**Methods:** Four soil sampling areas within the hospital and in the surrounding environment have been identified. Zone A corresponds to an outdoor relaxation area within the hospital, zone B consists of flowerbeds in front of the hospital entrance, zone C (inside) corresponds to pots containing trees below the hospital reception area and finally zone D corresponds to an outdoor area at level -1 below the zone A. From January 2019, external samples were taken using a homemade dust vane sensor equipped with 2 wipes, placed on a terrace at second floor, to capture the prevailing wind. Dust aspiration was also achieved using a filter placed at the end of a vacuum cleaner, towards the emergency door of the department of hematology, in the corridor along zone A, then in hematology consultation department. Soils and dust (wipes and aspiration) samples were seeded on medium containing itraconazole and voriconazole (2mg/L). The usual weekly air samples taken in the hematology departments and corridors from January 2019 were also analyzed. Resistance of *A. fumigatus* growing on azole media was tested using EUCAST method and *cyp51A* gene of ARAf were sequenced.

**Results:** From the 85 soils samples, 137 colonies were isolated on azole medium from zones A, B and D, grew on azole media. Resistance of 90 *A. fumigatus* isolates was evaluated with EUCAST method. Other strains (zone B) were *Neosartorya fisheri*. Moreover, 71% (64/90) of *A. fumigatus* isolates were resistant to at least one azole. So far, 20 strains have been sequenced and all harbored the TR34/L98H mutation. 59 ARAf were found in pots containing tulips coming from Netherlands and 5 in soil pots containing trees. Three isolates were detected on azole media with dust vacuum and 2 (hematology department and corridor along zone A) were resistant with TR34/L98H mutation. No resistant strains were collected with the external sensor. Four ARAf with the TR34/L98H mutation were also isolated from air of corridors.

**Conclusion:** Patios containing Dutch tulips in particular, had a significant number of ARAf. On the other hand, samples taken upstream of the esplanades, in relation to the prevailing wind, did not present any ARAf. Additionally, ARAf were also found in hospital corridors and to emergency exits, but not in hematology departments. Genotyping analyses will be carried out to confirm that tulips can be a source of ARAf found inside the hospital.

P026

Triazole-resistant *Aspergillus fumigatus*: Evaluation of the Fungiplex® Aspergillus and Azole-R kits

S. Rocchi, E. Scherer, A. Laboissière, C. Léchenault-Bergerot, C. Godeau, A.-P. Bellanger, G. Reboux, L. Millon

Mycology - Umr6249 Cnrs Chrono-environnement, University Hospital Besançon, Besançon, France

**Objectives:** Cases of Aspergillosis with azole resistant *Aspergillus fumigatus* are increasingly described. Some resistance mechanisms, with two major mutations on the *cyp51A* gene (TR34/L98H and TR46/Y121F/T289A), are linked to the unintended impact of triazole fungicides in agriculture. At the University Hospital of Besançon, 216 resistant strains have been isolated since 2012 and 142 sequenced for beta-tubulin: 52% from professional environments, 28% from clinical samples, 10% from hospital corridors, 7% from the inside garden of the Hospital and 3% from patients' homes. Given the seriousness of resistant infections, it is important to detect cases of resistance as soon as possible. It is in this context that we proposed to test to Fungiplex ® (*Aspergillus* et Azole R) kits (Bruker).

**Methods:** The Fungiplex® *Aspergillus* Azole-R kit was retrospectively evaluated on DNA extracts from a collection for which *cyp51A* gene sequencing and genotyping was previously performed. According to the instructions, the Azole-R kit is made for the identification of azole resistance markers in *Aspergillus* positive species directly from serum, plasma and bronchoalveolar lavage. We are evaluating the kits for the routine testing of cystic fibrosis patients working from isolated strains and directly on sputum. We also are testing the performance of the kits to detect 1) *Aspergillus fumigatus* strains 2) resistant strains directly in soil samples. To do this, we worked on inoculated soils with increasing concentrations of resistant strains (from 5 to 50 000 spores/mL with 200µL sprayed on 250 mg of soil) and directly on soils containing resistant strains.
**Results:** 97 resistant strains previously characterized by sequencing techniques (beta-tubulin and cyp51A) were analyzed: 82 TR34/L98H, 2 TR34/L98H/S297T/F495I, 9 TR46/Y121F/T289A and 4 other resistant strains. Results showed that 99% of strains carrying one of the two mutations are detected. The first prospective results (22 resistant strains in Etest, analysed directly with the kits) made it possible to detect 22 *A. fumigatus* with the TR34/L98H mutation. Verification sequencing is in progress. Sputum analysis and tests with cryptic species are also in progress. Tests on inoculated soils show that resistant strains can be detected even for the smallest inoculated quantities (200 µL of solution concentrated at 5 spores/mL). Real soil tests provide a signal for *Aspergillus* but not for the TR34 and TR46 mutations. Internal reaction controls are also not detected, suggesting a problem with DNA extraction and inhibitors rather than poor kit performance. Different methods for extracting DNA directly from the soil are under evaluation.

**Conclusion:** The kits show ease of use and very good reliability on retrospective analyses with 99% of the strains tested validated. The performance of the *Aspergillus* kit will also be evaluated on cryptic species of *A. fumigatus* (Neosartorya fisherii, *A. oerlinghausenensis*, Neosartorya hiratsukae, *A. lentulus*). If performance is equally good in ongoing prospective analyses, these kits will be promising to improve early diagnosis.

P027

**Relation between hanseniasis and superficial mycoses: prevalence, fungal identification and antifungal susceptibility.**

M.L. Scroferneker¹, D. Heidrich², R. Vettorato²,³, L. Eidt⁴, A. Carvalho Ribeiro¹, D. Pagani⁵, A.H. Da Silva Hellwig², T. Amaro³

¹Department Of Microbiology, Immunology And Parasitology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, ²Postgraduate Program In Medicine: Medical Sciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, ³Serviço de Dermatologia do complexo Hospitalar Santa Casa de Misericórdia de Porto Alegre, Porto Alegre, Brazil, ⁴Ambulatório de Dermatologia Sanitária de Porto Alegre, Porto Alegre, Brazil, ⁵Postgraduate Program In Agricultural And Environmental Microbiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

**Objectives:** The aim of this study was to evaluate superficial mycoses in patients with hanseniasis in relation to the prevalence of fungal species causing mycoses and susceptibility to antifungals.

**Methods:** A cross-sectional study was carried out with patients who were seen between May and October 2017 at the Hanseniasis Service of the Sanitary Dermatology Ambulatory in the city of Porto Alegre, Brazil. The samples collected were submitted to direct (DME) and cultural (CME) mycological examinations at the G post of the Santa Clara Hospital in Porto Alegre, were identified by region sequencing specified for each genus of fungus and traced sensitivity profile to clinical antifungals using protocols M38-A2 and M27-A3 from the Clinical and Laboratory Standards Institute.

**Results:** A total of 91 hanseniasis patients were evaluated and 37 presented suspicion mycosis. Of these, 23 had DME positive and 14 CME cultures were identified, with eight dermatophytes (seven *Trichophyton interdigitale* and one *Epidermophyton floccosum*), two isolates of *Fusarium keratoplasticum*, two *Acremonium* sp., one *Candida albicans* and one *Arthrinium arundinis*. Three patients had claws, caused by hanseniasis, at the mycosis site, indicating a relation of the two diseases (3/5 patients with claw). Terbinafine presented the lowest minimum inhibitory concentrations (MICs) for dermatophyte isolates (0.0078-0.06 µg / mL), while fluconazole MICs were the highest (4-> 64µg / mL). *Fusarium keratoplasticum* and *Acremonium* sp. have higher MICs than all antifungal agents than dermatophytes.

**Conclusion:** This is the second onychomycosis caused by *Arthrinium arundinis* from the literature and showed low sensitivity to antifungals. Itraconazole had higher MICs for dermatophytes isolated from patients with hanseniasis (0.25-1µg / mL) than without the disease cited in the literature, indicating a relation of susceptibility to antifungals between mycosis and hanseniasis.

P028
**Prevalence of azole-resistant *Aspergillus fumigatus* isolates from respiratory specimens of patients from Lyon and exploration of resistance mechanisms**

L. Simon¹, R. Kramer², D. Dupont³, T. Déméautis⁴, H. Garnier⁵, B. Lina⁶, M. Rabodonirina⁷, M. Wallon³, F. Persat⁸, J. Menotti⁴

¹Parasitology And Medical Mycology, Hospices Civils de Lyon / CHU de Nice, Nice, France,
²Parasitology And Medical Mycology, And Virology, Hospices Civils de Lyon / EUPHEM, Lyon, France,
³Parasitology And Medical Mycology / Umr 5292, Université Lyon 1 / Hospices Civils de Lyon, Lyon, France,
⁴Parasitology And Medical Mycology / Ea7426 Inflammation And Immunity Of The Respiratory Epithelium, Université Lyon 1 / Hospices Civils de Lyon, Lyon, France,
⁵Parasitology And Medical Mycology, Hospices Civils de Lyon, Lyon, France,
⁶Virology / U1111, Université Lyon 1 / Hospices Civils de Lyon, Lyon, France,
⁷Parasitology And Medical Mycology / U1111, Université Lyon 1 / Hospices Civils de Lyon, Lyon, France,
⁸Parasitology And Medical Mycology, Université Lyon 1 / Hospices Civils de Lyon, Lyon, France

**Objectives:** Resistance of *Aspergillus fumigatus* strains to triazole antifungals is increasingly reported in Europe. The main mechanisms of resistance described are mutations in the cyp51A gene encoding a lanosterol 14-α-demethylase or its promoter. As few data are available in Southern France, our objective was to assess the burden of *Aspergillus* isolates with azole resistance from clinical specimens in Lyon University Hospitals

**Methods:** In this retrospective cross-sectional study, 203 consecutive *A. fumigatus* isolates were identified during a seven-month period from February to September 2017 from respiratory specimens from 182 patients attending the inpatient and outpatient wards of the Pulmonary Medicine Departments of Lyon University Hospitals. Morphological identification was confirmed by sequence analysis of the β-tubulin gene. Minimum inhibitory concentrations were determined using E-test reagent strips for itraconazole, voriconazole, posaconazole, and isavuconazole. Resistance was defined according to the 2018 EUCAST clinical breakpoints. The molecular resistance mechanisms were searched for by sequence analysis of the cyp51A gene and its promoter region, as well as by gene expression analysis of the cyp51 genes and genes encoding several efflux transporters.

**Results:** PCR and sequence analysis of the β-tubulin gene confirmed the identification of *Aspergillus fumigatus* for the 203 isolates. Four isolates presented with azole resistance: two isolates against itraconazole/posaconazole/isavuconazole and another two against all four triazoles. Out of these four strains, three presented silent polymorphisms in an intronic part of cyp51A and one presented simultaneously the F46Y, M172V and E427K mutations. No mutation was identified in the cyp51A promoter, but significant inductions of cyp51A and cyp51B gene expression were observed for all four isolates and three isolates, respectively. Significant inductions of atrF and crd1B gene expression were observed for two and three isolates, respectively. No significant induction of MDR1-4, MFS56 and M85 gene expression was observed.

**Conclusion:** The prevalence of azole resistance in our study population was 2.2% (95% CI 0.9-5.6). Only polymorphisms were found in the cyp51A gene and no mutation was found in its promoter. Nevertheless, significant inductions of the expression of the cyp51 genes and two genes encoding efflux transporters were evidenced, underlying the diversity of resistance mechanisms to be explored.

P029

**Antifungal activity of a mastoparan analog peptide: mode of action and in vivo evaluation in a Galleria mellonella model**

J. Singulani¹, M.C. Galeane¹, M. Dorisse Ramos¹, P.C. Gomes¹, C. Santos¹, B. Monson De Souza², M.S. Palma², A.M. Fusco Almeida¹, M.J. Soares Mendes Giannini¹

¹Clinical Analysis, UNESP, Araraquara, Brazil, ²Biology, UNESP (Sao Paulo State University), Rio Claro, Brazil

**Objectives:** The purpose of this work was to evaluate the antifungal activity and toxicity of a peptide analog of mastoparan class from wasps (MK58911) under both *in vitro* and *in vivo* conditions.
**Methods:** Firstly, MK58911 was tested against *Cryptococcus neoformans* ATCC 90112 using the microdilution susceptibility test according to the CLSI M27-A3 (2008). In addition, the mechanism of action on fungal membrane, death cell events (apoptosis and necrosis) and reactive oxygen species (ROS) was evaluated by flow cytometry. Secondly, the peptide toxicity tests on lung fibroblasts (MRC5) and glioblastoma cells (U87) were performed. Finally, the efficacy and toxicity of the peptide were evaluated *in vivo* using a *Galleria mellonella* model.

**Results:** The results showed a promising activity of the peptide with minimal concentration inhibitory (MIC) of 31.2 µg/mL and low toxicity in MRC and U87 cells (IC50 > 500 µg/mL). Taken together, these results demonstrated that the peptide has a high selectivity by fungal cells compared to mammal cells (selectivity index - SI > 16). The peptide presented a mechanism of action on plasma membrane, leading to death of the fungus mainly by the necrosis process and without production of reactive oxygen species. Finally, the peptide showed no toxic effects on *G. mellonella* and significantly enhanced the survival rates and decreased the fungal burden of larvae infected with *C. neoformans*. These effects were independent of an immunomodulatory activity.

**Conclusion:** The results showed the peptide as a potential new antifungal drug against cryptococcosis. "This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grants no. 2017/06658-9 and 2016/16212-5)."

P031

Screening for Triazole resistance in clinically significant *Aspergillus* species from Pakistan

A. Zafar, S. Moin, K. Jabeen, J. Farooqi, S. Laiq

Pathology And Laboratory Medicine, Aga Khan University, Karachi, Pakistan

**Objectives:** To determine the frequency of triazole (itraconazole, voriconazole, posaconazole) resistance in clinically significant *Aspergillus* species isolated at a tertiary care centre, Karachi, Pakistan.

**Methods:** A descriptive cross-sectional study was conducted in the Department of Pathology and Laboratory Medicine, Microbiology Section of the Aga Khan University Clinical Laboratories, Karachi, Pakistan from September 2016 to May 2019. One hundred and fourteen, clinically significant *Aspergillus* isolates [*A. fumigatus* (38; 33.3%), *A. flavus* (64; 56.1%), *A. niger* (9; 7.9%) and *A. terreus* (3; 2.6%)] were included in the study. They were assessed for their clinical significance. The clinical spectrum ranged from Invasive Aspergillosis (IA) (n = 25; 21.9%), further divided into proven invasive extrapulmonary aspergillosis (n = 8; 7%), proven invasive pulmonary aspergillosis (IPA) (n = 6; 5.3%), putative/probable IPA (n = 11; 9.6%) according to the AspICU and 2008 EORTC criteria, to Chronic Pulmonary Aspergillosis (CPA) (n = 58; 50.9%), Allergic Bronchopulmonary Aspergillosis (ABPA) (n = 4; 3.5%), Severe Asthma with Fungal Sensitization (SAFS) (n = 4; 3.5%) and saprophytic tracheobronchial aspergillosis (n = 23; 20.2%). Screening for triazole resistance was performed by antifungal agar screening method as described by Mortenssen et al. The minimum inhibitory concentration (MIC) of 41 representative isolates [*A. flavus* (n = 15; 13.2%), *A. fumigatus* (n = 15; 13.2%), *A. niger* (n = 8; 7%), *A. terreus* (n = 3; 2.6%)] representing a clinical spectrum of extrapulmonary IA (n = 7; 6.1%), IPA (n = 4; 3.5%) putative/probable IPA (5; 4.4%), CPA (n = 18; 15.8%), ABPA (n = 3; 2.6%), SAFS (n = 1; 0.9%), and saprophytic tracheobronchial aspergillosis (n = 3; 2.6%) were tested according to the CLSI broth microdilution method.

**Results:** All the isolates were categorized as triazole-susceptible based on the triazole antifungal agar screening. The MICs of the three azole antifungals for 41 representative isolates tested were found to be ≤1 ml/L and hence according to CLSI breakpoints, all the isolates tested were found to be triazole-susceptible. The MIC 90 of itraconazole, voriconazole and posaconazole of the representative aspergillus isolates was 1mg/L, 1mg/L and 0.5mg/L respectively.

**Conclusion:** This study may set precedence for future periodic antifungal resistance surveillance studies in our region on *Aspergillus* isolates causing invasive disease, as well as other syndromes requiring long term antifungal therapy.

P032
# Antifungal susceptibility profile of invasive Candida glabrata isolates (2009-2019) from a tertiary care hospital laboratory in Pakistan

J. Farooqi¹, S. Memon², S. Laiq¹, F. Naqvi¹, A. Zafar¹, **K. Jabeen**³

¹Pathology And Laboratory Medicine, Aga Khan University, Karachi-KHI, Pakistan, ²Microbiology, Karachi University, Karachi, Pakistan, ³Pathology And Laboratory Medicine, Aga Khan University, Karachi, Pakistan, Pakistan

**Objectives:** *Candida glabrata* invasive infections are increasingly associated with antifungal resistance and poor clinical outcomes. The objective of this study was to evaluate antifungal resistance and distribution of minimum inhibitory concentrations (MICs) against invasive *C. glabrata* isolates (200-2019) at Aga Khan University (AKU) at Karachi, Pakistan. This laboratory, through its network of satellite collection centers receives specimens from more than 100 major cities and towns of the country.

**Methods:** 162 *Candida glabrata* strains were isolated from blood (101), body fluids (26), pus and wounds (21) tissue (4) and others (10). Isolates were identified by conventional method using API 20C AUX, gross morphology on chromogenic and microscopic morphology on corn meal agar. MICs were determined using colorimetric broth microdilution (YeastOne Sensititre, Trek diagnostics). Susceptibilities were interpreted according to Clinical Laboratory standard Institute breakpoints mentioned in "Performance Standard for Antifungal Susceptibility Testing of Yeasts M60-ED1:2017".

**Results:** Out of 1917 archived invasive candida, 162 (8.4%) were *C. glabrata* 76% of these isolates were from patients admitted at AKU. Male to female ratio was 1.4 and 62% of the isolates were from ages 18-64 year. MIC<sub>90</sub> of these strains against fluconazole was 64 µg/ml, voriconazole 2µg/ml, itraconazole 1µg/ml and posaconazole 2µg/ml. Among echinocandins, MIC<sub>90</sub> was 0.12µg/ml for caspofungin, 0.06µg/ml for anidulafungin and 0.03µg/ml for micafungin. MIC<sub>90</sub> for amphotericin B was 0.5µg/ml.

**Conclusion:** Antifungal sensitivity testing for invasive candidiasis is essential in the face of emerging resistance as empiric therapy may not have reliable outcomes. Surveillance data of antifungal resistance among common *Candida* species with potential for developing antifungal resistance, like *C. glabrata*, should be monitored closely for identifying resistant strains in circulation.

P033

# Clinical implications of azole-resistant vs. azole-susceptible invasive aspergillosis in hematological malignancy (CLARITY) – a multicenter study

D. Seidel¹, O.A. Cornely², P. Köhler¹,³, J. Meis⁴, M. Zarrouk³, J. Salmanton-Garcia³, D. Arenz⁵, O. Blennow⁶, D. Buchheidt⁷, J.-J. Vehreschild³, N. Alakel⁸, A. Bergeron⁹, G. Desoubeaux¹⁰, I. Falces-Romero¹¹, N. Klimko¹², K. Lagrou¹³, C. Lass-Flörl¹⁴, Y. Le Govic¹⁵, J. Maertens¹⁶, A. Ostojic¹⁷, J. Prattes¹⁸, Z. Racil¹⁹, A. Reséndiz Sharpe¹⁶, E. Schalk²⁰, M. Stanzani²¹, J. Steinmann²², W. Melchers²³, M.J.G.T. Vehreschild²⁴, P.E. Verweij²³

¹Department I Of Internal Medicine, Ecmm Excellence Centre Of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), University Hospital Cologne, Cologne, Germany, ²Department I Of Internal Medicine, Ecmm Excellence Centre Of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), German Centre For Infection Research, Partner Site Bonn-cologne, Clinical Trials C, University Hospital Cologne, Cologne, Germany, ³Department I Of Internal Medicine, University Hospital of Cologne, Cologne, Germany, ⁴Department Of Medical Microbiology And Infectious Diseases, Excellence Center For Medical Mycology (ecmm), Canisius Wilhelmina Hospital, Nijmegen, Netherlands, ⁵Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), University of Cologne, Cologne, Germany, ⁶Karolinska University Hospital, Stockholm, Sweden, ⁷Mannheim University Hospital, Heidelberg University,, Mannheim, Germany, ⁸University Hospital Dresden, Dresden, Germany, ⁹Université Paris Diderot, APHP Saint-Louis Hospital, Paris, France, ¹⁰Cepr Inserm U1100, Université de Tours, Tours, France, ¹¹Hospital Universitario La Paz, Madrid, Spain, ¹²Department Of Clinical Mycology, Allergology And Immunology, North-Western State Medical University n.a. I.I. Mechnikov, Saint-Petersburg, Russian Federation, ¹³Clinical Bacteriology And Mycology, Katholieke Universiteit Leuven, Leuven, Belgium,
¹⁴Div Hygiene & Med. Microbiology, Med. Univ. Innsbruck, Innsbruck, Austria, ¹⁵Groupe d'Etude des Interactions Hôte Pathogène (GEIHP), Université d'Angers, Angers Cedex, France, ¹⁶KU Leuven, Leuven, Belgium, ¹⁷University Hospital Centre Zagreb, Zagreb, Croatia, ¹⁸Section Of Infectious Diseases And Tropical Medicine, Medical University of Graz, Graz, Austria, ¹⁹Department Of Internal Medicine - Hematology And Oncology, University Hospital Brno, Brno, Czech Republic, ²⁰Otto-von-Guericke University, Magdeburg, Germany, ²¹"Lorenzo e Ariosto Seràgnoli" S'Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy, ²²Institute Of Clinical Hygiene, Medical Microbiology And Clinical Infectiology, Klinikum Nuernberg, Nuremberg, Germany, ²³Medical Microbiology, RadboudUMC, Nijmegen, Netherlands, ²⁴Uniklinikum Frankfurt, Med. Klinik II, Infektiologie, Frankfurt, Germany

**Objectives:** In recent years, survival of patients with invasive aspergillosis (IA) has improved mainly due to availability of extended spectrum triazoles. These advances are jeopardized by the emergence of azole resistance in *Aspergillus fumigatus*, the most common causative pathogen of IA. Despite several studies suggesting high probability of azole treatment failure in patients with azole-resistant isolates, the clinical implications of azole-resistant IA compared to azole-susceptible IA remain unclear. Thus, we seek to describe the epidemiology and determine the efficacy of antifungal therapy in patients with documented azole-resistant IA compared to azole-sensitive IA in patients with hematological malignancy.

**Methods:** In patients with hematological malignancies, cases of proven or probable IA (EORTC/MSG 2008) caused by *A. fumigatus* are registered. Retrospective data are documented, comprising demographics, diagnosis, treatment, response and outcome. Participating sites provided susceptibility results or isolates. Provided isolates were analyzed in a central laboratory.

**Results:** Since January 2018, 51 sites in 15 countries worldwide enrolled 154 cases diagnosed with IA between 2010 and 2019, of which 23 (14.9%) had azole-resistant IA. Of 44 cases, the respective clinical fungal isolate was analyzed in the central laboratory. A mixed fungal infection was reported for 34 patients (22.1%), 1 (2.9%) in the azole-resistant group; most were related to non-*fumigatus* *Aspergillus* species (n = 12, 35.3%) and non-*Aspergillus* molds (n = 10, 29.4). Most patients were male (n = 98, 63.6%); 19 (82.6%) in the azole-resistant group, 79 (60.3%) in the azole-susceptible group. Age was documented in categories instead of the exact age. Median age group was 50-69 years in both groups (ranging from 7-11 to 70-89 years for azole-resistant cases, 1-12 months to 70-89 years for azole-susceptible cases). Underlying disease and survival are shown in the table.

