RESUMO: Estudos sobre a microbiota fúngica de répteis e anfíbios são necessários para melhor compreender as interações hospedeiro-microrganismo e o estabelecimento de doenças fúngicas nesses animais. No entanto, esses estudos são limitados. O objetivo da presente pesquisa foi identificar leveduras isoladas de répteis e anfíbios do bioma Caatinga e avaliar a produção de fatores de virulência, a sensibilidade a antifúngicos no crescimento planctônico e de biofilme e a patogenicidade de Candida famata. Vinte e nove isolados dos gêneros Candida, Cryptococcus e Rhodotorula foram identificados por métodos fenotípicos e/ou moleculares e a produção de enzimas hidrolíticas in vitro por esses gêneros de fungos foi avaliada. Além disso, foi avaliada a suscetibilidade de células planctônicas e biofilmes a azólicos e anfotericina B. A patogenicidade de C. famata, a espécie de levedura isolada mais prevalente, foi avaliada usando Caenorhabditis elegans. C. famata foi a levedura mais prevalente na microbiota de anfíbios e répteis. A produção de fosfolipase e protease foi observada em 18/29 e 11/29 dos isolados de levedura, respectivamente, enquanto 100% formaram biofilmes. O itraconazol apresentou altas concentrações inibitórias mínimas contra C. famata e C. tropicalis. A anfotericina B reduziu a biomassa e atividade metabólica dos biofilmes. C. famata induziu a mortalidade de C. elegans. Em conclusão, répteis e anfíbios são colonizados por leveduras capazes de produzir importantes fatores de virulência, especialmente por cepas de Candida spp. que apresentam baixa suscetibilidade a azólicos que podem resultar de desequilíbrio no ecossistema. Por fim, C. famata isolados desses animais apresentaram alta patogenicidade, mostrando a importância do estudo da microbiota fúngica de répteis e anfíbios.

ABSTRACT: Studies on the fungal microbiota of reptiles and amphibians are necessary to better understand of host-microbe interactions and the establishment of fungal disease in these animals. However, these studies are limited. The present researchidentified yeasts from free-ranging reptiles and amphibians from the Caatinga biome andevaluated the virulence factors production, the antifungal susceptibility in planktonic and biofilm growth and the pathogenicity of Candida famata isolates. Twenty-nine isolates of the genera Candida, Cryptococcus and Rhodotorula were identified by phenotypic and/or molecular methods and production of hydrolytic enzymes in vitro by these genera of fungi was evaluated. In addition, susceptibility of planktonic cells and biofilms to azoles and amphotericin B was evaluated. The pathogenicity of C. famata, the most prevalent yeast species isolated, was evaluated using Caenorhabditis elegans model. C. famata was the most prevalent yeast in amphibian and reptilian microbiota. Phospholipase and protease production was observed in 18/29 and 11/29 of the yeast isolates, respectively, while 100% formed biofilms. Itraconazole presented high minimal inhibitory concentrations against C. famata and C. tropicalis. Amphotericin B reduced the biomass and metabolic activity of biofilms. C. famata induced the mortality of C. elegans. In conclusion, reptiles and amphibians are colonized by yeasts capable of producing important virulence factors, especially by Candida spp. that present low susceptibility to azoles which may result from imbalances in ecosystem. Finally, C. famata isolated from these animals presented high pathogenicity, showing the importance of the study of reptile and amphibians fungal microbiota.

