Evaluation of Etest and macrodilution broth method for antifungal susceptibility testing of Candida sp strains isolated from oral cavities of AIDS patients

A comparison of the Etest and the reference broth macrodilution susceptibility test for fluconazole, ketoconazole, itraconazole and amphotericin B was performed with 59 of Candida species isolated from the oral cavities of AIDS patients. The Etest method was performed according to the manufacturer's instructions, and the reference method was performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines. Our data showed that there was a good correlation between the MICs obtained by the Etest and broth dilution methods. When only the MIC results at ± 2 dilutions for both methods were considered, the agreement rates were 90.4% for itraconazole, ketoconazole and amphotericin B and 84.6% for fluconazole of the C. albicans tested. In contrast, to the reference method, the Etest method classified as susceptible three fluconazole-resistant isolates and one itraconazole-resistant isolate, representing four very major errors. These results indicate that Etest could be considered useful for antifungal sensitivity evaluation of yeasts in clinical laboratories.

Plasmid profile in oral Fusobacterium nucleatum from humans and Cebus apella monkeys

Fusobacterium nucleatum is a strict anaerobe and is indigenous of the human oral cavity. This organism is commonly recovered from different monomicrobial and mixed infections in humans and animals. In this study, the plasmid profile, the plasmid stability and the penicillin-resistance association in oral F. nucleatum isolated from periodontal patients, healthy subjects and Cebus apella monkeys were evaluated. Forty-five F. nucleatum strains from patients, 38 from healthy subjects and seven from C. apella were identified and analyzed. Plasmid extraction was performed in all the isolated strains. These elements were found in 26.7% strains from patients and one strain from C. apella. Strains from healthy subjects did not show any plasmid. Most of strains showed two plasmid bands ranging from 4 to 16 Kb, but digestions with endonucleases showed that they belonged to a single plasmid. The plasmid profile was similar and stable in human and monkey strains. Also, plasmids were classified into three groups according to size. Two strains were positive to beta-lactamase production and no plasmid DNA-hybridization with a beta-lactamase gene probe was observed, suggesting a chromosomal resistance.

Electrophoretic protein patterns and numerical analysis of Candida albicans from the oral cavities of healthy children

The aim of this research was to evaluate the protein polymorphism degree among seventy-five C. albicans strains from healthy children oral cavities of five socioeconomic categories from eight schools (private and public) in Piracicaba city, São Paulo State, in order to identify C. albicans subspecies and their similarities in infantile population groups and to establish their possible dissemination route. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed with cold saline solution. The whole-cell proteins were extracted by cell disruption, using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with Coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrix and dendrogram were generated by using the Dice similarity coefficient and UPGMA algorithm, respectively, which made it possible to evaluate the similarity or intra-specific polymorphism degrees, based on whole-cell protein fingerprinting of C. albicans oral isolates. A total of 13 major phenons (clusters) were analyzed, according to their homogeneous (socioeconomic category and/or same school) and heterogeneous (distinct socioeconomic categories and/or schools) characteristics. Regarding to the social epidemiological aspect, the cluster composition showed higher similarities (0.788 < S D < 1.0) among C. albicans strains isolated from healthy children independent of their socioeconomic bases (high, medium, or low). Isolates of high similarity were not found in oral cavities from healthy children of social stratum A and D, B and D, or C and E. This may be explained by an absence of a dissemination route among these children. Geographically, some healthy children among identical and different schools (private and public) also are carriers of similar strains but such similarity was not found among other isolates from children from certain schools. These data may reflect a restricted dissemination route of these microorganisms in some groups of healthy scholars, which may be dependent of either socioeconomic categories or geographic site of each child. In contrast to the higher similarity, the lower similarity or higher polymorphism degree (0.499 < S D < 0.788) of protein profiles was shown in 23 (30.6%) C. albicans oral isolates. Considering the social epidemiological aspect, 42.1%, 41.7%, 26.6%, 23.5%, and 16.7% were isolates from children concerning to socioeconomic categories A, D, C, B, and E, respectively, and geographically, 63.6%, 50%, 33.3%, 33.3%, 30%, 25%, and 14.3% were isolates from children from schools LAE (Liceu Colégio Albert Einstein), MA (E.E.P.S.G. "Prof. Elias de Melo Ayres"), CS (E.E.P.G. "Prof. Carlos Sodero"), AV (Alphaville), HF (E.E.P.S.G. "Honorato Faustino), FMC (E.E.P.G. "Prof. Francisco Mariano da Costa"), and MEP (E.E.P.S.G. "Prof. Manasses Ephraim Pereira), respectively. Such results suggest a higher protein polymorphism degree among some strains isolated from healthy children independent of their socioeconomic strata or geographic sites. Complementary studies, involving healthy students and their families, teachers, servants, hygiene and nutritional habits must be done in order to establish the sources of such colonization patterns in population groups of healthy children. The whole-cell protein profile obtained by SDS-PAGE associated with computer-assisted numerical analysis may provide additional criteria for the taxonomic and epidemiological studies of C. albicans.

