Isolation of Candida spp. from denture-related stomatitis in Pará, Brazil

ABSTRACT The aim of this study was to isolate and identify Candida species from the oral cavity of denture wearers with denture-related stomatitis who were attended at the University Federal of Pará (Belém City, Pará State, Brazil). A total of 36 denture wearers with denture-related stomatitis were included, and type I (50%), type II (33%) and type III (17%) stomatitis were observed. Candida spp. were isolated from 89% of the cases and included five different Candida species. C. albicans was the most frequently recovered species (78% of the cases), followed by C. famata and C. tropicalis. We observed a significant association between Candida species isolation and unsatisfactory denture condition (p = 0.0017). Our results demonstrated the highly frequency of Candida species isolation in denture wearers with denture-related stomatitis and showed the relationship between these species and poor denture maintenance.

Biosynthesis, characterization and leishmanicidal activity of a biocomposite containing AgNPs-PVP-glucantime.

AIM: Development of functionalized nanocomposites containing AgNPs-PVP-Glucantime® to evaluate their leishmanicidal activity as a novel method for improving the pharmacological properties of the drug Glucantime® against extracellular promastigotes and intracellular amastigotes of Leishmania amazonensis in vitro to treat cutaneous leishmaniasis. MATERIALS & METHODS: The silver nanoparticles and nanocomposites prepared containing silver nanoparticles, polyvinylpyrrolidone and different amounts of Glucantime were characterized using transmission electron microscopy, x-ray diffraction, energy-dispersive x-ray spectroscopy and ζ potential analysis; in addition, the in vitro cytotoxicity was evaluated. RESULTS: The nanocomposites showed an inhibitory effect on the cellular viability of promastigote forms, with values of 47.06, 51.71 and 65.67% for nanocomposite1, nanocomposite2 and nanocomposite3, respectively, as well as a dose-dependent decrease in the infectivity index, with values of 33.33 and 23% for nanocomposite2 and nanocomposite3, respectively. CONCLUSION: The proposed nanocomposite reveals leishmanial activity and the absence of cytotoxicity in macrophages. Further investigations will be conducted in vivo.

Oral carriage of Candida species in HIV-infected patients during highly active antiretroviral therapy (HAART) in Belém, Brazil.

OBJECTIVE: To identify the oral carriage of Candida spp in patients infected by human immunodeficiency virus (HIV) and the possible correlation with clinical characteristics. STUDY DESIGN: Mucosal swab samples collected from 246 patients who were infected by HIV, did not have oral candidiasis, and were being treated with highly active antiretroviral therapy were analyzed. Yeast colonies that developed were identified by using the VITEK 2 automated system. RESULTS: Candida yeasts were present in 41.87% of the samples, and Candida albicans was the most prevalent (32.52%). Other identified Candida species were C tropicalis (4.88%), C parapsilosis (2.85%), C dubliniensis (0.81%), and C famata (0.81%). CONCLUSIONS: There was low rate of oral Candida carriage in patients infected by HIV who were on highly active antiretroviral therapy. A greater prevalence of C albicans than non-albicans Candida species was found at the species level. Prior candidiasis predicted the oral carriage of C albicans; however, it did not influence the carriage of non-albicans species. This is the first report of oral carriage of C famata in patients with HIV infection.

Immunodiagnosis of paracoccidioidomycosis due to Paracoccidioides brasiliensis using a latex test: detection of specific antibody anti-gp43 and specific antigen gp43.

