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

J Oral Pathol Med (2010) 39 188–194 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.     

Ectophosphatase activity in Candida albicans influences fungal adhesion: study between HIV‐positive and HIV‐negative isolates

Oral Diseases (2010) 16 , 431–438 Objective: This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV‐infected children (HIV+) and compares them with fifteen isolates from HIV‐negative children (HIV?), as well as the fungal adhesion to epithelial cells and medical records. Methods: The activities were measured in intact cells grown in BHI medium for 48 h at 37°C. Phosphatase activity was assayed at pH 5.5 using 4‐methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line. Results: Mean values of ectophosphatase activity were 610.27 ± 166.36 and 241.25 ± 78.96 picomoles 4‐methylumbelliferone/h/10⁷ cells for HIV+ and HIV? group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV? (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor. Conclusion: These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.     

The isolation,identification and molecular analysis of Candida spp. isolated from the oral cavities of patients with diabetes mellitus

Previous studies have shown a high incidence (77%) of isolation of Candida spp. from the oral cavities of patients with type 1 diabetes mellitus. The aim of the present study was to assess the prevalence of yeast in the oral cavities of patients suffering from type 1 and type 2 diabetes mellitus. The patients were classified according to the level of diabetic control (HbA1_c), and further stratified on the presence or absence of dental prosthesis. Oral rinse samples were assessed for the growth of yeast and the degree of colonization. Oral isolates were defined to the species level by both phenotypic and novel molecular methods. The overall proportion (60%) of diabetic patients who had Candida spp. isolated from the oral cavity was similar to that previously reported. Local oral factors, such as the presence of dentures, seemed to have a greater influence than diabetic status on the amount and species of Candida isolated from the oral cavities of diabetic patients. Diabetic patients with dentures had more non‐albicans Candida isolated from their mouths than dentate diabetic patients. Candida dubliniensis was isolated from diabetic patients and may have a predilection for dentate patients.     

Asymptomatic oral Candida albicans carriage in HIV-infection: frequency and predisposing factors

Many studies have focused on the epidemiology and pathogenesis of oral candidiasis in HIV infection. Little is known on the incidence and predisposing factors of asymptomatic oral Candida carriage in this setting, obviously an important issue in view of prophylaxis. To address this question. 261 consecutive HIV-infected individuals without clinical evidence of candidiasis were investigated. C. albicans was isolated from cultured oral cavity swabs of 63 subjects (24%). Colonization was significantly more frequent in IV drug users. CDC groups IV. and in subjects with lymphocytopenia. CD4+ cell depletion, or elevated beta-2 microglobulin. These data further suggest that oral candidiasis occurs in HIV infection as a result of C. albicans overgrowth and raise the question of primary antifungal prophylaxis in subjects with low CD4 counts and asymptomatic oral Candida carriage.     

Th1/Th2 cytokine profiles in saliva of HIV‐positive smokers with oropharyngeal candidiasis

Oropharyngeal candidiasis (OPC) is a common opportunistic infection among HIV‐positive individuals and often correlates with a CD4 cell number < 200 cells/µl. This study further examined the association of smoking and OPC in HIV‐positive persons. A strong association between smoking and OPC was seen in HIV‐positive individuals with ≥ 200 CD4 cells/µl. In HIV‐positive persons with ≥ 200 CD4 cells/µl, OPC⁺ smokers had lower gamma‐interferon (IFN‐γ) concentrations and a trend toward higher interleukin (IL)‐4 concentrations in whole saliva compared to OPC^– persons with ≥ 200 CD4 cells/µl, a cytokine profile consistent with that observed in HIV⁺OPC⁺ persons with < 200 CD4 cells/µl. These results suggest that premature OPC in HIV‐positive smokers is associated with altered oral host defence mechanisms that cannot be overcome by levels of systemic CD4 cells that are otherwise sufficient to protect against OPC.     