Abstract This study aimed to evaluate the in vitro antifungal activity of terpinen-4-ol, tyrosol, and β-lapachone against strains of Coccidioides posadasii in filamentous phase (n = 22) and Histoplasma capsulatum in both filamentous (n = 40) and yeast phases (n = 13), using the broth dilution methods as described by the Clinical and Laboratory Standards Institute, to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of these compounds. The mechanisms of action of these compounds were also investigated by analyzing their effect on cell membrane permeability and ergosterol synthesis. The MIC and MFCf these compounds against C. posadasii, mycelial H. capsulatum, and yeast-like H. capsulatum, were in the following ranges: 350-5720 µg/mL, 20-2860 µg/mL, and 40-1420 µg/mL, respectively for terpinen-4-ol; 250-4000 µg/mL, 30-2000 µg/mL, and 10-1000 µg/mL, respectively, for tyrosol; and 0.48-7.8 µg/mL, 0.25-16 µg/mL, and 0.125-4 µg/mL, respectively for β-lapachone. These compounds showed a decrease in MIC when the samples were subjected to osmotic stress, suggesting that the compounds acted on the fungal membrane. All the compounds were able to reduce the ergosterol content of the fungal strains. Finally, tyrosol was able to cause a leakage of intracellular molecules.

Abstract Recent studies have shown that some drugs that are not routinely used to treat fungal infections have antifungal activity, such as protease inhibitor antiretroviral drugs. This study investigated the in vitro susceptibility of Histoplasma capsulatum var. capsulatum to saquinavir and ritonavir, and its combination with the antifungal itraconazole. The susceptibility assay was performed according to Clinical and Laboratory Standards Institute guidelines. All strains were inhibited by the protease inhibitor antiretroviral drugs. Saquinavir showed minimum inhibitory concentrations ranging from 0.125 to 1 μg mL−1 for both phases, and ritonavir presented minimum inhibitory concentrations ranging from 0.0312 to 4 μg mL−1and from 0.0625 to 1 μg mL−1 for filamentous and yeast phase, respectively. Concerning the antifungal itraconazole, the minimum inhibitory concentration values ranged from 0.0019 to 0.125 μg mL−1 and from 0.0039 to 0.0312 μg mL−1 for the filamentous and yeast phase, respectively. The combination of saquinavir or ritonavir with itraconazole was synergistic against H. capsulatum, with a significant reduction in the minimum inhibitory concentrations of both drugs against the strains (p < 0.05). These data show an important in vitro synergy between protease inhibitors and itraconazole against the fungus H. capsulatum.

ABSTRACTThe antifungal activity of some statins against different fungal species has been reported. Thus, at the first moment, the in vitro antifungal activity of simvastatin, atorvastatin and pravastatin was tested againstCandida spp. and Cryptococcus spp. Then, in a second approach, considering that the best results were obtained for simvastatin, this drug was evaluated in combination with antifungal drugs against planktonic growth and tested against biofilms ofCandida spp. and Cryptococcus spp. Drug susceptibility testing was performed using the microdilution broth method, as described by the Clinical and Laboratory Standards Institute. The interaction between simvastatin and antifungals against planktonic cells was analyzed by calculating the fractional inhibitory concentration index. Regarding biofilm susceptibility, simvastatin was tested against growing biofilm and mature biofilm of one strain of each tested yeast species. Simvastatin showed inhibitory effect against Candida spp. andCryptococcus spp. with minimum inhibitory concentration values ranging from 15.6 to 1000 mg L-1 and from 62.5 to 1000 mg L-1, respectively. The combination of simvastatin with itraconazole and fluconazole showed synergism against Candidaspp. and Cryptococcus spp., while the combination of simvastatin with amphotericin B was synergistic only againstCryptococcus spp. Concerning the biofilm assays, simvastatin was able to inhibit both growing biofilm and mature biofilm ofCandida spp. and Cryptococcus spp. The present study showed that simvastatin inhibits planktonic cells and biofilms ofCandida and Cryptococcus species.

