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.

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.

Association of Candida Species Isolated From the Dental Plaque of HIV-infected Children and Prevalence of Early Carious Lesions.

PURPOSE: The purposes of this study were to: (1) quantify and identify Candida species (spp.) in the dental plaque of children infected with the human immunodeficiency virus (HIV) and compare with noninfected children; and (2) determine the association of Candida spp. with the presence of caries. METHODS: Seventy HIV-infected and 55 non-HIV-infected three- to 12-year-old children were examined to determine caries prevalence. After a visual inspection, supragingival plaque was collected from the cervical region using standard dental curettes. The material was transferred to microtubes and submitted for analysis to identify and quantify the presence of Candida spp. RESULTS: Candida spp. were more prevalent in the HIV-infected group (72.9 percent) than in the control group (20 percent), and the most prevalent specie was Candida albicans. Caries was found in 72.9 percent of the HIV-infected group and in 58.2 percent of the control group, but a significant difference was only found in the presence of active white spot lesions between the groups. CONCLUSIONS: The dental plaque of HIV-infected children was colonized by Candida species to a much greater extent than that of non-HIV-infected children, and this colonization was significantly associated with the prevalence of early carious lesions in enamel.

Biofilm of Candida albicans from oral cavity of an HIV-infected child: challenge on enamel microhardness.

OBJECTIVE: The aim of this study was to analyze the effect of C. albicans on enamel microhardness in vitro. STUDY DESIGN: Candida albicans was isolated from the oral mucosa (M) and dentin carious lesion (D) of an HIV+ child. Three groups of 12 enamel blocks each were placed in Petri plates (yeast carbon base agar/1% bovine serum albumin): G1, exposed to biofilm formed by C. albicans from M; G2, exposed to biofilm formed by C. albicans from D; G3, no biofilm. Three enamel blocks from each group were removed on days 3, 5, 8, and 10 after biofilm formation to measure the cross-sectional Knoop microhardness (CSMH) of the enamel areas, exposed and not exposed to biofilm. RESULTS: CSMH decreased in G1 and G2: in G1 on day 5, and in G2 on day 3 (analysis of variance: P < .05; Mann-Whitney test: P < .05), with a similar mean percentage reduction for both groups. CONCLUSIONS: Candida albicans can reduce enamel microhardness in vitro.

Differential collagenolytic activity of Candida albicans isolated from oral mucosa and dentinal carious lesions of HIV-infected children.

OBJECTIVE: The aim of this study was to compare type I collagen degradation by Candida albicans isolated from oral mucosa (M) and cavitated active dentinal caries (CAD) of HIV-infected children. STUDY DESIGN: To verify the proteolytic activity, the specimens were cultivated in brain-heart infusion medium and the supernatants were incubated in the presence or absence of type I collagen at 37°C for 12 hours and analyzed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Intensity of the bands on the gels was assessed by densitometric analysis using a scanner and images analyzed with software from Kodak Digital Science EDAS 120. RESULTS: Supernatants of all the C. albicans degraded type I collagen: that from M, on average, by 38.3% (SD 21.67) and that from CAD by 54% (SD 25.94; Wilcoxon test: P < .05). Predisposing factors had no association with the percentage of type I collagen degradation (Mann-Whitney test: P > .05). CONCLUSIONS: Candida albicans from different sites of the oral cavity of HIV-infected children has proteolytic activity for type I collagen.

Use of chlorhexidine gel (0.2%) to control gingivitis and candida species colonization in human immunodeficiency virus-infected children: a pilot study.