Oral cysticercosis: case report and review of the literature

Cysticercosis is a condition that occurs when man is infested by the larvae of Taenia solium, acting as an intermediate host instead of definitive. Oral cysticercosis is a rare event, and it represents a difficulty in clinical diagnosis. A case of oral cysticercosis in a 23-year-old white female who presented a painless swelling in the dorsal portion of the tongue is reported. An excisional biopsy was performed and histopathological examination revealed a cystic cavity containing the tapeworm.

Failure of nitric oxide production by macrophages and decrease in CD4+ T cells in oral paracoccidioidomycosis: possible mechanisms that permit local fungal multiplication

Paracoccidioidomycosis is a chronic granulomatous disease that induces a specific inflammatory and immune response. The participation of nitric oxide (NO), a product of the inducible nitric oxide synthase enzyme (iNOS), as an important fungicidal molecule against Paracoccidioides brasiliensis has been demonstrated. In order to further characterize the Oral Paracoccidioidomycosis (OP), we undertook an immunohistochemical study of iNOS+, CD45RO+, CD3+, CD8+, CD20+, CD68+ cells and mast cells. The samples were distributed in groups according to the number of viable fungi per mm². Our results showed weak immunolabeling for iNOS in the multinucleated giant cells (MNGC) and in most of the mononuclear (MN) cells, and the proportion of iNOS+ MN/MNGC cells in the OP were comparable to Control (clinically healthy oral tissues). Additionally, our analysis revealed a similarity in the number of CD4+ cells between the Control and the OP groups with higher numbers of fungi. These findings suggest that a low expression of iNOS and a decrease in the CD4+ T cells in OP may represent possible mechanisms that permit the local fungal multiplication and maintenance of active oral lesions.

Molecular identification of Candida dubliniensis isolated from oral lesions of HIV-positive and HIV-negative patients in São Paulo, Brazil

Candida dubliniensis is a new, recently described species of yeast. This emerging oral pathogen shares many phenotypic and biochemical characteristics with C. albicans, making it hard to differentiate between them, although they are genotypically distinct. In this study, PCR (Polymerase Chain Reaction) was used to investigate the presence of C. dubliniensis in samples in a culture collection, which had been isolated from HIV-positive and HIV-negative patients with oral erythematous candidiasis. From a total of 37 samples previously identified as C. albicans by the classical method, two samples of C. dubliniensis (5.4%) were found through the use of PCR. This study underscores the presence of C. dubliniensis, whose geographical and epidemiological distribution should be more fully investigated.

Genetic diversity of oral Fusobacterium nucleatum isolated from patients with different clinical conditions

The genetic diversity of 23 oral Fusobacterium nucleatum isolated from 15 periodontal patients, eight from seven healthy subjects, nine from nine AIDS patients and two from two Cebus apella monkeys were analyzed. EcoRI restricted the bacterial DNA and 28 ribotypes grouped from A to J groups were obtained. Isolates formed 24 ribotypes which were contained into A, B, C, D, E and F groups, and three reference strains and two clinical isolates of A. actinomycetemcomitans, and E. coli CDC formed four different ribotypes into the G, H, I and J groups. Moreover, from nine F. nucleatum from AIDS patients, six were ribotyped as group C and three as group D. By using ribotyping we distinguished F. nucleatum recovered from different sources. It is possible that isolates from AIDS patients may contain some phenotypic or genotypic factor did not observed in this study.

Asymptomatic oral carriage of Candida species in HIV-infected patients in the highly active antiretroviral therapy era

Oropharyngeal candidiasis is the most common opportunistic fungal infection in individuals infected with human immunodeficiency virus. CD4+ lymphocytes count and the quantification of viral RNA in blood plasma have been found to be the main markers of HIV disease progression. The present study was conducted to evaluate Candida sp. diversity in the oral cavity of HIV-infected patients and to determine whether there was association of CD4+ cell count and viral load with asymptomatic oral Candida carriage. Out of 99 HIV-positive patients studied, 62 (62.6%) had positive culture for Candida (oral carriage) and 37 patients (37.4%) had Candida negative culture (no oral carriage). The etiologic agents most common were C. albicans and C. tropicalis. The range of CD4+ was 6-2305 cells/mm³ in colonized patients and 3-839 cells/mm³ for non-colonized patients, while the viral load was 60-90016 copies/mL for colonized patients and 75-110488 copies/mL for non colonized patients. The viral load was undetectable in 15 colonized patients and in 12 non colonized patients. Our results showed that there was no significant difference of the variables CD4+ cell count and viral load between oral candida carriage and no oral candida carriage patients.

Comparison between E-test and CLSI broth microdilution method for antifungal susceptibility testing of Candida albicans oral isolates

Thirty Candida albicans isolated from oral candidosis patients and 30 C. albicans isolated from control individuals were studied. In vitro susceptibility tests were performed for amphotericin B, fluconazole, 5-flucytosine and itraconazole through the Clinical and Laboratorial Standards Institute (CLSI) reference method and E test system. The results obtained were analyzed and compared. MIC values were similar for the strains isolated from oral candidosis patients and control individuals. The agreement rate for the two methods was 66.67% for amphotericin B, 53.33% for fluconazole, 65% for flucytosine and 45% for itraconazole. According to our data, E test method could be an alternative to trial routine susceptibility testing due to its simplicity. However, it can not be considered a substitute for the CLSI reference method.

