Enzyme production by Candida albicans and Candida dubliniensis in periodontal HIV-positive patients receiving and not receiving antiretroviral therapy / Producción enzimática de Candida albicans y Candida dubliniensis de pacientes con periodontitis VIH+ con y sin tratamiento antirretroviral

ABSTRACT Candida dubliniensis (Cd) and Candida albicans (Ca) are the most frequently isolated yeasts in HIV+ patients. Some of the enzymes produced by these yeasts are considered virulence factors since they contribute to pathogenicity of Candida spp. The aim of the present study was to compare production of enzymes such as phospholipase (Ph), proteinase (P), and hemolysin (H) by Cd and Ca strains isolated from periodontal HIV-positive patients receiving and not receiving highly active antiretroviral therapy (HAART). Subgingival biofilm samples were obtained using paper points, and a sample of oral mucosa was taken using a swab. Phenotypic and molecular methods were used to isolate 39 strains of Candida, including 25 strains of Cd and 14 strains of Ca, obtained from 33 periodontal pocket samples and 6 oral mucosa samples collected from 15 HIV+ patients (8 receiving and 7 not receiving HAART). Malt egg-yolk agar, albumin agar and blood agar were used to evaluate pH, P and H production respectively. The strains were inoculated in duplicate and incubated at 37 ºC. Colony and halo diameters were measured. A greater proportion of Ca was observed in patients not receiving HAART, and a higher proportion of Cd was observed in those under HAART, Chi2 p< 0.001. Phospholipase production was observed in 92.9% percent of isolated Ca strains but in none of the isolated Cd strains. Proteinase production was high in Ca and Cd strains isolated from patients not receiving HAART. Hemolysin production was observed in all the studied strains, though it was significantly higher (p=0.04) in Ca and Cd strains isolated from patients not receiving HAART. To sum up, the proportion of Candida dubliniensis strains was highest in the subgingival biofilm of patients receiving HAART, and Cd strains were found to express fewer virulence factors than Ca strains.

RESUMEN Las levaduras más aisladas en pacientes VIH+ son Candida dubliniensis (Cd) y Candida albicans (Ca). Algunas de sus enzimas constituyen factores de virulencia ya que favorecen la diseminación tisular. El objetivo fue comparar la producción de enzimas como fosfolipasa (F), proteinasa (P) y hemolisina (H) en cepas de Cd y Ca aisladas de pacientes VIH+ tratados y no tratados con antirretrovirales (TARGA). Se realizó la toma del biofilm de placa subgingival con conos de papel y la muestra de la mucosa bucal con hisopo. Se aislaron y tipificaron por métodos fenotípicos y moleculares 39 cepas: 25 de Cd y 14 Ca, obtenidas 33 de bolsas periodontales y 6 de mucosa bucal de 15 pacientes VIH+ (8 con y 7 sin tratamiento). Se utilizó agar malta con yema de huevo, agar albúmina y agar sangre para demostrar la producción de F, P y H, respectivamente. Se inocularon por duplicado e incubaron a 37°C. Se midieron los diámetros de las colonias y los de hidrólisis alrededor de las mismas. Se observó mayor proporción de Ca en los pacientes sin tratamiento y mayor proporción de Cd en los con tratamiento; Chi2 p< 0.001. El 92,9% de las Ca estudiadas, fueron productoras de fosfolipasa. En tanto que ninguna Cd produjo la enzima. En cuanto a la producción de proteinasa se observa una alta producción tanto en las cepas de Ca, como en las Cd aisladas en los pacientes no tratados. Todas las cepas estudiadas produjeron hemolisina, observándose una diferencia estadísticamente significativa (p=0,04) en ambas especies a favor de la alta producción de la enzima en las cepas obtenidas de pacientes no tratados. Podemos concluir que en el biofilm subgingival, en los pacientes bajo TARGA, se aíslan mayor proporción de Candida dubliniensis las cuales expresan menos factores de virulencia.

Concordance analysis between different methodologies used for identification of oral isolates of Candida species / Análisis de concordancia de diferentes metodologías para la identificación de aislamientos orales de especies de Candida

Abstract Background: The yeasts species determination is fundamental not only for an accurate diagnosis but also for establishing a suitable patient treatment. We performed a concordance study of five methodologies for the species identification of oral isolates of Candida in Colombia. Methods: Sixty-seven Candida isolates were tested by; API® 20C-AUX, Vitek®2 Compact, Vitek®MS, Microflex® and a molecular test (panfungal PCR and sequencing). The commercial cost and processing time of the samples was done by graphical analysis. Results: Panfungal PCR differentiated 12 species of Candida, Vitek®MS and Microflex® methods identified 9 species, and API® 20C-AUX and Vitek®2 Compact methods identified 8 species each. Weighted Kappa (wK) showed a high agreement between Panfungal PCR, Vitek®MS, Microflex® and API® 20C-AUX (wK 0.62-0.93). The wK that involved the Vitek®2 Compact method presented moderate or good concordances compared with the other methods (wK 0.56-0.73). Methodologies based on MALDI TOF MS required 4 minutes to generate results and the Microflex® method had the lowest selling price. Conclusion: The methods evaluated showed high concordance in their results, being higher for the molecular methods and the methodologies based on MALDI TOF. The latter are faster and cheaper, presenting as promising alternatives for the routine identification of yeast species of the genus Candida.

