Chromoblastomycosis in the Amazon region, Brazil, caused by Fonsecaea pedrosoi, Fonsecaea nubica, and Rhinocladiella similis: Clinicopathology, susceptibility, and molecular identification.

Chromoblastomycosis is a chronic subcutaneous disease caused by human contact with melanized fungi occurring mainly in tropical and subtropical zones worldwide. This study assessed 12 patients with chromoblastomycosis from Rondônia, Brazil, Amazon region. In sum, 83.3% were men, 41.6% were from Monte Negro city, median age was 52.9 years, and median time to disease progression was 12.2 years. Lesions were located on the lower limbs (75%), and verruciform was prevalent form (66.6%). After 3 years of treatment with itraconazole, two patients were considered cured. The etiological agents were identified by the molecular sequence of the ribosomal internal transcribed spacer ITS1, 5.8S, and ITS2 region and ß-tubulin genes. Eight strains were identified as Fonsecaea pedrosoi, two were F. nubica, and two were Rhinocladiella similis. The antifungal activity of five drugs was evaluated, and the most active drug was terbinafine (range minimal inhibitory concentration [MIC] 0.015-0.12 µg/ml), itraconazole (range MIC 0.03-0.5 µg/ml) and voriconazole (range MIC 0.06-0.5 µg/ml). The highest MIC was 5-fluorocytosine (range MIC 2-32 µg/ml), and amphotericin B (range MIC 0.25-2 µg/ml). In conclusion, the present study expanded the epidemiological disease database and described for the first time F. nubica and R. similis as chromoblastomycosis agents in the Brazilian Amazon region. Our results confirmed the importance of using molecular methods to identify the melanized fungi and stimulate the recognition of the disease in other places where no cases have been reported.

Dermatophytosis and immunovirological status of HIV-infected and AIDS patients from Sao Paulo city, Brazil.

Over the past decades, more people became infected with human immunodeficiency virus (HIV) and developed acquired immunodeficiency syndrome (AIDS). Because of that the incidence of fungal infections rose dramatically. It happened because this virus can modify the course of fungal diseases, leading to altered clinical pictures. The aim of this study was to evaluate epidemiological and biological aspects of dermatophytosis in HIV-positive and AIDS patients living in the city of São Paulo, Brazil. A total of 84 (44 HIV-positive and 40 AIDS) patients were enrolled in this study. The patients were tested for dermatophyte infections, as well as for the CD4(+) /CD8(+) and HIV viral load counts. Tinea unguium was most frequently observed in AIDS patients, whereas Tinea pedis was mostly observed in HIV-positive patients. The most frequent dermatophyte species was Trichophyton rubrum. CD4(+) counts and CD4(+) /CD8(+) ratios were not associated with a higher risk for dermatophytosis. On the other hand, viral load higher than 100 000 copies/ml was associated with a higher frequency of dermatophytosis. The results suggest to that although dermatophytosis is common in HIV-positive and AIDS patients, the degree of immunosuppression does not seems to correlate with increased risk of this fungal infection. In addition, high viral load as a predictive risk factor for dermatophyte infection should be subject of further evaluations.

Skin mycoflora of Cebus primates kept in captivity and semicaptivity.

BACKGROUND: This study was performed to determine the fungal mycoflora in healthy tufted capuchins primates (Cebus sp) kept in captivity and semicaptivity to allow a more realistic interpretation on the basis of fungi isolated from their skin. Furthermore, we aimed at evaluating the potential risk of infection to humans by fungi perpetuated in the tegument of monkeys of this genus. METHODS: For the collection of skin material, the carpet method was used, followed by seeding in Sabouraud dextrose agar culture media, Mycosel agar, Dermatophyte Test Medium and Sabouraud agar supplemented with olive oil. RESULTS: Seventeen genera of fungi were detected, being three of them isolated only in the captivity animals (Acremonium - Cephalosporium, Phoma and Trichosporon). The genera of fungi with the higher frequencies were identified in the semicaptivity capuchins (Fusarium, Aspergillus and Penicillium). CONCLUSIONS: Many of the genera of fungi identified are potential pathogens for immune-compromised monkeys and humans.

Voriconazole, combined with amphotericin B, in the treatment for pulmonary cryptococcosis caused by C. neoformans (serotype A) in mice with severe combined immunodeficiency (SCID).

