Differential Recovery of Candida Species from Subgingival Sites in Human Immunodeficiency Virus-Positive and Healthy Children from Rio de Janeiro, Brazil

Abstract

The prevalence of subgingival Candida species was studied in 52 human immunodeficiency virus (HIV)-positive and 42 HIV-negative children. Candida was cultured from 22 (42.3%) and 3 (7.1%) HIV-infected and control children, respectively. C. albicans was the most common Candida species isolated from HIV-infected children, followed by C. dubliniensis, C. glabrata, and C. tropicalis. In the HIV-positive group, the prevalence of Candida isolation was significantly higher in children who presented with low CD4⁺-T-lymphocyte counts, elevated viral loads, and gingivitis.

Oral manifestations of human immunodeficiency virus (HIV) disease have been recognized since the onset of the pandemic and are an important part of the natural history of HIV disease (11). Oral candidiasis, which is frequently caused by the yeast Candida albicans, is one of the most common AIDS-defining fungal opportunistic infections in HIV-positive individuals (10). Periodontal diseases are characterized by an inflammatory, degenerative, and necrotic response in the gingival and underlying connective tissues elicited by microbial colonization in periodontal pockets (8). The periodontal alterations should be the result of an altered immune response in the periodontal tissues, with changes in cellular and humoral immune responses (1, 15) that allow different Candida species to colonize the subgingival environment (2, 9, 13, 20).

In light of the paucity of information regarding HIV-associated periodontal disease in children, the periodontal health and associated microbiology of these individuals is of major interest. The present investigation determined whether the subgingival Candida microflora of the HIV-infected children differed from that of healthy children and evaluated the influence of the children's gingival health and systemic condition on the prevalence of these microorganisms.

Fifty-two infants with a positive diagnostic of HIV infection and 42 healthy nonimmunocompromised control children were recruited, and informed consent was obtained. All the children had the same socioeconomic status and were attended at the Hospital Pediátrio, Instituto de Puericultura e Pediatria Martagão Gesteira, and the Clínica Odontopediátrica at the Faculdade de Odontologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. These 94 children ranged in age from 4 to 12 years. The mean ages (± standard deviations) were 7.6 ± 1.9 years (59.5% were female, and 40.5% were male) and 8.4 ± 2.3 years (28% were female, and 72% were male) for the control and HIV-infected groups, respectively. No statistical difference (P > 0.05) in ages was found between the two studied groups of children. The distribution of the HIV-infected children according to disease stage, as previously established by the criteria of the Centers for Disease Control and Prevention (CDC) (3), is summarized in Table 1. In our study, 45 (86.5%) of the HIV-infected children were taking antiretroviral drugs. In this population, combined therapy was the most frequent (62.2%) type of treatment used. The combination of proteolytic inhibitors and nucleoside analogs was the therapy for 27 (96.4%) HIV-infected children. Nevertheless, there were no significant relationships observed between the use of antiretroviral medication and candidal isolation (P > 0.05).

TABLE 1.

Distribution of the HIV-infected children according to CDC criteria

Immunological stage	No. (%) of patients in CDC disease stageb:				No. (%) of patients testeda
	N	A	B	C
1	2	2	1	1	6 (12.8)
2	1	1	5	6	13 (27.7)
3	1	0	6	21	28 (59.6)
Total	4 (8.5)	3 (6.4)	12 (25.5)	28 (59.6)	47 (100)

^aThe CDC classification was not available for five children.

^bN, without clinical diseases; A, light clinical diseases; B, mild clinical diseases; C, severe clinical diseases.

All study subjects were given oral examinations that included periodontal indices and measures, dental caries indices, and soft tissue findings, as well as crevicular fluid samples (12, 18). The medical data were obtained from the hospital records. Subgingival plaque samples were obtained using sterile paper points (2). Aliquots of undiluted samples (0.1 ml) were spread into agar plates containing CHROMagar Candida medium (BD Diagnostic Systems, Paris, France) for presumptive identification of Candida species. The yeast isolates were subsequently identified by morphological and biochemical characteristics (5, 19).