Resumen Introducción: La clasificación a nivel de especies de las levaduras del género Candida de origen clínico es fundamental para el diagnóstico y la instauración de un adecuado tratamiento para el paciente. Se realizó un estudio de concordancia de cinco metodologías usadas para la identificación de aislamientos orales de Candida spp en Colombia. Métodos: Sesenta y siete aislamientos de Candida spp fueron identificados a nivel de especie utilizando; API® 20 C AUX‚ Vitek® 2 Compact, MALDI TOF (Vitek® MS y Microflex®) y una prueba molecular, PCR Panfungal y secuenciación. Un análisis del costo comercial y tiempo de procesamiento de las muestras por cada método fue realizado mediante el análisis gráfico de ambas variables. Resultados: La PCR Panfungal y secuenciación diferenció 12 especies de Candida‚ los métodos Vitek® MS y Microflex® identificaron 9 especies y los métodos API® 20 C AUX y Vitek® 2 Compact identificaron 8 especies. El análisis de Kappa ponderado (wK) demostró una concordancia alta entre los métodos PCR Panfungal y secuenciación‚ Vitek® MS‚ Microflex® y API® 20 C AUX‚ concordancias agrupadas en las categorías buena y muy buena (wK 0.62 - 0.93); los Kp que involucraron el método Vitek® 2 Compact presentaron concordancias moderadas o buenas frente a los otros métodos (wK 0.56 - 0.73). Las metodologías basadas en MALDI TOF MS requirieron 4 minutos para generar un resultado y el método Microflex® fue el método que en nuestro medio presentó el menor precio de venta del servicio. Conclusión: Los métodos evaluados presentaron una alta concordancia en sus resultados‚ siendo más alta para los métodos moleculares y las metodologías basadas en MALDI TOF MS; estas últimas son metodologías más rápidas, económicas y precisas, las cuales se presentan como alternativas prometedoras para la identificación rutinaria de especies de levaduras del género Candida.

Factores relacionados a candidiasis oral en niños y adolescentes con VIH, caracterización de especies y susceptibilidad antifúngica / Factors related to oral candidiasis in HIV children and adolescents, species characterization and antifungal susceptibility

Resumen Introducción: Se desconocen los factores asociados a la candidiasis oral en población pediátrica con infección por VIH de los países en desarrollo. Objetivo: Identificar los factores asociados a la colonización por Candida, candidiasis oral y la susceptibilidad in vitro a antifúngicos, en niños y adolescentes con infección por VIH institucionalizados en la ciudad de Tijuana, México. Materiales y Métodos: Se examinó la cavidad oral de 30 niños y adolescentes con infección por VIH, se obtuvo una muestra de la mucosa oral para identificar las especies de Candida mediante cultivo y auxonograma. La susceptibilidad a los antifúngicos se determinó de acuerdo al CLSI. Los indicadores del estado inmunológico y falla virológica se clasificaron conforme a la OMS. Resultados: Se identificaron seis especies de Candida, 53% colonizantes y 47% causantes de candidiasis. Los factores asociados a candidiasis fueron alta carga viral (p = 0,001), menor recuento de LTCD4+ (p = 0,002) y esquema TARAA (p ≤ 0,014). La especie prevalente fue C. glabrata (33%); sin embargo, C. albicans (27%) fue más resistente a fluconazol (p = 0,001). Las especies resistentes a itraconazol se identificaron en esquemas que incluyen un INNTR (p = 0,041). Conclusiones: Los niños y adolescentes con infección por VIH institucionalizados mostraron una prevalencia elevada de Candida spp. colonizante y resistencia a los antifúngicos relacionada con los INNTR .

Background: Factors associated with candidiasis and colonization in HIV-positive children and adolescents in developing countries are not well understood. Aim: To identify the factors associated with oral Candida colonization and candidiasis in institutionalized HIV-positive children and adolescents in Tijuana, México, as well as the response of the isolates to antifungals. Materials and Methods: Sample of the oral mucosa of 30 HIV positive children and adolescents were obtained to isolate and identify Candida species by culture and metabolic profile. Antifungal drugs susceptibility was determined according to CLSI. Indicators of immunological and virologic failure were classified in accordance to WHO criteria. Results: Six Candida species were identified from oral mucosa, 53% colonizers and 47% in candidiasis. Factors associated with candidiasis and oral colonization were viral load (p = 0,001), CD4+ counts (p = 0,002) and HAART regimen (p ≤ 0,014). The most prevalent species was C. glabrata (33%), but C. albicans (27%) was more resistant to fluconazole (p = 0,001). Itraconazol resistant species were identified in regimens that include an NNRTI (p = 0,041). Conclusion: HIV-positive children and adolescents living in an orphanage showed high prevalence of colonizing Candida spp. and resistance to antifungals, related to NNRTI.

Clinical and laboratorial features of oral candidiasis in HIV-positive patients

Abstract INTRODUCTION We describe the clinical and laboratorial features of oral candidiasis in 66 HIV-positive patients. METHODS: Polymerase chain reaction-based techniques were performed for differentiation of Candida spp. isolated from patients at a public teaching hospital in Midwest Brazil. RESULTS: Oral lesions, mainly pseudomembranous, were significantly related to higher levels of immunosuppression. Of 45 Candida isolates, 66.7% were C. albicans. Most of the isolates were susceptible to the antifungal drugs tested. CONCLUSIONS: Oral lesions were associated with higher immunosuppression levels. Lower susceptibility to antifungals by non-albicans isolates supports the importance of surveillance studies using susceptibility tests to aid in the treatment.

Antifungal activity of linalool in cases of Candida spp. isolated from individuals with oral candidiasis / Atividade antifúngica do linalol em casos de Candida spp. isolados de indivíduos com candidíase oral

Abstract This study analyzed the antifungal activity of phytoconstituents from linalool on Candida spp. strains, in vitro, isolated from patients with clinical diagnoses of oral candidiasis associated with the use of a dental prosthesis. Biological samples were collected from 12 patients using complete dentures or removable partial dentures and who presented mucous with diffuse erythematous or stippled features, indicating a clinical diagnosis of candidiasis. To identify fungal colonies of the genus Candida, samples were plated onto CHROMagar Candida®. The antifungal activity of linalool, a monoterpene unsaturated constituent of basil oil, was performed using the broth microdilution technique. Then, the minimum inhibitory concentration (MIC), the two subsequent stronger concentrations and the positive controls were subcultured on Sabouraud Dextrose Agar plates to determine the minimum fungicidal concentration (MFC). The experiments were performed in triplicate and nystatin was used as a positive control in all tests. Diagnoses of oral candidiasis were verified in eight patients (66.6%) and the most prevalent fungal species was Candida albicans (37.5%), followed by Candida krusei (25.0%); and Candida tropicalis (4.2%). The best antifungal activity of linalool was observed on Candida tropicalis (MIC = 500 mg/mL), followed by Candida albicans (MIC = 1.000 mg/mL), and Candida krusei (MIC = 2.000 mg/mL).Under the study conditions and based on the results obtained, it can be concluded that the Candida strains tested were susceptible to linalool.

