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With increasing frequency, clinical and laboratory-based mycologists are consulted on invasive fungal diseases caused by rare fungal species. This review aims to give an overview of the management of invasive aspergillosis (IA) caused by non-fumigatus Aspergillus spp.—namely A. flavus, A. terreus, A. niger and A. nidulans—including diagnostic and therapeutic differences and similarities to A. fumigatus. A. flavus is the second most common Aspergillus spp. isolated in patients with IA and the predominant species in subtropical regions. Treatment is complicated by its intrinsic resistance against amphotericin B (AmB) and high minimum inhibitory concentrations (MIC) for voriconazole. A. nidulans has been frequently isolated in patients with long-term immunosuppression, mostly in patients with primary immunodeficiencies such as chronic granulomatous disease. It has been reported to disseminate more often than other Aspergillus spp. Innate resistance against AmB has been suggested but not yet proven, while MICs seem to be elevated. A. niger is more frequently reported in less severe infections such as otomycosis. Triazoles exhibit varying MICs and are therefore not strictly recommended as first-line treatment for IA caused by A. niger, while patient outcome seems to be more favorable when compared to IA due to other Aspergillus species. A. terreus-related infections have been reported increasingly as the cause of acute and chronic aspergillosis. A recent prospective international multicenter surveillance study showed Spain, Austria, and Israel to be the countries with the highest density of A. terreus species complex isolates collected. This species complex seems to cause dissemination more often and is intrinsically resistant to AmB. Non-fumigatus aspergillosis is difficult to manage due to complex patient histories, varying infection sites and potential intrinsic resistances to antifungals. Future investigational efforts should aim at amplifying the knowledge on specific diagnostic measures and their on-site availability, as well as defining optimal treatment strategies and outcomes of non-fumigatus aspergillosis. CA extern

OA Förderung 2023 Neutrophil granulocytes (NGs) are among the key players in the defense against Aspergillus fumigatus (A. fumigatus). To better elucidate a pathophysiological understanding of their role and functions, we applied a human cell model using NGs from healthy participants and septic patients to evaluate their inhibitory effects on the growth of A. fumigatus ex vivo. Conidia of A. fumigatus (ATCC® 204305) were co-incubated with NGs from healthy volunteers or septic patients for 16 h. A. fumigatus growth was measured by XTT assays with a plate reader. The inhibitory effect of NGs on 18 healthy volunteers revealed great heterogeneity. Additionally, growth inhibition was significantly stronger in the afternoon than the morning, due to potentially different cortisol levels. It is particularly interesting that the inhibitory effect of NGs was reduced in patients with sepsis compared to healthy controls. In addition, the magnitude of the NG-driven defense against A. fumigatus was highly variable among healthy volunteers. Moreover, daytime and corresponding cortisol levels also seem to have a strong influence. Most interestingly, preliminary experiments with NGs from septic patients point to a strongly diminished granulocytic defense against Aspergillus spp.

Introduction: Melanization and biofilm formation are major virulence factors in pathogenic melanized fungi, making them capable of causing recurrent and chronic infections resistant to treatment and immune response. We here evaluated the interplay between melanization, cell wall constitution, and biofilm formation in melanized fungi. Methods: Several biofilm-forming strains of C. neoformans (N = 10), A. fumigatus (N = 10), and E. dermatitidis (N = 26), including melanin-deficient mutants and their wild types were examined for their biofilm formation capability using crystal violet assay and confocal laser scan microscopy. Furthermore, dihydroxyphenylalanine (DOPA) as melanin inducer in Cryptococcus and tricyclazole (TCA) as DHN-melanin biosynthesis pathway inhibitor in E. dermatitidis and A. fumigatus were added to cultivation. To determine the effects of melanization on cell wall constitution, we analyzed cell wall electronegativity with zeta potential measurements and cell surface hydrophobicity with a microbial adhesion to hydrocarbons (MATH) assay. Results: Melanin-lacking-mutants formed less biofilm compared to their corresponding wild types. While L-DOPA strengthened biofilm formation in C. neoformans, TCA inhibited in E. dermatitidis and less in A. fumigatus. Correlation analysis between biofilm formation capability and the relative hydrophobicity of the cell surface showed A. fumigatus and C. neoformans strains with higher hydrophobicity are more likely to form biofilms than those with less hydrophobicity. Additionally, zeta potential values, having electronegativity, could be related to the forming of biofilms. Conclusion: The first results implicate that melanization in fungi increases the biofilm formation potential, and further that biofilm formation of human pathogenic fungi is positively associated with electronegativity and hydrophobicity of cell surfaces.