## Patient characteristics

<table><thead><tr><th>n (%)</th><th>Azole-resistant</th><th>Azole-susceptible</th><th>Total</th></tr></thead><tbody><tr><td></td><td>23 (14.9)</td><td>131 (85.1)</td><td>154 (100)</td></tr><tr><td colspan="4">Hematologic malignancy n (%)</td></tr><tr><td>Acute leukemia</td><td>11 (47.8)</td><td>61 (46.6)</td><td>72 (46.8)</td></tr><tr><td>Chronic leukemia</td><td>1 (4.3)</td><td>9 (6.9)</td><td>10 (6.5)</td></tr><tr><td>Lymphoma</td><td>3 (13.0)</td><td>32 (24.4)</td><td>35 (22.7)</td></tr><tr><td>Multiple myeloma</td><td>1 (4.3)</td><td>9 (6.9)</td><td>10 (6.5)</td></tr><tr><td>Other</td><td>7 (30.4)</td><td>20 (15.3)</td><td>27 (17.5)</td></tr><tr><td colspan="4">Survival status n (%)</td></tr><tr><td>Missing values</td><td>0 (0)</td><td>2 (1.5)</td><td>2 (1.3)</td></tr><tr><td>Deceased</td><td>18 (78.3)</td><td>68 (51.9)</td><td>86 (55.8)</td></tr><tr><td>Alive</td><td>5 (21.7)</td><td>61 (46.6)</td><td>66 (42.9)</td></tr></tbody></table>

**Conclusion:** A worldwide network of investigators contributes to the CLARITY registry study. Completion of recruitment and subsequent data analysis are planned for 2019. Further sites may be added if azole resistant cases are encountered.
# Sensitivity of isolated dermatophyte strains to antifungal drugs in the Russian Federation

M. Manoyan, V. Sokolov, A. Gursheva, N. Gabuzyan, A. Panin

Veterinary Mycology, The All-Russian State Center for Quality of Animal Medicines and Feeds, Moscow, Russian Federation

**Objectives:** The spread of dermatophytosis is a veterinary problem; the spread of dermatophytosis pathogens in the human population is a social problem. In the Russian Federation immunobiological preparations, as well as external agents in the form of suspensions or shampoos are used for a treatment of animals dermatophytosis. As a rule, such agents contain azoles (clotrimazole, miconazole, enilconazole) or terbinafine as an active substance. They are often used unsystematically and can trigger the emergence of resistant strains. Recently, there have been increasing reports about dermatophytoses strains, resistant to antifungal drugs: *M. canis* strains resistant to terbinafine and azoles, and *T. rubrum* strains resistant to terbinafine.

**Methods:** For comparison, the strains of dermatophytes - *M. canis* and *T. mentagrophytes*, earlier collected and freshly isolated from cats and dogs, were chosen. Collection strains, stored in a lyophilized form, were isolated from animals between 1970 and 2000. For the study there were selected ten strains of *M. canis* and *T. mentagrophytes*. Freshly isolated strains were collected from animals between 2015 and 2018, they also were stored in the laboratory's collection in a lyophilized form; we also selected ten strains of *M. canis* and *T. mentagrophytes*. The choice of antifungal drugs spectrum, for which MIC was determined, was dictated by the data on the use of antifungal drugs in the Russian Federation: for terbinafine there was evidence of resistant strains; ketoconazole and enilconazole were used for animal fungal infections treatment and prevention. Cultures were isolated on Saburo medium with chloramphenicol and cycloheximide, pure cultures for determining MIC were cultivated on Saburo medium without additives for 7 days at +28°C. A method based on EUCAST E.def 9.3.1 was used to determine the MIC (inoculum density was 1.0E+5 CFU/ml, the incubation time was 4days.

**Results:** MIC studies of terbinafine, ketoconazole and enilconazole for strains isolated between 1970 and 2000 showed following. One of ten *M. canis* strains was resistant to the action of terbinafine (MIC> 32 µg / ml), there was no detected any strain resistant to ketoconazole and enilconazole. None of the ten *T. mentagrophytes* strains was resistant to the action of terbinafine, ketoconazole or enilconazole. MIC studies of terbinafine, ketoconazole and enilconazole for strains isolated between 2015 and 2018 showed following. Four of the ten *M. canis* strains were resistant to the action of terbinafine (MIC> 32 µg / ml); one strain was resistant to the action of enilconazole (MIC> 16 µg / ml); and we did not find a ketoconazole resistant strain. Four of the ten *T. mentagrophytes* strains were resistant to the action of terbinafine (MIC> 32 µg / ml); one strain was resistant to ketoconazole (MIC> 16 µg / ml); 2 strains were resistant to the action of enilconazole (MIC> 16 µg / ml). We did not find strains of *M. canis* or *T. mentagrophytes*, resistant to two or three drugs.

**Conclusion:** The number of antifungal resistant dermatophyte strains was higher among those isolated for a later period. This may indicate the emergence of resistant dermatophytosis pathogens strains in the Russian Federation.

P035

# Evaluation of the in vitro susceptibility testing of dermatophytic isolates: preliminary comparison of four methods

A.-M. Markantonatou, K. Samaras, E. Zachrou, T.-A. Vyzantiadis

First Department Of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece

**Objectives:** Superficial infections caused by dermatophytes affect a high percentage of the population. Antifungal susceptibility testing (AST) of these pathogens may offer information about their susceptibility profiles, documentation of the appropriate treatment and reduction of the cost. However, there are two factors that could delay and affect the performance of the AST: the slow growth rate of these fungi and their poor sporulation. The proposed methods by the CLSI or the EUCAST are both laborious for the everyday routine. Though, there are alternative applications that
propose the use of an inoculum consisting of a conidia-mycelium mixture or even from plain mycelia, as well as the use of resazurin in order to facilitate the reading. The aim of this study was to compare these approaches to the EUCAST method in order to evaluate their performance.

**Methods:** Three alternative methods of dermatophytic AST were compared to the EUCAST proposed methodology for conidia forming moulds. The methods under evaluation were a) a fragmented mycelia method, b) the EUCAST method with the addition of resazurin sodium salt solution and c) the fragmented mycelia method with the addition of resazurin sodium salt solution. The susceptibility of twenty dermatophytic isolates (8 *Trichophyton interdigitale*, 6 *T. rubrum* and 6 *M. canis*) was tested against the antifungal agents of griseofulvin, terbinafine, fluconazole and itraconazole.

**Results:** After the measurement of the MICs by all four different methods, the essential agreement between them was calculated in percentages. Data analysis revealed sufficient overall essential agreement of the methods with the addition of resazurin to the initial "uncoloured" ones (98.5% and 97.2% for the EUCAST and the fragmented mycelia method respectively). The fragmented mycelia method exhibited a relatively sufficient overall essential agreement to the EUCAST method (88.9%) but not a satisfactory correlation between the MIC values. The mean MICs (by the EUCAST method, in µg/mL) for the twenty isolates were 1.78 for griseofulvin, 0.034 for terbinafine, 25.2 for fluconazole and 0.57 for itraconazole.

**Conclusion:** The addition of resazurin sodium salt solution facilitates the reading and provides a more objective evaluation. The fragmented mycelia method could serve as an alternative in cases of poor or no sporulating dermatophytes.

P037

**Distribution of azole minimal inhibitory concentration (MIC) values and Erg11 amino acid substitutions among *Candida auris* isolates from different geographic clades**

M. Kordalewska¹, K.R. Healey², C. Jimenez-Ortigosa¹, A. Singh³, B. Williamson², A. Wilk², I. Berrio⁴, A. Chowdhary³, D.S. Perlin¹

¹Center For Discovery And Innovation, Hackensack Meridian Health, Nutley, United States of America, ²Department Of Biology, William Paterson University, Wayne, United States of America, ³Department Of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India, ⁴Hospital General de Medellín “Luz Castro de Gutiérrez” ESE, Medellín, Colombia

**Objectives:** *Candida auris* isolates are often characterized with reduced susceptibility to fluconazole (FLC) and other azole class drugs: voriconazole (VRC), posaconazole (POS), itraconazole (ITC), and isavuconazole (IVC). Such a situation is extremely worrying, since azoles are a mainstay in the treatment of *Candida* infections and antifungals other than fluconazole might be unavailable in resource-limited countries. Moreover, no antifungal clinical breakpoints are available for *C. auris*. CDC provides guidance for *C. auris* MIC interpretation, based on information gathered for related *Candida* species and expert opinion. Tentative epidemiological cut off values (ECVs) were proposed only for *C. auris* isolates from India. The aim of this study was to analyze distribution of azole minimal inhibitory concentration (MIC) values for isolates belonging to different geographic clades together with the data regarding molecular resistance determinants.

**Methods:** A total of 108 *C. auris* isolates representing four geographic clades (I – South Asian; II – East Asian; III – South African; IV – South American) were investigated in this study. Antifungal susceptibility testing (AFST) with azoles (FLC, ITC, IVC, POS, VRC) was performed in accordance with CLSI document M27-A3. The *ERG11* gene, encoding the target for azole antifungal drugs, was amplified and sequenced. All identified *ERG11* alleles were cloned onto a low-copy vector (pRS416) and expressed within a haploid strain of *Saccharomyces cerevisiae* (BY4741). Azole susceptibilities were then determined for the transformed strains of *S. cerevisiae*.

**Results:** The results of *C. auris* AFST and *ERG11* sequencing are presented in Table 1. Expression of *ERG11* alleles in *S. cerevisiae* revealed that triazole resistance was induced only upon expression of pCauErg11-V125A/F126L, pCauErg11-Y132F, and pCauErg11-K143R, but not pCauErg11-WT, pCauErg11-K177R/N335S/E343D, pCauErg11-I466M, or pCauErg11-Y501H.
<figure><img src="image_23.png" /><figcaption>**Table 1.** Results of AFST and *ERG11* sequencing performed for 108 *C. auris* isolates belonging to four geographic clades: I – South Asian; II – East Asian; III – South African; IV – South American.</figcaption></figure>

<table><thead><tr><th rowspan="2">Clade</th><th rowspan="2">Number of isolates</th><th rowspan="2">Erg11</th><th colspan="5">MIC range [mg/l]</th></tr><tr><th>FLC</th><th>ITC</th><th>IVC</th><th>POS</th><th>VRC</th></tr></thead><tbody><tr><td rowspan="3">I</td><td>2</td><td>WT</td><td>4 - 8</td><td>0.03</td><td>0.03</td><td>&lt;0.03 - 0.03</td><td>0.03 - 0.06</td></tr><tr><td>23</td><td>Y132F</td><td>64 - &gt;128</td><td>0.5 - 2</td><td>0.5 - 16</td><td>0.25 - 0.5</td><td>4 - &gt;16</td></tr><tr><td>19</td><td>K143R</td><td>64 - &gt;128</td><td>0.25 - 2</td><td>0.5 - 2</td><td>0.25 - 0.5</td><td>0.5 - 1</td></tr><tr><td>II</td><td>4</td><td>WT</td><td>2 - 8</td><td>0.03 - 0.06</td><td>&lt;0.03</td><td>&lt;0.03 - 0.06</td><td>0.03</td></tr><tr><td>III</td><td>2</td><td>V125A/F126L</td><td>&gt;128</td><td>0.125 - 0.25</td><td>0.03 - 0.06</td><td>0.03 - 0.06</td><td>1 - 2</td></tr><tr><td rowspan="5">IV</td><td>35</td><td>WT (K177R/ N335S/E343D)</td><td>2 - 64</td><td>0.125 - 1</td><td>&lt;0.03 - 0.5</td><td>0.03 - 0.125</td><td>0.03 - 1</td></tr><tr><td>4</td><td>Y132F</td><td>&gt;128</td><td>1</td><td>0.25 - 0.5</td><td>0.25</td><td>4</td></tr><tr><td>1</td><td>K143R</td><td>32</td><td>0.5</td><td>0.03</td><td>0.25</td><td>0.125</td></tr><tr><td>17</td><td>I466M</td><td>4 - 32</td><td>0.06 - 1</td><td>0.03 - 1</td><td>0.03 - 0.5</td><td>0.06 - 0.5</td></tr><tr><td>1</td><td>Y501H</td><td>64</td><td>1</td><td>1</td><td>0.5</td><td>1</td></tr></tbody></table>

<figure><img src="image_24.png" /></figure>

**Conclusion:** Differences in the distribution of MIC values and resistance-conferring *ERG11* mutations between isolates from different geographic clades underscore the need for an extreme caution when categorizing isolates as sensitive/resistant on a basis on AFST results. Only V125A/F126L, Y132F and K143R Erg11 amino acid substitutions identified in *C. auris* isolates directly mediate resistance and may be used as diagnostic markers for *C. auris* azole resistance. Mechanisms other than *ERG11* mutations may also contribute to reduced azole susceptibility in *C. auris*, although this remains to be determined.

P038

**To study the profile of conventional and newer antifungal agents against dermatophytes in a tertiary care hospital in South India**

A.J. Kindo<fnref n="1" />, H. Veena<fnref n="1" />, A. Subramanian<fnref n="2" />, A. Krishnan<fnref n="2" />

**Objectives:** 1.Speciation of dermatophytes from the clinical isolates 2.To study the antimicrobial pattern of frequently used antifungal agents 3.To compare the antifungal profile with the newer antifungal agents

**Methods:** Clinically diagnosed dermatophytic patients were sampled for microscopy and culture, grown dermatophytes were subjected to phenotypic identification by conventional method. Antifungal susceptibility testing was done by broth microdilution method according to CLSI M38-A2 document guidelines. *Trichophyton mentagrophytes* ATCC 4439 was included in each run of susceptibility testing as a quality control. Final drug concentration in the microdilution plates ranged from 64 to 0.25 µg/ml for fluconazole and Amphotericin B and from 16 to 0.06 µg/ml for Itraconazole,Voriconazole, Griesofulvin ,Terbinafine, Sertaconazole,Luliconazole, Fenticonazole tested.

<footnote n="1">¹Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, India,</footnote>

<footnote n="2">²Dermatology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, India</footnote>
Results:

<table><caption>MIC RANGE FOR THE CONVENTIONAL AND NEWER ANTIFUNGAL AGENTS</caption><thead><tr><th>Species identified</th><th>Trichophyton mentagrophyte</th><th>Trichophyton rubrum</th><th>Epidermophyton floccosum</th><th>Microsoprum nanum</th></tr></thead><tbody><tr><td></td><td>n = 58</td><td>n=40</td><td>n =1</td><td>n =1</td></tr><tr><td>AmpB<br>(64-0.25µg/ml)</td><td>4-&gt;64 µg/ml</td><td>4-&gt;64 µg/ml</td><td>4 µg/ml</td><td>4 µg/ml</td></tr><tr><td>Fluconazole<br>(64-0.25µg/ml)</td><td>2-64 µg/ml</td><td>2-64 µg/ml</td><td>2 µg/ml</td><td>&gt;64 µg/ml</td></tr><tr><td>Itraconazole<br>(16-0.06µg/ml)</td><td>0.06-1 µg/ml-</td><td>0.06-16 µg/ml-</td><td>8 µg/ml</td><td>0.06 µg/ml</td></tr><tr><td>Voriconazole<br>(32-0.125µg/ml)</td><td>0.125-4 µg/ml</td><td>0.06-4 µg/ml-</td><td>0.125 µg/ml</td><td>0.5 µg/ml</td></tr><tr><td>Griesofulvin<br>(16-0.06µg/ml)</td><td>0.06-4 µg/ml</td><td>0.125-16µg/ml</td><td>0.25 µg/ml</td><td>0.5 µg/ml</td></tr><tr><td>Terbinafine<br>(16-0.06µg/ml)</td><td>0.06-8µg/ml</td><td>0.06-2µg/ml</td><td>0.06 µg/ml</td><td>0.06 µg/ml</td></tr><tr><td>Sertaconazole<br>(16-0.06µg/ml)</td><td>0.06-4µg/ml</td><td>0.06 µg/ml</td><td>0.06 µg/ml</td><td>0.06 µg/ml</td></tr><tr><td>Luliconazole<br>(16-0.06µg/ml)</td><td>0.06 µg/ml</td><td>0.06 µg/ml</td><td>0.06 µg/ml</td><td>0.06 µg/ml</td></tr><tr><td>Fenticonazole<br>(16-0.06µg/ml)</td><td>0.06 µg/ml</td><td>0.06 µg/ml</td><td>0.06 µg/ml</td><td>0.06 µg/ml</td></tr></tbody></table>

Out of the 100 clinical isolates tested, 58 of them were identified as *Trichophyton mentagrophytes*, 40 were *Trichophyton rubrum*, and the remaining two isolates were identified as *Epidermophyton floccosum* and *Microsoprum nanum*. Antifungal susceptibility testing was carried out for 100 isolates of *Trichophyton* species against 9 antifungal agents. All isolates showed detectable growth after 4-5days of incubation. MICS for *Trichophyton mentagrophytes* ATCC 4439 were within the established range.

**Conclusion:** In this study *Trichophyton mentagrophytes* was the most common cause of dermatophytosis. The most challenging task is not the diagnosis but the treatment, compliance and recurrence. Since it requires a long term treatment, the management with appropriate and responsive drug is the need of the hour. Currently this study suggests that the overall activity of *Luliconazole*, *Fenticonazole*, *Sertaconazole*, as topical agent and *Itraconazole*, *Voriconazole* (systemic) are significantly effective against dermatophytes. Current solution to this could be to maintain skin hygiene, prudent use of antifungals appropriate dosing and duration, performing antifungal susceptibility testing, usage of conventional drugs or topical keratolytic agents in combination with newer drugs in appropriate dosages and combination therapy.

P039

Terbinafine resistant Trichophyton mentagrophytes genotype VIII, Indian type, isolated in Finland

H. Järv¹, S. Uhrlass², T. Simkin¹, P. Nenoff², E. Alvarado Ramirez³, E. Chryssanthou³, M. Monod⁴

¹SYNLAB Estonia, Tallinn, Estonia, ²Laboratory For Medical Microbiology, Mölbis, Mölbis, Germany,

³Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden, ⁴Department Of Dermatology, Lausanne University Hospital, Chexbres, Switzerland

**Objectives:** Objective We are reporting first four isolates of *Trichophyton mentagrophytes* representing novel Indian genotype VIII in Finland potentially resistant to terbinafine. Our study points out the need for antifungal susceptibility testing of dermatophytes as a new issue in microbiology laboratories. The roles of classical mycological culture vs DNA-based diagnostics for laboratory diagnostics of dermatophytosis will be discussed.
**Methods:** Primary fungal cultures were made on Sabouraud dextrose and 2% malt extract agar. Identification of fungal isolates was made by micromorphological appearance in SYNLAB laboratory in Estonia and by sequencing the ITS region of rDNA and TEF1-alpha gene in clinical microbiology laboratory in Mölbis, Germany. The genotyping of squalene oxidase gene was made in dermatology department of Vaudoise, Lausanne, Switzerland. The minimal inhibitory concentration (MIC) to terbinafine was determined by broth microdilution method in microbiology laboratory of Karolinska Hospital, Sweden. MIC was detected spectrophotometrically after 5 days incubation at 28°C.

**Results:** We describe four cases of extensive *tinea cutis glabrae* caused by *T. mentagrophytes*. All cases were diagnosed from January to April of 2019 and occurred in geographically distinct regions of Finland. Three of four of *T. mentagrophytes* sub-type VIII (Indian sybtype) strains have high MIC values of terbinafine from 4 mg/L to >8 mg/L.

**Conclusion:** Dermatophytosis has taken epidemic dimensions in India during the last 5 to 6 years. Unusual clinical presentation of dermatophytosis and corticosteroid modified lesions are making diagnosis challenging. Indian experts panel ECTODERM India has published new treatment guidelines in 2018. Nenoff *et al* have demonstrated in a recently published study from different geographical locations from India that 93.25% of the dermatophytes isolated from patients suffering from dermatophytosis correspond to a special genotype (VIII) of *T. mentagrophytes*. Singh with co-workers have published data about alarming terbinafine resistance of *T. interdigitale* strains in India. *Trichophyton* spp isolates with Indian origin have demonstrated high MICs to terbinafine, up to 32 mg/L, and also to azoles. Terbinafine is considered to be the most effective drug for treatment of *Trichophyton* spp infections. In general, we know that pathogens and resistant strains follow the human migration routes. The lack of data about presence of this novel genotype in European countries is because most clinical laboratories do not use multilocus sequencing for routine identification of dermatophytes. SYNLAB Estonia uses classical culture-based diagnostics and DNA-based diagnostics (in house PCR) for dermatophytes, handling approx. 10000 sample per year. We are not able to identify 1/5 of dermatophytes on species level. Most of commercial diagnostic DNA panels for dermatophytes do not discriminate *T. mentagrophytes* and *T. interdigitale*. In the light of new data diagnostic laboratories need to put more effort to identify all dermatophytes on species level by molecular methods. It will help to map the changing epidemiological situation of dermatophytosis in Europe.

P040

**An emerging clone of *Candida tropicalis* bloodstream isolates with ERG11 gene mutations and overexpression conferring reduced activity of isavuconazole**

P.-Y. Chen, Y.-C. Chuang, U.-I. Wu, H.-Y. Sun, J.-T. Wang, W.-H. Sheng, Y.-C. Chen, S.-C. Chang
National Taiwan University Hospital, Taipei, Taiwan

**Objectives:** In Asia, in vitro activity of isavuconazole (ISA) against clinical *Candida* isolates are seldomly reported, especially a rising concern that azole-resistant *Candida tropicalis* is common (≥10%) in this region. Also, few studies characterized molecular mechanisms of reduced isavuconazole activity against *C. tropicalis*.

**Methods:** Sixty-four non-duplicated *C. tropicalis* bloodstream isolates were prospectively collected in National Taiwan University Hospital in 2017. In vitro susceptibility to antifungal agents was performed by using the microdilution colorimetric Sensititre YeastOne panel and the results were interpreted by the Clinical and Laboratory Standards Institute. ISA MICs were determined using the EUCAST E.def 7.3.1 method. Wild-type upper limit (wtUL) was defined as two two-fold dilutions above the MIC₅₀. Isolates with reduced ISA activity (RIA) were defined as their ISA MICs above wtUL. The *ERG11* gene was sequenced for all isolates, and the results were compared to the *ERG11* wild-type sequence from *C. tropicalis* reference strain ATCC 750 (Genbank accession M23673). Quantitative realtime reverse transcription-polymerase chain reaction (*ERG11*, *CDR1*, and *MDR1*) were performed. Multilocus sequencing types were analyzed by the eBURST to determine putative relationships between isolates.
**Results:** Among sixty-four *C. tropicalis* bloodstream isolates, the resistant/ non-wild type rates of fluconazole, voriconazole, posaconazole or itraconazole were 21.5%, 30.7%, 64.6%, and 13.9%, respectively. The proportions of RIA was 18.8% (n = 12). ISA MICs correlated with other azoles MICs by calculating with nonlinear regression fits. The R² values were in the following orders: posaconazole (0.74) < itraconazole (0.82) < voriconazole (0.86) < fluconazole (0.88). The mean percentage of ISA trailing was 27.4%. A total of 8 nucleotide mutations were detected in *ERG11* gene of all isolates. Only 2 (A395T/W and C461T/Y) belonged to missense mutations resulting in amino acid substitutions (Y132F and S154F). There were fourteen isolates with one or two these mutations all resistant to fluconazole. Twelve of them had RIA, all of which belonging to the same clone. Gene relative expression levels among three groups (isolates with low and high ISA trailing, and those with RIA) are shown **Figure**. Only *ERG11* expression levels demonstrated significant difference (*P* < 0.001), and neither did *CDR1* nor *MDR1* (*P* > 0.05). Those with RIA showed the highest mean *ERG11* relative expression level (3.51 ± 0.59 [S.D.]) with statistically significance, while the mean *ERG11* levels between high and low ISA trailing group were not statistically different (1.08 ± 0.69 and 0.93 ± 0.66, respectively).