Resumo Este estudo analisou a atividade antifúngica do fitoconstituinte linalol em cepas de Candida ssp, in vitro, isolados de pacientes com o diagnóstico clínico de candidíase oral associado ao uso de prótese dentária. As amostras biológicas foram coletadas de 12 pacientes portadores de próteses totais ou próteses parciais removíveis e que apresentavam características de mucosa eritematosa difusa ou pontilhadas, indicando um diagnóstico clínico de candidíase. Para identificar colônias de fungos do gênero Candida, as amostras foram semeadas em CHROMagar Candida®. A atividade antifúngica do linalol, um componente insaturado monoterpene de óleo de manjericão, foi realizada através da técnica de microdiluição em caldo. Em seguida, a concentração inibitória mínima (MIC), as duas concentrações consecutivas mais fortes e os controles positivos foram subcultivados em placas de Agar Sabouraud Dextrose para determinar a concentração fungicida mínima (MFC). Os experimentos foram realizados em triplicata e a nistatina foi usada como controle positivo em todos os testes. O diagnóstico de candidíase oral foi comprovado em oito pacientes (66,6%) e as espécies de fungos mais prevalentes foram Candida albicans (37,5%), seguido por Candida krusei (25,0%); e Candida tropicalis (4,2%). A melhor atividade antifúngica do linalol foi observada em Candida tropicalis (MIC = 500 mg/ml), seguido por Candida albicans (CIM = 1,000 mg/mL), e Candida krusei (CIM = 2,000 mg/mL). Sob as condições do estudo e com base nos resultados obtidos, pode-se concluir que as estirpes de Candida testadas foram susceptíveis a linalol.

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.

Oral colonization by Candida species in HIV-positive patients: association and antifungal susceptibility study / Colonização oral por espécies de Candida em pacientes HIV positivo: estudo de associação e suscetibilidade antifúngica

ABSTRACT Objective To investigate antifungal susceptibility and factors associated with oral colonization by Candida species in HIV-positive patients. Methods A prospective study based on convenience sampling of subjects recruited from a pool of confirmed HIV-positive individuals seen at a specialty outpatient service in Rondonópolis, Mato Grosso, Brazil). Oral swabs were collected from 197 patients. Candida species were identified by standard microbiological techniques (phenotypic and molecular methods). Antifungal susceptibility was investigated using the broth microdilution method. Results A total of 101 (51.3%) patients were Candida spp carriers. Candida albicans was the most prevalent species (80%). Patients aged 45 to 59 years (Prevalence ratios: 1.90; 95%CI: 1.57-6.31) and 60 years or older (Prevalence ratios: 4.43; 95%CI: 1.57-34.18) were at higher risk of oral colonization by Candida species. Resistance to fluconazole and ketoconazole, or to itraconazole, corresponded to 1% and 4%, respectively. Conclusion Age (45 years or older) was the only factor associated with oral colonization by Candida . Low rates of antifungal resistance to azoles were detected in yeast isolates obtained from HIV-positive patients. Findings of this study may contribute to proper therapeutic selection for oral candidiasis in HIV-positive patients.

RESUMO Objetivo Investigar a suscetibilidade a antifúngicos e os fatores associados à colonização oral por espécies de Candida isoladas de pacientes HIV positivo. Métodos Estudo prospectivo realizado com amostragem por conveniência de indivíduos HIV positivo, acompanhados por um serviço de atendimento especializado da cidade de Rondonópolis, Mato Grosso, Brasil. Foram coletados swabs orais de 197 pacientes. As espécies de Candida foram identificadas por técnicas microbiológicas fenotípicas padrão e por método molecular. A suscetibilidade antifúngica foi determinada pelo método de microdiluição em caldo. Resultados Cento e um (51,3%) pacientes foram colonizados por Candida spp. Candida albicans foi a espécie mais prevalente (80%). Identificou-se um maior risco de colonização oral por espécies de Candida em pacientes com idade entre 45 e 59 anos (razão de prevalência: 1,90; IC95%: 1,57-6,31) e 60 anos ou mais (razão de prevalência: 4,43; IC95%: 1,57-34,18). A resistência ao fluconazol e ao cetoconazol foi de 1% cada e de 4% ao itraconazol. Conclusão O único fator associado à colonização oral por espécies de Candida foi ter 45 anos ou mais. Identificamos baixa taxa de resistência antifúngica aos azóis entre as leveduras isoladas de pacientes HIV positivo. Estes achados podem contribuir para selecionar o tratamento da candidíase oral em pacientes HIV positivos.

Oral Candida spp carriage and periodontal diseases in HIV-infected patients in Ribeirão Preto, Brazil

ABSTRACT The majority of HIV-infected patients develop Candida spp-associated clinical oral lesions. Studies have shown that asymptomatic oral colonization of Candida spp may lead to oral lesions or become a source of disseminated infections. The aim of this study was to verify the effects of periodontal conditions on Candida spp prevalence and Candida spp carriage in the oral cavity of HIV-infected patients compared to non-infected patients. Twenty-five patients not infected with HIV and 48 HIV-infected patients were classified according to periodontal conditions as being periodontal healthy or with periodontal disease. Candida spp carriage and classification were performed in oral rinse samples. Viral load and CD4+ T lymphocyte (CD4+L) counts were performed in blood samples from HIV-infected patients. No differences in Candida spp prevalence related to HIV status or periodontal condition were detected. However, Candida spp carriage was increased in periodontally affected HIV-infected patients when compared to periodontally healthy HIV-infected patients (p= 0.04). Periodontally healthy HIV-infected patients presented Candida spp carriage in similar levels as healthy or periodontally affected non-HIV-infected patients. Candida spp carriage was correlated with CD4+L counting in HIV-infected patients. We concluded that periodontal disease is associated with increased Candida spp carriage in HIV-infected patients and may be a predisposing factor to clinical manifestations of candidiasis.

Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients

ABSTRACT The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species.

Extracellular phospholipase production of oral Candida albicans isolates from smokers, diabetics, asthmatics, denture wearers and healthy individuals following brief exposure to polyene, echinocandin and azole antimycotics

Abstract Objective Candida albicans is the primary causative agent of oral candidosis, and one of its key virulent attributes is considered to be its ability to produce extracellular phospholipases that facilitate cellular invasion. Oral candidosis can be treated with polyenes, and azoles, and the more recently introduced echinocandins. However, once administered, the intraoral concentration of these drugs tend to be sub-therapeutic and rather transient due to factors such as the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, the pathogenic yeasts may undergo a brief exposure to antifungal drugs. We, therefore, evaluated the phospholipase production of oral C. albicans isolates following brief exposure to sub-therapeutic concentrations of the foregoing antifungals. Materials and methods Fifty C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sub-therapeutic concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for one hour. Thereafter the drugs were removed and the phospholipase production was determined by a plate assay using an egg yolk-agar medium. Results The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 10.65, 12.14, 11.45 and 6.40% following exposure to nystatin, amphotericin B, caspofungin and ketoconazole, respectively. This suppression was not significant following exposure to fluconazole. Conclusions Despite the sub-therapeutic, intra oral, bioavailability of polyenes, echinocandins and ketoconazole, they are likely to produce a persistent antifungal effect by suppressing phospholipase production, which is a key virulent attribute of this common pathogenic yeast.

Colonización por Candida spp. en sujetos diabéticos y no diabéticos / Colonization by Candida spp. in diabetic and non-diabetic individuals in the north of Colombia

Introducción: el género Candida puede encontrarse colonizando el cuerpo humano, pero también puede provocar infecciones de carácter oportunista. La diabetes es considerada comúnmente un factor de riesgo para la infecciones candidósicas, dadas sus características asociadas a inmunosupresión, hiperglucemia, problemas de microvascularización, entre otros. Usualmente, para que se inicie un proceso infeccioso, es necesaria la colonización; por tanto, es importante conocer su frecuencia de portación en grupos con factores de riesgo asociados. Objetivo: determinar los porcentajes de colonización por Candida spp. en la cavidad oral y la zona interdigital del pie en pacientes diabéticos y no diabéticos. Métodos: el estudio, de carácter descriptivo, se realizó en un grupo de 85 pacientes del norte de Colombia: 46 diabéticos y 39 no diabéticos. La identificación de los aislamientos se realizó por pruebas fenotípicas, que incluyeron CHROMagar, microscopía, prueba de tubo germinal, prueba de clamidosporas y zimograma. Resultados: en los pacientes diabéticos se encontró una frecuencia de colonización por Candida spp. en la cavidad oral de 23,9 por ciento, y en el pie, de 56,52 por ciento; mientras que, para los sujetos no diabéticos, la frecuencia de colonización en la cavidad oral fue del 33,3 por ciento y en el pie del 43,58 por ciento. Adicionalmente, en el grupo de diabéticos se encontró que la frecuencia de colonización por la especie Candida albicans en la cavidad oral fue de 45,5 por ciento, mientras que en el pie fue del 11,5 por ciento. Conclusiones: los resultados obtenidos sugieren que las condiciones locales del ambiente de donde se aísla Candida spp. pueden influir en los niveles de portación y la variedad de especies, lo que podría ser más determinante que la condición de base(AU)

Introduction: Candida genus may colonize the human body but also may cause opportunistic infections. Diabetes is frequently considered a risk factor for Candida infections, given its characteristics associated to immunosuppresion, hyperglycemia, microvascularization problems and others. Generally, colonization is necessary for the starting of an infectious process; therefore, it is important to know its frequency of carriage in groups with associated risk factors. Objective: to determine the percentages of Candida spp. colonization in the oral cavity and the inter-toe area in diabetic and nondiabetic patients. Methods: the descriptive study was carried out in a group of 85 patients from the north of Colombia, that is, 46 diabetics and 39 non diabetics. The isolates were identified through phenotypical tests including CHROMagar, microscopy, germ tube test, Chlamydospore test and zymogram. Results: it was found in diabetic patients that the frequency of colonization by Candida spp. in the oral cavity was 23.9 percent and in the foot was 56.52 percent whereas in non-diabetic patients, the frequency of colonization in the oral cavity was 33.3 percent and in the foot was 43.58 percent. Additionally, it was found in the group of diabetic subjects that the frequency of colonization by Candida albicans was 45.5 percent in the oral cavity whereas that of the foot was 11.5 percent. Conclusions: the achieved results indicate that the local environmental conditions where the Candida spp. is isolated may have an impact on the levels of carriage and the variety of species, which could be more determining than the underlying condition(AU)

RELATED FACTORS FOR COLONIZATION BY Candida SPECIES IN THE ORAL CAVITY OF HIV-INFECTED INDIVIDUALS / Fatores relacionados a colonização da cavidade bucal de indivíduos portadores do HIV por espécies de Candida

The colonization of the oral cavity is a prerequisite to the development of oropharyngeal candidiasis. Aims: The aims of this study were: to evaluate colonization and quantify Candida spp. in the oral cavity; to determine the predisposing factors for colonization; and to correlate the levels of CD4+ cells and viral load with the yeast count of colony forming units per milliliter (CFU/mL) in HIV-positive individuals treated at a University Hospital. Saliva samples were collected from 147 HIV patients and were plated on Sabouraud Dextrose Agar (SDA) and chromogenic agar, and incubated at 30 ºC for 72 h. Colonies with similar morphology in both media were counted and the result expressed in CFU/mL. Results: Of the 147 HIV patients, 89 had positive cultures for Candida spp., with a total of 111 isolates, of which C. albicans was the most frequent species (67.6%), and the mean of colonies counted was 8.8 × 10³ CFU/mL. The main predisposing factors for oral colonization by Candida spp. were the use of antibiotics and oral prostheses. The use of reverse transcriptase inhibitors appears to have a greater protective effect for colonization. A low CD4+ T lymphocyte count is associated with a higher density of yeast in the saliva of HIV patients.