Este estudo teve como objetivo avaliar a atividade antifúngica de extratos de M. oleifera frente a fungos isolados de camarões, cultivados em água doce, e testar a toxicidade dos extratos em larvas de Macrobrachium amazonicum. Os extratos etanólicos de vagens, sementes, folhas, caules e flores e o extrato clorofórmico de flores de M. oleifera foram testados contra 14 cepas de Candida spp. e 10 cepas de Hortaea werneckii isolados da água de cultivo e do trato digestório de M. amazonicum. A atividade antifúngica foi determinada por microdiluição, com base nos documentos M27-A3 e M38-A2 do CLSI. A toxicidade foi avaliada por exposição das larvas de M. amazonicum a concentrações entre 10-1000 mg mL-1 dos extratos, realizando contagem de larvas mortas (CL50), após 24 horas. Os melhores resultados foram verificados com o extrato clorofórmico de flores, agindo frente a todas as cepas testadas, com concentrações inibitórias mínimas variando entre 0,019-2,5 mg mL-1. O extrato etanólico de folhas, flores e sementes agiu ante 22/24, 21/24 e 20/24 cepas, respectivamente. O extrato de vagens foi eficaz contra cepas de Candida spp. (14/24) e o extrato de caule apenas contra quatro cepas de H. werneckii (4/24). Os extratos de sementes, flores (fração clorofórmica), caules e folhas apresentaram baixa ou nenhuma toxicidade, enquanto que extratos de vagens e flores (fração etanólica) apresentaram toxicidade moderada. Assim, observou-se atividade antifúngica dos extratos em Candida spp . e H. werneckii com uma ampla margem de segurança para as larvas de M. amazonicum, demonstrando ser promissor para o manejo sustentável dos efluentes do cultivo de M. amazonicum

This study aimed to evaluate the antifungal activity of M. oleifera extracts against fungi isolated from farmed prawns and test the toxicity of the extracts on larvae of Macrobrachium amazonicum. The ethanol extracts of pods, seeds, leaves, stems and flowers and chloroform extract of flowers of M. oleifera were tested against 14 strains of Candida spp. and 10 strains of Hortaea werneckii isolated from farming water and the digestive tract of M. amazonicum. Antifungal activity was determined by microdilution, based on the M27-A3 and M38-A2 CLSI documents. Toxicity was evaluated by exposing larvae of M. amazonicum at concentrations between 10-1000mg mL-1, counting dead larvae (CL50) after 24 hours. The best results were verified with the chloroform extract of flowers, acting against all tested strains, with MICs ranging from 0.019 to 2.5 mg mL-1. Ethanol extracts of leaves, flowers and seeds acted against 22/24, 21/24 and 20/24 strains, respectively. The extract of pods was only effective against strains of Candida spp. (14/24) and extract of stem only against four strains of H. werneckii (4/24). Extracts of seeds, flowers (chloroform fraction), stems and leaves showed low or no toxicity, whereas extracts of pods and flowers (ethanol fraction) showed moderate toxicity. Thus, the antifungal activity of these extracts agaisnt Candida spp. and H. werneckii was observed, a wide margin of safety for larvae of M. amazonicum, demonstrating to be promising for the sustainable management of effluents from M. amazonicum farming

Este estudo teve como objetivo avaliar o papel do camarão Macrobrachium amazonicum como carreador de Candida spp. do ambiente aquático, por meio da análise da correlação entre Candida spp. isoladas desse camarão e do seu ambiente (água de superfície e sedimento) através das técnicas de M13-PCR fingerprinting e RAPD-PCR. Para tanto, 27 cepas de Candida spp. foram avaliadas. Essas cepas foram recuperadas a partir do trato gastrointestinal de M. amazonicum adultos (7/27), oriundos da lagoa do Catú, Ceará, Brasil, e do meio aquático (águas superficiais e sedimentos) desse lago (20/27). A comparação molecular entre as cepas de camarões e o ambiente aquático foi conduzida por M13-PCR fingerprinting e RAPD-PCR, utilizando os iniciadores M13 e OPQ16, respectivamente. A análise molecular revelou semelhanças entre os padrões de bandas de oito isolados de Candida com o iniciador M13 e 11 isolados com o primer OPQ16, indicando que elas estão presentes no trato digestivo de M. amazonicum e no ambiente aquático, onde esses camarões habitam. Portanto, essa espécie de camarão pode ser usada como sentinela para monitoramento ambiental através da recuperação de Candida spp. do ambiente aquático, a partir do seu trato gastrointestinal