BACKGROUND: Paracoccidioidomycosis (PCM) is a life-threatening systemic disease and is a neglected public health problem in many endemic regions of Latin America. Though several diagnostic methods are available, almost all of them present with some limitations. METHOD/PRINCIPLE FINDINGS: A latex immunoassay using sensitized latex particles (SLPs) with gp43 antigen, the immunodominant antigen of Paracoccidioides brasiliensis, or the monoclonal antibody mAb17c (anti-gp43) was evaluated for antibody or antigen detection in sera, cerebrospinal fluid (CSF), and bronchoalveolar lavage (BAL) from patients with PCM due to P. brasiliensis. The gp43-SLPs performed optimally to detect specific antibodies with high levels of sensitivity (98.46%, 95% CI 91.7-100.0), specificity (93.94%, 95% CI 87.3-97.7), and positive (91.4%) and negative (98.9%) predictive values. In addition, we propose the use of mAb17c-SLPs to detect circulating gp43, which would be particularly important in patients with immune deficiencies who fail to produce normal levels of immunoglobulins, achieving good levels of sensitivity (96.92%, 95% CI 89.3-99.6), specificity (88.89%, 95% CI 81.0-94.3), and positive (85.1%) and negative (97.8%) predictive values. Very good agreement between latex tests and double immune diffusion was observed for gp43-SLPs (k = 0.924) and mAb17c-SLPs (k = 0.850), which reinforces the usefulness of our tests for the rapid diagnosis of PCM in less than 10 minutes. Minor cross-reactivity occurred with sera from patients with other fungal infections. We successfully detected antigens and antibodies from CSF and BAL samples. In addition, the latex test was useful for monitoring PCM patients receiving therapy. CONCLUSIONS/SIGNIFICANCE: The high diagnostic accuracy, low cost, reduced assay time, and simplicity of this new latex test offer the potential to be commercialized and makes it an attractive diagnostic assay for use not only in clinics and medical mycology laboratories, but mainly in remote locations with limited laboratory infrastructure and/or minimally trained community health workers.

Effects of Rosmarinus officinalis essential oil on germ tube formation by Candida albicans isolated from denture wearers.

INTRODUCTION: The aim of this study was to investigate the effects of Rosmarinus officinalis essential oil on germ tube formation by Candida albicans isolated from denture wearers. METHODS: Ten C. albicans isolates recovered from denture wearers were tested using 10% fetal bovine serum with or without 4% R. officinalis essential oil. RESULTS: The essential oil from R. officinalis completely inhibited germ tube formation in the investigated C. albicans isolates. CONCLUSIONS: The results demonstrate that the essential oil of R. officinalis modulates C. albicans pathogenicity through its primary virulence factor (i.e., germ tube formation was suppressed).

Dermatophytosis diagnosed at the Evandro Chagas Institute, Pará, Brazil

Dermatophytosis is caused by a dermatophyte fungus that affects the stratum corneum and keratinized tissue. Dermatophyte fungus has been reported worldwide as the causative agent of dermatophytosis, but the etio-epidemiological aspects of these mycoses in the state of Pará remain unknown. The purpose of this study was to describe the etio-epidemiological profile of dermatophytosis diagnosed in patients at the Evandro Chagas Institute from May 2005 to June 2006. A total of 494 patients were admitted, and their samples were collected, submitted for direct microscopic examination using 20% KOH and cultured in Sabouraud and Mycosel medium. The identification was based in macro and microscopic characteristics. Direct examinations were positive in 13% (66/494) of the patients, and agent isolation by cultivation of the biological sample was successful in 4% (20/494), with a high prevalence of T. mentagrophytes (40%; 8/20). Dermatophytosis was more frequent in women (58%; 38/66). Fifty-two percent (21/38) of the cases were children with an average age of 8 years. The most frequent clinical presentation was Tinea corporis (55%, 36/66). For the cases in which the dermatophyte agent was not isolated, we discuss the factors that may be interfering with isolation. Tinea corporis occurred more frequently observed when T. mentagrophytes and T. rubrum were the major etiologic agents.

Occurrence of Paracoccidioides lutzii in the Amazon region: description of two cases.

Paracoccidioidomycosis (PCM), the most important human systemic mycosis in Latin America, is known to be caused by at least four different phylogenetic lineages within the Paracoccidioides brasiliensis complex, including S1, PS2, PS3, and Pb01-like group. Herein, we describe two cases of PCM in patients native from the Amazon region. The disease was originally thought to have been caused by P. brasiliensis. Despite the severity of the cases, sera from the patients were negative in immunodiffusion tests using the standard exoantigen from P. brasiliensis B-339. However, a positive response was recorded with an autologous preparation of Paracoccidioides lutzii exoantigen. A phylogenetic approach based on the gp43 and ARF loci revealed high similarity between our clinical isolates and the Pb01-like group. The occurrence of PCM caused by P. lutzii in the Brazilian Amazon (Pará State) was thus proven. The incidence of PCM caused by P. lutzii may be underestimated in northern Brazil.

Effect of serum sample inactivation on the performance of latex agglutination test for paracoccidioidomycosis serodiagnosis.