Cryptococcosis is a subacute or chronic systemic mycosis with a cosmopolitan nature, caused by yeast of the genus Cryptococcus neoformans. The model of systemic cryptococcosis in mice with severe combined immunodeficiency (SCID) is useful for immunological and therapeutic study of the disease in immunodeficient hosts. Amphotericin B, fluconazole and flucytosine are the drugs most commonly used to treat cryptococcosis. Voriconazole is a triazole with high bioavailability, large distribution volume, and excellent penetration of the central nervous system. The objective of this study was to evaluate treatment with amphotericin B (AMB), voriconazole (VRC), and AMB, used in combination with VRC, of experimental pulmonary cryptococcosis in a murine model (SCID). The animals were inoculated intravenously (iv) with a solution containing 3.0 × 10(5) viable cells of C. neoformans ATCC 90112, (serotype A). Treatments were performed with amphotericin B (1.5 mg/kg/day), voriconazole (40.0 mg/kg/day) and AMB (1.5 mg/kg/day) combined with VRC (40.0 mg/kg/day); began 1 day after the initial infection; were daily; and lasted 15 days. Evaluations were performed using analysis of the survival curve and isolation of yeast in the lung tissue. There was a significant increase in survival in groups treated with AMB combined with VRC, compared with the untreated group and groups receiving other treatments (P < 0.05). In the group treated only with VRC and AMB combined with VRC, there was a significant reduction (P < 0.05) in the isolation of C. neoformans in lung tissue. Amphotericin B combined with voriconazole may be an effective alternative to increasing survival and may reduce yeast in the lung tissue of mice with pulmonary cryptococcosis and SCID.

Isolation of Microsporum gypseum in soil samples from different geographical regions of brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains.

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Extracellular proteolytic activity and molecular analysis of Microsporum canis strains isolated from symptomatic and asymptomatic cats.

Microsporum canis is the main zoophylic dermatophyte in dogs and cats, and it is also an important zoonotic agent. The literature showed that cats are asymptomatic carriers of M. canis. This is apparently due to host resistance and/or the presence of strains with lower virulence. This study was aimed to evaluate the keratinolytic, elastinolytic and collagenolytic activities of M. canis strains and their relationship with symptomatic and asymptomatic cats. In addition, these strains were analysed by RFLP. The strains isolated from cats with clinical dermatophytosis had higher keratinase and elastase activity than those isolated from asymptomatic animals (p minus than 0.05). There were not differences in RFLP patterns based on Hind III digestion.

Virulence profile of strains of Cryptococcus neoformans var. grubii evaluated by experimental infection in BALB/c mice and correlation with exoenzyme activity.

To evaluate the virulence profile of strains of Cryptococcus neoformans var. grubii, 62 strains of this yeast were inoculated into BALB/c mice. It was found that 69 % of the strains were significantly more lethal to the mice and were recovered from a higher percentage (60 %) of the organs compared with the other 31 % of the strains, which were recovered from 35 % of organs tested. Those strains that provoked higher death rates were also recovered from the central nervous system at a higher rate (84 %) than the less lethal strains (32 %). This finding led to an investigation of the factors that enhanced the capacity for neurological infection and death of the animals. The results of this study suggested that environmental strains present different degrees of virulence. The correlation of exoenzyme production before and after inoculation and between the groups of mice indicated that exoenzyme production had no influence on differences in virulence among the strains studied.

Effectiveness of house dust mite acaricide tri-n-butyl tin maleate on carpets, fabrics and mattress foam: a standardization of methodology.

The aim of this study was to determine the effectiveness of the acaricide tri-n-butyl tin maleate, industrially applied to samples of carpets, mattress foam, and fabrics used for furniture upholstery, soft toys and shoe uppers. Approximately 100 adult house dust mites of the species Dermatophagoides pteronyssinus were inoculated into a Petri dish containing the sample (a piece of carpet, mattress foam, or fabric), treated with the acaricide, randomly collected. Mite-maintenance culture medium was added on top of each sample. After one, two, three, seven and 30 days of incubation at 25 degrees C and 75% relative humidity, each dish was examined using a 40X stereoscopic microscope (40X). One hundred percent acaricide effectiveness was obtained in treated materials by the end of the 30th-day postinoculation period, under optimal conditions for mite maintenance.

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

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

Cryptococcus neoformans strains isolated from church towers in Rio de Janeiro City, RJ, Brazil.

