgroups of the children from the study, according to oral visible biofilm index

Biofilm classification	Oral visible biofilm index			Relationship between Candida isolation and biofilm
	HIV +	HIV −	p value (Fisher’s exact test)	HIV +	HIV −	p value (Fisher’s exact test)
No biofilm	4	10	0.1478	2/4	4/10	1.0
Thin scattered biofilm	14	24	0.0631	11/14	12/24	0.1006
Mature biofilm	32	16	0.0025	23/32	5/16	0.0123

HIV + (HIV-infected children); HIV − (non-infected children).

Table 4.

Multiple Candida isolation from the oral cavity of both HIV + and HIV − children and its relation with the biofilm index

Candida isolates	HIV +				HIV −
	n	Oral visible biofilm index			n	Oral visible biofilm index
		No biofilm	Thin scattered biofilm	Mature biofilm		No biofilm	Thin scattered biofilm	Mature biofilm
C. albicans + C. parapsilosis	4		2	2	4	2		2
C. albicans + C. famata	3			3	0
C. albicans + C. tropicalis	2		1	1	1		1
C. albicans + C. guilliermondii	2	1		1	0
C. albicans + C. glabrata	0				2		1	1
C. tropicalis + C. dubliniensis	1			1	0
C. famata + C. parapsilosis	1		1		0
C. albicans + C. famata + C. guilliermondii	1		1		0
C. albicans + C. famata + C. parapsilosis	1		1		0
C. parapsilosis + C. pelliculosa + C. lusitaniae	1		1		0
C. parapsilosis + C. tropicalis + C. dubliniensis	1			1	0
C. tropicalis + C. dubliniensis + C. famata + C. glabrata	1	1			0
Total	18	2	7	9	7	2	2	3

Table 5.

Density of Candida isolation in different subgroups of children according to caries experience

Caries experience	Caries index			Relationship between Candida isolation and caries experience
	HIV +	HIV −	p value (Fisher’s exact test)	HIV +	HIV −	p value (Fisher’s exact test)
No caries	7	12	0.308	6/7	8/12	0.6027
dmft + DMFT between 1 and 7	15	27	0.0253	7/15	9/27	0.5113
dmft + DMFT ≥ 8	28	11	< 0.00001	23/28	4/11	0.017

HIV + (HIV-infected children); HIV − (non-infected children).

dmft decayed, missing, and filled deciduous teeth decayed, DMFT decayed, missing, and filled permanent teeth

Thirty-two (64%) of the HIV-infected children exhibited mature biofilm in comparison to 16 (32%) of the non-infected children. Moreover, the occurrence of oral Candida was twofold higher in the HIV-infected group (Table 3). A similar pattern occurred for caries experience (Table 5).

The multiple Candida isolation were not statistically correlated with oral biofilm index regardless of the biofilm maturation in both HIV + and HIV − groups (Table 4). Correlation according to Fisher’s exact test is as follows: biofilm and no biofilm p = 0.5485; thin biofilm and no biofilm p = 0.5301; mature biofilm and no biofilm p = 0.5467; and thin biofilm and mature biofilm p = 1.

The oral Candida count, oral biofilm index, and caries index (dmft + DMFT ≥ 8) were significantly higher in the HIV-infected group than the non-infected group. Blignaut and van Heerden [42] demonstrated the presence of genetically identical strains of C. albicans on both oral soft tissue and carious dentin in children with HIV, raising the hypothesis that caries can harbor the yeast, which, at some stage, may result in infection in immunocompromised individuals. The isolation of Candida species in plaque biofilm from children affected with severe childhood caries is frequent [43, 44], and this correlation may be stronger in children with HIV, since Candida has been reported to be higher in such individuals, as demonstrated in the present study.

Levels of oral Candida in children can serve as a parameter for epidemiologic studies addressing dental caries because of the association with supragingival plaque and the caries index [40, 42–44]. Collagenolytic protease secreted by C. albicans is capable of degrading human dentinal collagen, and its secretion may be significant in the pathogenesis of dentinal caries [44, 45]. This is in agreement with statements made by Cerqueira et al. [17] and indicates that cavitated lesions can serve as a niche for the colonization and proliferation of Candida species in children with HIV [46], underscoring the importance of dental care programs for such children [4].