RESUMO A colonização da cavidade oral pode ser considerada um pré-requisito para o desenvolvimento de candidíase orofaríngea. Os objetivos deste estudo foram: avaliar e quantificar espécies de Candidaisoladas da cavidade oral, para determinar os fatores predisponentes para a colonização, e correlacionar os níveis de células CD4+ e carga viral em indivíduos HIV-positivos atendidos em um hospital universitário. Foram coletadas amostras de saliva de 147 pacientes portadores do HIV, as quais foram semeadas em Ágar Sabouraud Dextrose (ASD) e ágar cromogênico e incubadas a 30 °C por 72 horas. As colônias com morfologia semelhante em ambos os meios foram contadas e o resultado expresso em unidade formadora de colônias por mililitro (UFC/mL). Dos 147 pacientes HIV positivos, 89 apresentaram culturas positivas para Candidaspp., totalizando 111 isolados, e C. albicansfoi a espécie mais frequente (67,6%). A contagem média de colônias foi de 8.8 × 10³ UFC/mL. Os principais fatores predisponentes para colonização oral por Candidaspp. foram a utilização de antibióticos e de próteses orais. O uso de antirretroviral da classe de inibidores da transcriptase reversa pareceu ter maior efeito protetor para a colonização. Baixa contagem de linfócitos T CD4+ está relacionada com maior densidade de leveduras na saliva de indivíduos HIV positivos.

Factors associated with time free of oral candidiasis in children living with HIV/AIDS, São Paulo, Brazil / Fatores associados ao tempo livre de candidíase oral em crianças vivendo com HIV/AIDS, São Paulo, Brasil / Factores asociados al tiempo libre y la candidiasis oral en niños que viven con VIH/SIDA, São Paulo, Brasil

In clinical practice, recurrence of thrush is common in children living with HIV/AIDS. The aim of this study was to determine the factors associated with time spent free of oral candidiasis using survival analysis for recurrent events. A retrospective cohort study was carried out with 287 children treated between 1985 and 2009 at a reference center in the city of São Paulo, Brazil. The Prentice, Williams and Peterson model for recurrent events was used for the investigation of factors associated with the time free of oral candidiasis. The following factors were associated with the time patients were free of oral candidiasis: moderate immunodepression (HR = 2.5; p = 0.005), severe immunodepression (HR = 3.5; p < 0.001), anemia (HR = 3.3; p < 0.001), malnutrition (HR = 2.6; p = 0.004), hospitalization (HR = 2.2; p < 0.001), monotherapy (HR = 0.5; p = 0.006), dual therapy (HR = 0.3; p < 0.001) and triple therapy/highly active antiretroviral therapy (HR = 0.1; p < 0.001). The method analyzed in the present study proved useful for the investigation of recurrent events in patients living with HIV/AIDS.

A recorrência da candidíase oral em crianças vivendo com HIV/AIDS é um acontecimento muito comum na prática clínica. O objetivo foi verificar os fatores associados ao tempo livre de candidíase oral, utilizando técnica de análise de sobrevida para eventos recorrentes. Estudo de coorte retrospectivo com 287 crianças, atendidas entre 1985 e 2009, em um serviço de saúde de São Paulo, Brasil. Foi utilizado o modelo marginal para eventos recorrentes de Prentice, Williams e Peterson para investigação dos fatores associados ao tempo livre de candidíase oral. Imunodepressão moderada (HR = 2,5; p = 0,005) ou grave (HR = 3,5; p < 0,001), anemia (HR = 3,3; p < 0,001), desnutrição (HR = 2,6; p = 0,004) e internação (HR = 2,2; p < 0,001), monoterapia (HR = 0,5; p = 0,006), terapia dupla (HR = 0,3; p < 0,001) e terapia tripla/HAART (HR = 0,1; p < 0,001) foram associados ao tempo livre de candidíase oral. A metodologia apresentada neste artigo pode ser bastante útil em pesquisas na área de HIV/AIDS, quando pretende-se estudar eventos com comportamento de recorrência.

La repetición de candidiasis oral en los niños que viven con VIH/SIDA es muy común en la práctica clínica. El objetivo fue verificar los factores asociados al tiempo libre y la candidiasis oral, usando la técnica de análisis de supervivencia para eventos recurrentes. Se realizó un estudio de cohorte retrospectiva con 287 niños que visitaron entre 1985 y 2009 un servicio de salud de São Paulo, Brasil. Se usó el modelo marginal para eventos recurrentes de Prentice, Williams y Peterson, con el fin de investigar los factores asociados. Moderada inmunodepresión (HR = 2,5; p = 0,005) o grave (HR = 3,5; p < 0,001), anemia (HR = 3,3; p < 0,001), desnutrición (HR = 2,6; p = 0,004) y hospitalización (HR = 2,2; p < 0,001), monoterapia (HR = 0,5; p = 0, 006), terapia dual (HR = 0,3; p < 0,001) y triple terapia/TARGA (HR = 0,1; p < 0,001) se asociaron al tiempo libre y la candidiasis oral. La metodología presentada en este artículo puede ser útil para la investigación en el área de VIH/SIDA, cuando se pretenda estudiar el comportamiento de la repetición del evento.