**Figure**

<figure><img src="image_25.png" /></figure>

**Conclusion:** *C. tropicalis* bloodstream isolates in our report showed an emerging clone with pan-azole resistance, including RIA. Fluconazole MICs best correlated to ISA MICs. Combined *ERG11* gene missense mutations and *ERG11* overexpression conferred RIA.

P041

Comparative genomics of *Aspergillus fumigatus* and the influence of agriculture on ecology and azole resistance

A.E. Barber¹,²,³, J. Born⁴, T. Sae-Ong², I. Schliebner⁴, K. Kang², G. Walther²,³, G. Panagiotou², H.B. Deising⁴, O. Kurzai¹

¹University of Wuerzburg, Wuerzburg, Germany, ²Leibniz HKI Jena, Jena, Germany, ³National Reference Center for Invasive Fungal Infections, Jena, Germany, ⁴Martin Luther University Haale-Wittenberg, Halle (Saale), Germany

**Objectives:** *Aspergillus fumigatus* is an environmentally ubiquitous saprophyte capable of causing life-threatening invasive infections and allergic bronchopulmonary disease and current management strategies rely on the azoles. Unfortunately, over the last decade there has been a global emergence in resistance to the azoles in *A. fumigatus* and the dominant resistance mechanism is of environmental origin, suggesting that resistance is emerging through a selective pressure applied by the widespread usage of azoles in agriculture.
**Methods:** To examine the link between the use of azoles in agriculture and the emergence of clinical resistance, systematic soil sampling was performed over a three year period on seven conventional agricultural sites in Germany utilizing azole fungicides and on six organic sites withholding these compounds. Conventional sites were evaluated both before and after azole application. We also performed WGS on 250 environmental and 50 clinical isolates.

**Results:** In total, 2875 soil samples were analyzed between 2016 and 2018. We observed a high degree of variation in the abundance of *A. fumigatus* across sites, however, fields with high *A. fumigatus* density tended to be consistently so from year to year. Strikingly, we observed a significant reduction in the abundance of *A. fumigatus* on conventional fields following azole treatment – a finding that was not repeated on an organic agriculture control field – indicating that the application of azoles is imposing a bottleneck on *A. fumigatus*. We detected a low overall resistance frequency amongst agricultural isolates, with only 1-5% of isolates from 2016-2018 showing medical azole resistance – a rate lower than the 15-20% resistance frequency observed by the German National Reference Center for Invasive Fungal Infections during the same time period. Susceptibility to commonly-applied agricultural azoles was also assessed and isolates resistant to medical azoles almost always had elevated MICs to these agricultural azoles, suggesting cross resistance. Importantly, we observed an increased tolerance to both agricultural and medical azoles in the samples taken after the growing season and application of azoles. At the genome level, isolates from different regions and types of agriculture did not cluster separately, indicating a lack of population structure in the fungus. Comparison of environmental isolates with clinical isolates revealed several subgroups present in the environment that were not represented among clinical samples. We also observed differences in the distribution of resistance mutations between environmental and clinical isolates. Resistant environmental isolates were exclusively either wild type at the *cyp51a* loci or carried the environmentally-derived TR34/L98H mutation. Clinical isolates, in contrast, showed a much wider range mutations, including substitutions at positions F219, M220, S297, F495, and G448. Ongoing work is focused on defining fungal determinants critical for human infection as well as genetic mechanisms associated with *cyp51a*-independent azole resistance.

**Conclusion:** We observed a marked reduction in *A. fumigatus* fields following azole application and increased azole tolerance after the growing season and azole exposure. No population structure was observed among environmental isolates, but whole genome phylogeny identified genetic backgrounds enriched for among clinical isolates.

P042

**MIC distributions for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine and anidulafungin and thirty-five uncommon pathogenic yeast species from the UK determined using the CLSI broth microdilution method**

A. Borman, J. Muller, J. Walsh-Quantick, A. Szekely, Z. Patterson, M. Palmer, M. Fraser, E. Johnson
Public Health England UK National Mycology Reference Laboratory, Bristol, United Kingdom

**Objectives:** Epidemiological cut-off values and clinical interpretive breakpoints have been developed for a number of antifungal agents with the most common *Candida* species which account for the majority of infections due to pathogenic yeasts species. However, less common species for which susceptibility data are limited are increasingly reported in high risk patients and breakthrough infections.

**Methods:** The UK National Mycology Reference Laboratory performs routine antifungal susceptibility testing of clinical isolates of pathogenic yeast submitted from across the United Kingdom. Between 2002 and 2016, in excess of 32 000 isolates representing 94 different yeast species were referred to the laboratory. Here we present antifungal susceptibility profiles generated over this 15 year period for amphotericin B, fluconazole, voriconazole, itraconazole, anidulafungin and flucytosine against 35 species of uncommon yeast. Minimum inhibitory concentrations (MICs) were obtained using CLSI broth microdilution methodologies, and MIC data was interpreted against epidemiological cut-off values and clinical breakpoints developed with *Candida albicans*, in order to identify species with unusually skewed MIC distributions that potentially indicate resistance.
**Results:** Potential resistance to at least one antifungal agent (>10% of isolates with MICs greater than the epidemiological cut-off or clinical breakpoint) was evidenced for 29/35 species examined here. Four species exhibited elevated MICs with all of the triazole antifungal drugs against which they were tested, and 21 species exhibited antifungal resistance to agents from at least 2 different classes of antifungal agent.

**Conclusion:** The current study highlights the importance of rapid and accurate yeast identification, and provides data to aid clinicians in deciding which antifungal regimens are appropriate when confronted with infections with rarer yeasts.

P043

Case-series of *Candida albicans* and *glabrata* echinocandin-resistant candidemia in Switzerland

A.T. Coste¹, J. Li¹, N. Khanna², D. Goldenberger³, C. Garzoni⁴, Z. Zhender⁵, K. Boggian⁶, D. Neofytos⁷,
A. Riat⁸, D. Bachmann¹, D. Sanglard¹, F. Lamoth⁹,¹⁰

¹Microbiology Institute, University Hospital Lausanne, University of Lausanne, Lausanne, Switzerland, ²Division Of Infectious Diseases And Hospital Epidemiology, University Hospital of Basel, Basel, Switzerland, ³University Hospital Basel, Clinical Microbiology Laboratory Medicine, Basel, Switzerland, ⁴Riva Caccia 1b, Lugano, Switzerland, ⁵SYNLAB Suisse SA, Bioggio, Switzerland, ⁶Department Of Infectious Diseases, Cantonal Hospital of Saint Gallen, Saint Gallen, Switzerland, ⁷Infectious Diseases, University Hospital of Geneva, Geneva, Switzerland, ⁸Geneva University Hospitals, Service of Laboratory Medicine, Geneva, Switzerland, ⁹Microbiology Institute, University Hospital Lausanne, Lausanne, Switzerland, ¹⁰Infectious Diseases Service, CHUV, Lausanne, Switzerland

**Objectives:** Echinocandin drugs (anidulafungin, caspofungin, micafungin) represent first-line therapy of candidemia. Echinocandin resistance, due to hotspot mutations in the target *FKS* gene, has emerged among *C. glabrata* and *C. albicans*, which is associated with poor outcome. Therefore, national surveillance programs of echinocandin resistance are mandatory. The aims of this study were: i) to assess the epidemiology of echinocandin resistance among *C. albicans/glabrata* in Switzerland, ii) to describe the characteristics of candidemic episodes due to echinocandin-resistant isolates, and iii) to assess the efficacy of high-dose echinocandin against echinocandin-resistant *C. albicans* in an invertebrate model of infection.

**Methods:** Cases of candidemia attributed to *C. albicans/glabrata* resistant to echinocandins (CLSI criteria) were identified by: i) screening of a collection of *Candida* bloodstream isolates from the Fungal Infection Network of Switzerland (FUNGINOS) including all candidemic episodes from 26 hospitals in Switzerland from 2004 to 2013, and ii) screening of the microbiology databases of the most important university hospitals from 2014 to 2018. Echinocandin-resistant *Candida* isolates were sent to the reference laboratory for antifungal susceptibility testing (CLSI protocol) and sequencing of *FKS* hotspots. Clinical data of candidemia were collected. In order to assess a possible efficacy of high-dose echinocandins against these isolates, we tested the efficacy of escalating anidulafungin doses in a *Galleria mellonella* model of invasive candidiasis with a *C. albicans* *FKS* mutant (S645P) compared to the wild-type SC5314 strain.

**Results:** Seven cases of candidemia due to echinocandin-resistant *C. albicans/glabrata* were identified: 2 from the 2004-2013 cohort (incidence 0.1%) and 5 from the microbiological database screening from 2014 to 2018. Four infections were due to *C. albicans* and 3 to *C. glabrata*. Candidemia originated from urine (2 cases), intravascular catheter (2), gastro-intestinal tract (1) and primary bloodstream infection (1). All patients received previous echinocandin therapy with a median duration of 18 days (range 11-60). Overall mortality was 43%. One patient (*C. glabrata* infection) was cured by caspofungin therapy despite *in vitro* resistance. Minimal inhibitory concentrations (MIC) ranges for anidulafungin, caspofungin and micafungin were: 2-4, 1-16 and 1-4 µg/mL, respectively. The following hotspot mutations were identified in *C. albicans*: S645P (*n* = 3) and R1361G (*n* = 1). Among *C. glabrata*, 2 isolates displayed the S663P mutation in *FKS2*, while no hotspot *FKS* mutation was recovered from one pan-echinocandin resistant isolate. Because anidulafungin displayed some *in vitro* activity against *C. albicans* S645P mutant (MIC 2 µg/mL), we assessed its *in vivo* activity in a *Galleria mellonella* model of
infection (Figure 1). While a dose-dependent response was observed in *C. albicans* wild-type strain-
infect ed larvae, no effect was observed against the *C. albicans* FKS mutant despite high anidulafungin
doses
(12
mg/kg).

<figure><img src="image_26.png" /><figcaption>**Figure 1 :** *Anidulafungin dose-response against Candida albicans systemic infection in Galleria mellonella*</figcaption></figure>

Larvae were infected with 5 x10⁵ candida cells in a volume of 40 µl except the PBS groups (in grey). Two hours later animals were injected again with 40 µl of anidulafungin as indicated, except the PBS groups (darker colors). For each groups n=15.

**Conclusion:** Our analysis suggests an increase in echinocandin resistance among *C. albicans/glabrata* in Switzerland with 5 cases identified within the last 5 years, while only two cases were reported in a cohort including all candidemias from 2004 to 2013. Previous echinocandin exposure was observed in all cases. Our invertebrate model suggests that high-dose echinocandins are ineffective against *C. albicans* FKS mutants despite some level of *in vitro* activity.

P044

*Aspergillus* isolates from patients with chronic pulmonary aspergillosis mycologically and clinically resistant to azole antifungals are sensitive to Ibrexafungerp (SCY-078)

R. Rautemaa-Richardson<sup>1,2</sup>, C. Moore<sup>1,2</sup>, K. Rawson<sup>2</sup>, L. Novak-Frazer<sup>1,2</sup>, D. Angulo<sup>3</sup>, S. Barat<sup>3</sup>, M.D. Richardson<sup>1,2</sup>

¹Division Of Infection, Immunity And Respiratory Medicine, University of Manchester, Manchester, United Kingdom, ²Mycology Reference Centre, Excellence Center For Medical Mycology (ECMM), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom, ³Scynexis Inc, New Jersey, United States of America

**Objectives:** Ibrexafungerp (formerly MK-3118, SCY-078) is a novel and structurally distinct triterpenoid glucan synthase inhibitor whose oral availability and efficacy has already been demonstrated against a wide spectrum of *Candida* species. Currently the drug is being evaluated for efficacy in invasive and chronic pulmonary aspergillosis. Ibrexafungerp has been shown to be effective *in vitro* and *in vivo* against a broad range of *Aspergillus* species, including drug-resistant strains. *In vitro* activity is an important efficacy indicator of therapeutic success or failure. The objective of this study was to determine the activity of Ibrexafungerp against a panel of *Aspergillus* strains isolated from the respiratory tract of patients unresponsive to or failing azole therapy, and which were previously shown to be resistant to one or more azole antifungals (itraconazole, voriconazole, posaconazole and isavuconazole) using the EUCAST E.Def 10.1 standard.

**Methods:** Patients failing azole therapy for chronic pulmonary aspergillosis were identified in the weekly departmental multi-disciplinary team meetings and their isolates selected for Ibrexafungerp sensitivity testing. Twenty-two *Aspergillus fumigatus* complex, three *A. flavus* complex and one *A. niger* complex strains with varying degrees of resistance to one or more azole antifungals were tested by measuring the minimum effective concentration (MEC) for Ibrexafungerp following the EUCAST E.Def 10.1 standard. Where required, isolates were sequenced (using primers to the internal transcribed spacers (ITS), beta-tubulin (bt) and calmodulin (cal) genes) to identify to the species level.

**Results:** The Ibrexafungerp MEC range for the 26 *Aspergillus* spp. isolates was 0.008 to 0.25 mg/l. Isolates that were deemed to be resistant to all azoles had an Ibrexafungerp MEC of 0.015 to 0.25 mg/l. One isolate that was resistant to all azoles and amphotericin B was sensitive to Ibrexafungerp (MEC 0.125 mg/l).

**Conclusion:** The exquisite *in vitro* activity of Ibrexafungerp against *Aspergillus fumigatus*, *A. flavus* and *A. niger* isolated from patients with chronic pulmonary aspergillosis suggests that this new
compound has potential for treating patients with this condition and similar manifestations of pulmonary aspergillosis.

P045

**Analysis of the susceptibility pattern among Aspergillus isolates against the commonly used Antifungal agents in a tertiary care centre in South India.**

U. Almas Fathima¹, A.J. Kindo¹, S. Prasanna Kumar², M. Thirunarayan³, V. Lakshmi Sree⁴

¹Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, India, ²Ent, Sri Ramachandra Institute of Higher Education and Research, Chennai, India, ³Microbiology, Apollo Hospitals, Chennai, India, ⁴Microbiology, Apollo Speciality Hospital, Chennai, India

**Objectives:** To do *in vitro* susceptibility testing and analyse the difference in the Antifungal susceptibility profile of *Aspergillus* isolates by Broth Microdilution method.

**Methods:** *Aspergillus* grown from various clinical samples (Ear swab, Bronchial wash, Endotracheal Aspiration, Paranasal sinus, BAL, Sputum) was subcultured on Sabouraud's Dextrose Agar/Oat meal Agar. Tease mount/Slide culture was done to study the morphological features of the hyphae, size, shape and arrangement of the conidia. Antifungal susceptibility testing(AFST) was done according to CLSI guidelines M38-A2 2008 documented reference method for Broth Microdilution Antifungal susceptibility of Filamentous fungi. Antifungal stock solutions of AmphotericinB, Voriconazole, Posaconazole, Itraconazole and Caspofungin was prepared in di-methyl sulfoxide and stored at -70° C. RPMI medium was prepared, adjusted to pH 7 and filter sterilized. Sterility was checked and stored at 4° C. Dilution of the stock solution was done as per the guidelines. Inoculum was prepared by adjusting conidial suspension in sterile distilled water to 0.5 McFarland Turbidity standard; to 100 µl of drug solution, 100 µl of inoculum in RPMI 1640 medium was added. Test micro-titre plate was incubated for 48hrs at 35° C. Growth and sterility control wells were included in each test. AFST-READING RESULT: For AmphotericinB, Voriconazole, Posaconazole, Itraconazole - MIC is the first well without visible growth. For Echinocandins, eg. Caspofungin- MEC is the point (lowest value) where a visible change in growth as compared to the positive growth is noted.

**Results:** A total of 30 *Aspergillus* isolates were collected-*Aspergillus niger* (14), *Aspergillus flavus* (9), *Aspergillus fumigatus* (6) & *Aspergillus terreus* (1). The result was compiled based on mean MIC values of 21 isolates as follows.

<figure><img src="image_27.png" /></figure>

The MIC values of other isolates and Amphotericin B is yet to be completed.

**Conclusion:** The common *Aspergillus* species was *Aspergillus niger* which was obtained from Ear swab. The susceptibility profile showed high MIC for Itraconazole in case of *Aspergillus niger* and
high MIC for Itraconazole, Voriconazole and Posaconazole in case of *Aspergillus flavus*. For *Aspergillus fumigatus* no such high MIC was recorded. In case of *Aspergillus terreus*, being resistant in nature the low MIC is recorded only with Caspofungin. Due to less susceptibility of *Aspergillus* to the common Antifungal agents, it is important to perform antifungal susceptibility testing to reduce the morbidity and mortality in patients especially with Invasive Aspergillosis.

P046

CRISPR-Cas9 as a tool box to investigate antifungal resistance

F. Morio¹, L. Lombardi², U. Binder³, C. Loge¹, E. Robert¹, C. Lass-Flörl³, G. Butler², P. Le Pape¹

¹Ea1155 Iicimed, Nantes University Hospital, NANTES, France, ²School Of Biomolecular And Biomedical Science, Conway Institute, University College Dublin, Dublin, Ireland, ³Department Of Hygiene, Microbiology And Public Health, Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Innsbruck, Austria

**Objectives:** Antifungal resistance in human pathogenic fungi is increasingly reported and a public health issue. Deciphering the mechanisms underlying drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi. Over the last decade, the groundbreaking discovery of Clustered Regularly Interspaced Short Palindromic Repeats and the associate protein Cas9 (CRISPR-Cas9) revolutionized genome editing. We evaluated the potential of a CRISPR-Cas9 method, as a tool to perform precise genome editing of the *ERG11* gene in the context of azole resistance.

**Methods:** A new plasmid-based and marker-less CRISPR-Cas9 genome-editing method, recently developed for *Candida parapsilosis* (1), was used to investigate the potential contribution to azole resistance of two different single nucleotide polymorphisms (G1126A and G1372A), leading to amino acid substitutions (L376I and G458S, respectively) in the *ERG11* gene, identified in an azole-resistant clinical isolate of *Candida orthopsilosis*. These mutations were introduced by homology directed recombination (HDR) using a donor template into a fluconazole-susceptible *C. orthopsilosis* background isolate. *In vitro* susceptibility of the transformants displaying the expected mutations (two clones for each engineered mutation) to azoles was then performed.

**Results:** This approach allowed us to engineer both mutations in the fluconazole-susceptible background isolate. Though engineering L376I was straightforward, editing G458S required the addition of silent mutations within the core of the Cas9 recognition site to prevent Cas9 from recutting again. Eventually, *in vitro* susceptibility testing of the corresponding transformants demonstrated that G458S, but not L376I, confers resistance to fluconazole (MIC increased by 8-fold by introducing the homozygous mutation) and also affected voriconazole susceptibility. Importantly, after 3 passages on YPD at 30°C, transformants were not resistant to nourseothricin (selection marker) anymore, showing the rapid loss of the plasmid containing all the CRISPR-Cas9 components, thus proving that it did not integrate in the genome.

**Conclusion:** Our work supports the contribution of G458S but not L376I in fluconazole and voriconazole resistance in *C. orthopsilosis* (2). Besides these findings, this study highlights CRISPR-Cas9 genome editing as a powerful tool to streamline the study of antifungal resistance in human pathogenic fungi. Though critical points should be considered (e.g. HDR drops with cut-to-mutation distance), this approach paves the way for an easier screening approach of any SNP, occurring in any gene suspected to be involved in antifungal resistance. 1-Lombardi *et al.*, Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple *Candida* Species. **mSphere. 2019** Mar 13;4(2). pii: e00125-19. doi: 10.1128/mSphere.00125-19. 2-Morio *et al.*, Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in *Candida orthopsilosis*. **J Antimicrob Chemother. 2019** May 18. pii: dkz204. doi: 10.1093/jac/dkz204.

P047

Changes in the lipid microenvironment of β-(1,3)-D-glucan synthase of *Aspergillus fumigatus* is a
new clinically important echinocandin resistance mechanism
S. Satish¹, C. Jimenez-Ortigosa¹, Y. Zhao¹, M.H. Lee¹, E. Dolgov¹, T. Kruger², S. Park¹, D. Denning³, O. Kniemeyer², A. Brakhage², D.S. Perlin¹

¹Center For Discovery And Innovation, Hackensack Meridian Health, Nutley, United States of America, ²Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany, ³University of Manchester, Manchester, United Kingdom

**Objectives:** *Aspergillus fumigatus* is a leading cause of opportunistic invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high Minimum Effective Concentration (MEC) *A. fumigatus* clinical isolates from patients failing echinocandin therapy. Echinocandin resistance in *Candida* is known to arise from amino acid substitutions in β-(1,3)-D-glucan synthase encoded by the *fks1* gene. Yet, these clinical isolates did not contain mutations in *fks1*, indicating an undefined resistance mechanism. The objective of this study was to explore a novel mechanism of echinocandin resistance independent of *fks1* mutations.

**Methods:** To explore this new mechanism, we used a lab-derived strain, RG101, which does not contain *fks1* mutations but is resistant to caspofungin (CAS). Inhibition of glucan synthase enzyme isolated from RG101 grown in the absence and presence of CAS was determined *in vitro* using radioactive substrate and IC₅₀ (drug concentration required for 50% reduction of activity) values were measured. Post-translational modifications (PTMs) potentially leading to resistance was explored by evaluating enzyme derived from RG101 grown in the absence and presence of CAS (1 µg/mL) using nano LC-MS/MS. A comprehensive lipidomics analysis was also performed to compare lipid profiles of glucan synthase containing fractions derived from RG101 grown in the absence and presence of CAS (1 µg/mL) using LC-MS/MS. To measure Reactive Oxygen Species (ROS) levels induced by different echinocandins, a 2',7'-dichlorofluorescin diacetate (DCFDA)-based fluorescence assay was used, and ROS levels measured after one hour of drug exposure.

**Results:** Glucan synthase isolated from RG101 was fully sensitive to echinocandins. Yet, exposure of RG101 to CAS during growth yielded a modified enzyme that was drug insensitive (4 log-orders) in kinetic inhibition assays, and this insensitivity was also observed for enzymes isolated from clinical isolates. To understand this alteration, we analyzed whole enzyme post-translational modifications, but found none linked to resistance. However, analysis of the lipid microenvironment of CAS-induced resistant enzyme revealed a prominent increase in abundance of dihydrospHINGOSINE (DhSph) and phytoSPHINGOSINE (PhSph). Exogenous addition of DhSph and PhSph to sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrial-derived ROS, and dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance.

**Conclusion:** We conclude that CAS-induces cellular stress, promoting formation of ROS, triggering an alteration in the composition of plasma membrane lipids within the local environment of glucan synthase, rendering it insensitive to echinocandins. We have discovered a new mechanism of resistance in *A. fumigatus* independent of the well-characterized *FKS* mutation mechanism observed in *Candida*. This study also identifies an off-target effect of CAS, ROS production in *A. fumigatus*, and integrates oxidative stress and sphingolipid alterations into a novel mechanism of resistance. This stress-induced response has implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.