Molecular characterization of Candida spp. isolated from the oral cavities of patients from diverse clinical settings

Infections by Candida spp. have increased in medical importance over the past few decades. Our understanding of species identification, commensalisms, pathogenicity, person-to-person spread, and the development of antifungal resistance within specific strains has been greatly enhanced by the utilization of molecular epidemiological methodology. The aim of the current research was to assess the quantity, species and molecular characterization of oral yeast isolates from well-defined cohorts of immunocompetent patients from a diverse range of clinical settings. Oral rinse samples were assessed for the growth of yeast and degree of colonization. Isolates were defined to the species level by both phenotypic and molecular methods and strains were further genotypically subtyped. Significant variation was shown to exist in the number, species and genotypic subgroups of yeast isolated from the oral cavity in different patient groups. This variation could be attributed to the local oral conditions unique to these patient groups.     

健康老年人口腔念珠菌与义齿修复的相关研究

目的 研究健康老年人群中口腔念珠菌的定植状况,分析义齿修复对老年人口腔念珠菌定植的影响。方法 212例健康老年人分为4组：A1组（男性,有义齿）,B1组（男性,无义齿）,A2组（女性,有义齿）和B2组（女性,无义齿）。标准含潄法取样,样本接种于沙堡琼脂培养基培养念珠菌,CHROMagar CandidaTM鉴定培养基鉴定白色念珠菌,碳水化合物同化反应鉴定体系鉴定念珠菌菌种。培养基中念珠菌菌落计数为每个样本的念珠菌检出强度。比较4组健康老年人念珠菌检出率和检出强度有无统计学差异。结果 212例老年人中116例检出念珠菌,检出率为54.72％。检出念珠菌包括白色念珠菌、近平滑念珠菌、克柔念珠菌、热带念珠菌等10个菌种。A1、B1、A2和B2组念珠菌检出率分别为66.67%,36.07%,64.15%和56.00%;白色念珠菌检出率分别为56.25%,21.31%,56.60%和38.00%。A1组念珠菌、白色念珠菌检出率高于B1组（P<0.05）。高念珠菌强度者在A1、A2组所占比例分别较B1、
B2组高（P<0.05）。结论 健康老年人口腔总念珠菌及白色念珠菌检出率和检出强度增高与义齿修复相关。老年人
口腔念珠菌检出率和检出强度的差异主要是白色念珠菌检出率和检出强度的差异所造成。     

In vitro evaluation of virulence attributes of Candida spp. isolated from patients affected by diabetes mellitus

BACKGROUND: Diabetes mellitus is a common disease found worldwide and it has been previously suggested that oral candidal infections may be more frequent or severe in patients with this disease. Systemic and local factors may influence the balance between the host and yeasts, and favour the transformation of Candida isolates from commensal to pathogenic microorganisms. Candida species have developed specific virulence mechanisms that confer the ability to colonise host surfaces, to invade deeper host tissue, or to evade host defences. Few studies have investigated the expression of the virulence attributes of oral Candida isolates in patients with diabetes mellitus. MATERIAL AND METHODS: The in vitro extracellular proteinase production and the in vitro ability to adhere to fibronectin of 229 Candida isolates of two geographic different groups of patients with diabetes mellitus and of healthy subjects were assessed. RESULTS: Candida isolates of patients with diabetes mellitus expressed a higher ability to adhere than those of healthy subjects. Higher levels of adhesion were also recorded in patients with a lower oral Candida colonisation. No differences were observed in the in vitro expression of extracellular proteinase of Candida isolates of patients with diabetes mellitus and those of non-diabetic subjects. Isolates of patients with type 2 diabetes mellitus expressed greater levels of proteinase than isolates of type 1 diabetes mellitus. CONCLUSIONS: Diabetes mellitus could be considered as an additional variable that may influence not only oral Candida carriage but also the ability of isolates to enhance the expression of virulence attributes.     