Oral Candida spp carriers: its prevalence in patients with type 2 Diabetes Mellitus / Portadores de Candida spp na cavidade oral: a sua prevalência em pacientes com diabetes mellitus tipo 2

BACKGROUND: Prevalence of oral candidiasis in diabetic patients is 13.7-64%. Candida albicans was the most frequently isolated species (75-86.5%). OBJECTIVE: To obtain the prevalence of Candida carriers among patients with type 2 diabetes mellitus to identify the species of the yeast. Study design: It is an open, observational, descriptive, cross-sectional, and prospective study. METHODS: We included voluntary patients from the National Diabetes Marathon and performed a blood glucose measurement, sialometry test, Gram-stained exfoliative cytology, and culture on Sabouraud dextrose agar and CHROMagar Candida TM. Results were analyzed using descriptive statistics. RESULTS: We examined 141 patients (mean age 57 years): 103 women (73%) and 38 men (26.9%). Exfoliative cytology was positive in 32 cases (23 with oral lesions); 78 had oral lesions but no Candida (93.9%). Candida was isolated in 58 patients (41.1%), 21 (45.6 %) had blood glucose greater than 126 mg/dl, and 37 (38.9%) had less than 126 mg/dl. The most frequent species was C. albicans (82.7%). Forty-two Candida carriers had salivary flow greater than 20 mm (72.4%), and 16 (27.5%) had hyposalivation. Candida was isolated in 25 of 79 patients with dental prosthesis (31.6%), 9 of 15 were smokers (60%), and 22 of 71 had symptoms (30.9%). CONCLUSIONS: Prevalence of oral Candida carriers in patients with type 2 diabetes mellitus in Mexico was similar to that found in other countries; exfoliative cytology was effective in finding Candida; salivary flow rate, use of prosthesis, and presence of oral lesions and symptoms were similar in oral Candida carriers and negative patients. Most smokers were Candida carriers. .

FUNDAMENTOS: A prevalência de candidíase oral em pacientes diabéticos é de 13,7- 64%. A espécie mais frequentemente isolada é Candida albicans(75-86,5%). OBJETIVO: Obter a prevalência de portadores de Candida em pacientes com diabetes mellitus tipo 2 para identificar as espécies da levedura. Desenho do estudo: Aberto, observacional, descritivo, transversal e prospectivo. MÉTODOS: Incluímos pacientes voluntários dentro da Maratona Nacional do Diabetes e realizamos medida da glicose sanguínea, teste sialométrico, coloração de Gram da citologia esfoliativa, cultura em ágar Sabouraud dextrose e CHROMagar Candida TM. Os resultados foram analisados com estatística descritiva. RESULTADOS: Foram examinados 141 pacientes (média de idade de 57 anos), 103 mulheres (73%) e 38 homens (26,9%). A citologia esfoliativa foi positiva em 32 casos (23 com lesão oral), 78 tinham lesão oral, mas não tinham Candida (93,9%). Candida foi isolado em 58 pacientes (41,1%), 21(45,6%) tiveram glicemia superior a 126 mg / dl e 37 (38,9%) tiveram glicemia inferior a 126 mg / dl. A espécie mais frequente foi C. albicans (82,7%). 42 portadores de Candida tinham fluxo salivar maior que 20 mm(72,4%) e 16(27,5%) tinham hiposalivação. Candida foi isolado em 25 de 79 pacientes com prótese dentária (31,6%), 9 de 15 fumantes (60%), e 22 de 71 com sintomas (30,9%). CONCLUSÕES: A prevalência de portadores de Candida oral em pacientes com diabetes mellitus tipo 2 no México foi semelhante a outros países; citologia esfoliativa foi efetiva em encontrar Candida; fluxo salivar, uso de prótese, presença de lesão oral ...

Effectiveness of mechanical brushing with different denture cleansing agents in reducing in vitro Candida albicans biofilm viability

The adhesion of Candida albicans to surfaces is the prerequisite for occurrence of denture stomatitis, a common disease diagnosed among denture wearers. A routine of denture cleansing is essential to prevent biofilm formation and the onset of this infection. The aim of this study was to investigate the effectiveness of combining brushing and cleansing agents in killing C. albicans biofilm. Disks of acrylic resin were made, sterilized, and inoculated with C. albicans (10(7) cfu/mL). After incubation (37°C/48 h), specimens were randomly assigned to 10 experimental groups (n=9): 5 subjected to brushing with distilled water or cleansing agents - dentifrice slurry, 2% chlorhexidine gluconate (CHX), 1% sodium hypochlorite (NaOCl), and Polident fresh cleanse® (combined method) - and 4 exposed to the cleansing agents without brushing (immersion). Non-cleansed specimens were used as positive controls. The viability of cells was evaluated by XTT reduction method. Results were analyzed by Mann-Whitney and Kruskal-Wallis tests (α=0.05). The combined method was significantly more effective (p<0.0001) in reducing biofilm viability than the immersion. Brushing with CHX and NaOCl resulted in 100% removal of the biofilm. Immersion in the agents reduced significantly (p<0.0001) the biofilm viability, with CHX being the most effective (p<0.0001). The use of the combined method of brushing with cleansing agents is an effective method to reduce C. albicans biofilm, being CHX and NaOCl the most effective solutions.

