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On-line version ISSN 1678-9946
Rev. Inst. Med. trop. S. Paulo vol.54 no.1 São Paulo Jan./Feb. 2012
Isolamento e teste de susceptibilidade aos antifúngicos de Trichosporon asahii no Ceará, Brasil
Everardo Albuquerque MenezesI; José Arlécio de Sousa MarinhoI; Maria Rozzelê F. AngeloII; Maria da Conceição dos Santos Oliveira CunhaI; Francisco Afrânio CunhaI; Antônio Alexandre de Vasconcelos JúniorI
ILaboratory of Microbiology of Yeasts (LMY) Department of Clinical and Toxicological Analysis, College of Pharmacy, Federal University of Ceará. Tel. +55 85 33668266
IIGeneral Hospital of Fortaleza
Trichosporon spp. are yeasts capable of causing invasive disease, which mainly affect immunocompromised patients. A clinical strain of T. asahii was isolated from the blood cultures of patients admitted to the General Hospital of Fortaleza. Susceptibility tests were conducted by disk diffusion and broth microdilution. The isolated strain of T. asahii was resistant to fluconazole. The patient used amphotericin B and caspofungin in order to facilitate the microbiological cure. It was the first isolation and identification of T. asahii in blood culture in Ceará, Brazil.
Key words: T. asahii; Fluconazole; Amphotericin B; Caspofungin.
Trichosporon spp. são leveduras capazes de causar doença invasiva, que afetam principalmente pacientes imunocomprometidos. Uma cepa clínica de T. asahii foi isolada em hemocultura de paciente internado no Hospital Geral de Fortaleza. Os testes de suscetibilidade foram realizados por difusão em disco e microdiluição em caldo. A cepa isolada do T. asahii foi resistente ao fluconazol, o paciente fez uso de anfotericina B e caspofungina então a cura microbiológica ocorreu. Foi o primeiro isolamento e identificação de T. asahii em hemocultura no Ceará, Brasil.
Trichosporon are yeasts that belong to the basidiomycetes group, and may be found to be present in the external environment, primarily in the soil, but also in water, air, and organic substrates. The use of molecular biological techniques has led to the reorganization of the genus, with a proposed new classification that consists of 17 species and five varieties13. T. asahii and T. mucoides are associated with deep infections, while T. asteroides, T. ovoides and T. cutaneum are responsible for white piedra and other superficial infections. T. inkin is responsible for both superficial and deep infections, and cases of infection with T. pullulans have recently been reported1.
T. asahii is part of the cutaneous fungal microbiota present in humans. T. asahii of cutaneous origin may be one of the principal ways through which deep-seated trichosporonosis is acquired, whereas T. asahii taken from its natural environment is not associated with this infection15.
At present, there is little data on the susceptibility of T. asahii to antifungal agents. The fungus is known to exhibit variable susceptibility to amphotericin B (MIC 0.01 to 4 µg/mL), and some studies revealed relative resistance to this agent4. The triazoles exhibit greater in-vitro activity when compared with amphotericin B, and some authors recommend combined treatment with the latter. Other in-vitro studies reported greater fungicidal action by triazoles such as voriconazole, posaconazole and ravuconazole11.
Reports of isolation of T. asahii from blood cultures are rare11, in Ceará they are unknown. Very little is known about the susceptibility of these strains - mainly to fluconazole. The aim of this study was to isolate, identify and determine the susceptibility profile of one strain of Trichosporon spp isolated from a blood culture at the General Hospital of Fortaleza.
MATERIALS AND METHODS
The A.M.N. patient, male, 31 years old, underlying acute myeloid leukemia; had a fever and nosocomial infection, was a neutropenic, used chemotherapy drugs and antibiotics (rifampicin, imipenem and vancomycin), had a negative blood culture for bacteria and therefore used a prophylactic fluconazole. The patient underwent a chest drain, which was probably the source of the contamination. The blood sample was sent to the Laboratory, inoculated on potato dextrose agar and left to be incubated at 35 °C for 24 hours. T. asahii was isolated in three collections. The following indicators were used for identification: an API 20C AUX® kit for yeast identification (BioMérieux, Marcy-l'Étoile, France), fermentation and carbohydrate assimilation, susceptibility to cycloheximide, capacity for growth at different temperatures, growth in a chromogenic medium and micromorphology on a medium of rice agar Tween-80. The chromogenic medium was used for preliminary identification. Testing was carried out on a rice agar Tween-80, and we observed: the production of true hyphae with rectangular arthroconidia and the absence of appressoria that are characteristic of this species. This was additionally done with sugar assimilation tests.