At the time of collection, none of the subjects demonstrated clinical signs of classical oral candidiasis. However, six (11.5%) of the 52 HIV-positive children presented linear gingival erythema, which is a distinct fiery red band along the margin of the gingivae and probably has a candidal etiology (17). The prevalence of gingivitis was significantly higher in the HIV-infected group (89.4%) than in the healthy children (40.5%) (P < 0.05).

In the groups of 52 HIV-infected and 42 uninfected children, 22 (42.3%) and 3 (7.1%) presented positive cultures for Candida, respectively. A statistically significant difference was found between the two groups analyzed with respect to the prevalence of Candida isolation (P < 0.05). C. albicans was the most commonly recovered species isolated from both HIV-positive (n = 20) and HIV-negative (n = 3) infants. In the HIV-infected children, we also sampled three distinct non-albicans Candida species: C. dubliniensis (n = 3), C. glabrata (n = 1), and C. tropicalis (n = 1). Additionally, two Candida species (C. albicans plus C. dubliniensis, C. albicans plus C. glabrata, and C. albicans plus C. tropicalis) were coisolated from three (5.8%) HIV-infected patients, whereas none of the control subjects were populated with more than one yeast species.

We showed for the first time the presence of C. dubliniensis in the subgingival sites of HIV-positive children. Although C. albicans is the most common etiologic agent of oral candidosis, C. dubliniensis has emerged as another pathogen, noted for its in vitro potential for azole resistance and its enhanced in vitro adherence to human buccal epithelial cells (6, 9). Subgingival fungal infection may participate in the pathogenesis of destructive periodontal disease in HIV-infected populations (7, 16), which may also occur in an infant population. Moreover, the frequency of yeasts isolation was correlated positively with the seriousness of the gingival condition in the HIV-infected group, since 95% of infants who presented with Candida had inflammation in the gingivae. Interestingly, we also observed that all children positive for C. dubliniensis were classified as C3 and C2, which correspond to CDC clinical classifications of AIDS (3).

Several investigators have attempted to evaluate the relationship between the prevalence of Candida spp., and consequently between the development of oral candidiasis, and the most widely used indicator of progressive immune dysfunction, CD4⁺T-lymphocyte numbers (4, 5, 8, 13, 14, 18). In our investigations, the occurrence of cultivable Candida species was detected at all levels of CD4⁺-lymphocyte counts in HIV-positive children (Table 2). Moreover, we observed a positive correlation between the prevalence of oral Candida isolation and a decrease in CD4⁺T-lymphocytes numbers in peripheral blood (P < 0.05) (Table 2), as well as an increase in plasma viral loads (P < 0.05) (Table 2). The plasma viral load is a direct indicator of the total number of virus-producing cells in an infected person. Patients with higher plasma viral loads may develop AIDS in a shorter time because greater virus production more quickly exhausts the host's capacity to replenish destroyed CD4⁺ T lymphocytes (14). For instance, among the HIV-infected patients positive for Candida, a total of 65% had viral loads higher than 30,000 copies/ml and 52.4% had CD4⁺T-lymphocyte counts lower than 15%.

TABLE 2.

Candidal carriage and relationship to CD4⁺ counts and viral loads in the HIV-positive children

Systemic conditions	No. (%) of children for whom Candida spp. isolation was:
	+	−
CD4+-T-lymphocyte counts (%)
≤15a	11	6
15-24	5	7
≥25a	5	16
No. (%) of patients testedb	21 (42)	29 (58)
Plasma RNA viral loads (copies ml−1)
0-500	0	3
501-3,000	2	7
3,001-10,000	2	5
10,001-30,000	3	8
>30,000	13	6
No. (%) of patients testedc	20 (40.8)	29 (59.2)

^aWith the chi-square test, a significant difference in results was found at a P value of <0.05.

^bCD4⁺-lymphocyte counts were not available for two children.

^cViral loads were not available for three children.

Collectively, our results showed that Candida spp. may be present in gingival crevices of children, mainly children infected with HIV. We also observed a strong correlation between the isolation of Candida species and immunosuppression in HIV-infected children. This fact supports the idea of the necessity of more investigations about the real influence of these pathogens in an established progression and the prognostic value of periodontal manifestations in HIV-infected children.