This study aimed to evaluate the role of the Amazon River prawn, Macrobrachium amazonicum, as carrier of Candida spp., by analyzing the correlation between Candida spp. from these prawns and their environment (surface water and sediment), through M13-PCR fingerprinting and RAPD-PCR. For this purpose, 27 strains of Candida spp. were evaluated. These strains were recovered from the gastrointestinal tract of adult M. amazonicum (7/27) from Catú Lake, Ceará State, Brazil and from the aquatic environment (surface water and sediment) of this lake (20/27). Molecular comparison between the strains from prawns and the aquatic environment was conducted by M13-PCR fingerprinting and RAPD-PCR, utilizing the primers M13 and OPQ16, respectively. The molecular analysis revealed similarities between the band patterns of eight Candida isolates with the primer M13 and 11 isolates with the primer OPQ16, indicating that the same strains are present in the digestive tract of M. amazonicum and in the aquatic environment where these prawns inhabit. Therefore, these prawns can be used as sentinels for environmental monitoring through the recovery of Candida spp. from the aquatic environment in their gastrointestinal tract

Coccidioidomycosis is a systemic mycosis with a variable clinical presentation. Misdiagnosis of coccidioidomycosis as bacterial pneumopathy leads to inappropriate prescription of antibiotics and delayed diagnosis. This report describes an outbreak among armadillo hunters in northeastern Brazil in which an initial diagnosis of bacterial pneumonia was later confirmed as coccidioidomycosis caused by Coccidioides posadasii. Thus, this mycosis should be considered as an alternative diagnosis in patients reporting symptoms of pneumonia, even if these symptoms are only presented for a short period, who are from areas considered endemic for this disease.

The aim of the present study was to evaluate the effect of cotrimoxazole on the in vitro susceptibility of Coccidioides posadasii strains to antifungals. A total of 18 strains of C. posadasii isolated in Brazil were evaluated in this study. The assays were performed in accordance with the Clinical and Laboratory Standards Institute guidelines and the combinations were tested using the checkerboard method. The minimum inhibitory concentrations were reduced by 11, 2.4, 4.3 and 3.5 times for amphotericin B, itraconazole, fluconazole and voriconazole, respectively. Moreover, it was seen that cotrimoxazole itself inhibited C. posadasii strains in vitro. The impairment of folic acid synthesis may be a potential antifungal target for C. posadasii.

O aumento da incidência das infecções fúngicas, bem como o registro crescente de resistência e falha terapêutica, têm impulsionado a realização de estudos de prospecção de fitoquímicos com propriedades antifúngicas. Diante do exposto, o presente estudo investigou o potencial antifúngico de extratos de Baccharis ligustrina, B. schultzii, Croton jacobinensis, Licania rigida, Moringa oleifera, Vernonia sp. e V. brasiliana, e de óleos essenciais de Lippia alba (Quimiotipos 1, 2, 3 e 4) e Ocimum gratissimum. Inicialmente, foi realizada uma avaliação qualitativa da atividade antifúngica de cada amostra por meio do método de difusão em ágar, frente a cepas de Candida albicans e Microsporum canis, mostrando que apenas os extratos de M. oleifera (MLF-C) e Vernonia sp. (TVS-H) apresentaram atividade frente a C. albicans e M. canis, com halos de inibição =10mm. Também foram determinadas a concentração inibitória mínima (CIM), frente a 12 cepas de C. albicans e M. canis, e a toxicidade aguda de MLF-C e TVS-H, através de protocolos do Clinical and Laboratory Standards Institute (CLSI) e ensaio com Artemia sp., respectivamente. A CIM (80%) de MLF-C e TVS-H variou de 0,156 a 2,5mg mL-1 frente C. albicans e de 0,039 a 1,25 e 0,039 a 0,625mg mL-1 para M. canis, respectivamente. A CIM (100%) de MLF-C e TVS-H variou de 0,625 a 2,5mg mL-1 frente C. albicans é de 0,039 a 2,5 e 0,078 a 1,25mg mL-1 para M. canis, respectivamente. As doses letais (DL50) para o MLF-C e TVS-H foram de 201,09 e 204,17mg mL-1, respectivamente, sendo, portanto, demonstrada a baixa toxicidade desses extratos. Os extratos de M. oileifera e Vernonia sp. apresentaram atividade antifúngica frente cepas de C. albicans e M. canis, abrindo a perspectiva de estudos para caracterização dos seus componentes bioativos.