P048

**In vitro activity of fluconazole in combination with magnolol against *Candida auris***

I. De-La-Fuente¹, A. Matilla-Gutiérrez¹, A. Guridi¹, E. Sevillano¹, J. Pemán², E. Eraso¹, G. Quindós¹

¹Immunology, Microbiology And Parasitology, University of the Basque Country (UPV/EHU), Bilbao, Spain, ²Servicio De Microbiología, Hospital Universitario y Politécnico La Fe, Valencia, Spain

**Objectives:** *Candida auris* is an emerging pathogen that causes outbreaks worldwide. This fungus is responsible for severe infections such as invasive candidiasis that present a high overall mortality. Treatment of these infections suppose a clinical challenge due to the high levels of multidrug-resistance in *C. auris*, which makes the finding of new therapeutic alternatives an important issue.
Combination of fluconazole with different natural compounds has shown to be effective against fluconazole-resistant *Candida* isolates. For this reason, the aim of this study was to assess the *in vitro* interaction between fluconazole and magnolol, a polyphenol extracted from *Magnolia officinalis*, or epigallocatechin gallate (EGCG), a polyphenol obtained from *Camellia sinensis*, against clinical isolates of *C. auris*.

**Methods:** We studied 21 *C. auris* isolates from different clinical specimens (seven from blood, seven from oropharynx and six from urine). *Candida parapsilosis* ATCC 22019 and *Candida krusei* ATCC 6258 were used as quality controls. Different fluconazole combinations with magnolol or EGCG were studied. Fluconazole concentrations were selected according to the minimum inhibitory concentration (MIC) determined by the European Committee on Antibiotic Susceptibility Testing (EUCAST) EDef 7.3.1 document. MIC was defined as the lowest drug concentration causing 50% growth reduction after 24 h of incubation. Magnolol and EGCG concentrations ranged from 0.125 to 64 µg/mL. The interaction between fluconazole and magnolol or EGCG were assessed by a checkerboard microdilution method. *In vitro* interactions of drug combinations were interpreted in terms of fractional inhibitory concentration index (FICI) as follows: FICI ≤ 0.5, synergistic; 0.5 < FICI ≤ 1, additive; 1 < FICI ≤ 4, indifferent and FICI > 4 antagonistic.

**Results:** Fluconazole MIC range against *C. auris* was 64 to > 64 µg/mL and magnolol MIC range 16 to > 64 µg/mL; however, combination of both drugs reduced the concentrations necessary for inducing 50% reduction in the growth of the isolates. The effect of the combination of fluconazole with magnolol against seven isolates (33.3%) was interpreted as additive. The effect against the other 14 isolates was interpreted as indifferent, although the fluconazole MICs decreased to 1-16 µg/mL. The combination of fluconazole with EGCG was indifferent against all isolates and did not have any influence in the MICs of fluconazole.

**Conclusion:** The combination of fluconazole with magnolol significantly reduced the fluconazole MICs against all of the *C. auris* isolates, showing additive effect in seven of them. The combination of fluconazole with EGCG showed indifferent effect. **Funding:** This work was co-supported by the Consejería de Educación, Universidades e Investigación (GIC15/78 IT-990-16) of Gobierno Vasco-Eusko Jaurlaritza.

P049

**Resistance to medical importance antifungals in environmental yeasts**

M.L. Scroferneker¹, D. Pagani², C. C. Tavares De Souza², A. M. Vieira³, A.H. Da Silva Hellwig⁴, I. Da S. Rios³, D. Heidrich⁴, A. M. Fuentefria², P. Valente Da Silva²

¹Department Of Microbiology, Immunology And Parasitology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, ²Postgraduate Program In Agricultural And Environmental Microbiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, ³Federal University of Rio Grande do Sul, Porto Alegre, Brazil, ⁴Postgraduate Program In Medicine: Medical Sciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

**Objectives:** The aim of the study was to investigate the presence of potentially pathogenic yeasts from a lagoon in South America.

**Methods:** The lagoon was sampled in the summer of 2019. The water samples were inoculated in acidified YM agar medium (1% glucose, 0.5 % peptone, 0.3 % malt extract, 0.3 % yeast extract, 2 % agar) and incubated at 25 °C for 30 days. Representative yeast colonies were selected and stored in YM medium with 30 % of glycerol at -20 °C and in GYMP agar medium (2 % glucose, 2 % malt extract, 0.5 % yeast extract, 0.2 % monobasic sodium phosphate, 2 % agar) agar slants coated with mineral oil, maintained at 4 °C. We tested 8 environmental yeast strains. The antifungal susceptibility test was performed according to CLSI protocol, to select yeasts with possible resistance to clinical importance antifungals. The yeasts were tested in the antifungal concentrations established for *Candida* species against fluconazole, caspofungin and amphotericin B.

**Results:** Four isolates had MIC ≥ 64 µg/mL to fluconazole, none of them were susceptible to caspofungin, with MIC ≥ 2 µg/mL. To amphotericin B, six strains had MIC ≥ 1µg/mL. Three isolates were considered resistant to all tested antifungals, following the guidelines for *Candida* species.
**Conclusion:** The preliminary findings feature this lagoon environment as a possible resistance genes reservoir and fungal infection source. Further studies are needed to have a better understanding of this environment and the fungal resistance.

P051

A natural substitution in FksB Hot Spot 2 of *Saprochaete capitata* probably related to reduced susceptibility to echinocandins

P. Menéndez-Manjón¹, I. Arrieta-Aguirre¹, M.S. Cuétara², L. López-Soria³, J.C. García-Ruiz⁴, M.D. Moragues¹

¹Enfermería I, Universidad del País Vasco UPV/EHU, Leioa, Spain, ²Microbiología, Hospital Severo Ochoa, Leganés, Madrid, Spain, ³Microbiología, Hospital Universitario Cruces, Barakaldo, Spain, ⁴Biocruces Health Research Institute, Hospital Universitario Cruces, Barakaldo, Spain

**Objectives:** *Saprochaete capitata* is an emerging opportunistic pathogen reported in patients with haematological malignancies. Antifungal clinical breakpoints are not established and therapy remains empirical. *S. capitata* shows reduced susceptibility to echinocandins, which inhibit the enzyme β-1,3-D-glucan synthase (Fks) involved in the cell wall synthesis. Resistance to echinocandins is linked to mutations in two highly conserved regions of Fks, Hot Spot 1 (HS1) and Hot Spot 2 (HS2). We already reported a natural substitution F-to-L in Fks-HS1 that might be linked to this fungus low susceptibility to echinocandins (Arrieta-Aguirre et al., 2018). Mutations in HS2 have also been related to reduced susceptibility to echinocandins. Therefore, we resolved to sequence HS2 of *S. capitata* and search for mutations that could be related to echinocandin resistance.

**Methods:** DNA of 11 strains of *S. capitata* was extracted by mechanical cell disruption and purified with the DNeasy Plant Mini Kit (Qiagen). Since HS1 and HS2 are around 2500 bp apart, we applied a primer walking strategy of smaller fragments sequencing to locate them. With a specific primer from the HS1 sequence and a degenerate primer designed for the HS2 region, we obtained an amplicon for one of the strains that allowed us to design downstream specific primers that led us to HS2. Finally, we designed specific primers to sequence HS2 in the rest of the strains and the sequences were compared with other fungal species.

**Results:** The alignment of Fks-HS2 of *S. capitata* and other species revealed a natural substitution R-to-Q in the fourth position of HS2. The recently published genome of *S. capitata* reveals two Fks, FksA and B. Our sequence corresponds to FksB, which is more similar to Fks2 than Fks1 of other species. In *C. glabrata*, when R in the fourth position of Fks2-HS2 is modified it results in a deep decrease in the susceptibility to echinocandins (Jensen, 2016). Therefore, the natural substitution R-to-Q in FksB-HS2 of *S. capitata* could be related to resistance.

**Conclusion:** *S. capitata* has at least two natural substitutions (F-to-L in Fks-HS1 and R-to-Q in Fks-HS2) that may be involved in its reduced susceptibility to echinocandins. However, the effect of both substitutions still needs to be verified empirically. This work contributes to understanding the molecular basis of antifungal resistance of this fungus and may help to improve its treatment. Financial support: Grupos de Investigación Consolidados of the Basque Government (GIC15/103; IT913-16) and Grant FPI of the Basque Government to PM-M.

P052

Fluconazole resistance in *Candida tropicalis* blood stream isolates collected from 16 Brazilian Medical Centers

A. Melo¹, D. Da Matta¹, L. Favarello¹, C. Oliveira², T. Sukiennik³, F. Telles⁴, M. Nucci⁵, A. Colombo¹

¹Medicine, Federal University of São Paulo, São Paulo, Brazil, ²Medicine, Hospital Universitário do Oeste do Paraná, Cascavel, PR, Cascavel, Brazil, ³Medicine, Santa Casa de Porto Alegre, Porto Alegre, RS, Porto Alegre, Brazil, ⁴Medicine, Hospital de Clínicas da Universidade Federal do Paraná UFPR, Curitiba PR, Curitiba, Brazil, ⁵Medicine, Hospital Universitário da Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ, Rio de Janeiro, Brazil

**Objectives:** *C. tropicalis* is highly prevalent among episodes of candidemia in Latin America. The progressive use of fluconazole and other triazoles in regimens of prophylaxis and empirical therapy
in high-risk patients has led to selective pressure and emergence of secondary resistance among *Candida* spp. The aim of this study was to check for temporal trends in the rates of azole resistance among *C. tropicalis* blood stream isolates collected from patients assisted at 16 Brazilian medical centers at any time within 2007 and 2018.

**Methods:** We selected isolates of *C. tropicalis* stored at the culture collection of Laboratório Especial de Micologia (LEMI) – Universidade Federal de São Paulo, Brazil, that were obtained from candidemic patients admitted in 16 medical centers along the period of study. The isolates were identified at species level by *Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry* (MALDI TOF MS). *In vitro* antifungal susceptibility tests to fluconazole and voriconazole were evaluated by the Clinical & Laboratory Standards Institute (CLSI) microbroth method (documents M27-A3 and M27-S4). For quality control of in vitro assays we used the reference strains *C. parapsilosis* ATCC 22019, *C. krusei* ATCC 6258 and *C. tropicalis* ATCC 750.

**Results:** In order to check for historical trends of azole resistance among *C. tropicalis* isolates, we compared the MIC values generated with fluconazole and voriconazole against 200 blood stream isolates collected at any time within two different periods: P1 (2007-2012) versus P2 (2013-2018). Statistical differences were checked by student T Test. After testing all 200 isolates, 3.5% of *C. tropicalis* strains were considered to be non-susceptible to azoles. As illustrated below, we found resistance rates of *C. tropicalis* against fluconazole of 1.9% and 3.2% in P1 and P2, respectively (p>0.05). In regard to voriconazole, resistance rates of 0 and 1% were documented in P1 and P2, respectively (p>0.05).

**Conclusion:** After testing 200 blood stream isolates of *C. tropicalis* we identified a rate of 3.5% of isolates exhibiting limited susceptibility (SDD/R) to fluconazole and/or voriconazole. Otherwise, we were not able to demonstrate any historical trend in terms of increasing rates of azole resistance among *C. tropicalis* isolates collected along the 2 periods of study.

P053

## Evaluation of spectrophotometric reading of EUCAST antifungal susceptibility testing of *Aspergillus fumigatus*

G. Jan<fnref n="1" />, L. Kristensen<fnref n="2" />, I. Bach Bentsen<fnref n="2" />, M. Nielsen<fnref n="2" />

**Objectives:** The rise in azole resistance in *Aspergillus fumigatus*<fnref n="1" /> highlights the importance of antifungal susceptibility testing. While azole containing agar plates have been shown to have excellent sensitivity and specificity in detecting azole resistance<fnref n="2" />, access to MIC testing is still important. As commercially available methods have serious limitations reference broth microdilution is the only feasible option at present. The EUCAST method for antifungal susceptibility testing of moulds relies on visual reading of MICs which is laborious and time-consuming. A recent study³ suggests that spectrophotometric determination of MICs of azoles may improve objectivity and allow automatisation of the EUCAST E Def 9.3 method. The scope of this study is to evaluate the spectrophotometric metod in our center using seven antifungal drugs.

**Methods:** 22 clinical isolates from *A. fumigatus* species complex were selected for the study. 9 isolates were resistant to at least one azole while 13 were wild type. All isolates were tested against itraconazole, voriconazole, isavuconazole, posaconazole, amphotericin B, natamycin and terbinafine using EUCAST E Def 9.3. Visually determined MICs were read at complete inhibition of growth. Spectrophotometric reading was performed using a Thermo Multiskan FC microplate reader with a 492 nm filter. The OD values of the negative control wells were subtracted from all wells and MICs were determined as the lowest concentration corresponding to either 5% growth compared to the drug free control or as 0.02 OD values above background.

Spectrophotometrically determined MICs were compared to visually determined MICs. For all drugs the quantitative agreement within 0, 1 and 2 two-fold dilutions was calculated. For itraconazole, voriconazole, isavuconazole, posaconazole and amphotericin B categorical agreement was calculated using EUCAST Clinical breakpoints for fungi v. 9.0.

<footnote n="1">¹Department Of Clinical Microbiology, Aarhus University Hospital, Aarhus N, Denmark,</footnote>

<footnote n="2">²Department Of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark</footnote>
## Results:

<caption>Table 1</caption>

Results

<table><thead><tr><th rowspan="2">Drug</th><th colspan="2">Visual MIC (mg/L)</th><th colspan="2">Spectrophotometric MIC (mg/L)</th><th colspan="3">Agreement within two-fold dilution (%)</th><th colspan="4">Categorial agreement and minor error (mE), major error (ME) and very major error (vME) (%)</th></tr><tr><th>Range</th><th>MIC90</th><th>Range</th><th>MIC90</th><th>±0</th><th>±1</th><th>±2</th><th>Agreement</th><th>mE</th><th>ME</th><th>vME</th></tr></thead><tbody><tr><td rowspan="6">Endpoint: 2% growth</td><td>Itraconazole</td><td>0,125 - >8</td><td>>8</td><td>0,125 - >8</td><td>>8</td><td>82</td><td>100</td><td>100</td><td>91</td><td>9%</td><td>0</td><td>0</td></tr><tr><td>Voriconazole</td><td>0,25 - >16</td><td>8</td><td>0,25 - >16</td><td>8</td><td>77</td><td>100</td><td>100</td><td>86</td><td>14</td><td>0</td><td>0</td></tr><tr><td>Isavuconazole</td><td>1 - >16</td><td>16</td><td>1 - >16</td><td>16</td><td>77</td><td>95</td><td>100</td><td>86</td><td>14</td><td>0</td><td>0</td></tr><tr><td>Posaconazole</td><td>0,016 - 1</td><td>1</td><td>0,016 - 1</td><td>1</td><td>64</td><td>100</td><td>100</td><td>91</td><td>9</td><td>0</td><td>0</td></tr><tr><td>AmphotericinB</td><td>0,25 - 1</td><td>0,5</td><td>0,125 - 2</td><td>0,5</td><td>82</td><td>100</td><td>100</td><td>95</td><td>5</td><td>0</td><td>0</td></tr><tr><td>Natamycin</td><td>2 - 8</td><td>4</td><td>2 - 8</td><td>4</td><td>73</td><td>100</td><td>100</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td rowspan="6">Endpoint: 0.02 OD/MB</td><td>Terbinafine</td><td>1 - >16</td><td>16</td><td>1 - >16</td><td>>16</td><td>59</td><td>95</td><td>100</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Itraconazole</td><td>0,125 - >8</td><td>>8</td><td>0,125 - >8</td><td>>8</td><td>64</td><td>95</td><td>95</td><td>91</td><td>9</td><td>0</td><td>0</td></tr><tr><td>Voriconazole</td><td>0,25 - >16</td><td>8</td><td>0,25 - >16</td><td>4</td><td>50</td><td>95</td><td>100</td><td>91</td><td>9</td><td>0</td><td>0</td></tr><tr><td>Isavuconazole</td><td>1 - >16</td><td>16</td><td>0,5 - >16</td><td>8</td><td>50</td><td>95</td><td>100</td><td>82</td><td>18</td><td>0</td><td>0</td></tr><tr><td>Posaconazole</td><td>0,016 - 1</td><td>1</td><td>0,016 - 1</td><td>0,5</td><td>45</td><td>91</td><td>100</td><td>68</td><td>27</td><td>0</td><td>5</td></tr><tr><td>AmphotericinB</td><td>0,25 - 1</td><td>0,5</td><td>0,124 - 2</td><td>0,5</td><td>68</td><td>100</td><td>100</td><td>95</td><td>5</td><td>0</td><td>0</td></tr><tr><td></td><td>Natamycin</td><td>2 - 8</td><td>4</td><td>2 - 8</td><td>2</td><td>18</td><td>100</td><td>100</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td></td><td>Terbinafine</td><td>1 - >16</td><td>16</td><td>1 - >8</td><td>4</td><td>14</td><td>64</td><td>95</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr></tbody></table>

NA = Not applicable

Results are shown in Table 1. When using the 5% growth endpoint we found a high level of essential agreement (95-100%) for all drugs and a high level of categorical agreement (86-95%) for itraconazole, voriconazole, isavuconazole, posaconazole and amphotericin B. Of importance no major or very major errors was observed. When using the 0.02 OD values above background endpoint however we found only 64% essential agreement for terbinafine and a categorical agreement of 68% for posaconazole including 5% very major errors.

**Conclusion:** Spectrophotometric determination of MICs using the EUCAST E-Def 9.3 for A. fumigatus species complex appears to be a promising method for all tested drugs. Highest agreement between visual and spectrophotometric MICs is obtained using the 5% growth endpoint. References 1. van der Linden J et al. Prospective Multicenter International Surveillance of Azole Resistance in Aspergillus fumigatus. Emerg Infect Dis. 2015;21(6):1041-1044. 2. Arendrup MC et al; Multicentre validation of 4-well azole agar plates as a screening method for detection of clinically relevant azole-resistant Aspergillus fumigatus, Journal of Antimicrobial Chemotherapy, Volume 72, Issue 12, Pages 3325-3333 3. Meletiadis, J. et al., Spectrophotometric reading of EUCAST antifungal susceptibility testing of Aspergillus fumigatus, Clinical Microbiology and Infection, Volume 23, Issue 2, 98 - 103

## P054

### Molecular mechanism and frequency of olorofim resistance in *Aspergillus fumigatus*

J. Buil¹, J. Oliver², D. Law², M. Tehupeiory-Kooreman¹, J. Rex², M. Hokken¹, W. Melchers¹, M. Birch², P.E. Verweij¹

¹Medical Microbiology, Radboudumc, Nijmegen, Netherlands, ²F2G Ltd, Manchester, United Kingdom

**Objectives:** Olorofim (OLO) is a new antifungal agent with a novel mechanism of action, targeting dihydroorotate dehydrogenase (DHODH) in the *de novo* pyrimidine biosynthesis pathway. It is active against both azole-susceptible and azole-resistant strains of *Aspergillus fumigatus*. Thus, OLO may be an important treatment option in patients with *Aspergillus* disease, including azole-resistant cases. Azole resistance selection occurs in >10% of patients with chronic pulmonary aspergillosis during itraconazole (ITZ) therapy. In this study, we analysed the frequency of resistance mutation induction of OLO in *A. fumigatus* in three different strains using two different methods. The underlying OLO resistance mechanism was also investigated.

**Methods:** Method 1: From two *A. fumigatus* isolates, AZN 8196 and V139-36, 10 single colonies were separately inoculated onto Sabouraud agar and incubated at 37°C for 96h. From each culture 1 x 10⁸ conidia were applied to 6 RPMI agar plates containing either 0.5 mg/L OLO or 8 mg/L ITZ. Plates
were incubated at 37°C for up to 7 days. Colonies growing on drug-containing plates were subcultured on RPMI agar containing 0.5 mg/L OLO or 8 mg/L ITZ to confirm resistance. Method 2: Spore stocks of *A. fumigatus* strain Af293 were prepared and inoculated onto yeast nitrogen base with glucose agar (YNBG) containing 0.25 mg/L OLO. A total of 8 x 10⁹ spores were inoculated into 12 x 100 ml YNBG-OLO agar plates that were subsequently incubated for 5 days at 35°C. Colonies growing on drug-containing plates were subcultured on YNBG-OLO to confirm resistance. The mean rate of resistance was calculated for each isolate. The *pyrE* gene which codes for DHODH was sequenced for initial OLO-susceptible strains and -resistant progeny isolates and analysed for mutations.

**Results:** Method 1: The resistance rate of OLO was 1 in 5 x 10⁷ (frequency of 2 x 10⁻⁸) for AZN 8196 compared to 1 in 5 x 10⁶ (2 x 10⁻⁷) for ITZ. For V139-36 the resistance rate of OLO was 1 in 9 x 10⁷ (1.1 x 10⁻⁸) while the rate for ITZ was 1 in 8 x 10⁶ (1.3 x 10⁻⁷) Method 2: Resistance to OLO in Af293 occurred at a rate of 1 in 6 x 10⁸ (frequency of 1.7 x 10⁻⁹). Sequencing of *pyrE* revealed a hotspot for resistance mutations in the *pyrE* gene for OLO resistance in *A. fumigatus* at locus Gly119. Four amino acid substitutions were found; G119V, G119C, G119S and G119A. A single OLO-resistant mutant was obtained that carried the wild type DHODH sequence, but this mutant was slow growing compared with parent.

**Conclusion:** We demonstrate that OLO resistance can be selected in *A. fumigatus* and can be mediated by mutations in the *pyrE* gene at locus G119. The frequency of resistance varied from approximately 2 x 10⁻⁸ to 1.7 x 10⁻⁹ for spontaneous mutations. The resistance rate of OLO was 5 to 10-fold lower compared to ITZ. The low resistance rate found in these experiments indicates that resistance selection may be less frequent in patients with *Aspergillus* diseases treated with OLO.

P055

Comparison of killing activity of rezafungin, anidulafungin, caspofungin and micafungin against
*Candida auris* in the presence and absence of serum

Z. Tóth¹, L. Forgács¹, J.B. Locke², G. Kardos¹, F. Nagy¹, R. Kovács¹, A. Borman³, L. Majoros¹

¹Department Of Medical Microbiology, University of Debrecen, Debrecen, Hungary, ²Cidara Therapeutics, San Diego, United States of America, ³Public Health England UK National Mycology Reference Laboratory, Bristol, United Kingdom

**Objectives:** *Candida auris* is an emerging, difficult-to-treat multiresistant pathogen against which echinocandins are the recommended standard-of-care treatment. Rezafungin is a next-generation echinocandin with similar *in vitro* activity to existing echinocandins yet it attains much higher *in vivo* concentrations and exposures due to its extended half-life and front-loaded dosing paradigm. Because *in vitro* killing data with existing echinocandins are limited against *C. auris* and are lacking for rezafungin, we compared rezafungin to anidulafungin, caspofungin, and micafungin in time-kill assays against *C. auris* isolates in standard RPMI-1640 medium and also investigated the impact of serum on *in vitro* killing trends.

**Methods:** Two *C. auris* clinical isolates from each clade (Japanese/Korean, South Asian/Indian and South African, obtained from the National Mycology Reference Laboratory, UK) were tested. Both South African isolates were autoaggregative. MICs in RPMI-1640 +/-50% human serum were determined using the standard broth macrodilution method (CLSI M27 Ed4). Time-kill studies with the four echinocandins were performed from 0.25 to 32 mg/L in both media, and killing rates were compared. Positive *k* values indicate killing; negative values indicate growth.