A adesão de Candida albicans às superfícies é o primeiro passo para o desenvolvimento da estomatite protética, uma infecção frequente diagnosticada entre os usuários de próteses. Uma adequada higienização é essencial para prevenir a formação de biofilme microbiano e o início desta infecção. O objetivo deste estudo foi avaliar a efetividade da escovação com diferentes soluções na eliminação de biofilme de C. albicans. Para isso, discos de resina acrílica foram confeccionados, esterilizados e inoculados com uma suspensão de 10(7) células/mL de C. albicans. Após incubação (37°C/48 h), os espécimes foram aleatoriamente divididos em 10 grupos experimentais (n=9): 5 submetidos à escovação com água ou agentes de limpeza (água destilada, dentifrício, digluconato de clorexidina (CHX) a 2%, hipoclorito de sódio (NaOCl) a 1% e Polident fresh cleanse®) e 4 apenas imersos nos agentes de limpeza. Espécimes não submetidos à higienização foram utilizados como controle positivo. A viabilidade celular foi verificada pelo teste de redução do XTT. Os resultados obtidos foram analisados pelos testes de Mann-Whitney e Kruskal-Wallis (α=0,05). A escovação com todos os agentes de limpeza apresentou redução significativamente superior (p<0,0001) na viabilidade do biofilme quando comparada à exposição dos espécimes às soluções. Escovação com CHX a 2% e NaOCl a 1% resultaram em 100% de inativação do biofilme. A exposição aos agentes de limpeza resultou em redução significativa (p<0,0001) na viabilidade celular, com CHX a 2% sendo o mais efetivo (p<0,0001). A utilização de agentes de limpeza em associação ao método de escovação provou ser efetivo para reduzir biofilme C. albicans, sendo as soluções de CHX e NaOCl as mais efetivas.

Oral colonization by yeasts in HIV-positive patients in Brazil / Colonização oral por leveduras em pacientes HIV-positivos no Brasil

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 percent) followed by non-albicans Candida species (43.73 percent), Trichosporon mucoides (3.12 percent) and Kodamaea ohmeri (1.56 percent). Oral colonization by association of different species was observed in 42 percent 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.

INTRODUÇÃO: Em pacientes infectados pelo HIV, a colonização da cavidade bucal por leveduras patogênicas pode levar ao desenvolvimento de fungemias. No presente estudo, avaliamos a prevalência de leveduras na cavidade bucal de pacientes HIV-positivos e verificamos se as espécies isoladas foram enzimaticamente ativas. Além disso, as espécies identificadas foram testadas quanto à suscetibilidade a antifúngicos. MÉTODOS: Amostras de saliva e de candidose orofaríngea foram coletadas de 60 pacientes soropositivos para HIV e identificados pelo sistema API20C. A atividade enzimática foi avaliada pela produção de proteinase e fosfolipase. A suscetibilidade a antifúngicos foi determinada utilizando o método de microdiluição em caldo. RESULTADOS: As espécies mais comumente isoladas foram C. albicans (51,56 por cento), seguido por espécies de Candida não-albicans (43,73 por cento), Trichosporon mucoides (3,12 por cento) e Kodamaea ohmeri (1,56 por cento). A colonização bucal por associação de diferentes espécies foi observada em 42 por cento dos pacientes. A atividade enzimática foi verificada na maioria das espécies isoladas, com exceção de C. glabrata, C. lusitaniae e C. guilliermondii. Resistência ao fluconazol e anfotericina B foi observada em isolados de C. albicans, C. glabrata, C. parapsilosis, C. krusei, e K. ohmeri. CONCLUSÃO: Os pacientes HIV-positivos são colonizados por espécies únicas ou múltiplas de levedura que ocasionalmente são resistentes ao fluconazol ou anfotericina B.

Multi-species biofilm of Candida albicans and non-Candida albicans Candida species on acrylic substrate

OBJECTIVE: In polymicrobial biofilms bacteria extensively interact with Candida species, but the interaction among the different species of the Candida is yet to be completely evaluated. In the present study, the difference in biofilm formation ability of clinical isolates of four species of Candida in both single-species and multi-species combinations on the surface of dental acrylic resin strips was evaluated. MATERIAL AND METHODS: The species of Candida, isolated from multiple species oral candidiasis of the neutropenic patients, were used for the experiment. Organisms were cultured on Sabouraud dextrose broth with 8 percent glucose (SDB). Biofilm production on the acrylic resins strips was determined by crystal violet assay. Student's t-test and ANOVA were used to compare in vitro biofilm formation for the individual species of Candida and its different multi-species combinations. RESULTS: In the present study, differences between the mean values of the biofilm-forming ability of individual species (C. glabrata>C. krusei>C. tropicalis>C. albicans) and in its multi-species' combinations (the highest for C. albicans with C. glabrata and the lowest for all the four species combination) were reported. CONCLUSIONS: The findings of this study showed that biofilm-forming ability was found greater for non-Candida albicans Candida species (NCAC) than for C. albicans species with intra-species variation. Presence of C. albicans in multi-species biofilms increased, whereas; C. tropicalis decreased the biofilm production with all other NCAC species.

Susceptibilidad a la nistatina de aislamientos bucales de Candida y su correlación con la respuesta al tratamiento / Susceptibility to nystatin of oral Candida isolates and its correlation with the response to treatment

INTRODUCCIÓN: la candidiasis bucofaríngea es un marcador precoz de progresión a sida en los pacientes seropositivos al VIH sin tratamiento antirretroviral, así como un indicador de no adherencia al tratamiento antirretroviral o su posible fracaso. OBJETIVO: determinar la susceptibilidad frente a nistatina de las cepas de Candida aisladas de la cavidad bucal de pacientes seropositivos al VIH, y correlacionar los resultados con la respuesta al tratamiento. MÉTODOS: se realizó un estudio de susceptibilidad in vitro frente a nistatina para 104 cepas aisladas de lesiones de candidiasis bucofaríngea de 97 pacientes VIH/sida que habían participado, como voluntarios, en un ensayo clínico para evaluar la respuesta a cuatro drogas. De los 104 aislamientos, 58 fueron obtenidos antes de iniciar los esquemas de tratamientos y 46 una vez finalizado estos. Se procedió según el micrométodo el documento M27-A3 del Clinical and Laboratory Standards Institute. RESULTADOS: todas las cepas se comportaron como sensibles a la nistatina, con valores de concentración mínima inhibitoria (CMI) menores que 16 µg/mL, independientemente del momento de su aislamiento y de la droga administrada a los pacientes. En el grupo de pacientes tratados con nistatina, los rangos de CMI80 (1-2 µg/mL), las medias geométricas y los valores acumulativos de la droga frente a los aislamientos de Candida albicans obtenidos antes de iniciar el tratamiento, resultaron similares a los obtenidos frente a las cepas recobradas al finalizar este. De todas las especies identificadas, C. lusitaniae fue la que alcanzó el valor absoluto de CMI más elevado, 4 µg/mL. CONCLUSIONES: la correlación entre la evolución clínica de los pacientes y los resultados de los estudios de susceptibilidad de sus aislamientos in vitro, fue buena para los pacientes con evolución favorable, pero no para predecir posibles fracasos terapéuticos.