Cryptococcosis has been a significant cause of morbidity and mortality in patients with Aids. Many reservoirs of the agent Cryptococcus neoformans have been reported, but the ecology of this yeast must be elucidated in order to establish surveillance programs and to prevent infections. The objective of this study was to evaluate the presence of C. neoformans in Rio de Janeiro City, RJ, Brazil. Ten churches were selected for sampling and detection of the yeast collecting pigeon dropping, air samples from church towers and neighboring areas during one year. The data demonstrated that C. neoformans has been present in every church selected and was present in 37.8% of 219 pigeon dropping samples. As well as, the yeast was isolated from soil, insects, eggs, pigeon nests and feathers. Fifteen air samples (4.9%) were positive. The growth on C.G.B. medium showed that all strains belonged to C. neoformans var. neoformans, with 98.8% of the strains belonging to serotype A.

Yeasts of the genital region of patients attending the dermatology service at Hospital São Paulo, Brazil.

BACKGROUND: The knowledge of the diversity of yeasts that make up the skin microbiota of human beings is essential for the efficient monitoring of infections to which a person may be predisposed. AIMS: This study identified yeasts comprising the genital skin microbiota of patients attending the Dermatology Service at the Hospital São Paulo-UNIFESP, Brazil. METHODS: Samples were collected from the genital region of each patient and cultured on Sabouraud dextrose agar. Individual colonies were carefully transferred to tubes daily. Yeasts were identified based on classical methodologies and confirmed using a commercial kit. RESULTS: Eighty-three patients were included in the study. Approximately 80% were women and 20% were men. The average age was 55 years. Hypertension, diabetes, kidney transplant and AIDS were the main underlying diseases reported by the patients. The most prevalent yeasts were Candida parapsilosis (36.1%), Rhodotorula mucilaginosa (9.2%), Rhodotorula glutinis (8.3%), Candida tropicalis (5.5%) and Trichosporon inkin (1.8%). Approximately 78% of the isolates were obtained in pure cultures. Trichosporon inkin was isolated only from women, in contrast to literature describing a high prevalence in males. CONCLUSIONS: Our results suggest that Candida albicans is not the main yeast found on genital skin as previously thought, and opportunistic pathogens such as C. parapsilosis, C. tropicalis, Rhodotorula spp. and T. inkin make up the genital skin microbiota, representing a risk for infection in immunocompromised subjects. These results also indicate that women are carriers of T. inkin, the etiological agent of white piedra and trichosporonosis.

Upper respiratory symptoms associated with aging of the ventilation system in artificially ventilated offices in São Paulo, Brazil.

BACKGROUND: The increase of work-related respiratory complaints in artificially ventilated buildings has multiple causes, and the role of allergen exposure and symptoms is still controversial. STUDY OBJECTIVES: To analyze the risk factors and the association of work-related symptoms with allergen exposure and different conditions of the same air conditioning system in São Paulo, Brazil. DESIGN: Workers were classified according to characteristics of the air conditioning system: the first group (group 1) with ventilation machinery and ducts with > 20 years of use, the second group (group 2) with ventilation machinery with > 20 years of use and ventilation ducts with < 2 years of use, and the third group (group 3) with ventilation machinery and ducts with < 2 years of use. Logistic regression was performed to check the associations between air conditioning groups, allergen exposure (fungi, mites, animal dander, and cockroach), and symptoms. RESULTS: There was a higher prevalence of building-related worsening of respiratory symptoms (p = 0.004; odds ratio [OR], 8.53) and symptoms of rhinoconjunctivitis (p = 0.01; OR, 8.49) in group 1. There was a lower relative humidity (p = 0.05) and nonsignificant lower temperature in group 1, when compared to the other groups. The viable mold spores totals were higher outdoors than in the indoor samples (n = 45, p = 0.017). There were higher levels of Der p 1 in group 2 (p = 0.032). All allergen levels were considered low. CONCLUSION: There was a strong association of building-related upper-airway symptoms with places having ventilation systems with > 20 years of use.

Fungi allergens produced by solid-state fermentation process: optimization and allergen characterization.

Allergenic extracts were produced from Drechslera (Helminthosporium) monoceras biomass cultured by solid-state fermentation using wheat bran as the substrate. The main fermentation variables were selected by statistical design, and the optimized biomass yield (1.43 mg/[g of dry substrate d]) was obtained at pH 9.5 and 45.8% moisture. The allergenic extracts were produced from crude extract by protein precipitation and polyphenol removal. Proteins in the range of 16-160 kDa were identified in the extracts. Their reactions in patients were characterized by in vivo cutaneous tests (positive in 40% of the atopic patients) and by dot-blotting assays.

Candida spp. isolated from inpatients, the environment, and health practitioners in the Pediatric Unit at the Universitary Hospital of the Jundiaí Medical College, State of São Paulo, Brazil.