The isolation,identification and molecular analysis of Candida spp. isolated from the oral cavities of patients with diabetes mellitus

Previous studies have shown a high incidence (77%) of isolation of Candida spp. from the oral cavities of patients with type 1 diabetes mellitus. The aim of the present study was to assess the prevalence of yeast in the oral cavities of patients suffering from type 1 and type 2 diabetes mellitus. The patients were classified according to the level of diabetic control (HbA1c), and further stratified on the presence or absence of dental prosthesis. Oral rinse samples were assessed for the growth of yeast and the degree of colonization. Oral isolates were defined to the species level by both phenotypic and novel molecular methods. The overall proportion (60%) of diabetic patients who had Candida spp. isolated from the oral cavity was similar to that previously reported. Local oral factors, such as the presence of dentures, seemed to have a greater influence than diabetic status on the amount and species of Candida isolated from the oral cavities of diabetic patients. Diabetic patients with dentures had more non-albicans Candida isolated from their mouths than dentate diabetic patients. Candida dubliniensis was isolated from diabetic patients and may have a predilection for dentate patients.     

Biotypes of oral Candida albicans isolated from AIDS patients and HIV-free subjects in Thailand

This study was conducted to examine biotypes and antifungal susceptibility patterns of oral Candida albicans isolated from HIV-infected patients, HIV-free patients with candidiasis and healthy subjects. All isolates were biotyped using a typing system based on enzyme profiles, carbohydrate assimilation patterns and boric acid resistance. Thirty-eight biotypes were found amongst 218 oral C. albicans isolates. The major biotype found was A1S, which accounted for 32.6% of all isolates, and this biotype was the most common in all groups. There was a greater variety of biotypes of C. albicans in the HIV-infected group than in the other groups; however, there was no statistically significant difference between the groups. The minimum inhibitory concentrations (MICs) of a total of 118 isolates were determined for amphotericin B and for ketoconazole using the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method and the E-test. When the antifungal susceptibility patterns among the groups were compared, a statistically significant difference was found only with amphotericin B. The median MIC of amphotericin B in the HIV-infected group was higher than in the healthy group (P=0.013, NCCLS method; P=0.002, E-test). However, this difference in sensitivity was not restricted to any sub-type investigated. Our results showed that the biotype patterns of C. albicans isolates that colonize HIV-infected patients are similar to those of HIV-free subjects, and there is no relationship between antifungal susceptibility patterns and the biotypes.     

各年龄段儿童口腔中念珠菌的分布研究

目的 研究以白色念珠菌为代表的念珠菌群在不同年龄阶段儿童口腔中的分布情况。方法 对4个不同年龄阶段的儿童口腔中的念珠菌群进行研究，颊黏膜拭子取样，鉴定培养基培养鉴定，比较培养法和PCR法对其中一组样本的白色念珠菌检出率的影响。结果念珠菌的检出率分别是：新生儿7．5％，乳牙列儿童组70％，混合牙列儿童组56．36％，恒牙列儿童组49．12％；白色念珠菌的比例各有不同，PCR方法比培养法更加敏感。结论 各个年龄阶段儿童口腔中均有念珠菌的检出，但检出率不同，白色念珠菌是主要成分。     

In vitro activity of a monoclonal killer anti‐idiotypic antibody and a synthetic killer peptide against oral isolates of Candida spp. differently susceptible to conventional antifungals

Background/aims: A monoclonal killer anti‐idiotypic antibody (mAbK10) and a synthetic killer peptide, acting as internal images of a microbicidal, wide‐spectrum yeast killer toxin (KT) have been recently shown to express candidacidal in vitro and an in vivo therapeutic activity against experimental mucosal and systemic candidosis models caused by a reference strain of Candida albicans (10S). Material and methods: The in vitro candidacidal activity of mAbK10 and synthetic killer peptide was compared using a colony forming unit assay against a large number of isolates of different Candida spp., obtained from oral saliva of adult diabetic (type 1 and 2) and nondiabetic subjects from Parma (Italy) and London (UK). Results: Both the KT‐mimics exerted a strong dose‐dependent candidacidal activity, probably mediated by the interaction with β‐glucan KT receptors on target yeast cells, against all the tested strains, regardless of their species and pattern of resistance to conventional antifungal agents. Conclusions: These observations open new perspectives in the design and production of candidacidal compounds whose mechanism reflects that exerted in nature by killer yeasts.     