**Results:** MIC values were higher in 50% serum than in RPMI-1640 alone (Table 1). In RPMI-1640, at 1xMIC or higher concentrations all four echinocandins showed only fungistatic effect. The highest *k* value with rezafungin (1.34 1/h) was at 32 mg/L against isolate 209. However, none of the echinocandins produced any CFU decrease against aggregating isolates (*k* values in cases of isolates 2 and 204 were always negative). Similar results were found in cases of caspofungin and micafungin against isolates 12, 15 and 27, and isolate 15, respectively. Re-growth was frequently observed for all four echinocandins. In 50% serum, growth rates were significantly lower. At 1xMIC or higher concentrations, echinocandins showed concentration-dependent killing activity, however, this CFU
reduction never reached the fungicidal threshold. The highest *k* values were 0.34, 0.33, 0.34 and 0.29 1/h for rezafungin, anidulafungin, caspofungin and micafungin, respectively. However, *k* values were always negative at 1-32 mg/L, in the case of isolate 2 (an autoaggregative isolate) for all echinocandins and in cases of isolates 27 and 204 for caspofungin. Table 1. MIC values for rezafungin and echinocandin comparators against *C. auris* strains in the presence and absence of 50% serum

<table><thead><tr><th rowspan="2">Clade</th><th rowspan="2">Isolate</th><th colspan="4">MIC values in RPMI/RPMI + 50% serum (mg/L)</th></tr><tr><th>rezafungin</th><th>anidulafungin</th><th>caspofungin</th><th>micafungin</th></tr></thead><tbody><tr><td rowspan="2">South Asia/ India</td><td>12</td><td>0.03-0.06/0.5</td><td>0.12/0.5-1</td><td>1/0.5-1</td><td>0.12-0.25/1</td></tr><tr><td>27</td><td>0.12/1</td><td>0.12/2</td><td>0.5/2</td><td>0.25/2</td></tr><tr><td rowspan="2">Japan/Korea</td><td>209</td><td>0.06/0.5</td><td>0.03/0.5-1</td><td>0.25/0.5</td><td>0.12/2-4</td></tr><tr><td>15</td><td>0.03/0.25-0.5</td><td>0.03-0.06/1</td><td>0.5-1/1</td><td>0.12/2</td></tr><tr><td rowspan="2">South Africa</td><td>204</td><td>0.06/0.25-0.5</td><td>0.03/1</td><td>0.25/0.5-1</td><td>0.12/2</td></tr><tr><td>2</td><td>0.12/1-2</td><td>0.03/1-2</td><td>0.5/1</td><td>0.25/2-4</td></tr></tbody></table>

**Conclusion:** Killing activity in RPMI-1640 alone was less consistently positive than in 50% serum and only fungistatic activity was detected. Aggregating isolates were less susceptible to echinocandins than non-aggregating isolates. Rezafungin showed the same or better activity than anidulafungin and micafungin at clinically attainable concentrations. The trend towards stronger killing activity in the presence of serum helps to account for the disconnect between the modest time-kill activity of echinocandins *in vitro* versus their strong efficacy against *C. auris* *in vivo*, as rezafungin has previously demonstrated in animal models.

P056

Frequency of paradoxical and trailing effects with rezafungin, anidulafungin, caspofungin and micafungin against Candida species

L. Forgács¹, Z. Tóth¹, J.B. Locke², F. Nagy¹, B. Balázs¹, E. Prépost¹, G. Kardos¹, R. Kovács¹, A. Szekely³, A. Borman³, L. Majoros¹

¹Department Of Medical Microbiology, University of Debrecen, Debrecen, Hungary, ²Cidara Therapeutics, San Diego, United States of America, ³Public Health England UK National Mycology Reference Laboratory, Bristol, United Kingdom

**Objectives:** Currently, echinocandins are the first-line agents for treatment of invasive *Candida* infections. Echinocandins demonstrate excellent efficacy and fungicidal activity against *Candida* species *in vivo*. However, echinocandins can exhibit some disconnected growth phenomena *in vitro*, such as paradoxical effect (PE) where activity decreases at higher drug concentrations and the trailing effect (TE) where complete growth inhibition is never achieved or occurs at dilutions well above the 50% endpoint used to determine MIC values. PE and TE can significantly alter the determination/interpretation of MIC values and impact other *in vitro* assays. Rezafungin is a next-generation echinocandin capable of attaining high concentrations and exposures *in vivo*, exceeding those measured for the three approved echinocandins, due to its long half-life and front-loaded dosing regimen. Given the relevance of evaluating higher drug concentrations for rezafungin and that rezafungin PE/TE trends have not yet been characterized, herein we compared the occurrence of PE and TE for rezafungin with data generated in parallel for caspofungin, micafungin and anidulafungin against clinically important *Candida* species.

**Methods:** We tested 365 non-duplicate Hungarian clinical isolates belonging to 13 species (see Table). Isolates were collected between 2005 and 2018 and were identified with MALDI Biotyper (Bruker, Bremen, Germany). *C. auris* were obtained from National Mycology Reference Laboratory, UK. MICs were determined by BMD method according to CLSI (M27 Ed4) in RPMI-1640. Concentration ranges of antifungals were 0.06-32 mg/L. PE was defined as visible growth occurring at higher but not at lower supra-MIC concentrations. TE was defined when yeasts show reduced but observable growth in supra-MIC concentrations.
**Results:** The percentages of isolates showing PE or TE after 48 hours are shown in the Table. Cumulative percentage of PE plus TE was the highest for caspofungin (47.7%), followed by anidulafungin (35.9%), micafungin (25.8%) and rezafungin (17.7%). PE was observed most frequently for caspofungin (31.4%) but was uncommon for rezafungin (3.1%). PE with caspofungin, micafungin or anidulafungin occurred most frequently in cases of *C. tropicalis*, *C. albicans*, *C. orthopsilosis* and *C. inconspicua* but was never found in case of *C. krusei*. The majority of *C. auris* and *C. dubliniensis* isolates showed TE for all four echinocandins. Table. Percentages of *Candida* spp. isolates demonstrating paradoxical effect (PE) or trailing effect (TE)

<table><thead><tr><th rowspan="2">Species (n)</th><th colspan="2">rezafungin</th><th colspan="2">anidulafungin</th><th colspan="2">micafungin</th><th colspan="2">caspofungin</th></tr><tr><th>PE</th><th>TE</th><th>PE</th><th>TE</th><th>PE</th><th>TE</th><th>PE</th><th>TE</th></tr></thead><tbody><tr><td><i>C. albicans</i> (100)</td><td>5</td><td>7</td><td>33</td><td>6</td><td>7</td><td>1</td><td>48</td><td>3</td></tr><tr><td><i>C. tropicalis</i> (50)</td><td>12</td><td>46</td><td>32</td><td>40</td><td>62</td><td>14</td><td>46</td><td>42</td></tr><tr><td><i>C. glabrata</i> (30)</td><td></td><td></td><td>23.8</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><i>C. parapsilosis</i> (26)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>76.9</td><td>11.5</td></tr><tr><td><i>C. krusei</i> (30)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><i>C. auris</i> (16)</td><td></td><td>50</td><td></td><td></td><td>63</td><td></td><td>56</td><td>100</td></tr><tr><td><i>C. kefyr</i> (16)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>12.5</td><td></td></tr><tr><td><i>C. dubliniensis</i> (22)</td><td></td><td>59.1</td><td>4.5</td><td>59.1</td><td></td><td>68.2</td><td>18.2</td><td>54.5</td></tr><tr><td><i>C. lusitaniae</i> (27)</td><td></td><td></td><td></td><td></td><td>3.7</td><td></td><td>3.7</td><td></td></tr><tr><td><i>C. guilliermondii</i> (16)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>6.3</td><td>75</td></tr><tr><td><i>C. orthopsilosis</i> (8)</td><td></td><td></td><td>12.5</td><td></td><td>75</td><td></td><td>62.5</td><td>25</td></tr><tr><td><i>C. metapsilosis</i> (11)</td><td></td><td></td><td>27.2</td><td>9.1</td><td>27.2</td><td>27.2</td><td>36.4</td><td>9.1</td></tr><tr><td><i>C. inconspicua</i> (13)</td><td></td><td></td><td>76.9</td><td>15.4</td><td>61.6</td><td></td><td>38.5</td><td>30.8</td></tr></tbody><tfoot><tr><td><b>Total (365)</b></td><td><b>3.1</b></td><td><b>14.6</b></td><td><b>21</b></td><td><b>14.9</b></td><td><b>15.8</b></td><td><b>10</b></td><td><b>31.4</b></td><td><b>16.3</b></td></tr></tfoot></table>

**Conclusion:** PE or TE was widely observed among *Candida* species and were echinocandin- and species-dependent. PE was frequently found with caspofungin, micafungin and anidulafungin but not with rezafungin. This study is the first to characterize PE and TE trends for rezafungin and helps to inform future *in vitro* work with this novel echinocandin.

P057

In vitro antifungal susceptibility testing of cryptic species of *Aspergillus*: a multi-centre study

S. Imbert¹, A.C. Normand², F. Gabriel³, C. Bonnal⁴, L. Lachaud⁵, D. Costa⁶, S. Cassaing⁷, L. Hasseine⁸, L. Kristensen⁹, C. Schuttler¹⁰, H. Raberin¹¹, S. Brun¹², R. Piarroux¹, A. Fekkar¹

¹Laboratoire De Parasitologie-mycologie, APHP La Pitié-Salpêtrière, PARIS, France, ²Ap-hp, Groupe Hospitalier Pitié-salpêtrière, Service de Parasitologie Mycologie, Paris, France, ³Parasitology Mycology, CHU de Bordeaux, Bordeaux, France, ⁴Laboratoire De Parasitologie Mycologie, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, PARIS, France, ⁵Laboratoire De Parasitologie-mycologie, CHU de Montpellier, Montpellier, France, ⁶Laboratoire De Parasitologie-mycologie, CHU de Rouen, Rouen, France, ⁷CHU de Toulouse, Toulouse, France, ⁸Laboratoire De Parasitologie Mycologie, CHU DE NICE, NICE, France, ⁹Department Of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark, ¹⁰Laboratoire BioParisOuest, Levallois Peret, France, ¹¹CHU Saint Etienne, Saint Etienne, France, ¹²Parasitology-mycology, Avicenne Hospital, Bobigny, France

**Objectives:** In recent years, *Aspergillus* species taxonomy has been revolutionized with the introduction of the concepts of cryptic species and species complex. However, these “new” species remain usually rare and their characteristics, including antifungal susceptibility, are little known. Thus, the aim of this study was to collect a large number of isolates per cryptic species and to assess their antifungal susceptibility.

**Methods:** From September 2017, users of the MSI application, a free online MALDI-TOF mass-spectrometry database, were asked to send to our University Hospital, isolates they identified as cryptic species of *Aspergillus*. Identification at the species level was confirmed by sequencing a part of beta-tubulin and calmodulin genes. Antifungal susceptibility for azole drugs (itraconazole, voriconazole, posaconazole and isavuconazole) and amphotericin B was assessed by EUCAST broth microdilution reference method.
**Results:** During the study period, 192 isolates were collected from 11 centers in France and Denmark and were assessed for antifungal susceptibility. According to sequence-based identification, isolates corresponded to 43 species, belonging to 6 species complexes. *Nidulantes* species complex was the most represented (53 isolates, 12 species), followed by the species complexes *Circumdati* (43 isolates, 10 species) and *Fumigati* (40 isolates, 8 species). The last species complexes represented were *Usti* (23 isolates, 4 species), *Flavi* (19 isolates, 5 species) and *Terrei* (14 isolates, 4 species). Regarding the 3 main species complexes, diversity in antifungal susceptibility between cryptic species was observed. Indeed, inside the *Fumigati* complex *A. thermomutatus*, *A. lentulus*, *A. udagawae*, *A. fischeri* and (15, 7, 3 and 3 isolates respectively) showed pan-azole high minimal inhibitory concentrations (MICs), whereas *A. hiratsukae* (8 isolates) showed low MICs. All these species had low MICs for amphotericin B, except *A. lentulus* with MICs ³⁴ mg/L. Regarding the *Circumdati* species complex, *A. westerdijkiae* (12 isolates) showed the highest MICs for amphotericin B (> 16 mg/L), whereas *A. sclerotiorum* (17 isolates) showed lower MICs (MIC₅₀ = 4 mg/L). In contrast *A. sclerotiorum* showed pan-azole high MICs, whereas azole MICs where much lower for *A. westerdijkiae*. Finally, inside the *Nidulantes* complex, all species showed low MICs for all drugs, except *A. creberand* *A. unguis* (6 and 2 isolates respectively) which showed high MICs (> 8 mg/L) for itraconazole only. MICs regarding the 3 other species complexes studied are shown in table 1.

<table><caption>Table 1: MIC50 for the main species identified in the study</caption><thead><tr><th rowspan="2">Section</th><th rowspan="2">Cryptic species</th><th colspan="5">MIC50 (mg/l)</th></tr><tr><th>Itraconazole</th><th>Voriconazole</th><th>Posaconazole</th><th>Isavuconazole</th><th>Amphotericin B</th></tr></thead><tbody><tr><td rowspan="2">Circumdati</td><td>A. sclerotiorum (n=17)</td><td>>8</td><td>4</td><td>2</td><td>4</td><td>2</td></tr><tr><td>A. westerdijkiae (n=12)</td><td>0.5</td><td>0.5</td><td>0.5</td><td>0.5</td><td>>16</td></tr><tr><td>Flavi</td><td>A. alliaceus (n=12)</td><td>0.125</td><td>0.25</td><td>0.06</td><td>0.5</td><td>>16</td></tr><tr><td rowspan="5">Fumigati</td><td>A. thermomutatus (n=15)</td><td>>8</td><td>4</td><td>0.5</td><td>2</td><td>0.5</td></tr><tr><td>A. hiratsukae (n=8)</td><td>0.5</td><td>1</td><td>0.25</td><td>0.5</td><td>0.5</td></tr><tr><td>A. lentulus (n=7)</td><td>>8</td><td>4</td><td>0.5</td><td>2</td><td>4</td></tr><tr><td>A. udagawae (n=3)</td><td>>8</td><td>4</td><td>0.5</td><td>2</td><td>2</td></tr><tr><td>A. fischeri (n=3)</td><td>>8</td><td>2</td><td>0.25</td><td>2</td><td>1</td></tr><tr><td rowspan="5">Nidulantes</td><td>A. latus (n=26)</td><td>0.5</td><td>0.5</td><td>0.25</td><td>0.25</td><td>0.5</td></tr><tr><td>A. spinulosporus (n=5)</td><td>0.25</td><td>0.5</td><td>0.25</td><td>0.25</td><td>0.5</td></tr><tr><td>A. creber (n=6)</td><td>>8</td><td>1</td><td>0.5</td><td>0.5</td><td>1</td></tr><tr><td>A. jensenii (n=5)</td><td>0.5</td><td>1</td><td>0.5</td><td>1</td><td>1</td></tr><tr><td>A. unguis (n=2)</td><td>>8</td><td>0.5</td><td>0.5</td><td>0.5</td><td>0.5</td></tr><tr><td>Terrei</td><td>A. alabamensis (n=8)</td><td>0.25</td><td>2</td><td>0.125</td><td>1</td><td>2</td></tr><tr><td>Usti</td><td>A. calidoustus (n=18)</td><td>>8</td><td>8</td><td>>8</td><td>4</td><td>1</td></tr></tbody></table>

**Conclusion:** This study brings evidence on the diversity of antifungal susceptibility pattern inside the same species complex. However, including more isolates is needed to confirm these preliminary results. Nevertheless, this underline the importance of an accurate and easy-to-perform identification of *Aspergillus* at the species level for the management of *Aspergillus* diseases and the conduct of epidemiological studies.

P058

## Species identification, *in vitro* antifungal susceptibility testing and human pathogenicity of Fusarium species: a European multicenter study

S. Imbert<fnref n="1" />, A.C. Normand<fnref n="1" />, S. Brun², S. Ranque³, L. Hasseine⁴, D. Costa⁵, S. Cassaing⁶, E. Chryssanthou⁷, C. Bonnal⁸, L. Delhaes⁹, E. Rubio¹⁰, N. Bourgeois¹¹, C. Schuttler¹², J. Guitard¹³, M. Sautour¹⁴, G. Jost¹⁵, M. Brandenberger¹⁶, L. Kristensen¹⁷, B. Sendid¹⁸, C. Gronfors Seeth¹⁹, E. De Laere²⁰, M. Hendrickx²¹, V. Sainte Rose<fnref n="1" />, R. Piarroux<fnref n="1" />, A. Fekkar<fnref n="1" />

<footnote n="1">¹Laboratoire De Parasitologie-mycologie, APHP La Pitié Salpétrière, PARIS, France, ²Parasitology-mycology, Avicenne Hospital, Bobigny, France, ³IHU-Méditerranée Infection, Marseille, France, ⁴Laboratoire De Parasitologie Mycologie, CHU DE NICE, NICE, France, ⁵Laboratoire De Parasitologie-mycologie, CHU de Rouen, Rouen, France, ⁶CHU de Toulouse, Toulouse, France, ⁷Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden, ⁸Laboratoire De Parasitologie Mycologie, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, PARIS, France, ⁹Laboratoire De Parasitologie-mycologie, CHU de Bordeaux, Bordeaux, France, ¹⁰Barcelona hospital, Barcelona, Spain, ¹¹Laboratoire De Parasitologie-mycologie, CHU de Montpellier, Montpellier,</footnote>
France, ¹²Laboratoire BioParisOuest, Levallois Peret, France, ¹³Parasitology-mycology, APHP, hôpital St Antoine, Paris, France, ¹⁴Laboratoire Parasitology Mycology, CHU Dijon, Dijon, France, ¹⁵Dianalabs, Geneve, Switzerland, ¹⁶synlab, Lucerne, Switzerland, ¹⁷Department Of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark, ¹⁸Laboratory Of Mycology And Parasitology, University Hospital of Lille, Lille Cedex, France, ¹⁹Clinical Microbiology, Karolinska University Hospital, karolinska Institutet, Stockholm, Sweden, ²⁰AZ Roeselare, Roeselare, Belgium, ²¹Mycology, Scientific Institute of Public Health, Brussels, Belgium

**Objectives:** Fungi belonging to the genus *Fusarium* are responsible of a large number of infections that may be difficult to treat because of a low susceptibility to antifungal drugs available. However, in recent years, fungal taxonomy has been revolutionized with the introduction of the concepts of cryptic species and species complex. This is particularly true for the genus *Fusarium*, which taxonomy remains partially unclear. In consequence, pathogenicity and antifungal susceptibility specific to each *Fusarium* species is largely unknown. Thus, the aim of this study was to collect a large number of isolates per species, to collect clinical data on their pathogenicity and to assess their antifungal susceptibility.

**Methods:** During 1 year (January-December 2018), users of the MSI application, a free online MALDI-TOF mass-spectrometry database, were asked when they identified a *Fusarium* sp. to send prospectively to our University Hospital laboratory the isolates and to collect clinical data. Identification at the species level was confirmed by sequencing a part of *TEF1α* gene. Antifungal susceptibility for azole drugs (voriconazole, posaconazole and isavuconazole), terbinafin and amphotericin B was assessed by measuring minimal inhibitory concentrations (MICs) by EUCAST broth microdilution reference method.

**Results:** During the study period, 145 isolates from 20 centers in 6 European countries were collected. According to sequence-based identification, 23 species were identified and were distributed in 6 species complexes (SC) whose main ones are *Fusarium oxysporum* SC (FOSC, 54 isolates), *Fusarium solani* SC (FSSC, 48 isolates) and *Fusarium fujikoroi* SC (FFSC, 36 isolates). Regarding these 3 main species complexes, minimal inhibitory concentrations which inhibited 50% of our isolates (MIC50) were high for all azole drugs, FSSC showing the highest MICs for voriconazole. For amphotericin B, MIC50 were not high (1 mg/l) and no differences were observed between complexes. Finally, terbinafine susceptibility seemed to be related to the species complex. Indeed, FOSC and FSSC showed the highest MICs (MIC50 = 16 mg/l) whereas FFSC showed lower MICs (MIC50 = 2 mg/l). MICs results are shown in Table 1.

<table><caption>Table 1: MICs results for the 145 *Fusarium* isolates studied</caption><thead><tr><th></th><th></th><th>Terbinafin<br>(mg/L)</th><th>Voriconazole<br>(mg/L)</th><th>Posaconazole<br>(mg/L)</th><th>Isavuconazole<br>(mg/L)</th><th>Amphotericin B<br>(mg/L)</th></tr></thead><tbody><tr><td rowspan="3">FOCS (N=54)</td><td>Geometric mean</td><td>11</td><td>6</td><td>6,8</td><td>10,6</td><td>1,1</td></tr><tr><td>MIC50</td><td>16</td><td>4</td><td>8</td><td>16</td><td>1</td></tr><tr><td>Range</td><td>[0,5-16]</td><td>[1-16]</td><td>[0,5-8]</td><td>[1-16]</td><td>[0,25-16]</td></tr><tr><td rowspan="3">FSSC (N=48)</td><td>Geometric mean</td><td>16</td><td>14,3</td><td>8</td><td>16</td><td>1,1</td></tr><tr><td>MIC50</td><td>16</td><td>16</td><td>8</td><td>16</td><td>1</td></tr><tr><td>Range</td><td>[16-16]</td><td>[4-16]</td><td>[8-8]</td><td>[16-16]</td><td>[0,125-16]</td></tr><tr><td rowspan="3">FFCS (N=36)</td><td>Geometric mean</td><td>1,9</td><td>6,1</td><td>5</td><td>12,3</td><td>1,1</td></tr><tr><td>MIC50</td><td>2</td><td>8</td><td>8</td><td>16</td><td>1</td></tr><tr><td>Range</td><td>[0,5-16]</td><td>[2-16]</td><td>[0,5-8]</td><td>[2-16]</td><td>[0,25-16]</td></tr><tr><td>FDCS (N=4)</td><td>Range</td><td>[1-16]</td><td>[4-16]</td><td>[8-8]</td><td>[16-16]</td><td>[0,5-4]</td></tr><tr><td>FICS (N=2)</td><td>Range</td><td>[1-16]</td><td>[1-4]</td><td>[0,5-8]</td><td>[4-16]</td><td>[1-1]</td></tr><tr><td>FRCS (N=1)</td><td>MIC</td><td>2</td><td>8</td><td>8</td><td>16</td><td>8</td></tr></tbody></table>

Among isolates collected, clinical data were available for 117 isolates. They were responsible for 11 invasive/disseminated infections, 19 keratitis and 40 superficial infections (34 onychomycosis and 6 intertrigo). Sixteen isolates were considered as pulmonary airways colonization, whereas 31 were considered as culture contaminants. The three main species complexes identified in the study were represented in each category.
**Conclusion:** This study brings evidence on the diversity of *Fusarium* species implicated in human diseases and their differences in terms of *in vitro* antifungal susceptibility, mainly for terbinafin. This underlines the importance of an accurate and easy identification of *Fusarium* species, at least at the complex level, to improve therapeutic management of *Fusarium*-related diseases.

P059

## Tacrolimus and not cyclosporin A or sirolimus interacts synergistically *in vitro* with isavuconazole against *Aspergillus* species

P. Schwarz¹,², E. Dannaoui³,⁴

¹Department Of Internal Medicine - Respiratory And Critical Care Medicine, University Hospital Marburg, Marburg, Germany, ²Center For Invasive Mycoses And Antifungals, Philipps University Marburg, Marburg, Germany, ³Hôpital Européen Georges Pompidou, Laboratoire de Parasitologie-Mycologie, Paris, France, ⁴Working Group Dynamyc, Faculté de Médecine, Hôpital Henri Mondor, Créteil, France

**Objectives:** Invasive aspergillosis is a devastating disease in immunocompromised patients. It mostly affects patients with hematological malignancies, especially those with severe and prolonged neutropenia, but is also encountered in solid organ transplant recipients. Immunosuppressive agents are used as anti-rejection drugs in organ transplant patients, but besides their immunosuppressive activity, these drugs also possess intrinsic antifungal activity. The aim of the present study was to assess the *in vitro* interaction of the broad-spectrum azole isavuconazole with the calcineurin inhibitors (e.g. tacrolimus or cyclosporin A) or the mTOR pathway inhibitor (e.g. sirolimus) against the most important *Aspergillus* species responsible for human disease.