The increase in the incidence of fungal infections and the frequent report of resistance and therapeutic failure has promoted the performance of phytochemical screening for compounds with antifungal properties. Based on this, the present study investigated the antifungal potential of extracts of Baccharis ligustrina, B. schultzii, Croton jacobinensis, Licania rigida, Moringa oleifera, Vernonia sp. and V. brasiliana and of essential oils of Lippia alba (Chemotypes 1, 2, 3 and 4) and Ocimum gratissimum. Initially, a qualitative evaluation of the antifungal activity of each vegetal sample against strains of Candida albicans and Microsporum canis, through the agar diffusion method, was performed. Extracts of M. oleifera (MLF-C) and Vernonia sp. (TVS-H) presented activity against C. albicans and M. canis with inhibition halos =10mm. Then, minimum inhibitory concentrations (MICs) for MLF-C and TVS-H against 12 strains of C. albicans and M. canis were determined through the methodology established by the Clinical and Laboratory Standards Institute (CLSI), and acute toxicity tests against Artemia sp. were performed for both extracts. MICs (80%) for MLF-C and TVS-H varied from 0.156 to 2.5mg mL-1 against C. albicans and from 0.039 to 1.25mg mL-1 and 0.039 to 0.625mg mL-1 against M. canis, respectively. MICs (100%) for MLF-C and TVS-H varied from 0.625 to 2.5mg mL-1 for C. albicans and from 0.039 to 2.5mg mL-1 and 0.078 to 1.25mg mL-1 against M. canis, respectively. Lethal doses (DL50) of MLF-C and TVS-H were 201.09 and 204.17mg mL-1, respectively, being, therefore, demonstrated the low toxicity of these extracts. M. oleifera and Vernonia sp. extracts presented in vitro antifungal activity against C. albicans and M. canis, creating perspectives for the development of studies on the characterizations of their bioactive components.

O gênero Candida é composto por leveduras que vivem como comensais na microbiota de homens e animais. Em geral, não causam nenhum dano aos seus hospedeiros, entretanto, em virtude de desequilíbrios nas defesas química, física e imunológica, esses microrganismos podem se tornar patogênicos. Infecções por Candida spp. são pouco frequentes na Medicina Veterinária no entanto, nos últimos anos, tem sido observado aumento considerável de relatos de enfermidades causadas por essas leveduras, acometendo diferentes animais. Várias espécies do gênero são implicadas em quadros infecciosos, sendo a C. albicans a principal delas, seguida por C. tropicalis e C. parapsilosis. Considerando-se o potencial patogênico do gênero Candida, aliado ao surgimento de cepas resistentes a derivados azólicos, in vitro, o presente trabalho se propôs a realizar detalhada revisão de literatura, abordando os aspectos clínico-laboratoriais, etiológicos e terapêuticos da candidose na Medicina Veterinária.

The Candida genus is composed by yeasts that live as commmensal on human and animals' microbiota. In general, they do not cause any damage to their hosts. However, due instability on chemical, physical and immunological defenses, these microorganisms can become pathogens. Candida spp. infections are rare on Veterinary Medicine. However, on the last years, a considerable raise of illness caused by these yeasts has been related on varied animal species. Several species of this genus has been mentioned as responsible for infectious diseases in animals, being C. albicans the main of them, followed by C. tropicalis and C. parapsilosis. Considering the pathogenic role of the genus Candida, allied to the emerging of resistant strains to the azole derivatives, in vitro, the present research proposed to perform a detailed review, approaching clinic-laboratorial, etiologic and therapeutic aspects of the candidosis on Veterinary medicine.