Paracoccidioidomycosis is diagnosed from the direct observation of the causative agent, but serology can facilitate and decrease the time required for diagnosis. The objective of this study was to determine the influence of serum sample inactivation on the performance of the latex agglutination test (LAT) for detecting antibodies against Paracoccidioides brasiliensis. The sensitivity of LAT from inactivated or non-inactivated samples was 73% and 83%, respectively and the LAT selectivity was 79% and 90%, respectively. The LAT evaluated here was no more specific than the double-immunodiffusion assay. We suggest the investigation of other methods for improving the LAT, such as the use of deglycosylated antigen.

Effect of serum sample inactivation on the performance of latex agglutination test for paracoccidioidomycosis serodiagnosis

Paracoccidioidomycosis is diagnosed from the direct observation of the causative agent, but serology can facilitate and decrease the time required for diagnosis. The objective of this study was to determine the influence of serum sample inactivation on the performance of the latex agglutination test (LAT) for detecting antibodies against Paracoccidioides brasiliensis. The sensitivity of LAT from inactivated or non-inactivated samples was 73% and 83%, respectively and the LAT selectivity was 79% and 90%, respectively. The LAT evaluated here was no more specific than the double-immunodiffusion assay. We suggest the investigation of other methods for improving the LAT, such as the use of deglycosylated antigen.

Effects of pretreating serum samples on the performance of a latex agglutination test for serodiagnosis of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a fungal disease caused by Paracoccidioides brasiliensis, and Brazil is one of the principal countries where it is endemic. Diagnosis is based on the observation of budding P. brasiliensis yeast in clinical specimens from patients; however, the sensitivity of the visualization of fungi is low, indicating that serological tests are used for early diagnosis. The double-immunodiffusion test (ID) is the "gold standard" test for serology in PCM, although the execution of this test requires the availability of laboratorial infrastructure. We report the improved performance of a latex agglutination test (LAT) by pretreating 30 serum samples from PCM patients and 71 controls (histoplasmosis and aspergillosis patients, patients with bacterial infections, and normal human sera) with a dilution buffer incubated at 37°C for 30 min. The sensitivity and specificity of the LAT test in the nonpretreated samples were 73% and 79%, respectively. However, when samples were pretreated, the sensitivity and specificity of the test increased to 90%. In this study, we did not observe cross-reactivity with histoplasmosis patient sera, but some reactions to sera from patients with aspergillosis and bacterial infections were noted. Normal human sera were not reactive in our tests. These results indicate the need for the elimination of heterologous reactions so that we can adequately use this method for screening cases of PCM.

Differentiation between Candida albicans and Candida dubliniensis using hypertonic Sabouraud broth and tobacco agar.

INTRODUCTION: Opportunistic fungal infections in immunocompromised hosts are caused by Candida species, and the majority of such infections are due to Candida albicans. However, the emerging pathogen Candida dubliniensis demonstrates several phenotypic characteristics in common with C. albicans, such as production of germ tubes and chlamydospores, calling attention to the development of stable resistance to fluconazole in vitro. The aim of this study was to evaluate the performance of biochemistry identification in the differentiating between C. albicans and C. dubliniensis, by phenotyping of yeast identified as C. albicans. METHODS: Seventy-nine isolates identified as C. albicans by the API system ID 32C were grown on Sabouraud dextrose agar at 30°C for 24-48h and then inoculated on hypertonic Sabouraud broth and tobacco agar. RESULTS: Our results showed that 17 (21.5%) isolates were growth-inhibited on hypertonic Sabouraud broth, a phenotypic trait inconsistent with C. albicans in this medium. However, the results observed on tobacco agar showed that only 9 (11.4%) of the growth-inhibited isolates produced characteristic colonies of C. dubliniensis (rough colonies, yellowish-brown with abundant fragments of hyphae and chlamydospores). CONCLUSIONS: The results suggest that this method is a simple tool for screening C. albicans and non-albicans yeast and for verification of automated identification.