**Methods:** A panel of 30 Aspergillus isolates belonging to 5 species responsible for human invasive aspergillosis was used for the experiments (10 *Aspergillus fumigatus*, 5 *Aspergillus flavus*, 5 *Aspergillus terreus*, 5 *Aspergillus nidulans*, and 5 *Aspergillus niger*). Combinations of isavuconazole with cyclosporine A, tacrolimus or sirolimus were tested by a broth microdilution checkerboard technique based on the EUCAST reference methodology. Plates were read spectrophotometrically, and 90% of inhibition was used as an endpoint for both, the drugs alone and in combination. Results were interpreted with the fractional inhibitory concentration index (FICI). Drug interactions were defined as synergistic (FICI≤0.5), indifferent (FICI>0.5≤4) or antagonistic (FICI>4).

**Results:** Isavuconazole MICs ranged from 0.25 to 16 µg/ml. When tested alone, immunosuppressive drugs showed poor activity with MICs < 16 µg/ml for only 2, and 3 isolates for tacrolimus and cyclosporin A, respectively. Combinations of isavuconazole with tacrolimus, cyclosporine A or sirolimus were synergistic for 53, 20, and 10% of the tested isolates, respectively. There were some differences between species (Table). Antagonism was never seen.

<table><thead><tr><th rowspan="2">Species (number of isolates)</th><th colspan="3">percentage of synergistic interaction with</th></tr><tr><th>tacrolimus</th><th>cyclosporin A</th><th>sirolimus</th></tr></thead><tbody><tr><td><i>Aspergillus fumigatus</i> (10)</td><td>40</td><td>0</td><td>0</td></tr><tr><td><i>Aspergillus flavus</i> (5)</td><td>60</td><td>0</td><td>40</td></tr><tr><td><i>Aspergillus terreus</i> (5)</td><td>80</td><td>40</td><td>20</td></tr><tr><td><i>Aspergillus nidulans</i> (5)</td><td>20</td><td>0</td><td>0</td></tr><tr><td><i>Aspergillus niger</i> (5)</td><td>80</td><td>80</td><td>0</td></tr><tr><td>All (30)</td><td>53</td><td>20</td><td>10</td></tr></tbody></table>

**Conclusion:** Calcineurin is a key enzyme in the calcium-dependent signal transduction pathways of eukaryotes. Antifungal drug resistance and virulence of several pathogenic fungi, including *Aspergillus* species, is regulated by this pathway. Results of the present study showed synergistic interactions between isavuconazole and tacrolimus and underline the role of the calcineurin pathway as a target for the development of new antifungal drugs.

P060
**Synergistic antifungal effect of fluconazole combined with pure culture extract of *Candida albicans* against *Trichophyton mentagrophytes***

T. Lemes¹, B. De Almeida², M. Caetano³, C.R. Polaquini⁴, L.O. Regasini⁵, T. Maschio-Lima³, N. Brizzotti¹,⁶, E. Castilho⁶, J.P. Siqueira⁷, M. Marques⁶, M. Almeida⁸

¹Microbiologia, Unesp, São José do Rio Preto, Brazil, ²Unesp, São José do Rio Preto, Brazil, ³UNESP, São José do Rio Preto, Brazil, ⁴São Paulo State University - Institute of Biosciences, Humanities and Exact Sciences (UNESP - IBILCE), São José do Rio Preto, Brazil, ⁵Chemistry And Environmental Sciences, UNESP (São Paulo State University), São José do Rio Preto, Brazil, ⁶FAMERP, São José do Rio Preto, Brazil, ⁷Infeciosas Disease, FAMERP, São José do Rio Preto, Brazil, ⁸Infectious Disease, Medical School, São José do Rio Preto, Brazil

**Objectives:** The present study evaluated the synergistic interaction between a pure culture extract of *Candida albicans* and fluconazole against *Trichophyton mentagrophytes*.

**Methods:** This study used clinical strains of *C. albicans* from the Laboratory of Microbiology of the Medical School in São José do Rio Preto (FAMERP), Brazil. A 500-mL Inoculum prepared in Sabouraud Dextrose Broth was filtered through a 0.2 µm millipore membrane and separated using ethyl acetate as a counter-phase. The ethyl acetate phase was completely dried using a rotary evaporator and subsequently solubilized in sterile distilled water with 10% dimethyl sulfoxide (DMSO). Minimal Inhibitory Concentration (MIC) tests were performed for *T. mentagrophytes* strains following the Clinical and Laboratory Standards Institute (CLSI) M38-A2 guidelines. After obtaining the extract's MIC, a checkerboard trial with fluconazole was performed to evaluate the synergistic interaction with the extract based on the calculation of the fractional inhibitory concentration index (ICIF) = (MIC fluconazole in the mix / MIC fluconazole alone) + MIC extract in the mix / MIC extract isolated). The synergistic interaction was classified using the method described by Kumar et al., where values ≤ 0.5 indicate significant interactions.

**Results:** The results obtained for the *C. albicans* extract's MIC against the *T. mentagrophytes* strain and fluconazole in isolation were 1000 µg/mL and 16 µg/mL, respectively. However, when the extract was used in combination with fluconazole, the MIC value was 0,001 µg/mL and of fluconazole it was 2 µg/mL with an ICIF value of 0,01.

**Conclusion:** The extract of *C. albicans* in isolation shows antifungal activity against *T. mentagrophytes*, which may influence the laboratory diagnosis of mixed infections. Furthermore, the action of this extract is greater in association with an azole derivative, thus proving synergy. In the future, the isolation and identification of extract compounds may allow new therapeutic approaches in the control of fungal infections.

P061

**Screening of antifungal susceptibility of *Malassezia pachydermatis* isolates from dogs using disk diffusion test**

S. Hadina¹, I. Benvin², Z. Štritof¹, J. Habuš¹, V. Stevanović¹, M. Perharić¹, K. Martinković¹, V. Mojčec¹, L. Barbić¹, Z. Milas¹, V. Starešina¹, N. Turk¹, L. Pinter¹

¹Department Of Microbiology And Infectious Diseases With Clinic, Faculty of Veterinary Medicine, The University of Zagreb, Zagreb, Croatia, ²Faculty of Veterinary Medicine, The University of Zagreb, Zagreb, Croatia

**Objectives:** *Malassezia pachydermatis* is lipophilic yeast commonly seen in skin and ear diseases of dogs. Lately, different studies are describing the appearance of reduced susceptibility of *M. pachydermatis* strains. In addition, reports about its zoonotic potential and emerged number of *Malassezia* yeast resistance in human medicine, highlight the need for study of its antifungal susceptibility. The problem of surveillance of potential resistance is lying in no established reference method for antifungal susceptibility testing and no interpretative criteria for this yeast. Various studies used different methodologies and interpreted obtained results based on different recommendations. Modified microdilution method is considered the most accurate, but at the same time more expensive and technically demanding to perform in clinical laboratories. Recent study demonstrated good correlation of results obtained by modified CLSI M44-A2 disk diffusion and
broth microdilution method, indicating its suitability for *in vitro* susceptibility testing of *M. pachydermatis* in epidemiological studies (Pasquetti et al., 2015.). The aim of this study was to screen antifungal susceptibility of *M. pachydermatis* for miconazole (MCZ) and clotrimazole (CTZ) using disk diffusion method.

**Methods:** A total of 96 isolates of *M. pachydermatis* were recovered from animals with clinical signs of otitis. Identification was based on their ability to grow on Sabouraud dextrose agar and typical macroscopic and microscopic appearance. Two antifungal tablets containing most commonly azole derivatives used for topical therapy were tested: MCZ and CTZ (10 µg, Neo-Sensitabs, Rosco, Denmark). Disk diffusion test was performed using Mueller-Hinton agar supplemented with 2% glucose and 0.5 µg/l methylene blue. Inoculum of *M. pachydermatis* was prepared in sterile saline from 3 days old cultures, with addition of the lipid source, containing approximately 1-5x10⁷ CFU/mL. Readings were performed 72 hours after incubation at 37°C, and interpreted according to guidelines provided by manufacturer.

**Results:** Out of 96 tested, all isolates demonstrated good susceptibility to CTZ with variations of inhibition zones from 34 to 73 mm (mean ± SD: 56 ± 8,06 mm). Susceptibility testing against MCZ resulted with the range of inhibition zones from 14 to 65 mm (mean ± SD: 41 ± 6,77 mm) and good sensitivity of 95 strains, while one isolate demonstrated intermediate sensitivity. Comparison of the size of the zones showed that CTZ had larger zones of inhibition than MCZ indicating its higher antifungal activity.

**Conclusion:** Screening of antifungal susceptibility of *M. pachydermatis* using disk diffusion method resulted with identification of one strain with reduced susceptibility to MCZ. However, taking into consideration that there are no standardized interpretative criteria for *Malassezia* yeast, follow up study using broth microdilution assay is needed, in order to check if this isolate could be categorized as potentially resistant. Reference: Pasquetti, M., E. Chiavassa, P. Tizzani, P. Danesi, A. Peano (2015): Agar diffusion procedures for susceptibility testing of *Malassezia pachydermatis*: Evaluation of Mueller-Hinton agar plus 2% glucose and 0.5 µg/mL methylene blue as the test medium. *Mycopathologia* 180, 153-158.

P062

Susceptibility to voriconazole of *Aspergillus* spp. strains, isolated from patients in Russia

N. Vasilyeva<fnref n="1" />, I. Vybornova<fnref n="2" />, T. Bogomolova<fnref n="1" />

**Objectives:** Azole resistance of aspergillosis etiologic agents is an emerging problem in Europe and other parts of the world. The aim of the study: to determine susceptibility to voriconazole of *Aspergillus* spp. strains isolated from patients with aspergillosis in Saint Petersburg, Russia.

**Methods:** Fungal species identification was done using morphological and molecular methods. A total of 230 strains of *Aspergillus* spp. isolated from biological samples of patients with different clinical forms of aspergillosis (invasive, chronic and allergic) in Saint Petersburg during 2014 – 2019 yrs. have been tested by standard disk diffusion method according to CLSI M51A Document. For 34 strains MICs of voriconazole have been determined according to EUCAST Document E Def 9.3.1.

**Results:** Species distribution among *Aspergillus* spp. isolates was as follows: *A. fumigatus* – 68%, *A. niger* – 16%, *A. flavus* – 13%, rare species – 3% (*A. terreus* – 4 strains, *A. calidoustus* – 2, *A. ustus* – 1, *A. sydowii* – 1). By disk diffusion method no voriconazole resistant isolates were found among common species: *A. fumigatus*, *A. niger*, *A. flavus*. Only 4 resistant strains were revealed among rare species: *A. terreus* – 1, *A. calidoustus* – 2, *A. ustus* – 1 (inhibition zone diameter < 17 mm). Voriconazole MICs for *A. fumigatus*, *A. flavus*, *A. niger* strains ranged from 0,125 to 0,5 mg/L. Only *A. calidoustus* showed high MIC – 4 mg/L.

<footnote n="1">¹Kaschkin Research Institute Of Medical Mycology. Department Of Medical Microbiology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation,</footnote>

<footnote n="2">²Kaschkin Research Institute Of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation</footnote>
**Conclusion:** Resistance to voriconazole among etiologic agents of aspergillosis in Saint Petersburg, Russia was found only among rare fungal species (*A. calidoustus*, *A. ustus*, *A. terreus*). Future surveillance is necessary to monitor antifungal resistance.

P064

Antifungal susceptibility testing of *Malassezia furfur*

T. Bogomolova¹, T. Bogdanova², N. Vasilyeva¹, A. Alexeyev¹, V. Spiridonova³

¹Kaschkin Research Institute Of Medical Mycology. Department Of Medical Microbiology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation,
²Department Of Medical Microbiology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ³Kaschkin Research Institute Of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation

**Objectives:** *Malassezia* spp. may be etiologic agents of superficial and invasive mycoses. There are no standard protocols for evaluation of antifungal susceptibility of these yeasts. The aim of the study was to determine *Malassezia furfur* isolates susceptibility to antifungal agents by two different methods.

**Methods:** Disk diffusion method CLSI M44A2 was modified by addition of lipids to the Muller-Hinton agar. Commercial test panels Sensititre Yeast One were also supplemented by fatty acid RPMI 1640.

**Results:** We tested 11 strains of *M. furfur* isolated from blood of pediatric patients. Both methods showed resistance to fluconazole and voriconazole. By Sensititre Yeast One panels the following MICs were obtained: amphoterecin B – 1 mg/L, fluconazole > 64 mg/L, caspofungin >8 mg/L, voriconazole – 1-8 mg/L, anidulafungin >8 mg/L, micafungin >8 mg/L, 5-fluorocytozne -> 256 mg/L, itraconazole - < 0,015, mg/L, pozaconazole – 0,03-0,06 mg/L.

**Conclusion:** Disk diffusion method may be used for screening antifungal resistance in *Malassezia* spp. isolates. Itraconazole and pozaconazole demonstrated highest activity against *M. furfur*.

P065

Evaluation of the efficacy of antifungals against invasive aspergillosis in an invertebrate animal model

J. Sana¹, É. Melloul¹, J. Guillot¹, F. Botterel¹,², E. Dannaoui¹,³

¹⁸ Rue Du General Sarrail, DYNAMYC, Creteil, France, ²Laboratoire De Parasitologie-mycologie, APHP Hôpital Henri Mondor, Créteil, France, ³Hôpital Européen Georges Pompidou, Laboratoire de Parasitologie-Mycologie, Paris, France

**Objectives:** Evaluation of new therapeutic strategies for the treatment of invasive aspergillosis is needed due to emergence of azole-resistance in *Aspergillus fumigatus*. Animal models in rodents are generally used but are costly and their use is limited by ethical considerations. For these reasons, evaluation of antifungal efficacy in mini-host models such as *Galleria mellonella* is a promising alternative. The aim of our study was to develop an invasive *Aspergillus* infection in larvae of *Galleria mellonella* to evaluate the efficacy of antifungals.

**Methods:** Three clinical strains of *Aspergillus fumigatus* (HEGP064, HEGP4017 and HEGP2666) have been used. Minimum inhibitory concentration for voriconazole (VRZ) was 0.5, 0.5 and 8µg/mL for HEGP064, HEGP4017 and HEGP2666 respectively. Inoculum finding experiments (with spore suspensions at 10⁵ to 10⁸ conidia/mL) have been performed to determine the concentration that gave 90% mortality at seven days post-infection (LD₉₀). The presence of an invasive infection has been demonstrated by direct examination of tissues stained with Gomori-Grocott (GG) and by histopathology (H&S staining). In a first set of experiments, groups of 10 larvae were infected with the LD₉₀ and treated with monotherapy by voriconazole (VRZ, [Vfend®] at 0.5, 1, 2, 4, and 8 µg/larva), amphotericin B (AMB, [Fungizone®], or caspofungin (CSP, [Cancidas®] at 0.2, 0.4 and 0.8 µg/larva). Subsequently, HEGP4017 or HEGP2666 infected larvae were treated after two hours by VRZ at
4µg/ml alone and in combination with CSP at 4, 2 or 1µg/larvae. The mortality was estimated daily for 7 days.

**Results:** After infection, an invasive aspergillosis was obtained as shown by the presence of hyphae in tissues. The LD₉₀ values for the HEGP064, HEGP4017 and HEGP2666 strains were 4.38×10⁷, 9.59×10⁷ and 1×10⁸ CFU/ml respectively. In infected and untreated groups, mortality was at least 90% on day 7 post-infection. The mortality decreased with increasing doses of amphotericin B. At 4 µg/larvae, the mortality was on average 20% for the 3 strains. In animals treated with VRZ, the mortality rates on day 7 post-infection were 35%, 40% and 60% for HEGP064 and were 55%, 65% and 95% for HEGP4017 and 85%, 95% and 95% for 8, 4 and 2 µg/larva, respectively (p = 0.0001 compared to control). VRZ at 8, 4 and 2 µg/larva was statistically more effective for the treatment of larvae infected with HEGP064 or HEGP4017 compared to those infected by HEGP2666. Administration of VRZ 4 did not decreased, significantly, the mortality of HEGP2666 infected larvae (p = 0.304) in contrast to those infected by HEGP4017 (p = 0.0082). The bitherapy VRZ 4µg/larvae and CSP 4µg/larvae lead to a more significant decrease in mortality compared to monotherapy (VRZ 4µg/larvae) and control group infected by HEGP4017(p = 0.026) and HEGP2666 (p = 0.005) strain.

**Conclusion:** *G. mellonella* is a reliable model for inducing invasive aspergillosis. There was a clear dose-response when animals were treated with VRZ and AMB. This model could be used for screening and evaluation of different therapeutic strategies against *Aspergillus spp*.

P066

**Analysis of the efflux-pump activity against resistant *Sporothrix schenckii* complex isolates**

M.L. Scroferneker¹, T.R. Pinheiro², G. Seibert², A.L.R. Poletto³, J.V. Prade³, C.D.O. Stopiglia³

¹Department Of Microbiology, Immunology And Parasitology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, ²Universidade Federal do Pampa, Uruguaiana, Brazil, ³Universidade Federal do Pampa, Uruguaiana, RS, Brazil

**Objectives:** The present study aims to assess the efflux-pump activity in the resistant *Sporothrix schenckii* complex strains.

**Methods:** The minimum inhibitory concentration (MIC) of itraconazole (0.03125-16 µg/mL) was determined through broth microdilution method, as described by protocol M38-A2 of the *Clinical and Laboratory Standards Institute* (CLSI, 2008). Ten clinical isolates of *Sporothrix schenckii* complex were prepared from 7-day-old cultures grown on potato dextrose agar at 28°C. The inocula were adjusted to a final concentration of 0.5–2.5×10³ cells/mL. The microdilution plates were incubated at 35°C for 72h. The MIC of itraconazole was defined as the lowest drug concentration capable of inhibiting total of growth. For the analysis of the efflux-pump activity, the resistant *Sporothrix schenckii* complex strains (04/10) were tested against two efflux-pump inhibitors, promethazine and verapamil by broth microdilution method. Posteriorly, susceptibility test was performed with itraconazole and sub-inhibitory concentrations of promethazine (MIC/8 = 24 µg/mL) and verapamil (MIC/8 = 100µMol).

**Results:** There were no reduction of MIC for itraconazole in the presence of the verapamil and promethazine inhibitors.

**Conclusion:** Thus, the present study suggests that the mechanism of resistance of the isolated evaluated isn't the presence of efflux pumps.

P067

**Terbinafine resistant *Trichophyton mentagrophytes* ITS genotype VIII from India in Germany**

P. Nenoff¹, A. Süß², A. Ludes³, A. Schuller⁴, E. Fischer⁵, S. Dessoi⁶, W. Hofmann⁶, S. Schmidt⁷, S.B. Verma⁸, M. Monod⁹, K. Salamin⁹, C. Krueger¹, A. Ebert¹, S. Uhrlass¹

¹Partnership Dr. C. Krueger & Prof. P. Nenoff, Laboratory of medical microbiology, Roetha OT Moelbis, Germany, ²Hautärztin, Gemeinschaftspraxis Allgemeinmedizin und Dermatologie, Wittlich, Germany, ³Allgemeinmedizinische Praxis Dr. med. Alfons Ludes und Thorsten Koech, Leiwen, Germany, ⁴Hautarztpraxis, Halle/Saale, Germany, ⁵Hautarztpraxis Dr. med. Gunhild Kratzsch, Leipzig, Germany, ⁶Hautzentrum Nordwest, Frankfurt am Main, Germany, ⁷Hautarztpraxis im MVZ Friedrichstadt, Dresden, Germany, ⁸"Nirvan" and "In Skin" Clinics,
Vadodara, India, ⁹Dermatology Service, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

**Case Report: Objectives:** In India, a dramatic increase of chronic recalcitrant dermatophytoses due to *Trichophyton* (T.) *mentagrophytes* genotype VIII is on the rise. Due to travelling and migration, globalization of the mostly terbinafine resistant strains of the causative agent from India to other parts of the world is assumed. First strains of *T. mentagrophytes* genotype VIII have reached Germany, now.

**Patients:** 1) A 6 months-old-female infant from Bahrain visiting Germany with her family for a holiday was seen for extensive dermatophytosis of the back, buttocks, chest and groins. Topical treatment by terbinafine for over 2 months was not successful. 2) A 28 years old male from Libya, living for 3 years in Germany, suffered from tinea cruris and tinea faciei involving the left upper and lower eyelids. Treatment by oral fluconazole and by terbinafine failed. His German girlfriend was affected by the dermatomycosis, too. Her child was free of signs of a dermatophytosis. The patient had no contact to India and Indians or Arabs, he did not visit Libya in the last years. The man, however, regularly goes to the gym. 3) After a trip to Saudi Arabia, a pregnant German woman suffered from tinea cruris and tinea corporis (trunk). Her husband was affected, too. 4) An Indian male, living in Germany, was treated because of a dermatomycosis of the buttocks and groins by oral terbinafine, without improvement. 5) A couple from Iraq, living for long time in Germany, suffered from chronic recalcitrant dermatophytosis of the groins for at least 1.5 years. Repeated topical treatments by „Combo Creams“ failed. Antifungal topical preparations (ciclopirox olamine, sertaconazole) for 5-6 weeks acted very slowly, progression of the dermatomycoses lead to therapy break-off. Oral itraconazole 200 mg daily was started. **Results:** Mycological examination of skin scrapings from all 5 patients revealed the detection of *T. mentagrophytes*. Four out of the five dermatophyte isolates were subject to sequencing of the "internal transcribed spacer" (ITS) region of the fungal rDNA. All these 4 strains belonged to the newly described genotype VIII of *T. mentagrophytes*. The until now investigated point mutations revealed for patient 1) TTC/TTA mutation of the squalene epoxidase (SQLE) gene which is closely associated with Phe³⁹⁷Leu amino acid substitution of the enzyme. *T. mentagrophytes* isolates of patient 2) and 3) were *in vitro* sensitive to terbinafine. The *T. mentagrophytes* strain of patient 3) showed GCT/ACT mutation (Ala⁴⁴⁸Thr). The above mentioned Phe³⁹⁷Leu and Ala⁴⁴⁸Thr substitutions are indicative of *in vitro* resistance of the dermatophyte against terbinafine. **Conclusion:** Transmission of the here found Indian ITS genotype VIII of *T. mentagrophytes* to other countries due to globalization is a serious issue to be considered. Moreover, a significant percentage of these Indian *T. mentagrophytes* strains are resistant to terbinafine both *in vitro* and due to point mutations in the SQLE gene. Some strains are also found to be partially resistant against itraconazole and voriconazole. The here described patients represent the first reports on an infection due to terbinafine-resistant *T. mentagrophytes* of the ITS genotype VIII from India in Germany.

P068

**Comparison of two antifungal susceptibility tests for *Malassezia* yeasts**

A. Abdillah¹, A. Packeu², S. Khelaifia³, M. Hendrickx⁴, S. Ranque¹

¹Vitrome, Aix Marseille Univ, IRD, AP-HM, SSA, Marseille, France, ²Mycology & Aerobiology, Sciensano, Brussels, Belgium, ³Culturomics, IHU Méditerranée Infection, Marseille, France, ⁴Service Of Mycology And Aerobiology, Bccm/ihem Fungal Collection, Sciensano, Brussels, Belgium

**Objectives:** The lipid-dependent *Malassezia* spp. yeast are members of the normal human skin microbiota that are occasionally involved in various skin diseases or in blood stream infections in immunocompromised and neonates patients. A better knowledge of the antifungal susceptibility testing (AFST) profiles of these yeast is pivotal to enhance antifungal treatment strategies. This study aimed to compare two broth microdilution assays for the *in vitro* AFST of *Malassezia* spp.