INTRODUCTION: This study aimed to isolate and identify Candida spp. from the environment, health practitioners, and patients with the presumptive diagnosis of candidiasis in the Pediatric Unit at the Universitary Hospital of the Jundiaí Medical College, to verify the production of enzymes regarded as virulence factors, and to determine how susceptible the isolated samples from patients with candidiasis are to antifungal agents. METHODS: Between March and November of 2008 a total of 283 samples were taken randomly from the environment and from the hands of health staff, and samples of all the suspected cases of Candida spp. hospital-acquired infection were collected and selected by the Infection Control Committee. The material was processed and the yeast genus Candida was isolated and identified by physiological, microscopic, and macroscopic attributes. RESULTS: The incidence of Candida spp. in the environment and employees was 19.2%. The most frequent species were C. parapsilosis and C. tropicalis among the workers, C. guilliermondii and C. tropicalis in the air, C. lusitanae on the contact surfaces, and C. tropicalis and C. guilliermondii in the climate control equipment. The college hospital had 320 admissions, of which 13 (4%) presented Candida spp. infections; three of them died, two being victims of a C. tropicalis infection and the remaining one of C. albicans. All the Candida spp. in the isolates evidenced sensitivity to amphotericin B, nystatin, and fluconazole. CONCLUSIONS: The increase in the rate of hospital-acquired infections caused by Candida spp. indicates the need to take larger measures regarding recurrent control of the environment.

Malassezia spp. in acoustic meatus of bats (Molossus molossus) of the Amazon Region, Brazil.

The yeasts of the Malassezia genus are opportunistic microorganisms and can cause human and animal infections. They are commonly isolated from the skin and auricular canal of mammalians, mainly dogs and cats. The present study was aimed to isolate Malassezia spp. from the acoustic meatus of bats (Molossus molossus) in the Montenegro region, "Rondônia", Brazil. From a total of 30 bats studied Malassezia spp. were isolated in 24 (80%) animals, the breakdown by species being as follows (one Malassezia sp. per bat, N = 24): 15 (62.5%) M. pachydermatis, 5 (20.8%) M. furfur, 3 (12.5%) M. globosa and 1 (4.2%) M. sympodialis. This study establishes a new host and anatomic place for Malassezia spp., as it presents the first report ever of the isolation of this genus of yeasts in the acoustic meatus of bats.

Morphogenesis and growth kinetics of Fusarium verticillioides.

The present study aimed to analyze the growth kinetics and morphogenesis of toxigenic Fusarium verticillioides strains. Growth curves based on mycelial dry weight measured after 24, 48, and 96 h and every 4 days for a period of 60 days were obtained for each strain. The morphogenesis of this fungus was studied during its developmental stages through the fluorescent method Fluorecein Diacetate-FDA and Ethidium Bromide-EB. The growth curves of the strains usually followed a homogeneous pattern comparable to the ideal growth curve. Using the fluorescence method, non-viable cells showed a light bright red coloration and viable cells presented green fluorescence with three fluorescent patterns. The present results showed that the morphogenesis of F. verticillioides is an asynchronous process characterized by the presence of a wide variety of fungal forms until 50 days of culture. The method is very useful to demonstrate the F. verticillioides growth stages as well as the perfect differentiation between viable and non-viable cell.

Candida spp. isolated from inpatients, the environment, and health practitioners in the pediatric unit at the Universitary Hospital of the Jundiaí Medical College, state of São Paulo, Brazil / Candida spp. isoladas de pacientes internados, profissionais da saúde e de ambiente na unidade de pediatria do Hospital Universitário da Faculdade de Medicina de Jundiaí, estado de São Paulo, Brasil

INTRODUCTION: This study aimed to isolate and identify Candida spp. from the environment, health practitioners, and patients with the presumptive diagnosis of candidiasis in the Pediatric Unit at the Universitary Hospital of the Jundiaí Medical College, to verify the production of enzymes regarded as virulence factors, and to determine how susceptible the isolated samples from patients with candidiasis are to antifungal agents. METHODS: Between March and November of 2008 a total of 283 samples were taken randomly from the environment and from the hands of health staff, and samples of all the suspected cases of Candida spp. hospital-acquired infection were collected and selected by the Infection Control Committee. The material was processed and the yeast genus Candida was isolated and identified by physiological, microscopic, and macroscopic attributes. RESULTS: The incidence of Candida spp. in the environment and employees was 19.2%. The most frequent species were C. parapsilosis and C. tropicalis among the workers, C. guilliermondii and C. tropicalis in the air, C. lusitanae on the contact surfaces, and C. tropicalis and C. guilliermondii in the climate control equipment. The college hospital had 320 admissions, of which 13 (4%) presented Candida spp. infections; three of them died, two being victims of a C. tropicalis infection and the remaining one of C. albicans. All the Candida spp. in the isolates evidenced sensitivity to amphotericin B, nystatin, and fluconazole. CONCLUSIONS: The increase in the rate of hospital-acquired infections caused by Candida spp. indicates the need to take larger measures regarding recurrent control of the environment.