Onychomycosis is a fungal infection of nails caused by dermatophytes, yeasts and moulds, accounting for about 50% of onychopathies. A high frequency of onychomycosis caused by Candida species has been reported during the last few years in northeast Brazil, as well as in other regions of the world. A clinical diagnosis of onychomycosis needs to be confirmed through laboratory exams. We evaluated the importance of serial repetition of direct microscopic exams and fungal culture for the diagnosis of onychomycosis in the city of Fortaleza, Ceará, in northeast Brazil. We first made a retrospective study of 127 patients with onychomycosis, identifying the fungi that had been isolated from fingernails and toenails. We then made a prospective study of 120 patients, who were submitted to three successive mycological examinations. Ungual residues were scraped off and directly examined with a microscope and fungal cultures were made. In the retrospective study, in which only one sample was analyzed, the incidence of onychomycosis was 25.0%. In our prospective study, in which we had data from successive mycological examinations, 37.8% had onychomycosis. The most commonly isolated fungi in both studies were yeasts from the genera Candida, especially C albicans, C. parapsilosis and C. tropicalis. We found a high proportion of onychomycosis caused by Candida species. We also concluded that serial repetition of direct microscopic examination and fungal culture, with intervals of 2-5 days improved the diagnosis of onychomycosis. We suggest that this laboratorial strategy is necessary for accurate diagnosis of this type of mycosis, especially when the standard procedures fail to diagnose fungal infection, despite strong clinical suspicion.

O presente estudo objetivou desenvolver uma análise retrospectiva de casos de candidemia em hospital brasileiro na cidade de Fortaleza, Ceará. Um total de 50 hemoculturas foram analisadas de 40 pacientes com quadros de candidemia. O diagnóstico micológico foi baseado na análise morfológica e bioquímica e os dados dos pacientes foram coletados das histórias clínicas. As espécies mais freqüentes foram Candida parapsilosis (n = 18), seguida por C. albicans (n = 14), C. tropicalis (n = 8), C. guillermondii (n = 6), C. glabrata (n = 2) e Candida spp. (n = 2). Um estudo descritivo foi realizado com apenas 21 pacientes os quais possuíam dados clínicos completos. Os episódios de candidemia aconteceram em oito pacientes do sexo masculino e 13 do feminino. Os fatores de risco implicados em candidemia foram antibioticoterapia prévia, uso de cateter venoso central, nutrição parenteral, sondagem gástrica e ventilação mecânica. A morte aconteceu em 13 dos 21 pacientes com candidemia. Este estudo demonstrou a emergência de candidemia causada por C. parapsilosis em um hospital brasileiro na cidade de Fortaleza, Ceará.

The aim of this study was to perform a retrospective analysis of cases of candidemia in a Brazilian hospital in the city of Fortaleza, Ceará. A total of 50 blood cultures were analyzed from 40 candidemic patients. The mycological diagnosis was based on the phenotypical analysis and the patients' data were recorded in appropriate files. The most frequent species were Candida parapsilosis (n = 18), followed by C. albicans (n = 14), C. tropicalis (n = 8), C. guillermondii (n = 6), C. glabrata (n = 2), and Candida spp. (n = 2). A detailed descriptive study was undertaken with 21 patients whose medical records were complete. The candidemia episodes occurred in eight male patients and 13 female patients. The most representative risk factors implicated in candidemia were prior antibiotic therapy, central venous catheters, parenteral nutrition, gastric probes and mechanical ventilation. Death occurred in 13 of the 21-candidemic patients. This study demonstrated the emergence of candidemia caused by C. parapsilosis in a Brazilian hospital in the city of Fortaleza, Ceará.