Poster-Abstract Introduction: Invasive Candida infections are of public concern and could possibly act as a health threat to the community. Clinical and epidemiological data indicate a change of species spectra and resistance patterns in recent years. To monitor these dynamics and the distribution of certain pathogens, we established the first German surveillance network in 2020. Objectives: The surveillance network will monitor the species distribution and the development of antifungal drug resistance for invasive yeast infections in Germany. Materials and Methods: The network consists of ten German microbiological labs which collect relevant Candida species derived from blood cultures implicating invasive bloodstream infection. Epidemiological and resistance data are consequently curated by the NRZMyk. Data were assessed by Date and place of specimen, species ID, method of species ID, antimicrobial susceptibility testing (AST) results of a diverse range of antifungals and method of AST. Results: In the first two years, data sets of around 966 strains and 21 different species could be preserved (514 in 2020 and 452 in 2021). All Species-IDs were confirmed by MALDI-TOF. Around 5000 MIC-values were evaluated. In both years, ~6% of the isolates show secondary resistance against ≥1 antifungal substance (according to either EUCAST or CLSI). MDR-resistant strains were found. Interestingly, not all phenotypic echinocandin-resistant isolates harbour target gene mutations and lose reduced susceptibility upon retesting. Epidemiological data is currently assessed by each participating partner focussing on incidence and prevalence of candidemia. Conclusion: Data collection through this national surveillance network and a database with open access to all participating partners enables systematic research projects, epidemiological studies and active surveillance concerning the prevalence of fungal bloodstream infection and emergence of antifungal drug resistance.

Poster-Abstract Objectives: Infection with Candida spp. have been an increasing threat to hospital patients worldwide. Whereas risk factors for the most common species—C. albicans and C. glabrata—are well described, little is known on risk factors for C. krusei fungemia. Patients and Methods: We retrospectively compared patient’s data with at least one C. krusei or C. albicans positive blood culture at Essen University Hospital between 2008 and 2020. Relevant categories consisted of age, gender, underlying condition, central venous catheters (CVC), steroids, leukopenia

Introduction: Invasive fungal infections with azole-resistant A. fumigatus isolates (ARAF) increased recently. These infections have become a significant threat in clinical settings. A factor known to contribute to increased resistances is the biofilm formation capability. Developing novel strategies for treatment of infections with ARAF has become crucial. Here, the activity of the imidazole luliconazole (LLCZ) against ARAF biofilms was investigated in vitro. Methods: The activity of LLCZ was assessed in a crystal violet and in an XTT assay. LLCZ concentrations of the previously identified MICs (according to EUCAST) were applied to ARAF biofilms at different formation stages, both in concentrations below the MIC (sub-MIC) and above the MIC (supra-MIC). Additionally, LLCZ activity was visualized in confocal laser scanning microscopy. Results: Biofilm formation was mostly inhibited in the pre-adhesion stages (0 h). Sub-MIC concentrations of LLCZ were sufficient for the disruption of early-stage biofilm formation (0 h and 4 h). In contrast, mature biofilms (24 h) were more resistant towards LLCZ treatment. These 24 h old biofilms could be only partially inhibited by LLCZ application of 256-times the strain specific MIC. Conclusion: In conclusion, LLCZ showed activity against early biofilms of ARAF when applied in concentrations below the MIC, while a substantially higher concentration of LLCZ is required for the inhibition of mature biofilms.

This is the first study comparing three commercially available PCR assays for the detection of Aspergillus DNA from respiratory specimen of immunocompromised patients and the presence of cyp51A gene mutations. Bronchoalveolar lavages (BALs, N = 103) from patients with haematological/oncological underlying diseases were retrospectively investigated. The performance of three PCR assays, namely MycoGENIE®Aspergillus fumigatus Real-Time PCR Kit (Adamtech), Fungiplex®Aspergillus Azole-R IVD Real-Time PCR Kit (Bruker Daltonik GmbH) and AsperGenius® (PathoNostics B.V.), were evaluated. All patients were categorised following current EORTC/MSG criteria, with exclusion of the PCR-results. From the 11 invasive pulmonary aspergillosis (IPA) probable samples, eight were detected with MycoGENIE®, resulting in a sensitivity of 80% and a specificity of 73%. Furthermore, Fungiplex® resulted in six positive BALs with a sensitivity of 60% and a specificity of 91% and AsperGenius® in seven positive BAL samples, with a sensitivity of 64% and a specificity of 97%. No proven IPA was detected. One isolate showed phenotypically an azole-resistance, which was also detected in each of the tested PCR assays with the mutation in TR34. The here tested PCR assays were capable of reliably detecting A. fumigatus DNA, as well as differentiation of the common cyp51A gene mutations. However, evaluation on the AsperGenius® assay revealed a low risk of false positive results.