INTRODUÇÃO: Isolar e identificar Candida spp. do ambiente, dos profissionais de saúde e de pacientes com diagnóstico presuntivo de candidíase, em Unidade de Pediatria do Hospital Universitário da Faculdade de Medicina de Jundiaí, verificar a produção de enzimas consideradas fatores de virulência e identificar a resistência das cepas isoladas dos pacientes aos antifúngicos. MÉTODOS: Foram feitas 283 coletas no período entre março e novembro de 2008 de forma randomizada do ambiente, das mãos de profissionais de saúde e de todos os pacientes com casos suspeitos de infecção hospitalar por Candida spp., triados pelo Comitê de Infecção Hospitalar. Todo o material coletado foi processado e as leveduras do gênero Candida foram isoladas e identificadas pelas características macroscópicas, microscópicas e fisiológicas. RESULTADOS: A incidência de Candida spp. no meio-ambiente e funcionários foi de 19,2%. Destes, as espécies mais frequentes entre os funcionários foram C. parapsilosis e C. tropicalis; no ar ambiente, C. guilliermondii e C. tropicalis; nas superfícies dos ambientes, C. lusitanae e nos sistemas de climatização, C. tropicalis e C. guilliermondii. No período de estudo, foram feitas 320 internações, e 13 (4%) apresentaram infecção hospitalar por Candida spp, sendo que 3 pacientes foram a óbito, dois apresentando infecção por C. tropicalis e um por C. albicans. Todas as espécies de Candida spp. isoladas dos pacientes foram sensíveis à anfotericina B, nistatina e fluconazol. CONCLUSÕES: Verificamos um aumento crescente de infecções hospitalares por Candida não albicans, o que sugere a necessidade de maiores precauções em relação a um controle ambiental destas espécies.

Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Genotyping by RAPD-PCR analyses of Malassezia furfur strains from pityriasis versicolor and seborrhoeic dermatitis patients.

Malassezia furfur is lypophilic yeast commonly associate with dermatological disorders. In the present work, we described the isolation of 47 M. furfur strains from three groups of patients: pityriasis versicolor (21 isolates), seborrhoeic dermatitis (15 isolates) and seborrhoeic dermatitis of the HIV positive patients (11 isolates). To investigate the identity of the strains at molecular level, DNA genomic of M. furfur strains were prepared and used to RAPD-PCR analyses. RAPD assay were carried out using two decamer primers and bands pattern generated were analyzed by an Unweighted Pair-Group Method (UPGMA). Dendrogram established a distinct differentiation between M. furfur isolates from pityriasis versicolor and seborrhoeic dermatitis patients with or without AIDS. We concluded that RAPD typing presented a high discriminatory power between strains studied in this work and can be applied in epidemiological investigation of skin disease causing by M. furfur.

Fluorescent method for studying the morphogenesis and viability of dermatophyte cells.

The dermatophyte Microsporum canis is commonly isolated from human and animal infection. The morphogenesis of this fungus was studied during its developmental stages through the fluorescent method Fluorescein Diacetate and Ethidium Bromide. To this end, 50 microl dermatophyte suspension were transferred onto cellophane wrapping esterilized discs (2.5 cm of diameter) placed over the surface of Sabouraud dextrose agar on Petri dishes and incubated at 25 degrees C for 30 days. Every 60 minutes during the first 24 hours and every 12 hours for next 29 days, one disc was transferred onto glass slide, covered with equal volumes of freshly prepared fluorescein diacetate (FDA) and ethidium bromide (EB) solution, mounted with a coverslip and incubated in the dark for 30 minutes, at 25 degrees C. Each preparation was then examined on a fluorescent microscope. M. canis presented well defined growth stages: (1) tumescence of cells; (2) germination; (3) development of hyphae; (4) production of conidia and (5) tumescence and formation of arthroconidiae. Using the fluorescent method, non viable cells showed a light bright red coloration and viable cells presented green fluorescence. The principal morphological changes have occurred between the 3rd until the 18th day of culture. The method is very useful to demonstrate the dermatophyte growth stages as well as the perfect differentiation between viable and non viable cells.