PURPOSE: The purpose of this study was to evaluate chlorhexidine to control gingivitis and Candida species (spp.) in children infected with the human immunodeficiency virus (HIV) and their acceptance of the therapy. METHODS: Twenty-six HIV+ children were selected, and oral exam-established biofilm, gingival indexes, and stimulated saliva were collected for Candida ssp. identification. The children brushed their teeth for 21 days with chlorhexidine gel (0.2%). Salivary samples, biofilm, and gingival indexes were collected after 21-days and again 35 days after ceasing gel use. The children answered a questionnaire about the therapy. RESULTS: All children tested positive for Candida and gingivitis. After 21 days, Candida counts and gingivitis decreased in 25 and 26 children, respectively. Mean reduction was approximately 68% for Candida spp. and 74% for gingivitis. Thirty-five days after discontinuing gel use, gingivitis and Candida spp. increased in 13 and 16 patients, respectively. Considering the Candida spp., the heavy growth was lower in the first re-evaluation. Candida albicans was the most frequent species. Approximately 85% did not experience inconvenience with the gel, and approximately 48% thought it was good for tooth-brushing. CONCLUSION: Chlorhexidine therapy may be an option to treat and pre- vent gingivitis and reduce yeast counts in children infected with HIV.

Oral Candida colonization and its relation with predisposing factors in HIV-infected children and their uninfected siblings in Brazil: the era of highly active antiretroviral therapy.

OBJECTIVES: To evaluate predisposing factors such as orofacial manifestations, immunosuppression status and antiretroviral therapy in relation to oral colonization by Candida spp. in Brazilian HIV-infected children and their uninfected siblings in the era of highly active antiretroviral therapy (HAART). METHODS: Whole stimulated saliva was collected from 65 HIV-infected children (HIV+) and 40 uninfected siblings (HIV-), followed by assessment of orofacial manifestation, caries indexes and the number of cavitated dentinal carious teeth (CDT). The salivary samples were cultured and the colonies were counted. After which they were identified by sugar assimilation and fermentation (API 20C). Data was analyzed using chi-square, Mann-Whitney, Spearman tests and logistic regression. RESULTS: Regarding positive growth, HIV+ presented 80% (52/65) and HIV- 57.5% (23/40) (P = 0.013). Absence of antiretroviral therapy and HAART increased the probability of Candida isolation (P < 0.05). Mean CD4%, immune-status and history of recurrent oral candidiasis (OC) had no influence on Candida isolation. Mixed Candida spp. cultures were observed in HIV+ (40%) and HIV- (52%): C. albicans was more frequently found in both groups, with a higher prevalence in HIV+ (P = 0.05); other non-albicans species were isolated in HIV+ and HIV-. Low prevalence of orofacial manifestations was observed in HIV+ (10.7% of OC). There was an association between means of CDT and Candida growth (P < 0.05) and a positive correlation between number of CDT and Candida cfu-counts in HIV+ and HIV-. Mean CD4% and immune-status had no influence on Candida isolation. Absence of antiretroviral therapy and HAART increased the probability of Candida isolation (P < 0.05). CONCLUSIONS: The HIV infected children had a significantly higher prevalence of oral Candida spp. compared to their uninfected siblings. Absence of HAART and presence of dentinal carious teeth increased significantly Candida spp. colonization in these children.

Associations among the use of highly active antiretroviral therapy, oral candidiasis, oral Candida species and salivary immunoglobulin A in HIV-infected children.

OBJECTIVES: The aim was to examine the impact of antiretroviral therapy on the prevalence of oral candidiasis, recovery of oral Candida spp. , and salivary levels of total secretory immunoglobulin A (SIgA) and Candida-specific SIgA in human immunodeficiency virus (HIV)-infected children. STUDY DESIGN: Sixty-six HIV+ and 40 HIV- children were cross-sectionally examined for the presence of oral lesions. Whole stimulated saliva samples were collected for the identification of Candida spp. using culture and measurement of total and specific SIgA using enzyme-linked immunosorbent assay (ELISA). RESULTS: The HIV+ children had a higher prevalence of oral candidiasis (P < .05), higher frequency of detection of Candida spp. (P < .05), and higher levels of total (P < .05) and Candida-specific SIgA (P < .001) than the HIV- children. Among the HIV+ subjects, antiretroviral users had lower viral loads (P < .001) and lower levels of Candida spp. (P < .05) and total SIgA (P < .05) compared with antiretroviral nonusers. CONCLUSIONS: The use of antiretroviral therapy was associated with decreases in the prevalence of oral candidiasis. This diminished exposure to Candida spp. was accompanied by decreases in levels of total and Candida-specific SIgA.

Effect of suramin on the human pathogen Candida albicans: implications on the fungal development and virulence.

