Virulence Attributes and Antifungal Susceptibility Profile of Opportunistic Fungi Isolated from Ophthalmic Infections.

Investigations of both virulence factors and antifungal susceptibility profiles are crucial for understanding the pathogenesis and prognosis of ophthalmic mycoses. In this study, we investigated the in vitro antifungal susceptibility of amphotericin B (AMB), voriconazole (VRC), and natamycin (NAT) against a set of 50 fungal isolates obtained from patients with ocular mycoses using the Clinical and Laboratory Standards Institute broth microdilution method. In addition, putative virulence factor, such as secretory phospholipases and proteinases, and biofilm formation activity were analyzed. The geometric means (GMs) of the minimum inhibitory concentrations (MICs) of the antifungals across all isolates were the following (in increasing order): VRC (0.70 µg/mL), AMB (0.81 µg/mL), and NAT (1.05 µg/mL). The highest activity against 14 Aspergillus strains was exhibited by VRC (GM MIC: 0.10 µg/mL), followed by AMB and NAT (GM MICs: 0.21 and 0.27 µg/mL), respectively. However, for 12 Fusarium spp., the GM MIC of VRC (2.66) was higher than those of NAT and AMB (GM MICs 1.3 and 0.8 µg/mL, respectively). Proteinase and phospholipase activity were observed in 30 % and 42 % of the isolates, respectively, whereas only 8 % of the isolates were able to produce biofilms. Phospholipase activity was observed in all Fusarium isolates, but not in any of the Aspergillus isolates. In contrast, biofilm-forming capability was detected in 25 % of the Fusarium isolates, but none of the Aspergillus isolates. The differences in the MICs of AMB, VRC, and NAT, biofilm-forming ability and proteinase and phospholipase activities among the isolates were not significant (p > 0.05). Overall, our study suggests no significant correlation between the antifungal susceptibility profiles and virulence attributes of ocular fungal isolates.

Time-Kill Kinetics and In Vitro Antifungal Susceptibility of Non-fumigatus Aspergillus Species Isolated from Patients with Ocular Mycoses.

Aspergillus species can cause ocular morbidity and blindness, and thus, appropriate antifungal therapy is needed. We investigated the in vitro activity of itraconazole, voriconazole, posaconazole, caspofungin, anidulafungin, and amphotericin B against 14 Aspergillus isolates obtained from patients with ocular mycoses, using the CLSI reference broth microdilution methodology. In addition, time-kill assays were performed, exposing each isolate separately to 1-, 4-, and 16-fold concentrations above the minimum inhibitory concentration (MIC) of each antifungal agent. A sigmoid maximum-effect (E max) model was used to fit the time-kill curve data. The drug effect was further evaluated by measuring an increase/decrease in the killing rate of the tested isolates. The MICs of amphotericin B, itraconazole, voriconazole, and posaconazole were 0.5-1.0, 1.0, 0.5-1.0, and 0.25 µg/ml for A. brasiliensis, A. niger, and A. tubingensis isolates, respectively, and 2.0-4.0, 0.5, 1.0 for A. flavus, and 0.12-0.25 µg/ml for A. nomius isolates, respectively. A. calidoustus had the highest MIC range for the azoles (4.0-16.0 µg/ml) among all isolates tested. The minimum effective concentrations of caspofungin and anidulafungin were ≤0.03-0.5 µg/ml and ≤0.03 µg/ml for all isolates, respectively. Posaconazole demonstrated maximal killing rates (E(max) = 0.63 h(-1), r(2) = 0.71) against 14 ocular Aspergillus isolates, followed by amphotericin B (E(max) = 0.39 h(-1), r(2) = 0.87), voriconazole (E(max) = 0.35 h(-1), r(2) = 0.098), and itraconazole (E(max) = 0.01 h(-1), r(2) = 0.98). Overall, the antifungal susceptibility of the non-fumigatus Aspergillus isolates tested was species and antifungal agent dependent. Analysis of the kinetic growth assays, along with consideration of the killing rates, revealed that posaconazole was the most effective antifungal against all of the isolates.

Infrequent Production of Xanthomegnin by Fungal Strains Recovered from Patients with Ocular Mycoses.

Mycotoxins are putative virulence factors of fungi that play an important role in the pathogenesis of fungal infections. Mycotoxin production has been used as a diagnostic marker for the early diagnosis of fungal diseases. Using high-performance liquid chromatography, we investigated whether the fungal strains recovered from eye tissue samples obtained from patients with ocular mycoses produced the mycotoxin xanthomegnin. We tested 62 well-characterized strains of fungi, including Aspergillus spp. (n = 14), Exophiala spp. (n = 9), Fusarium spp. (n = 15), and several molds (n = 24). All isolates were identified to the species level using PCR and DNA sequencing of rRNA genes. We detected xanthomegnin activity (0.02 µg/ml) in one of the three Aspergillus flavus strains. However, we were unable to detect xanthomegnin in any of the other 61 fungal strains. Our result suggests that xanthomegnin production was infrequent in fungal strains recovered from patients with ocular mycoses.

Antifungal susceptibility of ocular fungal pathogens recovered from around the world against itraconazole, voriconazole, amphotericin B, and caspofungin.

Although fungal infections of the eye are rare, they create an intractable clinical problem in ophthalmology because of the limited number of intravitreal and systemic therapeutic options. In this investigation, the in vitro efficacies of itraconazole (ITR), voriconazole (VOR), amphotericin B (AMB), and caspofungin (CAS) against 29 globally-collected ocular fungal isolates were assessed, following the standards that are outlined in the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document. AMB [Geometric Mean (GM) MIC (Minimum Inhibitory Concentration): 0.49 µg/ml] was the most active drug, followed by VOR, CAS, and ITR (GM MICs: 0.52, 1.07, and 2.86 µg/ml, respectively). For the Exophiala strains (n = 8), VOR was the most active drug, followed by AMB, ITR, and CAS (GM MICS: 0.21, 0.27, and 1.09 µg/ml, respectively). ITR had no activity against Fusarium spp. (n = 9; GM MIC: 32 µg/ml), but AMB was found to be the most effective antifungal against the tested members of this genus, followed by CAS and VOR (GM MICs: 0.86, 1.59, and 2.72 µg/ml, respectively). These data should be used to design future targeted clinical efficacy trials. We also report on several fungal species that are rarely encountered in the clinical laboratory, for which little information about drug sensitivities was previously available.