Aspects related to biofilm production and antifungal susceptibility of clinically relevant yeasts of the genus Trichosporon.

Trichosporonosis corresponds to a systemic fungal disease that leads to high mortality rates and is frequently associated with medical devices. It affects immunosuppressed patients in particular and is strongly linked to acquired human immunodeficiency, organ and tissue transplants, and malignant hematologic diseases such as leukemia and lymphomas. Trichosporon infections have been increasingly reported worldwide; however, little information is available either about their characteristics or the causative microorganism. Thus, the aims of the present study were: to investigate 59 yeasts of the genus Trichosporon by verifying the biofilm formation capacity of isolates; to analyze the susceptibility patterns of planktonic cells against the antifungals fluconazole, itraconazole, amphotericin-B, voriconazole, and caspofungin by comparing European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution technique with the commercial method Etest; and to assess the susceptibility patterns of biofilm cells (sessile) against the same antifungals through broth microdilution. The ability to form biofilm on the surface of polystyrene plates was noted for all isolates, and 54.3% of samples were considered strong producers. Comparison between the antifungal susceptibility techniques evidenced that Etest showed higher and discordant minimum inhibitory concentrations (MICs) from those obtained by the microdilution method, especially for fluconazole, itraconazole, and caspofungin. Considering the susceptibility of biofilms, most species had high MIC50 and MIC90 against the tested antifungals, showing 4-to-66-fold higher concentrations for amphotericin B and 2-to-33-fold greater concentrations for caspofungin. These results highlight the importance of further studies with Trichosporon spp. for comparison between laboratory findings and in vivo response, considering both the susceptibility tests and the behavior of biofilm cells against drugs.

This study investigated 59 isolates of the medically important yeast Trichosporon in relation to their ability to form biofilms and the susceptibility of biofilms to antifungal agents. All isolates were able to produce biofilms and biofilms showed lower antifungal susceptibility.

Early clinical and microbiological predictors of outcome in hospitalized patients with cryptococcal meningitis.

BACKGROUND: Cryptococcal meningitis causes high mortality in immunocompromised and immunocompetent patients. The objective of this study was to identify early predictors of clinical outcome, available at the first days of hospitalization, in patients with cryptococcal meningitis in a tertiary center in Brazil. METHODS: Ninety-six cases of cryptococcal meningitis with clinical, epidemiological and laboratory data, and identification and antifungal susceptibility of the strains were analyzed. Quantitative CSF yeast counts were performed by direct microscopic exam with a Fuchs-Rosenthal cell counting chamber using an institutional protocol. Univariable and multiple analyses using logistic regression were performed to identify predictors, available at the beginning of hospitalization, of in-hospital mortality. Moreover, we performed a secondary analysis for a composite outcome defined by hospital mortality and intensive care unit transfer. RESULTS: The species and the antifungal susceptibility were not associated with the outcomes evaluated. The variables significantly associated with the mortality were age (OR = 1.08, 95% CI 1.02-1.15), the cerebrospinal fluid (CSF) yeasts count (OR = 1.65, 95% CI 1.20-2.27), systemic arterial hypertension (OR = 22.63, 95% CI 1.64-312.91) and neurological impairment identified by computed tomography (OR = 41.73, 95% CI 3.10-561.65). At the secondary analysis, CSF yeast count was also associated with the composite outcome, in addition to the culture of Cryptococcus spp. from bloodstream and cerebral toxoplasmosis. The associations were consistent with survival models evaluated. CONCLUSIONS: Age and CSF yeast count were independently associated with in-hospital mortality of patients with cryptococcal meningitis but Cryptococcus species identification and antifungal susceptibility were not associated with the outcomes. Quantitative CSF yeast counts used in this study can be evaluated and implemented in other low and middle-income settings.

A Rare Case of Aspergillus Mediastinitis After Coronary Artery Bypass Surgery: A Case Report and Literature Review.

BACKGROUND Mediastinitis is a serious complication after cardiac surgery; it is a deep sternal wound infection following sternotomy, with clinical evidence and/or microbiological involvement and sternal osteomyelitis. The most common pathogens are Staphylococcus spp (S. aureus), followed by gram-negative organisms. Establishing an etiological diagnosis of fungal mediastinitis is often a challenging issue, given the nonspecific clinical presentation. CASE REPORT A 74-year-old man was diagnosed with a three-vessel coronary artery disease in a university hospital. The patient had as clinical background hypertension, a body mass index (BMI) of 29.78 kg/m², and no diabetes mellitus. After an uneventful coronary artery bypass surgery, he presented clinical and radiological mediastinitis manifestations on the 9th postoperative day. He was treated with a range of antibiotics, with no clinical improvement until the 33rd postoperative day. Then, mediastinal fluid and biopsied tissue were collected and he was started on voriconazole due to growing Aspergillus spp. On the 93rd postoperative day, he had clinical improvement and, after several exams, was released from the hospital. We present the first report of Aspergillus fumigatus mediastinitis after cardiac surgery in Brazil, successfully treated with voriconazole. CONCLUSIONS Aspergillus infection should be considered in the differential diagnosis of mediastinitis after coronary surgery, especially in a clinical case of unexplained sepsis, negative blood culture, and no clinical improvement despite antibiotic therapy. This case report highlights that the mediastinal fluid and biopsy tissue culture can be useful for the diagnosis of fungal mediastinitis.