Differentiation between Candida albicans and Candida dubliniensis using hypertonic Sabouraud broth and tobacco agar / Diferenciação entre Candida albicans e Candida dubliniensis usando caldo Sabouraud hipertônico e ágar Tabaco

INTRODUCTION: Opportunistic fungal infections in immunocompromised hosts are caused by Candida species, and the majority of such infections are due to Candida albicans. However, the emerging pathogen Candida dubliniensis demonstrates several phenotypic characteristics in common with C. albicans, such as production of germ tubes and chlamydospores, calling attention to the development of stable resistance to fluconazole in vitro. The aim of this study was to evaluate the performance of biochemistry identification in the differentiating between C. albicans and C. dubliniensis, by phenotyping of yeast identified as C. albicans. METHODS: Seventy-nine isolates identified as C. albicans by the API system ID 32C were grown on Sabouraud dextrose agar at 30°C for 24-48h and then inoculated on hypertonic Sabouraud broth and tobacco agar. RESULTS: Our results showed that 17 (21.5%) isolates were growth-inhibited on hypertonic Sabouraud broth, a phenotypic trait inconsistent with C. albicans in this medium. However, the results observed on tobacco agar showed that only 9 (11.4%) of the growth-inhibited isolates produced characteristic colonies of C. dubliniensis (rough colonies, yellowish-brown with abundant fragments of hyphae and chlamydospores). CONCLUSIONS: The results suggest that this method is a simple tool for screening C. albicans and non-albicans yeast and for verification of automated identification.

INTRODUÇÃO: Infecções fúngicas oportunistas em hospedeiros imunocomprometidos são causadas por espécies de Candida, cuja maioria das infecções se deve a Candida albicans. Entretanto, o patógeno emergente Candida dubliniensis demonstra várias características fenotípicas em comum com C. albicans, tais como produção de tubo germinativo e clamidósporos, solicitando atenção por desenvolver resistência in vitro estável ao fluconazol. O objetivo do presente estudo foi avaliar a performance da identificação bioquímica na diferenciação entre C. albicans e Candida dubliniensis, analisando fenotipicamente leveduras previamente identificadas como C. albicans. MÉTODOS: Setenta e oito isolados identificados como C. albicans pelo sistema API ID 32C foram cultivados em ágar Sabouraud dextrose a 30°C por 24-48h e em seguida inoculados em caldo hipertônico Sabouraud e agar tabaco. RESULTADOS: Nossos resultados mostraram que 17 (21,5%) isolados tiveram o crescimento inibido no caldo hipertônico Sabouraud, característica fenotípica inconsistente para C. albicans neste meio de cultura. Entretanto, os resultados observados em ágar tabaco mostraram que somente 9 (11,4%) dos isolados inibidos produziram colônias características de C. dubliniensis (colônias rugosas, marrom-amarelada com fragmentos de hifas e abundantes clamidósporos). CONCLUSÕES: Os resultados obtidos sugerem que este é um instrumento simples para triagem entre leveduras de C. albicans e não-albicans, bem como confirmação de identificação automatizada.

Development and evaluation of a latex agglutination test for the serodiagnosis of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. It is caused by the dimorphic fungus Paracoccidioides brasiliensis. The immunodiffusion (ID) test is one of the most widely used techniques for PCM serologic diagnosis due to the simplicity and low costs of its execution. However, it requires trained and qualified people to execute it. The purpose of this study was to evaluate a latex particle agglutination (LA) test for the detection of anti-P. brasiliensis antibodies by using pooled crude exoantigens from the fungus. Fifty-one serum samples obtained from patients with PCM were tested. Positivity was observed in 84% (43/51) of these patients, and the agglutination patterns varied from small clumps with a cloudy background to large clumps with a clear background. The antibody titer reactivity ranged from 1:2 to 1:64. Cross-reactivity was observed in sera from patients with aspergillosis, histoplasmosis, and nonfungal disease. Serum samples obtained from healthy donors were not reactive. The sensitivity and specificity of the LA test were 84% and 81%, respectively. When comparing the LA test with the double-immunodiffusion test, we found an agreement of 92%. Further work is needed to improve the performance of the LA assay before it can be proposed as a reliable diagnostic tool, mainly in laboratories with little infrastructure.

Diagnosis of Paracoccidioidomycosis by detection of antigen and antibody in bronchoalveolar lavage fluids.

Paracoccidioidomycosis (PCM) is a systemic infection caused by the fungus Paracoccidioides brasiliensis and is believed to be the leading cause of fungal pulmonary infection. In this study, we used an inhibition enzyme-linked immunosorbent assay to diagnose pulmonary PCM based on the detection of 43-kDa and 70-kDa molecules in bronchoalveolar lavage fluids. The results were compared with results obtained by classical methods for antibody detection.

Diagnosis of neuroparacoccidioidomycosis by detection of circulating antigen and antibody in cerebrospinal fluid.

