With voriconazole (VRC) being approved as the first choice in treating invasive aspergillosis (IA) and its increasing use in treatment, a VRC-resistant strain of Aspergillus flavus , the second leading cause of IA after Aspergillus fumigatus , has emerged. The VRC-resistant strain of A. flavus was isolated for the first time from the surgical lung specimen of an IA patient with no response to VRC therapy. In order to ascertain the mechanism of VRC resistance, the azole target enzyme genes in this strain of A. flavus were cloned and sequenced, and 4 mutations generating amino acid residue substitutions were found in the cyp51C gene. To further determine the role of this mutated gene for VRC resistance in A. flavus , an Agrobacterium tumefaciens -mediated gene replacement approach was applied. Consequently, the mutated cyp51C gene from this A. flavus strain was proven to confer the VRC resistance. Finally, to discern the one out of the four mutations in the cyp51C gene that is responsible for contributing to VRC resistance, a site-directed gene mutagenesis procedure combined with a gene replacement method was performed. As a result, the T788G missense mutation in the cyp51C gene was identified as responsible for VRC resistance in A. flavus . These findings indicated that the detection of this mutation in A. flavus could serve as an indicator for physicians to avoid the use of VRC during IA treatment. Further comprehensive surveillance for antifungal susceptibility, as well as intensive study on the mechanism of azole resistance in A. flavus causing IA, would be required to fully understand this mechanism.

We investigated the antifungal effect of non-thermal plasma, as well as its combination with common antifungal drugs, against Candida biofilms. A direct current atmospheric pressure He/O₂ (2%) plasma microjet (PMJ) was used to treat Candida biofilms in a 96-well plate. Inactivation efficacies of the biofilms were evaluated by XTT assay and counting colony forming units (CFUs). Morphological properties of the biofilms were evaluated by Scanning Electron Microscope (SEM). The sessile minimal inhibitory concentrations (SMICs) of fluconazole, amphotericin B, and caspofungin for the biofilms were also tested. Electron Spin Resonance (ESR) spectroscopy was used to detect the reactive oxygen species (ROS) generated directly and indirectly by PMJ. The Candida biofilms were completely inactivated after 1 min PMJ treatment, where severely deformed fungal elements were observed in SEM images. The SMICs of the tested antifungal drugs for the plasma-treated biofilms were decreased by 2–6 folds of dilution, compared to those of the untreated controls. ROS such as hydroxyl radical (^•OH), superoxide anion radical (^•O₂^-) and singlet molecular oxygen (¹O₂) were detected by ESR. We hence conclude that He/O₂ (2%) plasma alone, as well as in combination with common antifungal drugs, is able to inactivate Candida biofilms rapidly. The generation of ROS is believed to be one of the underlying mechanisms for the fungicidal activity of plasma.

The emergence of resistant Aspergillus spp. is increasing worldwide. Long-term susceptibility surveillance for clinically isolated Aspergillus spp. strains is warranted for understanding the dynamic change in susceptibility and monitoring the emergence of resistance. Additionally, neither clinical breakpoints (CBPs) nor epidemiological cutoff values (ECVs) for Aspergillus spp. in China have been established. In this study, we performed a 20-year antifungal susceptibility surveillance for 706 isolates of Aspergillus spp. in a clinical laboratory at Peking University First Hospital from 1999 to 2019; and in vitro antifungal susceptibility to triazoles, caspofungin, and amphotericin B was determined by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. It was observed that Aspergillus fumigatus was the most common species, followed by Aspergillus flavus and Aspergillus terreus. Forty isolates (5.7%), including A. fumigatus, A. flavus, A. terreus, Aspergillus niger, and Aspergillus nidulans, were classified as non-wild type (non-WT). Importantly, multidrug resistance was observed among A. flavus, A. terreus, and A. niger isolates. Cyp51A mutations were characterized for 19 non-WT A. fumigatus isolates, and TR₃₄/L98H/S297T/F495I was the most prevalent mutation during the 20-year surveillance period. The overall resistance trend of A. fumigatus increased over 20 years in China. Furthermore, based on ECV establishment principles, proposed ECVs for A. fumigatus and A. flavus were established using gathered minimum inhibitory concentration (MIC)/minimum effective concentration (MEC) data. Consequently, all the proposed ECVs were identical to the CLSI ECVs, with the exception of itraconazole against A. flavus, resulting in a decrease in the non-WT rate from 6.0 to 0.6%.

Aspergillus fumigatus is the most common cause of invasive fungal mold infections in immunocompromised individuals. Current antifungal treatment relies heavily on the triazole antifungals which inhibit fungal Erg11/Cyp51 activity and subsequent ergosterol biosynthesis.