**Methods:** A total of 19 strains of the BCCM /IHEM collection, including 4 *Malassezia* species: (n = 6) *M. furfur*, (n = 4) *M. pachydermatis*, (n = 5) *M. sympodialis* and (n = 4) *M. slooffiae* were tested against ketoconazole, voriconazole, terbinafine and fluconazole using the broth microdilution method (CLSI - M27-A2). Two different media were used as diluent, modified Dixon broth (mDIXB) and FastFung
broth, a new medium derived from the Schaedler agar. MIC values were visually read after 48 h of incubation at 30°C. The essential agreement (EA) (±1 or 2 dilution) of the MIC values between both media was estimated. The inter-laboratory reproducibility between Marseille and Brussels was tested.

**Results:** Ketoconazole, voriconazole and terbinafine MICs (mg/l) were low, with MIC90 values of 0.06 to 0.5, 0.03 to 0.125, and 0.125, in mDIXB, and of 0.03 to 0.25, 0.03 to 0.125, and 0.06 to 1, in FastFung broth, respectively. Fluconazole displayed the highest MIC values. The EAs varied from 84.2 to 100% for ±1 dilution while EAs of 100% were observed for ±2 dilution. The inter-laboratory EAs (±2 dilutions) ranged from 73.8 to 100% and 94.7 to 100% in mDIXB or FastFung broth, respectively.

**Conclusion:** Our findings showed that both media performed similarly, with relatively higher EAs (>60%), except for terbinafine. The FastFung broth can be used as alternative medium for *Malassezia in vitro* AFTS.

P075

The effect of farnesol on *Candida auris* biofilms, virulence and azole susceptibility

F. Nagy¹, Z. Tóth¹, L. Forgács¹, A. Borman², L. Majoros¹, B. Balázs¹, R. Kovács¹

¹Department Of Medical Microbiology, University of Debrecen, Debrecen, Hungary, ²Public Health England UK National Mycology Reference Laboratory, Bristol, United Kingdom

**Objectives:** Farnesol is a fungal quorum-sensing molecule, which has a pivotal role in regulation of fungal morphogenesis. The aim of our study was to examine the effect of farnesol on biofilms, azole susceptibility (fluconazole, voriconazole, itraconazole, posaconazole, isavuconazole) against *Candida auris* clinical isolates as well as *in vivo* tissue fungal burden in a murine bloodstream infection model.

**Methods:** The effect of farnesol on *C. auris* cells was tested in three experiments: i) biofilm forming ability of cells pre-treated with farnesol for 24-hours prior to biofilm formation, ii) biofilm forming ability of cells continuously exposed to farnesol for 24-hours during biofilm formation, iii) effect of farnesol on one-day-old biofilms. Farnesol concentrations tested were 10, 50, 100, 300 µM in all experiments. Metabolic activity of sessile cells was determined at 0, 2, 4, 6, 8, 10, 12 and 24 hours using XTT-assay. One-way ANOVA with Dunnett's post-testing was used to analyze the metabolic activity change exerted by different farnesol concentrations compared to untreated control. Interactions between farnesol and azoles were examined against one-day-old biofilms by broth microdilution checkerboard method. The tested concentrations were 1.17-300 µM and 0.125-512 mg/L for farnesol and azoles, respectively. Fractional inhibitory concentration index (FICI) was used to assess the nature of interactions. Synergy was specified if the FICI was ≤0.5, between >0.5 and 4 was indifferent and as antagonistic if the FICI was >4. MICs were determined as at least 50% reduction of metabolic activity compared to control in case of individual drugs and isoeffective combinations. Farnesol pre-exposed (75 µM) cells were inoculated intravenously into immuncompromised female BALB/c mice to examine the day six fungal burden in kidneys, heart and liver.

**Results:** The one-day-long farnesol pre-exposure (50-300µM) led to formation of biofilms with significant metabolic activity inhibition after 24 hours (p<0.01). Biofilms formed under farnesol exposure showed significantly decreased metabolic activity in first the 12, 12, 10 and 8 hours for 300, 100, 50 and 10 µM farnesol, respectively (p<0.05-0.001). In case of one-day old biofilms treated with farnesol after formation, only the highest farnesol concentration caused significant inhibition at 24 hours (p<0.001). The median MIC values of azoles and farnesol against sessile *C. auris* clinical isolates are showed in Table 1. Based on FICI values, farnesol was synergistic with all tested azoles (FICIs ranged from 0.061 to 0.375) (Table 1). Fungal tissue burdens caused by farnesol pre-exposed *C. auris* were significantly higher in all examined organs compared to organs infected by farnesol-unexposed fungal cells at sixth day postinoculation.
<table><caption>**Table 1** Minimum inhibitory concentration of fluconazole (FLU), voriconazole (VOR), itraconazole (ITRA), posaconazole (POSA) and isavuconazole (ISA) alone and in combination with farnesol (FAR) against *Candida auris* (Ca) biofilms (sMIC). Furthermore, *in vitro* interactions by fractional inhibitory concentration index (FICI) determination of fluconazole, voriconazole, itraconazole, posaconazole and isavuconazole in combination with farnesol against *Candida auris* biofilms.</caption><thead><tr><th rowspan="3">Isolates</th><th colspan="4">Median sMIC values</th><th colspan="2">Interaction analysis</th></tr><tr><th colspan="2">sMIC alone</th><th colspan="2">sMIC in combination</th><th rowspan="2">Median FICI</th><th rowspan="2">Type of interaction</th></tr><tr><th>FLU (mg/L) &gt;512<sup>a</sup></th><th>FAR (µM) 300 &gt;512<sup>a</sup></th><th>FLU (mg/L) 64 &gt;512<sup>a</sup></th><th>FAR (µM) 75 &gt;512<sup>a</sup></th></tr></thead><tbody><tr><td>Ca1 Ca2</td><td>VOR (mg/L) 64 &gt;512<sup>a</sup></td><td>FAR (µM) 150 &gt;512<sup>a</sup></td><td>VOR (mg/L) 0.5 &gt;512<sup>a</sup></td><td>FAR (µM) 4.69 &gt;512<sup>a</sup></td><td>0.375 0.35</td><td>Synergy Synergy</td></tr><tr><td>Ca1 Ca2</td><td>VOR (mg/L) 64 &gt;512<sup>a</sup></td><td>FAR (µM) 150 &gt;512<sup>a</sup></td><td>VOR (mg/L) 0.5 &gt;512<sup>a</sup></td><td>FAR (µM) 4.69 &gt;512<sup>a</sup></td><td>0.093 0.061</td><td>Synergy Synergy</td></tr><tr><td>Ca1 Ca2</td><td>ITRA (mg/L) 16 &gt;512<sup>a</sup></td><td>FAR (µM) 300 &gt;512<sup>a</sup></td><td>ITRA (mg/L) 0.5 &gt;512<sup>a</sup></td><td>FAR (µM) 4.69 &gt;512<sup>a</sup></td><td>0.155 0.140</td><td>Synergy Synergy</td></tr><tr><td>Ca1 Ca2</td><td>POSA (mg/L) 16 &gt;512<sup>a</sup></td><td>FAR (µM) 150 &gt;512<sup>a</sup></td><td>POSA (mg/L) 0.25 &gt;512<sup>a</sup></td><td>FAR (µM) 2.34 &gt;512<sup>a</sup></td><td>0.062 0.062</td><td>Synergy Synergy</td></tr><tr><td>Ca1 Ca2</td><td>ISA (mg/L) 4 &gt;512<sup>a</sup></td><td>FAR (µM) 300 &gt;512<sup>a</sup></td><td>ISA (mg/L) 0.125 &gt;512<sup>a</sup></td><td>FAR (µM) 9.38 &gt;512<sup>a</sup></td><td>0.091 0.062</td><td>Synergy Synergy</td></tr><tr><td>Ca1 Ca2</td><td>ISA (mg/L) 8 &gt;512<sup>a</sup></td><td>FAR (µM) 300 &gt;512<sup>a</sup></td><td>ISA (mg/L) 0.125 &gt;512<sup>a</sup></td><td>FAR (µM) 18.75 &gt;512<sup>a</sup></td><td>0.091 0.062</td><td>Synergy Synergy</td></tr></tbody></table>

**Conclusion:** Farnesol inhibited the biofilm forming ability of *C. auris* and significantly reduced the metabolic activity of *C. auris* biofilms in a concentration-dependent manner; in addition, it showed a remarkable synergistic effect with azoles. On the other hand, farnesol pre-exposure led to increased tissue fungal burdens. In summary, though its systemic usage may be hindered by a potential enhancement of fungal virulence, farnesol may be a useful addition to therapy where biofilm formation is important, e.g. in catheter-lock therapy against *C. auris*.

P076

Transcriptional and physiological effect exerted by tyrosol on Candida parapsilosis cells

R. Kovács¹, Á. Jakab², Z. Tóth¹, F. Nagy¹, T. Emri², I. Pócsi², L. Majoros¹

¹Department Of Medical Microbiology, University of Debrecen, Debrecen, Hungary, ²Department Of Molecular Biotechnology And Microbiology, University of Debrecen, Debrecen, Hungary

**Objectives:** Tyrosol has a potent antifungal activity against *Candida* species; however, the background of its antifungal mechanism is still poorly understood. We examined the effect of tyrosol at supraphysiological concentration on growth, biofilm formation, redox homeostasis, virulence as well as on fluconazole susceptibility to reveal the background of its antifungal effect. To gain further insights into the physiological consequences of tyrosol treatment we examined the caused genome wide gene expression changes using RNA-Seq.

**Methods:** CLIB 214 *Candida parapsilosis* (sensu stricto) strain was used and 15 mM tyrosol concentration was applied to examine physiological and transcriptional changes. Following 4 hours incubation of *C. parapsilosis* precultures, we added 15 mM tyrosol to the cultures then growth was examined at 1-hour intervals by determination of cell density both by means of measuring of the absorbance (at 640 nm) and by counting the living cells. Reactive species were measured in the presence or absence of tyrosol by a technique that converts 2',7'dichlorofluorescin-diacetate to 2',7'-dichlorofluorescein. One-day-old biofilm formation ability in the presence of tyrosol was determined by quantitative CFU determination of adhered cells. Interaction between fluconazole and tyrosol was evaluated by two-dimensional broth microdilution chequerboard assay. Afterwards, the nature of interaction was analyzed using Fractional Inhibitory Concentration Index determination. Tyrosol induced effect on virulence was examined in immuncompromised systemic BALB/c mouse model. To obtain global transcriptome data regarding tyrosol treatment, high-throughput mRNA sequencing was performed on Illumina NextSeq sequencing platform.

**Results:** Fifteen mM tyrosol caused significant growth inhibition within two hours of the addition of tyrosol (p<0.01). Tyrosol increased the production of reactive oxygen species (p<0.001) and it was associated with elevated superoxide dismutase, glutathione peroxidase and catalase activities

<footnote><sup>a</sup> MIC is offscale at >512 mg/l, 1024 mg/l (one dilution higher than the highest tested concentration) was used for analysis</footnote>
(p<0.05). Biofilm formation ability was comparable in the presence or absence of 15 mM tyrosol in terms of living fungal cells. The interaction between fluconazole and tyrosol was antagonistic (FICI 4.5). Fifteen mM daily treatment decreased the fungal tissue burden in the kidneys compare with untreated control (p<0.01). Tyrosol exposure resulted in 1,462 differentially expressed genes. Out of them, 261 and 181 genes with at least 1.5-fold increase or decrease in expression, respectively, were selected for further investigations (Figure 1). Genes involved in ribosome biogenesis showed down-regulation, while genes related to later biofilm events, oxidative stress response and ethanol fermentation were up-regulated. In addition, tyrosol treatment up-regulated the expression of certain fluconazole efflux pump genes including *MDR1* and *CDR1* and down-regulated the *FAD2* and *FAD3* virulence genes belonging to desaturated fatty acid formation.

<table><tr><td><strong>Adhesion:</strong> ALS6<br/><strong>Phospholipase:</strong> PLB3<br/><strong>Biofilm:</strong> MKC1<br/><strong>Fatty acid metabolism:</strong> FAD2, FAD3</td></tr><tr><td><strong>Cell wall organization:</strong> RHD3, PGA30, IFF6</td></tr><tr><td><strong>Ribosome biogenesis</strong><br/>Small subunit genes: RPS3, RPS15, TIF5, YST1 etc.<br/>Large subunit genes: RPL5, RPL8B, RPL25, RPP2A etc.</td></tr><tr><td><strong>Transport</strong><br/>Amino acid: GAP1, GAP4, PUT4, DIP5, GNP3, MUP1, AGP3, ARG11, UGA1<br/><strong>Carbohydrate:</strong> HGT1, HGT10, HGT13, HGT14, HGT17, YOR1, NGT1, NAG3-4, HXT5<br/><strong>Lactate:</strong> JEN2, Sulfate:SUL2<br/><strong>Zinc:</strong> ZRT2, Iron: SIT1<br/><strong>Alcohol:</strong> HOL1<br/><strong>Nucleosid:</strong> NUP, FCY21</td></tr><tr><td><strong>Signal transduction:</strong> MKC1, RGS2, PDE2</td></tr></table>

<figure><img src="image_28.png" /></figure>

<table><tr><td><strong>Morphology:</strong> CZF1<br/><strong>Biofilm- Maturation:</strong> CZF1, RBT1, IFD6, ADH5, TEC1, HGC1<br/><strong>Biofilm- Dispersion:</strong> NRG1<br/><strong>Drug transporters:</strong> CDR1, MDR1, C1_00830W, CPAR2_405280, FCR1</td></tr><tr><td><strong>Cell wall organization:</strong> GPH1, EXG2, WSC1, XYL2, GPM2</td></tr><tr><td><strong>Glycolysis:</strong> PGII, PFK1, PFK2, TDH3, PGK1, GPM2, ENO1, CDC19<br/><strong>Fermentation:</strong> PDC11, PDC12, ADH1, ADH5, ADH7<br/><strong>Glycerol biosynthesis:</strong> RHR2</td></tr><tr><td><strong>Glutamate metabolism:</strong> GDH3</td></tr><tr><td><strong>Oxidative stress</strong><br/><strong>Antioxidant enzymes:</strong> CAT1, SOD4, CPAR2_803850, GST2, AHP1, GPX1<br/><strong>Regulation and other functions:</strong> CAP1, PST1, CIP1, GND1, MSN4, TAC1, SRR1, XBP1, POS5, C3_06860C_A</td></tr><tr><td><strong>Signal transduction:</strong> RIM101, MSB2, DPL1, SRR1, CPP1, PHO84</td></tr></table>

Figure 1 Up-regulated (red) and down-regulated (dark blue) genes were defined as differentially expressed genes (p<0.05) where log₂(FC)>0.585 or log₂(FC)<-0.585, respectively, and FC stands for fold change FPKM (fragments per kilobase per million mapped fragments) value.

**Conclusion:** Tyrosol exposure enhanced oxidative stress response and efflux pumps, while inhibited growth and ribosome biogenesis as well as virulence. Metabolism was modulated towards glycolysis and ethanol fermentation. Primer adherence was inhibited, and rather later biofilm events was enhanced. Our findings suggest that tyrosol may be a potential locally active and/or adjuvant agent in the development of alternative treatments targeting quorum-sensing in the future.

P077

## Nonyl 3,4-dihydroxybenzoate as a potent anti-biofilm compound for the treatment of dermatophytosis

C. Orlandi¹, R.H. Pires², N.M. Bila¹, A. Serafim-Pinto¹, J.L. Bonatti¹, L. Scorzoni¹, J. Singulani¹, C. Santos¹, C.R. Andrade³, P. Silva⁴, M. Chorilli⁴, L.O. Regasini⁵, A.M. Fusco Almeida¹, M.J. Soares Mendes Giannini¹

¹Clinical Analysis, UNESP (Sao Paulo State University), Araraquara, Brazil, ²Health Promotion, UNIFRAN (University of Franca)), Araraquara, Brazil, ³Physiology And Pathology, UNESP (Sao Paulo State University), ARARAQUARA, Brazil, ⁴Drugs And Pharmaceuticals, UNESP (Sao Paulo State University), Araraquara, Brazil, ⁵Chemistry And Environmental Sciences, UNESP (Sao Paulo State University), São José do Rio Preto, Brazil

**Objectives:** This study aimed to verify the *in vitro* susceptibility of nonyl 3,4-dihydroxybenzoate against planktonic cells and biofilms of dermatophytes. In addition, efficacy and toxicity were tested
in a murine model of dermatophytosis. The compound was incorporated into nanostructured lipid systems (NLS) in order to prove whether the incorporation was able to maintain its antifungal activity as well as, reduce toxicity in human cell lines and alternative models. In addition, the probable mechanism of action was evaluated.

**Methods:** The preformed biofilms and planktonic cells of *Trichophyton rubrum* and *T. mentagrophytes* were treated with different concentrations of synthetic antifungal drugs and nonyl 3,4-dihydroxybenzoate. The metabolic activities were determined using the XTT reduction assay. The effects of the treatments on biofilms structures were visualized by scanning electron microscopy (SEM). The efficacy of the compound was evaluated in wild type (WT) C57BL/6 mice by determining the fungal burden and histopathological analysis. Hepatic and renal toxicity were assessed by blood analysis in the equipment Vitros 250 chemistry analyzer. After incorporation into NLS, susceptibility tests were conducted according the document M38-A2, proposed by the CLSI (2008). The toxicity of incorporation was evaluated in HaCaT cell lines by the sulforhodamine B method and in *Caenorhabditis elegans* model. Finally, the damage of cell ultrastructure was evaluated by transmission electron microscopy (TEM).

**Results:** Both biofilms and planktonic cells were susceptible to nonyl compound in concentrations ranging from 125 – 250 mg/L. *T. rubrum* and *T. mentagrophytes* biofilms were significantly more resistant to fluconazole, griseofulvin and terbinafine than planktonic cells (p<0.05). Fluconazole, griseofulvin, terbinafine and nonyl were able to prevent biofilm formation in all reference strains and clinical isolates tested (p<0.05). SEM results revealed that the antifungal drugs caused minor or no damage to the structure of the biofilms, and in some cases, stimulated the development of resistance structures. In contrast, the biofilms treated with the nonyl compound were seriously damaged. The fungal burden was significantly reduced after treatment and histopathological analysis showed complete tissue regeneration. In addition, there was no systemic toxicity after treatment in mice. Nonyl incorporated to NSL formed by the surfactant Brij®98 Epikuron® (98-I) presented the best results of minimum inhibitory concentration (MIC), with values between 1.98 and 3.9 mg/L. Cytotoxicity tests indicated that the incorporation resulted in cell viability greater than 80% at all tested concentrations. These results corroborated with *in vivo* testing in *C. elegans* model, which indicated that the incorporation of nonyl significantly increased the survival of larvae. Finally, TEM results indicated that the compound might have more than one mechanism of action in the dermatophytes, causing enlargement of the vacuoles, besides derangement and formation of pores in the plasma membrane, similar to amphotericin B.

**Conclusion:** These findings show that nonyl 3,4-dihydroxybenzoate may be a promising antifungal. Funding: CNPq, Capes and FAPESP Process # 2018/02785-9; 2017/18388-6.

P078

Anti-dermatophyte biofilm activity of photodynamic therapy using 2-chalcone as photosensitizer

N.M. Bila¹,², C. Orlandi¹, L.C. Gonçalves¹, C.O. Vaso¹, C. Santos¹, P. Silva³, M. Chorilli³, L.O. Regasini⁴, F.L. Primo⁵, C.R. Fontana¹, A.M. Fusco Almeida¹, M.J. Soares Mendes Giannini¹

¹Clinical Analysis, UNESP (Sao Paulo State University), Araraquara, Brazil, ²Para-clinics, UEM (Eduardo Mondlane University), Maputo, Mozambique, ³Drugs And Pharmaceuticals, UNESP (Sao Paulo State University), Araraquara, Brazil, ⁴Chemistry And Environmental Sciences, UNESP (Sao Paulo State University), São José do Rio Preto, Brazil, ⁵Bioprocess And Biotechnology, UNESP (Sao Paulo State University), Araraquara, Brazil

**Objectives:** This study aimed to evaluate the anti-dermatophytic and anti-biofilm activity of 2-chalcone alone and combined with photodynamic therapy (PDT).

**Methods:** Strains of *Trichophyton rubrum*, *T. mentagrophytes* and one clinical isolate of *Microsporum canis* were used. Susceptibility tests were conducted according to the M38-A2 document proposed by CSLI. Planktonic cells, biofilms with 24 h (initial stage) and 72-96 h of growth (mature) were formed in RPMI-1640 medium and placed in contact with different concentrations of 2-chalcone (1-500 mg / L), fluconazole (1,25-512 mg / L) and terbinafine (0.002-32 mg / L). The metabolic activities after
the treatments were quantified using the tetrazolium salt (XTT) reduction method. The topographies of initial and mature biofilms with or without treatment were visualized by scanning electron microscopy (SEM). For the PDT assay, 2-chalcone was used a photosensitizer and a blue led as a source of light with a dose of 150 J.cm⁻².

**Results:** All the strains, in the planktonic form, were inhibited by treatment with 2-chalcone (MIC = 3,9 - 7.8 mg/L), terbinafine (MIC = 0.008 - 0.03 mg/L) and fluconazole (1-64 mg/L). The substance 2-chalcona eradicated the 24 hour biofilm of all strains tested at the concentration of 15.6 mg / L, terbinafine ranging from 0.06 to 16 mg / L, and fluconazole inhibited only the formation of *T. mentagrophytes* biofilm in the concentration of 32 mg / l. Regarding mature biofilms, only 2-chalcone was able to reduce the metabolic activities in the concentration of 31.2 mg/L. Mature biofilms were resistant to both antifungal drugs tested. When planktonic and biofilms (initial and mature) were treated with PDT using 2-chalcone as photosynthesizer, the inhibitory concentrations were reduced by 4 times (2 – 8 mg/L). The SEM of biofilms treated with 2-chalcone showed a total collapse of the cell walls, resulting from a probable extravasation of cytoplasmic content.

**Conclusion:** The substance 2-chalcone is a promising antifungal drug. Funding: CNPq, IBE process 150/2017, Capes and FAPESP Process # 2018/02785-9; 2017/18388-6.

P079

Antifungal activity of voriconazole and micafungin against the biofilms and planktonic cells of Candida albicans and Aspergillus fumigatus in vitro

D. Shi<fnref n="1" />, W. Liu²

¹Jining No. 1 People's Hospital, Jining, China, ²Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing, China

**Objectives:** *Candida albicans* and *Aspergillus fumigatus* are among the most common invasive opportunistic pathogens known to have high morbidity and mortality. Micafungin (MCF) and voriconazole (VOR) are the most commonly used first-line therapies for these fungi. However, to our knowledge, there are few data comparing the efficacy of MCF and VOR on biofilm and planktonic cells on both fungi.

**Methods:** We have carried out an *in vitro* study to evaluate the efficacy of VOR and MCF against biofilm development and inhibition of fungal viability within biofilm formed by these two pathogenic fungi.

**Results:** We find that, when compared with planktonic cells, the corresponding fungal biofilm magnificently decreases drug sensitivities. To inhibit more than 50% of cell viability within a mature biofilm, the concentration of each drug must increase by at least 5 to 10 MIC – depending on the specific drug and fungus combination. With specific regard to biofilm formation itself, we find that MCF is more effective than VOR against developing biofilm in *C. albicans*, whereas VOR is more effective at high concentration against developing biofilm in *A. fumigatus*. We also find that the earlier the therapeutic intervention, the greater the biofilm inhibition that will be achieved. The critical time window for biofilm inhibition is 12h for *A. fumigatus* and 8h for *C. albicans*; beyond those points in time safe and effective drug intervention is almost impossible.

**Conclusion:** Our results provide a few clinical hints that may help in choosing antifungal agents when treating common fungal infections.

P080

Contribution of Galleria mellonella model to understand interactions between Aspergillus fumigatus and Stenotrophomonas maltophilia: study of human, animal and environmental strains.