Candida albicans is an opportunistic pathogen that is of growing medical importance because it causes superficial, mucosal and systemic infections in susceptible individuals. Here, the effect of suramin, a polysulfonated naphthylurea derivative, on C. albicans development and virulence was evaluated. Firstly, it was demonstrated that suramin (500 microM) arrested its growth, showing a fungicidal action dependent on cell number. Suramin treatment caused profound changes in the yeast ultrastructure as shown by transmission electron microscopy. The more important changes were the enlargement of the fungi cytoplasmic vacuoles, the appearance of yeasts with an empty cytoplasm resembling ghost cells and a reduction in cell wall thickness. Suramin also blocked the transformation of yeast cells to the germ-tube and the interaction between C. albicans and epithelial cells. In order to ascertain that the action of suramin on C. albicans growth is a general feature instead of being strain-specific, the effects of suramin on 14 oral clinical strains isolated from healthy children and HIV-positive infants were analyzed. Interestingly, the strains of C. albicans isolated from HIV-positive patients were more resistant to suramin than strains isolated from healthy patients. Altogether, the results produced here show that suramin interfered with essential fungal processes, such as growth, differentiation and interaction with host cells.

Secretion of serine peptidase by a clinical strain of Candida albicans: influence of growth conditions and cleavage of human serum proteins and extracellular matrix components.

Candida albicans expresses a vast number of hydrolytic enzymes, playing roles in several phases of yeast-host interactions. Here, we identified two novel extracellular peptidase classes in C. albicans. Using gelatin-sodium dodecyl sulfate polyacrylamide gel electrophoresis two gelatinolytic activities were detected at physiological pH: a 60-kDa metallopeptidase, completely blocked by 1,10-phenanthroline, and a 50-kDa serine peptidase inhibited by phenylmethylsulfonyl fluoride. In an effort to establish a probable functional implication for these novel peptidase classes, we demonstrated that the 50-kDa secretory serine peptidase was active over a broad pH range (5.0-7.2) and was capable to hydrolyze some soluble human serum proteins and extracellular matrix components. Conversely, when this isolate was grown in yeast carbon base supplemented with bovine serum albumin, a secretory aspartyl peptidase activity was measured, instead of metallo- and serine peptidases, suggesting that distinct medium composition induces different expression of released peptidases in C. albicans. Additionally, we showed by quantitative proteolytic measurement, flow cytometry and immunoblotting assays that the brain heart infusion medium might repress the Sap1-3 production. Collectively, our results showed for the first time the capability of an extracellular proteolytic enzyme other than aspartic-type peptidases to cleave a broad spectrum of relevant host proteinaceous substrates by the human pathogen C. albicans.

Leishmania (Leishmania) amazonensis: differential expression of proteinases and cell-surface polypeptides in avirulent and virulent promastigotes.

A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania (Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE). The surface iodinated protein profiles showed two major polypeptides of 65-60 and 50-47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS-PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65-60, 52, and 50-47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65-60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. (L.) amazonensis.

Heterogeneity of metallo and serine extracellular proteinases in oral clinical isolates of Candida albicans in HIV-positive and healthy children from Rio de Janeiro, Brazil.

Candida yeasts frequently cause life-threatening systemic infections in immunocompromised hosts. In the present study, gelatin-SDS-PAGE analysis was used to characterize extracellular proteinases in 44 oral clinical isolates of Candida albicans from HIV-positive (29/50) and healthy children (15/50). Our survey indicates that these oral clinical isolates of C. albicans have complex extracellular proteolytic activity profiles, which illustrates the heterogeneity of this species. We showed four distinct proteolytic patterns composed of distinct serine (30-58 kDa) and metalloproteinase (64-95 kDa) activities, based on the inhibition profile with phenylmethylsulfonyl fluoride and 1,10-phenanthroline, respectively. This is the first report on secreted serine and metalloproteinases present in the culture supernatant fluids of C. albicans; however, we did not observe a significant correlation between proteolytic profile expressed by the C. albicans isolates from HIV-positive children and CD4(+) T cell count and plasma viral load.

