Candida albicans caused 44% of the overall candidemia episodes from 2006 to 2010 in our university tertiary care hospital. As different antifungal agents are used in therapy and also immunocompromised patients receive fluconazole prophylaxis in our institution, this study aimed to perform an antifungal susceptibility surveillance with the C.albicans bloodstream isolates and to characterize the fluconazole resistance in 2 non-blood C.albicans isolates by sequencing ERG11 gene. The study included 147 C. albicans bloodstream samples and 2 fluconazole resistant isolates: one from oral cavity (LIF 12560 fluconazole MIC: 8μg/mL) and one from esophageal cavity (LIF-E10 fluconazole MIC: 64μg/mL) of two different patients previously treated with oral fluconazole. The in vitro antifungal susceptibility to amphotericin B (AMB), 5-flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), caspofungin (CASP) was performed by broth microdilution methodology recommended by the Clinical and Laboratory Standards Institute documents (M27-A3 and M27-S4, CLSI). All blood isolates were classified as susceptible according to CLSI guidelines for all evaluated antifungal agents (MIC range: 0,125–1.00 μg/mL for AMB, ≤0.125–1.00 μg/mL for 5FC, ≤0.125–0.5 μg/mL for FLC, ≤0.015–0.125 μg/mL for ITC, ≤0.015–0.06 μg/mL for VRC and ≤0.015–0.125 μg/mL for CASP). In this study, we also amplified and sequenced the ERG11 gene of LIF 12560 and LIF-E10 C.albicans isolates. Six mutations encoding distinct amino acid substitutions were found (E116D, T128K, E266D, A298V, G448V and G464S) and these mutations were previously described as associated with fluconazole resistance. Despite the large consumption of antifungals in our institution, resistant blood isolates were not found over the trial period. Further studies should be conducted, but it may be that the very prolonged direct contact with the oral antifungal agent administered to the patient from which was isolated LIF E-10, may have contributed to the development of resistance.

The second cause of death among systemic mycoses, cryptococcosis treatment represents a challenge since that 5-flucytosine is not currently available in Brazil. Looking for alternatives, this study evaluated antifungal agents, alone and combined, correlating susceptibility to genotypes. Eighty Cryptococcus clinical isolates were genotyped by URA5 gene restriction fragment length polymorphism. Antifungal susceptibility was assessed following CLSI-M27A3 for amphotericin (AMB), 5-flucytosine (5FC), fluconazole (FCZ), voriconazole (VRZ), itraconazole (ITZ) and terbinafine (TRB). Drug interaction chequerboard assay evaluated: AMB + 5FC, AMB + FCZ, AMB + TRB and FCZ + TRB. Molecular typing divided isolates into 14 C. deuterogattii (VGII) and C. neoformans isolates were found to belong to genotype VNI (n = 62) and VNII (n = 4). C. neoformans VNII was significantly less susceptible than VNI (P = 0.0407) to AMB; C. deuterogattii was significantly less susceptible than VNI and VNII to VRZ (P < 0.0001). C. deuterogattii was less susceptible than C. neoformans VNI for FCZ (P = 0.0170), ITZ (P < 0.0001) and TRB (P = 0.0090). The combination FCZ + TRB showed 95.16% of synergistic effect against C. neoformans genotype VNI isolates and all combinations showed 100% of synergism against genotype VNII isolates, suggesting the relevance of cryptococcal genotyping as it is widely known that the various genotypes (now species) have significant impact in antifungal susceptibilities and clinical outcome. In difficult-to-treat cryptococcosis, terbinafine and different antifungal combinations might be alternatives to 5FC.

Candida species are present in the human body microbiota and, when there is an imbalance in the relationship between microbiota and host, these yeasts can become opportunistic pathogens, being Candida albicans the most frequently found species. The systemic candidiasis presents localized infectious symptoms and is able to spread to other organs through the haematogenous route. The fluconazole antifungal inhibits the synthesis of ergosterolan essential component of the fungal cell membraneand is the most common prophylactic choice when treating candidiasis. While some species are intrinsically resistant to fluconazole (C.krusei) or are more prone to develop resistance (C.glabrata), reports on the resistance of C.albicans to fluconazole are rare. However, this species is in fact able to develop such resistance mechanisms, including the mutations of the ERG11 gene, which codifies the production of the enzyme that acts in the biosynthesis of the ergosterol. This report aimed to perform an antifungal susceptibility surveillance study on C.albicans bloodstream samples, as well as investigating mutations in the ERG11 gene on two clinical C.albicans isolates (LIF-E10 and LIF 12560) that had previously presented in vitro resistance to fluconazole. This experiment was provided with 147 samples of C.albicans isolates from hemocultures that had been conducted in the Hospital and Clinics of the State University of Campinas from 2006 to 2010, as well as the LIF-E10 and LIF 12560 isolates, obtained, respectively, from oral cavity and esophageal cavity. The bloodstream isolates had been previously identified by conventional methods and Vitek ™ 2 Systems (bioMérieux), and were reactivated through Sabouraud dextrose agar cultures, being subsequently placed in identification panels in the BD Phoenix ™ equipment. The in vitro tests to susceptibility to antifungals micafungin, amphothericin B, 5-flucytosine, fluconazole, voriconazole, itraconazole and caspofungin were performed by the broth microdilution methodology in accordance with the Clinical and Laboratory Standards Institute (CLSI) documents M27-A3 and M27-S4. The screening of the ERG11 gene mutations of the LIF-E10 and LIF 12560 isolates was obtained by amplifying three genic regions through specific PCR primers ErgSec1A/1B, ErgSec2A/2B and ErgSec3A/3B. The resulting product was submitted to both sequencing and analysis of the DNA fragments, amplified by comparing them to the standard fluconazole-sensible sample C.albicans ATCC 90028. From the 147 clinical isolates that went through reactivation, 143 were recovered, whereas four did not present any growth. From the 143 submitted to reidentification in the BD Phoenix ™ equipment, 128 (89.5%) were identified as C.albicans, and 15 (10.5%) were identified as Candida dubliniensis. Since such result presented by the BD Phoenix ™ differed from the previous identification by the Vitek ™ 2, the sequencing was necessary (primers ITS4/ITS5 and NL1/NL4), resulting in the confirmation of the 15 isolates as belonging to the C.albicans spec...