Salivary IgA to cariogenic bacteria in HIV‐positive children and its correlation with caries prevalence and levels of cariogenic microorganisms

The interrelationship of HIV infection, dental caries and mucosal immune responses remains controversial. In our study population of 40 HIV‐infected and 40 healthy control children (ages 2–5 years) there was a significantly higher prevalence of dental caries in HIV‐infected children (P<0.05). The extent of caries correlated with the severity of HIV disease. To determine whether the immunosuppression that ensues after HIV infection could contribute to the increased caries prevalence, the concentrations of total IgA and IgA specific to cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus and Lactobacillus acidophilus) were determined in whole saliva by enzyme‐linked immunosorbent assay . Levels of the same bacteria were also quantified in saliva using checkerboard DNA–DNA hybridization. A significantly increased level of total salivary IgA was found in the HIV‐positive population (P < 0.05), but there were comparable titers of specific IgA to cariogenic bacteria in HIV‐positive and healthy controls. The microbiological assessment also demonstrated similar levels of cariogenic microorganisms in both groups. We conclude that HIV‐positive children appear to maintain the capacity to mount a mucosal immune response to cariogenic microorganisms, at least until late stages of disease.     

Cytokine profiles in parotid saliva from HIV‐1‐infected individuals: changes associated with opportunistic infections in the oral cavity

The purpose of this study was to quantitate levels of cytokines in parotid saliva of subjects infected with human immunodeficiency virus‐1 (HIV‐1) and to determine if the cytokine profiles differ in subjects with an oral opportunistic infection, i.e., candidiasis or oral hairy leukoplakia. Parotid saliva samples were obtained from HIV‐infected individuals with or without candidiasis or oral hairy leukoplakia and from healthy controls and were assessed by ELISA for levels of interleukin (IL)‐1, IL‐2, IL‐4, IL‐5, IL‐10, transforming growth factor‐β, tumor necrosis factor‐α and interferon (IFN)‐γ. Saliva from HIV‐infected subjects with oral candidiasis had significantly higher levels of IFN‐γ than that seen in HIV‐infected individuals with no oral disease and significantly higher levels of IL‐2, IL‐5 and IFN‐γ than saliva of healthy controls. No significant difference was seen in cytokine levels in saliva from HIV‐infected subjects with no oral infections and healthy controls. The HIV‐infected subjects with oral hairy leukoplakia displayed significantly higher levels of both IL‐1α and IFN‐γ compared with the HIV and no oral disease group and a higher level of IFN‐γ than seen in saliva from the healthy control group. In comparing cytokine levels from both HIV and oral disease groups, significant differences were detected in levels of IL‐5 and IL‐10. These results indicate that the profile of salivary cytokines is altered as a result of the oral opportunistic infection candidiasis or oral hairy leukoplakia and also by concurrent HIV infection.     

Clonal identity of Candida albicans in the oral cavity and the gastrointestinal tract of pre‐school children

The clonal relationship between oral and fecal Candida albicans isolated from children of pre‐school age was examined using RAPD analysis. Significantly higher levels of C. albicans were found in saliva, dental plaque, carious specimens and stools of 56 patients with severe caries as compared to 52 healthy control subjects. The highest prevalence was found in carious specimens and a strong correlation was observed between its presence in saliva, dental plaque, carious specimen and feces. RAPD analysis of isolates from 23 patients with simultaneous oral and fecal C. albicans revealed clonal counterparts present in both oral and stool samples in 15 cases; five patients harbored closely related strains; and three patients harbored unrelated strains. Our results demonstrate a strong correlation between oral and gastrointestinal C. albicans colonization. We assume that carious teeth may constitute an ecologic niche for C. albicans potentially responsible for recurrent oral and non‐oral candidiasis.