Relationship between plasma and red blood cell concentrations of quinine in Brazilian children with uncomplicated Plasmodium falciparummalaria on oral therapy

We determined the relationship between plasma and red blood cell concentrations of quinine in children with uncomplicated falciparum malaria from an endemic area of Amazonian region. Quinine was determined by high performance liquid chromatography with ultraviolet detection. In the steady state the ratio between plasma and red blood cell quinine concentration was 1.89 ± 1.25 ranging from 1.05 to 2.34. This result demonstrated that quinine do not concentrate in red blood cell of Brazilian children and characterize the absence of interracial difference in this relationship.

Differences in exoenzyme production and adherence ability of Candida spp. isolates from catheter, blood and oral cavity

Phospholipase and proteinase production and the ability of adhesion to buccal epithelial cells (BEC) of 112 Candida isolates originated from oral cavity of HIV infected patients and from blood and catheter of intensive care unit patients were investigated. The proteinase production was detected by inoculation into bovine serum albumin (BSA) agar and the phospholipase activity was performed using egg yolk emulsion. A yeast suspension of each test strain was incubated with buccal epithelial cells and the number of adherence yeast to epithelial cells was counted. A percentage of 88.1% and 55.9% of Candida albicans and 69.8% and 37.7% of non-albicans Candida isolates produced proteinase and phospholipase, respectively. Non-albicans Candida isolated from catheter were more proteolytic than C. albicans isolates. Blood isolates were more proteolytic than catheter and oral cavity isolates while oral cavity isolates produced more phospholipase than those from blood and catheter. C. albicans isolates from oral cavity and from catheter were more adherent to BEC than non-albicans Candida isolates, but the adhesion was not different among the three sources analyzed. The results indicated differences in the production of phospholipase and proteinase and in the ability of adhesion to BEC among Candida spp. isolates from different sources. This study suggests that the pathogenicity of Candida can be correlated with the infected site.

Oral cavity eumycetoma

Mycetoma is a pathological process in which eumycotic (fungal) or actinomycotic causative agents from exogenous source produce grains. It is a localized chronic and deforming infectious disease of subcutaneous tissue, skin and bones. We report the first case of eumycetoma of the oral cavity in world literature. CASE REPORT: A 43-year-old male patient, complaining of swelling and fistula in the hard palate. On examination, swelling of the anterior and middle hard palate, with fistula draining a dark liquid was observed. The panoramic radiograph showed extensive radiolucent area involving the region of teeth 21-26 and the computerized tomography showed communication with the nasal cavity, suggesting the diagnosis of periapical cyst. Surgery was performed to remove the lesion. Histopathological examination revealed purulent material with characteristic grain. Gram staining for bacteria was negative and Grocott-Gomori staining for the detection of fungi was positive, concluding the diagnosis of eumycetoma. The patient was treated with ketoconazole for nine months, and was considered cured at the end of treatment. CONCLUSION: Histopathological examination, using histochemical staining, and direct microscopic grains examination can provide the distinction between eumycetoma and actinomycetoma accurately.

Oral colonization by yeasts in HIV-positive patients in Brazil

INTRODUCTION: In HIV-infected patients, colonization of the oral cavity by potential pathogenic yeast may lead to development of systemic fungemia. We evaluated the prevalence of yeast in the oral cavity of Brazilian HIV-positive patients and verified whether or not the species characterized were enzymatically active. Furthermore, the species identified were tested for their susceptibility to antifungal treatment. METHODS: Patient saliva and oropharyngeal candidiasis samples were collected from 60 seropositive HIV patients and identified by the API20C system. Enzymatic activity was evaluated by the production of proteinase and phospholipase. Susceptibility to antifungal treatments were determined using the broth microdilution method. RESULTS: the most commonly isolated species were C. albicans (51.56%) followed by non-albicans Candida species (43.73%), Trichosporon mucoides (3.12%) and Kodamaea ohmeri (1.56%). Oral colonization by association of different species was observed in 42% of the patients. Enzymatic activity was verified in most of species isolated, except for C. glabrata, C. lusitaniae and C. guilliermondii. Resistance to Fluconazole and Amphotericin B was observed in isolates of C. albicans, C. glabrata, C. parapsilosis, C. krusei, and K. ohmeri. CONCLUSION: HIV-positive patients are orally colonized by single or multiple species of yeast that are occasionally resistant to Fluconazole or Amphotericin B.

Scedosporium apiospermum EUMYCETOMA SUCCESSFULLY TREATED WITH ORAL VORICONAZOLE: REPORT OF A CASE AND REVIEW OF THE BRAZILIAN REPORTS ON SCEDOSPORIOSIS

We describe a case of white-grain eumycetoma caused by Scedosporium apiospermum in an immunocompetent host that was successfully treated with oral voriconazole, and we review the Brazilian reports on scedosporiosis.