Infections Caused by Fusarium Species in Pediatric Cancer Patients and Review of Published Literature.

Fusarium species have emerged as responsible for a broad spectrum of infections, including superficial, locally invasive and disseminated ones, especially in the hospital environment. Since there are few reports of invasive and disseminated fusariosis in children, the aim of this study was to report four cases of nosocomial infection caused by this microorganism in children with cancer hospitalized in a public children's hospital located in Brazil. Two of these patients were female and two were male. All patients presented febrile neutropenia, while three patients had acute lymphocytic leukemia and one patient had Wilms' tumor as underlying disease. In two cases, fungi were isolated from blood and identified as Fusarium oxysporum species complex after phenotypic and genotypic studies, while in two other cases fungi were isolated from skin biopsies and identified as Fusarium solani species complex. One patient died 12 days after the onset of cutaneous lesions. All isolates, after susceptibility testing, presented high levels of minimum inhibitory concentration for itraconazole, voriconazole and amphotericin B. Considering the emergence of filamentous fungi as etiologic agents of nosocomial infections, health professionals should be aware of the problems these infections, especially fungal ones, may cause to debilitated patients.

DISSEMINATED FUNGAL INFECTION WITH ADRENAL INVOLVEMENT: REPORT OF TWO HIV NEGATIVE BRAZILIAN PATIENTS / Infecções fúngicas disseminadas com acometimento das supra-renais: descrição de dois pacientes brasileiros HIV-negativos

Paracoccidioidomycosis and histoplasmosis are systemic fungal infections endemic in Brazil. Disseminated clinical forms are uncommon in immunocompetent individuals. We describe two HIV-negative patients with disseminated fungal infections, paracoccidioidomycosis and histoplasmosis, who were diagnosed by biopsies of suprarenal lesions. Both were treated for a prolonged period with oral antifungal agents, and both showed favorable outcomes.

A paracoccidioidomicose e a histoplasmose são infecções fúngicas sistêmicas endêmicas no Brasil. As formas clínicas disseminadas são incomuns em pacientes imunocompetentes. Nós descrevemos dois pacientes HIV-negativos com infecções fúngicas disseminadas, paracoccidioidomicose e histoplasmose, que foram diagnosticadas por biópsias de lesões de supra-renal. Ambos foram tratados por períodos prolongados com antifúngicos orais, evoluindo com boa resposta terapêutica.

Nosocomial candidiasis in Rio de Janeiro State: Distribution and fluconazole susceptibility profile

One hundred and forty-one Candida species isolated from clinical specimens of hospitalized patients in Rio de Janeiro, Brazil, during 2002 to 2007, were analized in order to evaluate the distribution and susceptibility of these species to fluconazole. Candida albicans was the most frequent species (45.4%), followed by C. parapsilosis sensu lato (28.4%), C. tropicalis (14.2%), C. guilliermondii (6.4%), C. famata (2.8%), C. glabrata (1.4%), C. krusei (0.7%) and C. lambica (0.7%). The sources of fungal isolates were blood (47.5%), respiratory tract (17.7%), urinary tract (16.3%), skin and mucous membrane (7.1%), catheter (5.6%), feces (2.1%) and mitral valve tissue (0.7%). The susceptibility test was performed using the methodology of disk-diffusion in agar as recommended in the M44-A2 Document of the Clinical and Laboratory Standards Institute (CLSI). The majority of the clinical isolates (97.2%) was susceptible (S) to fluconazole, although three isolates (2.1%) were susceptible-dose dependent (S-DD) and one of them (0.7%) was resistant (R). The S-DD isolates were C. albicans, C. parapsilosis sensu lato and C. tropicalis. One isolate of C. krusei was resistant to fluconazole. This work documents the high susceptibility to fluconazole by Candida species isolated in Rio de Janeiro, Brazil.

DISSEMINATED FUNGAL INFECTION WITH ADRENAL INVOLVEMENT: REPORT OF TWO HIV NEGATIVE BRAZILIAN PATIENTS.

