Microdilution procedure for antifungal susceptibility testing of Paracoccidioides brasiliensis to amphotericin b and itraconazole

Received: February 10, 2009 Accepted: August 13, 2009 Abstract published online: August 24, 2009 Full paper published online: November 30, 2009 J Venom Anim Toxins incl Trop Dis. V.15, n.4, p.718-731, 2009. Original paper. ISSN 1678-9199. MICRODILUTION PROCEDURE FOR ANTIFUNGAL SUSCEPTIBILITY TESTING OF Paracoccidioides brasiliensis TO AMPHOTERICIN B AND ITRACONAZOLE Takahagi-Nakaira E (1), Sugizaki MF (1), Peraçoli MTS (1) (1) Department of Microbiology and Immunology, Botucatu Biosciences Institute, São Paulo State University, UNESP, Botucatu, São Paulo State, Brazil. ABSTRACT: In vitro tests employing microdilution to evaluate fungal susceptibility to antifungal drugs are already standardized for fermentative yeasts. However, studies on the susceptibility of dimorphic fungi such as Paracoccidioides brasiliensis employing this method are scarce. The present work introduced some modifications into antifungal susceptibility testing from the European Committee on Antimicrobial Susceptibility Testing (EUCAST), concerning broth medium and reading time, to determine minimal inhibitory concentration (MIC) of amphotericin B and itraconazole against Paracoccidioides brasiliensis. Yeast-like cells of P. brasiliensis (Pb18 strain) were tested for susceptibility to amphotericin B and itraconazole in RPMI 1640 medium, supplemented with 2% glucose and nitrogen source and incubated at 35°C. The MIC of amphotericin B and itraconazole against Pb18 were respectively 0.25 μg/mL and 0.002 μg/mL. The results of minimal fungicidal concentration (MFC) showed that amphotericin B at 0.25 μg/mL or higher concentrations displayed fungicidal activity against Pb18 while itraconazole at least 0.002 μg/mL has a fungistatic effect on P. brasiliensis. In conclusion, our results showed that the method employed in the present study is reproducible and reliable for testing the susceptibility of P. brasiliensis to antifungal drugs. KEY WORDS: amphotericin B, itraconazole, P. brasiliensis, antifungal susceptibility. CONFLICTS OF INTEREST: There is no conflict. FINANCIAL SOURCE: FAPESP. CORRESPONDENCE TO: MARIA TEREZINHA S. PERAÇOLI, Departamento de Microbiologia e Imunologia, Instituto de Biociências, UNESP, Botucatu, SP, 18618-970, Brasil. Fax: +55 14 38153744. Email: . Takahagi-Nakaira E et al. Microdilution procedure for antifungal susceptibility testing of Paracoccidioides brasiliensis to amphotericin B and itraconazole. J Venom Anim Toxins incl Trop Dis. 2009;15(4):719 INTRODUCTION Paracoccidioidomycosis (PCM), a deep mycosis endemic to Latin America, is caused by Paracoccidioides brasiliensis, a fungus that presents thermal dimorphism and develops as yeast at body temperature (1). The clinical manifestations of the infection are equivalent to a chronic granulomatous disease with involvement of the lungs, reticuloendothelial system, mucocutaneous areas and other organs (2). The fungus enters the human body through inhalation of propagules that can only establish infection once they undergo phase transition to yeast in the pulmonary alveolar epithelium (3). Patients with acute or chronic PCM present symptoms such as malaise, fever, anorexia or weight loss tending to cachexia. In the severe forms of PCM, immune system alterations have been described including hypergammaglobulinemia, high level of specific antibodies, eosinophilia, and depressed general and antigen-specific cellular immunity (4-6). The treatment of PCM is limited to the utilization of a few drugs including sulfonamides, amphotericin B and imidazole derivatives (ketoconazole, itraconazole and fluconazole). Since it was introduced for therapy, amphotericin B intravenous injection comprises an effective treatment that has been used for patients with severely disseminated mycosis (7, 8). This drug interacts with the plasma membrane of sensitive fungi, causing impairment of the barrier function and oxidative damage to the cell membrane. Its selectivity results from a higher affinity for the ergosterol component from fungal membranes than for cholesterol from mammalian cells (9, 10). The introduction of agents such as fluconazole and itraconazole (azole antifungal agents) has provided clinicians with less toxic oral alternatives to amphotericin B, marking a significant improvement in antifungal therapy (11). Although methodologies for in vitro susceptibility testing have been available since the early years of antifungal drug development, few studies on antifungal susceptibilities of P. brasiliensis strains have been performed (12-15). In vitro tests of P. brasiliensis susceptibility to antimycotic drugs are scarce and the results have not always been consistent due to the diversity of the techniques employed (13, 14, 1618). However, significant progress has been achieved in relation to in vitro susceptibility testing, which aids in the initiation and monitoring of antifungal therapy (19, 20). The only standardization to determine the MIC is the reference method of the Clinical and Laboratorial Standards Institute (CLSI), adopted for yeasts, which has not been standardized for P. brasiliensis (21). Takahagi-Nakaira E et al. Microdilution procedure for antifungal susceptibility testing of Paracoccidioides brasiliensis to amphotericin B and itraconazole. J Venom Anim Toxins incl Trop Dis. 2009;15(4):720 The EUCAST techniques are characterized by their high reliability and reproducibility, are essential features for identifying organisms unlikely to respond to particular antifungal treatments (22). This standard is based on the CLSI reference procedure described in the document M27A2 (21). In this investigation, we proposed a technique to determine the MIC of amphotericin B and itraconazole against P. brasiliensis using the standard method of EUCAST, which employs RPMI 1640 medium supplemented with 2% glucose for MIC determination by broth dilution of fermentative species of yeasts (22). Besides, we introduced a modification in this culture medium with the objective of providing a better growth of the fungus in microdilution plate wells and serial dilutions of antifungal agents. This alteration can contribute to the standardization of an adequate methodology for MIC determination of dimorphic fungi. MATERIALS AND METHODS Fungus Strain The P. brasiliensis strain (Pb18) was maintained in yeast cells at 35°C on RPMI 1640 without sodium bicarbonate (Gibco Laboratories, USA) and with L-glutamine (SigmaAldrich Inc., USA) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (Sigma) and supplemented with 2% glucose, 2% ammonia sulfate, 0.12% asparagine (RPMI-Pb) and 2% agar medium for the three days of culture. Yeast cells were washed and suspended in RPMI-Pb. In order to obtain individual cells, the fungal suspension was homogenized with glass beads in a vortex homogenizer (three cycles of ten seconds) and yeast cell viability was determined by phase-contrast microscopy (23, 24). Fungal suspensions containing more than 95% (...truncated)