Neuroparacoccidioidomycosis (neuroPCM) is the central nervous system infection by the fungus Paracoccidioides brasiliensis. Its diagnosis is a difficult task that depends on neuroimaging techniques such as computed tomography and magnetic resonance imaging. However, the detection of circulating P. brasiliensis antigens in body fluids by inhibition enzyme-linked immunosorbent assay (inh-ELISA) has provided encouraging results. In this study, 14 cerebrospinal fluid (CSF) and 11 serum samples of patients with neuroPCM were analyzed by inh-ELISA for detection of circulating glycoprotein antigens of 43 kDa (gp43) and 70 kDa (gp70). Circulating gp43 and gp70 antigens were detected in all CSF samples from patients with neuroPCM at mean concentrations of 19.3 and 6.8 mug/ml, respectively. In addition, both gp43 and gp70 antigens were detected in 10 of 11 serum samples analyzed at mean concentrations of 4.6 and 4.0 mug/ml, respectively. By immunodiffusion test, CSF samples were determined to be negative in 13 of 14 samples. The detection of anti-gp43 and anti-gp70 antibodies by conventional ELISA showed positive results for all CSF samples, with titers ranging from 1:50 to 1:51,200. Therefore, the high sensitivity of the inh-ELISA technique in detecting gp43 and gp70 antigens in the CSF of neuroPCM patients strongly indicates that this assay can be considered as a powerful diagnostic tool. In addition, the finding of anti-gp43 and anti-gp70 antibodies in CSF samples by conventional ELISA also seems to be a promising diagnostic method for this special modality of PCM.

Detection of Paracoccidioides brasiliensis gp70 circulating antigen and follow-up of patients undergoing antimycotic therapy.

Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Central and South America, is caused by the dimorphic fungus Paracoccidioides brasiliensis and has a high prevalence in Brazil. Glycoproteins of 43 and 70 kDa are the main antigenic compounds of P. brasiliensis and are recognized by Western blotting by 100 and 96% of PCM patient sera, respectively. In the present study, an inhibition enzyme-linked immunosorbent assay (ELISA) was used to detect gp70 in different biological samples from patients with PCM. gp70 was detected in 98.76% of 81 serum samples, with an average concentration of 8.19 microg/ml. The test was positive for 100% of the patients with the acute and chronic unifocal forms of PCM and 98.43% of the patients with the multifocal chronic form, with average concentrations of 11.86, 4.83, and 7.87 microg/ml, respectively. Bronchoalveolar lavage fluid from 23 patients with pulmonary unifocal PCM and 14 samples of cerebrospinal fluid from patients with neurological PCM were also tested for gp70 detection, with the test showing 100% sensitivity and 100% specificity, with mean gp70 concentrations of 7.5 and 6.78 microg/ml, respectively. To investigate the potential of gp70 detection by inhibition ELISA for the follow-up of PCM patients during antimycotic therapy with itraconazole (ITZ), the sera of 23 patients presenting with the chronic multifocal form of PCM were monitored at regular intervals of 1 month for 12 months. The results showed a decrease in circulating gp70 levels during treatment which paralleled the reduction in anti-P. brasiliensis antibody levels. The detection of P. brasiliensis gp70 from the biological fluids of patients suspected of having PCM proved to be a promising method for diagnosing infection and evaluating the efficacy of ITZ treatment.

Monitoring gp43 antigenemia in Paracoccidioidomycosis patients during therapy.

Paracoccidioidomycosis (PCM) is a systemic fungal disease that is particularly important among individuals living and working in rural areas of endemicity in Latin America. Detection of anti-Paracoccidioides brasiliensis antibodies is of limited value due to false-negative results. Detection of P. brasiliensis-gp43 circulating antigen is a practical approach for a specific diagnosis of the disease. In a previous study we described an inhibition enzyme-linked immunosorbent assay able to detect the 43-kDa P. brasiliensis antigen in sera of 100% of patients with the acute form of PCM and in 95.31 and 100% of patients with the chronic multifocal and unifocal forms of PCM. To investigate its potential application for the follow-up of PCM patients during treatment, antigen levels were monitored at regular intervals for up 8 to 12 months in serum samples from 23 patients. The results showed that treatment with itraconazole resulted in decreasing levels of circulating gp43 that were correlated with the reduction of anti-gp43 antibodies. It was also observed that by the end of 12 months of treatment gp43 levels were <5 microg/ml in all patients.