Paracoccidioidomycosis and histoplasmosis are systemic fungal infections endemic in Brazil. Disseminated clinical forms are uncommon in immunocompetent individuals. We describe two HIV-negative patients with disseminated fungal infections, paracoccidioidomycosis and histoplasmosis, who were diagnosed by biopsies of suprarenal lesions. Both were treated for a prolonged period with oral antifungal agents, and both showed favorable outcomes.

Vulvovaginal candidiasis in Mato Grosso, Brazil: pregnancy status, causative species and drugs tests

Causative agent in majority of VVC is Candida albicans, but infection due to non-C. albicans is common. Use of empiric antifungal therapy in Brazil due to syndromic management of vulvovaginitis could act as risk factor for increase resistance among VVC causative agents. From Mato Grosso patients, 160 with culture-proved among 404 women who had clinical symptoms of VVC, were enrolled in this study. 70 non-pregnant women and 90 pregnant women were included. Candida albicans was the most prevalent, representing 72.9 percent in the non-pregnant group and 92.3 percent in the pregnant group. Differences in species distribution were noted between the two groups, being C. parapsilosis the second more prevalent species among non-pregnant women. Susceptibility testing revealed high susceptibility to fluconazole (except for C. krusei), itraconazole, ketoconazole, and amphotericin B regardless the species (C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, C. krusei) analyzed.

Molecular typing and antifungal susceptibility of clinical sequential isolates of Cryptococcus neoformans from Sao Paulo State, Brazil.

The antifungal susceptibility profiles and the genetic variability of 83 sequential clinical isolates of Cryptococcus neoformans, including four Cryptococcus gattii isolates, obtained from 38 Sao Paulo AIDS patients with cryptococcal meningitis were assessed by electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis. The majority of the Cryptococcus neoformans isolates were highly susceptible to amphotericin B and fluconazole. Twenty percent of the minimum inhibitory concentration values for amphotericin B varied from 0.5 to 1 micro g mL(-1). For fluconazole, 22% occurred in the range 8-16 mug mL(-1). Sequential isolates from nine patients showed a trend towards lower susceptibility to fluconazole, flucytosine, itraconazole and amphotericin B. The results of molecular typing by electrophoretic karyotyping and RAPD analysis showed the presence of 22 electrophoretic karyotypes (EK) and 15 RAPD profiles that were highly correlated. Our results provided evidence for the occurrence of genetic changes in some strains associated with microevolution during the course of infection. We also observed both microevolution and simultaneous coinfection with two distinct Cryptococcus neoformans strains in one patient. In some patients, we found changed EK- and RAPD patterns in association with increased MIC values.

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (> 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.

Cryptococcus neoformans, pertencente à classe dos basidiomicetos, é um importante patógeno, principalmente em pacientes imunocomprometidos. Neste estudo, 47 isolados clínicos de C. neoformans de várias regiões do Estado de São Paulo foram avaliados quanto aos sorotipos e ao mating-type por PCR. A diversidade genética foi analisada por PCR-fingerprinting com a seqüência iniciadora específica para regiões microssatélite (GACA)4, RAPD com o iniciador 6 (Amersham Pharmacia Biotech) e por RFLP do gene PLB1 digerido com AvaI. Todos os isolados foram obtidos de pacientes HIV positivos e identificados como sorotipo A e MATalfa. A maioria dos isolados pertencia ao tipo molecular VNI (45/47) e apenas dois foram VNII quando analisados por PCR-fingerprinting e PCR-RFLP. Homogeneidade alta foi obtida com o iniciador (GACA)4, com a maioria dos isolados apresentando correlação alta (> 0.9). Os resultados do RAPD, por sua vez, revelaram maior heterogeneidade com número maior de perfis moleculares. Por PCR-RFLP, nenhum tipo molecular novo foi encontrado, realçando a idéia de que em genes conservados como PLB1, as diferenças podem ser resultantes de divergências evolutivas dentro do complexo C. neoformans, separando os isolados nos oito subtipos moleculares já estabelecidos. Nossos resultados fornecem novas informações sobre a epidemiologia molecular de C. neoformans na região sudeste do Brasil.

Neurocryptococcosis: diagnosis by PCR method.

Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

Neurocryptococcosis: diagnosis by PCR method

A detecção de Cryptococcus neoformans em líquor foi otimizada pela técnica de PCR. A amplificação foi realizada nas áreas ITS e 5,6S do RNA ribossomal (rRNA). Foram estudados 72 líquors obtidos de casos de pacientes com e sem AIDS. Os pacientes eram portadores de meningite criptocócica (n = 56) e meningite ocasionada por outros agentes (n = 16). Os resultados demonstraram que a técnica tem alta sensibilidade, superior a cultura (85,7) e ao teste da tinta da china (76,8). A técnica de PCR pode detectar 1 célula/mL de líquor e é altamente específica. A análise comparativa dos três métodos, tinta da china, cultura e PCR, demonstrou que o último é muito mais sensível e específico, podendo ser aplicável como importante recurso laboratorial no diagnóstico da neurocriptococose.

Biótipos, cariótipos e perfis de suscetibilidade a antifúngicos de amostras seriadas de Candida albicans de pacientes com candidíase bucal associada à síndrome de imunodeficiência adquirida / Biotyping, karyotyping and variation besides antifungal suceptibility of Candida albicans in patients with oropharyngeal candidiases associated to AIDS

Estuda a variaçäo de subtipos e suscetibilidade a fármacos antifúngicos de amostras de Candida albicans isoladas de 25 pacientes com candidíase bucal associada à AIDS. Foram coletadas três amostras seriadas de cada paciente, sendo a primeira antes do tratamento, a segunda após o início da terapia com fármaco antifúngico e a terceira durante esta e após remissäo clínica da lesäo bucal. As amostras, assim obtidas, foram submetidas à prova fenotípica de biotipagem de ODDS & ABBOTT (1980), morfotipagem e cariotipagem por eletroforese em campo pulsátil (CHEF-DR II, Bio-Rad, USA). O padräo de peso molecular de Saccharomyces cerevisiae foi usado como controle de qualidade da corrida eletroforética e para cálculo do peso molecular. O DNA das cepas foi contido em blocos de agarose para a eletroforese em gel. A tipagem das amostras revelou 14 morfotipos, 13 biótipos de ODDS & ABBOTT e 11 cariótipos. A maioria dos pacientes apresentavam um só biótipo (68,0 por cento) ou um só cariótipo (72,0 por cento) nas três amostras analisadas. Todos os cariótipos apresentaram bandas de DNA entre 850 kb e 2200 kb e um deles, agrupando 8 (10,7 por cento) cepas, também mostrou bandas em 680 kb. Os fármacos apresentaram boa atividade antifúngica in vitro contra as amostras estudadas, com valores de concentraçäo inibitória mínima (CIM) para anfotericina B situados entre 0,31ug ml-1 a 2,50 ug ml-1

Leveduroses na Síndrome de Imunodeficiência Adquirida (AIDS/SIDA) / Mycoses in the Acquired Immunodeficiency Syndrome (AIDS/SIDA)

Foram revistos, retrospectivamente, 174 resultados de exames micológicos de 78 pacientes com imunodeficiência adquirida (AIDS). Pesquisou-se, através dos prontuários desses casos, dados relativos a manifestaçöes clínicas compatíveis com micoses oportunistas, grupo de risco, doenças associadas e participaçäo dos fungos no óbito dos pacientes. As espécies encontradas nos diversos materiais biológicos estudados foram: Cryptococcus neoformans, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida famata, Kluyveromyces thermotolerans e Trichosporon beigelii.

Avaliação da prova de hemaglutinação passiva no diagnóstico da leptospirose humana em diversos períodos evolutivos da doença / Evaluation of passive hemagglutination test in the diagnosis of human leptospirosis in different evolutive periods of the illness

Foram realizadas provas de hemaglutinação passiva em amostras séricas de 189 pacientes com leptospirose, distribuídas quanto ao período decorrido após início dos sintomas. Os resultados foram comparados aos obtidos com a prova de referência, a soroaglutinação microscópica. A hemaglutinação passiva revelou, na primeira amostra examinada, maior número de resultados positivos, estatisticamente significante, a nível de 1%, no intervalo de 6 a 10 dias de doença, quando comparada à soroaglutinação. Nos demais intervalos, os valores diagnósticos das provas foram aceitos como iguais, Os resultados sugerem que a prova de hernaglutinação passiva pode ser empregada no acompanhamen to sorológico, como subsídio no estabelecimento do estágio da infecção leptospirótica (AU).

Avaliação da prova de hemaglutinação passiva no diagnóstico da leptospirose humana / Evaluation of passive hemagglutination test for human leptospirosis

A prova de hemaglutinação passiva foi avaliada em relação à soroaglutínação microscópica em 437 amostras de soros provenientes de 192 pacientes com quadro clínico sugestivo de leptospirose, internados no Hospital de Isolamento "Emílio Ribas", em São Paulo. A prova de hemaglutinação passiva apresentou associação significativa com a soroaglutinação microscópica, revelando-se, pois, uma prova alternativa de elevado valor diagnóstico (AU).