Herpetomonas samuelpessoai: dimethylsulfoxide-induced differentiation is influenced by proteinase expression.

In this study, we analyzed the influence of proteinase expression on the cellular differentiation of Herpetomonas samuelpessoai. Along cellular differentiation, which was induced by dimethylsulfoxide (DMSO), the trypanosomatids secreted several molecules with variable proteolytic activity. All of them were inhibited by 10 m M 1,10-phenanthroline, suggesting that they are zinc-metalloproteinases. Analysis of parasite extracts revealed the occurrence of a 63-kDa metalloproteinase and a 45-kDa cysteine proteinase. After extraction with Triton X-114 followed by water-detergent partition, the 63-kDa component was present in both aqueous and detergent phases, which indicated that this enzyme may be distributed over different cellular compartments including membrane domains. The 45-kDa component, however, presented hydrophilic properties and was predominantly expressed by DMSO non-treated parasites, suggesting that proteinases may be involved in the process of cellular differentiation in H. samuelpessoai. This was confirmed by the fact that a cysteine proteinase inhibitor abrogated parasite differentiation. The role of proteinases and their relevance in the differentiation of H. samuelpessoai are discussed.

Changes of sialomolecules during the dimethylsulfoxide-induced differentiation of Herpetomonas samuelpessoai.

The expression of sialoglycoconjugates during the dimethylsulfoxide (DMSO)-induced differentiation of Herpetomonas samuelpessoai was analyzed by flow cytometry and Western blotting using sialic acid-specific lectins. Parasites reacted strongly with Limax flavus (LFA) and Sambucus nigra (SNA) agglutinins, and only weakly with Maackia amurensis (MAA) lectin. However, analysis of crude protein extracts by Western blotting revealed that bands with molecular masses corresponding to 15 and 40 kDa are recognized by MAA, and that treatment with DMSO induced the expression of two additional polypeptides with molecular masses of 65 and 90 kDa. Profiles of binding to LFA were indistinguishable when protein extracts from control or differentiated cells were analyzed. SNA recognized a major molecule with 25 kDa in extracts from non-differentiated forms and two low-molecular-weight bands from differentiated cells. These results indicate that molecules containing alpha2,6 and alpha2,3 sialyl-galactosyl sequences are present in H. samuelpessoai, and that their biosynthesis and expression are influenced by DMSO-induced differentiation.

Detection of sialoglycomolecules in five plant trypanosomatids and in an insect phytophagous isolate.

The sialoglycoprotein profiles of five plant trypanosomatids (Phytomonas spp.) and of one flagellate (Herpetomonas sp.) isolated from the salivary gland of a phytophagous insect (Phthia picta) were analyzed by Western blotting using three distinct lectins (LFA, SNA and MAA), which recognize specifically sialic acid residues in glycoconjugates. All six flagellates presented at least one polypeptide recognized by the lectins, with the exception of Phytomonas françai, which did not show any reactivity with SNA agglutinin. Phytomonas serpens and P. françai showed the most distinct pattern of sialoglycoproteins. Phytomonas mcgheei, Herpetomonas sp. and the two other Phytomonas spp., isolated from latex, displayed an identical sialomolecule profile. We discuss the possible role of the sialoglycoproteins in the physiology of these trypanosomatids.

Activation of the glycosylphosphatidylinositol-anchored membrane proteinase upon release from Herpetomonas samuelpessoai by phospholipase C.

We have analyzed the effects of exogenous phospholipase C (PLC) on the cell-surface polypeptides and proteinases of Herpetomonas samuelpessoai grown in chemically defined conditions by SDS-PAGE gels. Live parasites treated with PLC released into the extracellular medium a complex profile of glycosylphosphatidylinositol (GPI)-anchored polypeptides ranging from 15 to 100 kDa, some of them with proteolytic activity. Two major metalloproteinases with apparent molecular masses of 63 and 115 kDa were observed after PLC hydrolysis. Interestingly, only the PLC-released soluble form of the 115-kDa metalloenzyme, and not the membrane-anchored form, displayed proteolytic activity, demonstrating that cleavage of the GPI anchor can lead to enzymatic activation.