The susceptibility of the T. asahii strain was evaluated by the disk diffusion method, in accordance with protocol M44-A of the Clinical and Laboratory Standards Institute (CLSI). Susceptibility to fluconazole and amphotericin B was also assessed through the broth microdilution method (protocol M27-A3) using RPMI, with the concentration of fluconazole ranging from 64 to 0.25 µg/mL, and the concentration of amphotericin B ranging from 16 to 0.03 µg/mL2,3,9. The strain was classified according to the inhibition halos on the discs: halo >19 mm (susceptible (S)); 14-19 mm (dose-dependent susceptible (DDS)); and < 14 mm (resistant (R))6,9,12. To test the microdilution for fluconazole, the breakpoints were: MIC susceptible ≤ 8 µg/mL; DDS = 16-32 µg/mL and resistant > 64 µg/mL; for amphotericin B: MIC susceptible ≤ 1 µg/mL; MIC resistant > 1 µg/mL. The test for each sample was repeated twice. The strains Candida albicans ATCC 10231, C. albicans ATCC 14053 and C. parapsilosis ATCC 22019 were all used as controls.
RESULTS AND DISCUSSION
Disseminated trichosporonosis is a severe opportunistic mycosis and has a high mortality rate. However, the clinical features of the patients with Trichosporon and Candida could not be distinguished. The overall in-hospital mortality rate of trichosporonosis was 56.1%8. In our study, the patient survived. The probable cause of infection was invasive medical procedures, however no epidemiological studies were undertaken to discover the origin from T. asahii.
The T. asahii strain was resistant to fluconazole by the disk diffusion and microdilution method with halo = 10 mm and MIC > 64 µg/mL respectively, and exhibited susceptibility to amphotericin B with halo = 14 mm and MIC = 0.06 µg/mL. When infected with T. asahii, immunocompromised patients can have a poor prognosis, and it is essential to identify and determine the susceptibility profile of the strain for the application of the appropriate therapy5.
The therapeutic response to new azoles such as voriconazole is quite promising. In a study of 101 strains of T. asahii isolated in Thailand, a low susceptibility to fluconazole and amphotericin B was observed, and voriconazole represented the best alternative8. In a study of 26 strains of T. asahii, high resistance to fluconazole was observed10. There is little data available regarding T. asahii's susceptibility to antifungal drugs in Brazil. The emergence of T. asahii with reduced susceptibility to common antifungal drugs is cause for concern14. In our study, resistance to fluconazole was detected, but the strain was susceptible to amphotericin B.
Recent studies demonstrate a synergy between caspofungin and amphotericin B. However, it is difficult to explain how the echinocandin MICs are lowered when the drug is together with amphotericin B7. In our study, the patient used amphotericin B together with caspofungin and was cured of the fungal infection. Caspofungin was used for 12 days and amphotericin was used for nine days and the T. asahii culture turned negative.
The most common underlying conditions related to trichosporonosis were hematological diseases, peritoneal dialysis and solid tumor. Trichosporonemia occurred in 115 out of the 154 (74.7%) patients and disseminated infection occurred in 78 out of the 154 (50.6%) cases. The majority of cases of trichosporonosis were reported in North American medical centers (33.9%), followed by Europe (27.6%) and Asia (23.3%). Only six cases were reported in South American institutions, including five cases in Brazil and one case in Argentina1. There are no studies on the cases and identification of T. asahii in blood cultures in Ceará, Brazil. Further studies with a higher number of strains may assist in understanding the mechanisms of resistance of this yeast.
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60430-370 Fortaleza, Ceará, Brasil.
Received: 01 June 2011
Accepted: 07 November 2011