The mean density of Candida isolation in the children with HIV was higher than 1200 CFUs/mL, but all children were free of oral candidiasis, which apparently indicates that there is no association between the Candida count and the clinical manifestation of candidiasis. This finding is not consistent with previously published data [47, 48], which identified salivary counts higher than 400 CFUs/mL as indicative of clinical candidiasis. This divergence may indicate the contributory role of other factors, such as salivary flow rate, pH, and composition, which may be other predisposing colonization factors [41]. Approximately 40 to 65% of non-infected children may harbor commensal Candida spp. without exhibiting any clinical signs or symptoms of mucosal disease [49, 50].

Regarding the correlation of Candida and systemic conditions of the children in the present study, the prevalence of Candida was inversely correlated with the CD4⁺ T lymphocyte count. In contrast, no correlation was found between the occurrence of oral Candida and the plasma RNA viral load (Table 6).

Table 6.

Candida carriage and relationship to CD4⁺ T lymphocyte counting and viral load in the HIV-infected children

Systemic conditions	No. (%) of children for whom Candida spp isolation was:
	Positive	Negative
CD4+ T lymphocyte counting (%)
CD4 ≥ 25a	14	10
15 < CD4 < 24	11	4
CD4 ≤ 15a	11	0
Plasma RNA viral load (copies/ml)
≤ 500	7	0
501–3000	4	6
3001–10,000	6	4
10,001–30,000	8	1
> 30,000	11	3
No. (%) of patients tested	36 (72)	14 (28)

^aWith the chi-square test, a significant difference in results was found with p value of 0.0146

The prevalence of fungi detected in saliva using the swabbing technique was higher in children with a CD4⁺ T count lower than 15%; the same pattern was seen by Sah et al. [10], in which oral Candida density carriage and CD4⁺ cells were significantly correlated. Resistance to mucosal candidiasis and the prevalence of colonizing Candida spp. appear to be largely dependent on cell-mediated immunity, the CD4⁺ T lymphocyte count, and viral load [3, 10, 51]. The present data showed that laboratory markers of disease progression, especially CD4⁺ lymphocyte counting, exerted an influence on the incidence of oral Candida in children with HIV with no clinical evidence of oral candidiasis.

The present study presented some limitations regarding the selection of the study participants, since HIV-infected children were outpatients who came to receive HIV treatment, while non-HIV children were healthy ones who came to receive dental care at a dental school; nevertheless, all children were assisted by a multiprofessional team, dentists included. Socioeconomic status and education of parents in each group were not considered, factors that can influence on the hygiene and nutrition of the children, thus influence on Candida carriage in the children. Additionally, Candida species were identified only by conventional methods (morphological, physiological, and biochemical characteristics).

The results of the present study suggest that oral Candida rate is correlated with HIV infection, oral biofilm index, caries experience, and CD4⁺ T lymphocyte count. Although the current literature reports the association between the prevalence of oral Candida and systemic conditions in HIV-infected children, little information about what may exert influence on Candida rate in those specific patients has been published, especially in Brazil. Controlling the factors associated with Candida rate may improve the patient’s quality of life by helping to control the Candida proliferation and installation of the oral candidiasis, an opportunistic infection.

Author contribution

EMMBC: designed study, performed the research, analyzed data, and wrote the paper; CMAM and PGSV: wrote the paper; MBP and CMB: performed research and analyzed data; ASC and RMAS: funding acquisition and conceived of study; ALSS: designed study and analyzed data.

Funding

The present study was financially supported by the following Brazilian agencies: CNPq, FAPERJ, and CAPES (financial code 001).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.

Declarations

Ethics approval

Approval of this study was obtained from the Ethics Committee of UFRJ Center for Collective Health Studies (certificate number: 073/99) at the Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. The present study is in accordance with the standards set forth in the Declaration of Helsinki principles of 1975, as revised in 2013 (http://ethics.iit.edu/ecodes/node/3931).

Consent to participate

A freely-given informed consent to participate in the study was signed by the parents or guardians of all children before their enrollment.

Consent for publication

The study participants were informed about the present research, and a written consent form was taken from the parents or guardians of all children before their enrollment.

Conflict of interest

The authors declare no competing interests.