INTRODUCTION: oropharyngeal candidiasis is an early marker of progression to AIDS in HIV-positive patients and an indicator of non-adherence of treatment or possible failure in patients undergoing anti-retroviral therapy. OBJECTIVE: to determine the in vitro susceptibility to nystatin in Candida strains isolated from the oral cavity of HIV-positive patients, and to correlate the results with the response to treatment. METHODS: a study of in vitro susceptibility to nystatin was conducted in 104 oral isolates from 97 HIV/AIDS patients, who participated as volunteers in a clinical trial to evaluate the response to four antifungal drugs. Fifty-eight of the 104 isolates were obtained before starting treatment and the remaining 46 at the end of therapy. Broth microdilution method was performed according to the document M27-A3 of the Clinical and Laboratory Standards Institute. RESULTS: one hundred percent of the strains were susceptible to nystatin, with minimum inhibitory concentration (MIC) values of <16 µg/mL, regardless of the time of isolation and of the drug administered to patients. In the group of patients treated with nystatin, the MIC80 ranges (1-2 µg/mL), geometric mean and cumulative values of the drug against Candida albicans isolates before starting treatment were similar to those obtained against strains recovered at the end. Among the identified species, C. lusitaniae reached the highest absolute MIC value (4 µg/mL). CONCLUSIONS: the correlation between the clinical evolution and the results of in vitro susceptibility tests was good in patients with a favorable outcome; however, it did not allow predicting possible treatment failure.

Candida dubliniensis does not show phospholipase activity: true or false? / Candida dubliniensis não evidencia atividade fosfolipásica: verdadeiro ou falso?

INTRODUCTION: The phospholipase activity in Candida albicans and Candida dubliniensis isolated from oral candidiasis cases were studied. METHODS: The phospholipase activity was evaluated in egg yolk agar. RESULTS: All the C. albicans isolates (n = 48) showed phospholipase activity (mean Pz = 0.66). However, none of the C. dubliniensis isolates (n = 24) showed this activity. CONCLUSIONS: The authors discuss whether these findings are a true characteristic of C. dubliniensis or a consequence of the methodology employed, which includes the possibility that NaCl may have inhibited the enzymatic activity of C. dubliniensis.

INTRODUÇÃO: Avaliou-se a atividade fosfolipásica em Candida albicans e Candida dubliniensis isoladas de casos de candidíase oral. MÉTODOS: A atividade de fosfolipase foi avaliada em ágar gema de ovo. RESULTADOS: Todos os isolados de C. albicans (nº = 48) evidenciaram atividade fosfolipásica (média Pz = 0.66). Todavia, nenhum isolado de C. dubliniensis (nº= 24) demonstrou esta atividade. CONCLUSÕES: Os autores discutem se estes achados são uma característica verdadeira de C. dubliniensis ou uma conseqüência da metodologia empregada, a qual inclui a possibilidade de que o NaCl seja inibidor da atividade enzimática de C. dubliniensis.

Candida albicans in patients with oronasal communication and obturator prostheses

Patients using obturator prostheses often present denture-induced stomatitis. In order to detect the presence of oral Candida albicans in patients with oronasal communications and to evaluate the effectiveness of a topical antifungal treatment, cytological smears obtained from the buccal and palatal mucosa of 10 adult patients, and from the nasal acrylic surface of their obturator prostheses were examined. A therapeutic protocol comprising the use of oral nystatin (Mycostatin®) and prosthesis disinfection with sodium hypochlorite was prescribed for all patients. Seven patients were positive for C. albicans in the mucosa, with 1 negative result for the prosthetic surface in this group of patients. Post-treatment evaluation revealed the absence of C. albicans on prosthesis surface and on the oral mucosa of all patients. The severity of the candidal infection was significantly higher in the palatal mucosa than in the buccal mucosa, but similar in the palatal mucosa and prosthesis surface, indicating that the mucosa underlying the prosthesis is more susceptible to infection. The therapeutic protocol was effective in all cases, which emphasizes the need for denture disinfection in order to avoid reinfection of the mucosa.

Os pacientes portadores de prótese obturadora freqüentemente apresentam estomatite protética. Com o objetivo de detectar a presença de Candida albicans oral em pacientes com comunicação oronasal e avaliar a eficácia de um tratamento tópico antifúngico foi realizada citologia esfoliativa da mucosa palatina e jugal e da superfície acrílica nasal da prótese obturadora. O protocolo terapêutico consistiu de nistatina (Mycostatin®) para tratamento da mucosa oral e uma solução de hipoclorito de sódio para desinfecção da prótese. Sete pacientes (70 por cento) apresentaram resultado positivo para C. albicans na mucosa, com um resultado negativo para a superfície protética neste grupo. A avaliação após o tratamento revelou ausência de C. albicans na mucosa oral de todos os pacientes, bem como na superfície protética. A infecção por C. albicans das mucosas jugal e palatina diferiram significantemente, enquanto que a mucosa palatina e a superfície protética apresentaram valores semelhantes. O grau de infecção da mucosa palatina foi significantemente maior quando comparado àquele da mucosa jugal e semelhante ao apresentado pela prótese, sugerindo que a mucosa subjacente à prótese é mais susceptível à infecção. O protocolo terapêutico foi efetivo em todos os casos, o que enfatiza a necessidade da desinfecção protética para se evitar a reinfecção da mucosa oral.