M.-F. Durieux<fnref n="1" />,², É. Melloul², L. Roisin², P.-L. Woerther²,³, V. Risco²,⁴, J. Guillot²,⁴, M.-L. Dardé<fnref n="1" />, E. Dannaoui⁵, J.-W. Decousser²,³, F. Botterel²,⁶

<footnote n="1">¹Hopital Dupuytren, Laboratoire de Parasitologie-Mycologie, Limoges, France, ²Dynamyc, Créteil,</footnote>
France, ³Microbiology, Hôpital Henri-Mondor, Créteil, France, ⁴École Nationale Vétérinaire D'alfort, Unité de Parasitologie-Mycologie, Maisons-Alfort, France, ⁵Hôpital Européen Georges Pompidou, Laboratoire de Parasitologie-Mycologie, Paris, France, ⁶Laboratoire De Parasitologie-mycologie, APHP Hôpital Henri Mondor, Créteil, France

**Objectives:** Clinical and environmental strains of *Aspergillus fumigatus* (Af) and *Stenotrophomonas maltophilia* (Sm) can colonize the respiratory tract of patients with chronic lung diseases such as cystic fibrosis, specially in biofilm form. The interactions between these two pathogens are not well known (1, 2). For a decade, insect model *Galleria mellonella* (Gm) can be used to screen microorganism pathogenicity. The aim of this study was to compare several associations of Af – Sm strains of different origin in a Gm model to determine the virulence of these associations.

**Methods:** An *in vivo* infection model with human, animal and environmental strains of Af and Sm, alone and associated, was developed using larvae of Gm. Several couples were tested using different strains of Sm and Af, especially human reference clinical strains (Af13073 and Sm13637). Virulence was analysed by survival curves at 7 days and histology method.

**Results:** Mortality rate with Af alone showed no significant difference between human, animal and environmental strains. For Sm, mortality rates showed variations depending on the strains. The association between reference clinical strains Sm13637 and Af13073 showed a synergistic effect in Gm (60% of mortality at 7 days) versus Sm 13637 alone (10% mortality) and Af13073 alone (30% mortality). This effect was not observed with the other co-infections. Gomori-Grocott coloration of histological sections showed fungal nodules disseminated in the larvae and a Gram coloration demonstrated the presence of the bacteria, validating our infection model (3).

**Conclusion:** For the first time, we described a coinfection of Af and Sm in a Gm model with an interesting synergistic effect between human clinical strains. This effect did not exist for environmental and animal strains showing a particular fitness between the human strains. These preliminary results provide complementary data to the *in vitro* data already obtained. The Gm model has demonstrated its relevance for the study of mixed infections, which remains to be improved (2). Indeed, the extracellular matrix could not be identified in Gm, which could validate the presence of mixed biofilm. (1, 2) Melloul et al. 2016 and 2018 (3) Société Française de Mycologie Médicale price of the best oral communication, 2019.

P081

Antifungal susceptibility of Lictheimia corymbifera biofilms to Amphotericin B formulations and Posaconazole

A. Vikelouda¹, M. Simitsopoulou¹, C. Antachopoulos¹, L. Skoura², A. Chatzimoschou¹, I. Stamouli¹,
E. Roilides¹

¹³rd Department Of Pediatrics, Aristotle University of Thessaloniki, Thessaloniki, Greece,
²Department Of Microbiology, Aristotle University, Thessaloniki, Greece

**Objectives:** Mucormycosis is an emerging life-threatening opportunistic fungal infection that affects patients with underlying immunosuppressive conditions, such as hematological malignancies, trauma, hematopoietic stem cell transplantation and diabetes mellitus. The ability of *Mucorales* to form biofilms, structures with reduced susceptibility to antifungal agents, has recently been demonstrated, *Lictheimia Corymbifera* is the second most common clinically significant *Mucorales* species and is related mostly to pulmonary and trauma infections. We investigated the antifungal activity of deoxycholate amphotericin B (D-AMB), liposomal amphotericin B (L-AMB), posaconazole (POS) against planktonic cells and mature biofilms of *Lictheimia corymbifera*. Additionally, we investigated the combinational activity of L-AMB with POS against mature biofilms.

**Methods:** Three *L. corymbifera* clinical strains, were isolated from bronchial secretions and incubated at 10^5 cfu/ml in 96-well microtiter plates at 37°C for 48h. Biofilm formation was assessed by 1% safranin staining and measured spectrophotometrically at 490 nm. In order to determine the MIC, two-fold dilutions of D-AMB, L-AMB and POS (0.007-256 mg/l) were incubated with biofilms or planktonic cells (2x10^5 cfu/ml) for 24h (n = 9). XTT metabolic reduction assay was used to assess biofilm damage compared to controls. MIC50 was determined as ≥50% biofilm damage. The activity
of the combination of L-AMB (0.5-32 mg/l) with POS (0.125-64 mg/l) against biofilms was determined using a checkerboard microdilution method (n = 10). Drug interactions were analyzed using Bliss independence model. The combination effect was defined as synergistic, antagonistic or indifferent when the observed biofilm damage was significantly higher, lower or equal to the expected damage, respectively.

**Results:** All *L. corymbifera* strains exhibited strong biofilm formation. Biofilm MIC50 of D-AMB, L-AMB and POS were 2, 4 and >256 mg/l, respectively, as compared to 0.007, 0.03 and 0.03 mg/l for planktonic cells. Synergistic effects were observed at 1-2 mg/l of L-AMB combined with 8-16 mg/l of POS (mean ΔE value of significant interactions:19% [range: 17% to 21%]; mean SE:1% [range: 1% to 3%]). None of the combinations exhibited antagonism.

**Conclusion:** *L. corymbifera* can produce strong biofilms that possess comparable susceptibility profiles to D-AMB and L-AMB, but are resistant to POS. The combination of L-AMB with POS at super-therapeutic concentration ranges exhibits synergistic activity against mature *L. corymbifera* biofilms.

P082

**Biofilm formation by Histoplasma capsulatum in different culture mediums and oxygen atmospheres**

L.C. Gonçalves<fnref n="1" />, C. Orlandi<fnref n="1" />, N.M. Bila<fnref n="1" />,², C.O. Vaso<fnref n="1" />, R.A. Moraes Da Silva<fnref n="1" />, M.L. Taylor³, M.J. Soares Mendes Giannini<fnref n="1" />, A.M. Fusco Almeida<fnref n="1" />

¹Clinical Analysis, UNESP (Sao Paulo State University), Araraquara, Brazil, ²Para-clinics, UEM (Eduardo Mondlane University), Maputo, Mozambique, ³Microbiology And Parasitology, UNAM (Autonomous University of Mexico), México, D.F., Mexico

**Objectives:** This work aimed to study *Histoplasma capsulatum* biofilms, by checking the influence of different culture media and oxygen atmospheres in the development of these communities.

**Methods:** The biofilm formation by two strains (ATCC 26029 - G186A and EH315) was characterized in different nutrient conditions [Brain Heart Infusion (BHI), Roswell Park Memorial Institute (RPMI) with 2% of glucose, Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and nutrient medium HAM-F12 (HAM-F12) supplemented with glucose (18.2 g / L), glutamic acid (1 g / L), HEPES (6 g / L), and L-cysteine (8.4 mg / L)] and atmospheres of oxygen (aerobic and microaerophilic). Optical microscopy techniques, XTT reduction assay, as well as crystal violet and safranin staining, and scanning electron microscopy (SEM) were performed.

**Results:** The results indicated that, although all the culture media stimulated the maturation of the communities, the nutrient-rich culture media, such as HAM-F12, provided a better biomass and extracellular matrix development when compared to the others. In addition, microaerophilic conditions were the most favorable than aerobic. The topographies observed in SEM showed yeasts surrounded at several points by an exuberant amount of extracellular matrix. However, the biofilms formed in BHI, RPMI and DMEM presented a reversal yeast for significant filamentation, which needs better investigation.

**Conclusion:** The results obtained so far represent significant advances for the field of biofilms and open new possibilities for the study of virulence and *Histoplasma*-host interaction. Funding: FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo Process # 2016/11836-0); CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico Process # 130018/2018-0) and Capes (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

P085

**Combined treatment of experimental paracoccidiodomycosis with Itraconazole and low-level LASER therapy**

E. Burger<fnref n="1" />, J.C. Grisolia<fnref n="1" />, L.A. Santos<fnref n="1" />, N.A. Dias<fnref n="1" />, L.C.C. Malaquias<fnref n="1" />, A.M. Rodrigues², Z.P.D. Camargo²

<footnote n="1">¹Department Of Microbiology And Immunology, Federal University of Alfenas, Alfenas, Brazil,</footnote>
²Department Of Microbiology, Immunology And Parasitology, Federal University of Sao Paulo, Alfenas, Brazil

**Objectives:** Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus *Paracoccidioides brasiliensis* (Pb), requiring long treatment, justifying studies that amplify the therapeutic options. Itraconazole (Itra) is effective for PCM and requires shorter therapy than other drugs. We propose to study a shorter treatment model with fewer side effects through the activation of neutrophils by the simultaneous administration of the antifungal drug Itraconazole and adjuvant therapy with Low Level LASER Therapy (LLLT) to provide better control in the early stages of the infection, leading to a more efficient and protective immune response, resulting in the control or cure of this disease.

**Methods:** We used a highly purified PMNs preparation by inoculating Pb in subcutaneous air pouches of mice. We evaluated the effect of Itraconazole (Itra) therapy and/or LLLT therapy on their size, cellular composition, mitochondrial activity, protein metabolism, production of reactive oxygen (ROS) and nitrogen (NO) species, production of IL-10, INF-γ and TNF-α cytokines, and on tissue architecture and number of viable fungi.

**Results:** The viability of air pouch cells was always greater than 70%, therefore, none of the treatments had adverse effects, and about 80% of the cells were PMN. Mitochondrial activity and cell counts differed significantly between LLLT-treated and controls and between mice treated with different Itra concentrations, with or without LLLT. Cells from 10mg/mL Itra-treated mice produced the highest levels of ROS followed by those of 3mg/mL. When Itra was associated with LLLT treatment, we found an increase in NO production in the 3mg/mL group. Total protein levels were higher when Itra was used, compared to the untreated group or to the group treated with LLLT alone, in which LLLT increased protein production in the 3mg/mL group and decreased in the 10mg/mL group. LLLT increased the production of cytokines IL-10, INF-γ and TNF-α, and the use of antifungal decreased their production at all doses, both using only Itra treatments and in combination with LLLT. The highest dose of Itra (50mg/mL) resulted in a significant reduction of air pouch size, volume and weight. The intensity of the inflammatory process and the number of vessels and neovas decreased when the concentration of Itra in combination with LLLT increased. The numbers of viable Pb were significantly reduced when Itra was administered in a dose-response way and more markedly when used in combination with LLLT.

**Conclusion:** Pb numbers decreased significantly when Itra was given at all concentrations, and this effect increased when used in combination with LLLT. Itra at 50 mg/mL caused a significant reduction in air pouch, indicating a dose-response control. Our results strongly suggest that the efficiency of Itra is enhanced by LLLT. Therefore, we could detect a possible additive or synergistic effect of PMNs, the antifungal drug Itra and LASER therapy, suggesting a new, more effective treatment for PCM. Grants: CNPq 305216/2016-3 and FAPEMIG APQ 012941-16. J.C.Grisolia and L.A. Santos are recipients of CAPES scholarships.

P086

**Clinical outcome of chronic pulmonary aspergillosis patients managed surgically**

F. Setianingrum<sup>1,2</sup>, R. Rautemaa-Richardson<sup>1,3,4</sup>, R. Shah<sup>4,5</sup>, D. Denning<sup>1,3,4</sup>

¹Division Of Infection, Immunity And Respiratory Medicine, University of Manchester, Manchester, United Kingdom, ²Department Of Parasitology, Universitas Indonesia, Jakarta, Indonesia, ³National Aspergillosis Centre, Manchester University NHS Foundation Trust, Manchester, United Kingdom, ⁴Manchester Academic Health Science Centre, Manchester, United Kingdom, ⁵Department Of Cardiology And Cardiothoracic Surgery, Manchester University NHS Foundation Trust, Wythenshawe Hospital, Manchester, United Kingdom

**Objectives:** Surgery is one of the treatment options for chronic pulmonary aspergillosis (CPA), especially for single aspergilloma, for those with recurrent haemoptysis despite bronchial artery embolisation or with the unilateral azole-resistant disease. This study aims to evaluate the outcomes of patients managed surgically and the correlation between *Aspergillus* Immunoglobulin G (IgG) levels and response to treatment.
**Methods:** This was a retrospective cohort study evaluating CPA patients who were treated at National Aspergillosis Centre, Manchester, UK over an 11-year period (2007-2018). Clinical variables including antifungal therapy and *Aspergillus*-IgG (Thermo Fisher/Phadia) were recorded and correlated with surgical success or relapse. *Aspergillus*-IgG were analysed based on 37 patients with complete serology database.

**Results:**

<figure><img src="image_29.png" /><figcaption>Fig 1. Kaplan-Meier cumulative survival plot representing relapse time for patients receiving antifungal in the pre perioperative and those who did not, p=0.002.</figcaption></figure>

61 patients with CPA underwent surgery with a mean duration of follow-up of 36 months. The mean age was 52.8 (± 14.3) years. The most common presenting symptoms pre-operatively were cough (n = 41, 67%), haemoptysis (n = 24, 39%), dyspnea (n = 23, 38%) and 5 patients (8%) were asymptomatic. Failed medical therapy (n = 31, 51%) and presumed malignancy (n = 23, 38%) were the most common indications for surgery. The most common sign of failed medical therapy was recurrent haemoptysis (n = 19, 31%). Twenty-six patients (43%) had single aspergilloma, 23 patients (38%) showed chronic cavitary pulmonary aspergillosis (CCPA) while 19 patients (31%) had one or more *Aspergillus* nodule. Main underlying diseases were chronic obstructive pulmonary disease (n = 19, 31%), asthma (n = 18, 30%), bronchiectasis (n = 14, 23%) and previous pneumothorax (n = 13, 21%). The procedures included lobectomy (n = 39, 64%), wedge resection (n = 17, 28%), segmentectomy (n = 3, 5%), pneumonectomy (n = 3, 5%) and decortication (n = 2, 3%). Main surgical complications were pleural infection (n = 7, 12%), prolonged air leak (n = 3, 5%) and persistent pneumothorax (n = 3, 5%). Assessed 6-8 months after surgery, 21 patients were asymptomatic (34%), 23 experienced cough (38%), 6 patients (10%) had haemoptysis, and the frequency of these symptoms reduced significantly after surgery (p< 0.05). Relapse occurred in 25 patients (41%) while the rest of the patients (n = 36, 59%) showed no sign of relapse. Haemoptysis, cough and dyspnea decreased significantly in the non-relapse group, whereas only haemoptysis was initially less frequent in relapse group (p<0.05). In this relapse group, 6 (24%) patients received antifungal before surgery compared to 23 (64%) in the non-relapse group (p = 0.002). Perioperative antifungal therapy was administered to 5 (20%) patients who relapsed and 19 (53%) who did not (p = 0.01). The median *Aspergillus*-IgG in the non-relapse group (n = 12, 32.4%) before surgery was 75 mg/L decreasing to 52 mg/L in the end of follow-up (p = 0.003). The median survival time (free-relapse) was 88 months in patients with antifungal therapy before surgery and 31 months in patients without antifungal therapy before surgery (p = 0.002).
**Conclusion:** Surgery in these selected CPA patients resulted in favourable outcomes with a decrease in clinical symptoms and the *Aspergillus*-IgG level of the non-relapse group of CPA. Pre and perioperative antifungal therapy reduced the likelihood of CPA relapse, but confounding factors may contribute to this. The diagnosis of CPA before surgery is important to optimise pre and perioperative antifungal therapy in patients to improve prognosis.

P087

**The all new, updated antifungal drug interaction database at www.aspergillus.org.uk The all new, updated antifungal drug interaction database at www.aspergillus.org.uk The all new, updated antifungal drug interaction database at www.aspergillus.org.uk**

S. Niazi-Ali¹, D. Denning²,³, G. Atherton²,³, M. Walczak³,⁴

¹Fungal Interaction Trust, Macclesfield, United Kingdom, ²University of Manchester, Manchester, United Kingdom, ³National Aspergillosis Centre, Manchester University NHS Foundation Trust, Manchester, United Kingdom, ⁴Manchester Academic Health Science Centre, Manchester University NHS Foundation Trust, Wythenshawe, United Kingdom

**Objectives:** A drug-drug interaction describes the influence of one drug upon another or the change in a drug's effect on the body when the drug is taken together with a second drug. A drug-drug interaction can delay, decrease, or enhance absorption or metabolism of either drug. The antifungal drug interaction database was first launched in 2012. It is available as web (www.aspergillus.org.uk/content/antifungal-drug-interactions) and app versions to allow information on potential drug interactions with antifungals with a version for patients and another for health professionals. New drugs are constantly being launched and approved. In this era, with the requirement of speed and accurate information due to pressures on all healthcare professionals, a new and updated database with apps (on Apple and Android platforms) has been created. This allows all of the above with the added benefit of real time updates in a more time efficient manner.

**Methods:** Horizon scanning documents were reviewed from 2017, 2018 and 2019 to date. Drugs that were not in the current database were identified and interactions determined using the Summary of Product Characteristics together with further exploration, as needed, with review of papers. CYP P450 and p-glycoprotein are the major interaction pathways where antifungal drugs are involved. However other pathways like OATP1B1 and BCRP were also explored, as there is new and emerging information on the effect of these pathways with drug-drug interactions.

**Results:**

<table><thead><tr><th></th><th>Itraconazole</th><th>Voriconazole</th><th>Fluconazole</th><th>Amphotericin B</th><th>Ambisome</th><th>Caspofungin</th><th>Terbinafine</th><th>Isavuconazole</th><th>Andidulafungin</th><th>Posaconazole</th><th>Micafungin</th><th>Total</th></tr></thead><tbody><tr><th>Severe</th><td>134</td><td>140</td><td>44</td><td>19</td><td>18</td><td>10</td><td>1</td><td>50</td><td>0</td><td>91</td><td>0</td><td>507</td></tr><tr><th>Moderate</th><td>158</td><td>179</td><td>171</td><td>125</td><td>125</td><td>38</td><td>10</td><td>81</td><td>0</td><td>189</td><td>4</td><td>1080</td></tr><tr><th>Mild</th><td>112</td><td>140</td><td>178</td><td>88</td><td>87</td><td>13</td><td>16</td><td>22</td><td>3</td><td>142</td><td>8</td><td>809</td></tr><tr><th>Unlikely</th><td>1082</td><td>1027</td><td>1093</td><td>1254</td><td>1256</td><td>1425</td><td>1459</td><td>1333</td><td>1483</td><td>1064</td><td>1474</td><td>13950</td></tr><tr><th>Total</th><td>1486</td><td>1486</td><td>1486</td><td>1486</td><td>1486</td><td>1486</td><td>1486</td><td>1486</td><td>1486</td><td>1486</td><td>1486</td><td>16346</td></tr></tbody></table>

134 new preparations were identified to April 2019 leading to updated information. 16 classed as red (severe interactions with some antifungals), 29 as amber (moderate interactions where co-administration is possible and guidance on any management required) and 89 as green (interaction unlikely). These were added to the database together with references to ensure the user is able to, refer and, find references with ease to validate their decisions. Of 16,324 possible interactions for drugs licensed by the end of 2018, 507 are potentially severe (3.1%), 1080 (6.6%) moderate and 809 mild interactions (4.9%). The antifungals with the least interactions are anidulafungin (n = 3 mild) and micafungin (n = 4 moderate & 8 mild) which contrasts with voriconazole with 459 interactions (140 severe) and itraconazole with 404 interactions (134 severe).

**Conclusion:** The new and revised version of the web and smartphone app-based database of these drug interactions with antifungal agents is now available. This will allow clinicians to effectively evaluate the use of antifungals with all the new drugs on the market and allow the best outcome for their patients. It will also enable them to make the most effective and evidence-based decisions for those drugs that are co-administered and require a little more clinical management to provide the most successful treatment for the wellbeing of their patients.
P088

**Posaconazole Serum Drug Levels Associated with Hypertension and Hypokalemia: The Syndrome of Pseudohyperaldosteronism**

M.-V. Nguyen¹, M. Davis¹, R. Wittenberg¹, I. Mchardy¹, J. Baddley¹, B. Young¹, A. Odermatt², G. Thompson¹

¹Infectious Disease, University of California at Davis, Davis, United States of America, ²Toxicology, University of Basel, Basel, Switzerland

**Objectives:** Posaconazole tablets are well tolerated and efficacious in the prophylaxis and treatment of aspergillosis, mucormycosis, and other invasive fungal infections. There have been case reports of posaconazole-induced pseudohyperaldosteronism, however, its occurrence and association with serum posaconazole drug levels has not previously been investigated.

**Methods:** In this single-center retrospective observational study, we examined the occurrence of posaconazole-induced pseudohyperaldosteronism (PIPH) in outpatients newly starting posaconazole and evaluated differences in serum posaconazole concentrations and clinical characteristics between those with and without this syndrome.

**Results:** Sixty-nine patients receiving posaconazole were included, of whom 16 (23.2%) met the definition of PIPH. Patients with PIPH were significantly older (61.1 vs 44.7 years, *P* = 0.007) and more frequently had hypertension prior to starting posaconazole (68.8% vs 32.1%, *P* = 0.009). Patients with PIPH had a significantly higher median serum posaconazole level than those without PIPH (3.0 vs 1.2 µg/mL, *P* < 0.0001). There was a positive correlation between serum posaconazole levels and changes in systolic blood pressure (*r* = .37, *P* = 0.01), a negative correlation between serum posaconazole levels and changes in serum potassium (*r* = -.39, *P* = 0.006), and a positive correlation between serum posaconazole levels and serum 11-deoxycortisol (*r* = .69, *P* < 0.0001).

**Conclusion:** Posaconazole is associated with secondary hypertension and hypokalemia, consistent with pseudohyperaldosteronism, and development is associated with higher serum posaconazole concentrations, older age, and baseline hypertension.

P089

**A case of primary joint infection caused by Candida pelliculosa**

S.-H. Kim¹, K.Y. Song², J.-H. Choi¹

¹Department Of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea, Republic of, ²Department Of Orthopaedic Surgery, College of Medicine, The Catholic University of Korea, Seoul, Korea, Republic of, Seoul, Korea, Republic of

**Case Report:** *Candida* joint infection is most often due to hematogenous seeding during an episode of candidemia in immunocompromised patients. Exogenous inoculation after trauma or surgical procedures also can lead to infection, but primary joint infection caused by *Candida* species is extremely rare. Until now, some *Candida* joint infections by *Candida albicans*, *Candida krusei*, *Candida lusitaniae*, *Candida parapsilosis*, and *Candida tropicalis* have been reported. Herein, we report the first case of primary knee joint infection caused by *Candida pelliculosa* without predisposing factors. A 75-year-old woman came to our hospital with a 3-year history of pain and swelling of the left knee, with the pain particularly aggravated for the past 1 month. The patient had undergone a left total knee arthroplasty (TKA) for incapacitating knee pain at a local hospital 32 months ago. At that time, *C. pelliculosa* was isolated from the tissue taken by intraoperative biopsy, but no antifungal treatment was performed. One year after TKA, *C. pelliculosa* was repeatedly isolated from left knee synovial fluid and the patient was treated with intravenous amphotericin B deoxycholate (0.5 mg/kg/day) for 4 weeks, then intravenous fluconazole (6 mg/kg/day) for 3 weeks, followed by oral fluconazole (4 mg/kg/day) for 2 weeks. However, progressive bone loss around prosthetic components was seen on follow-up radiographs and the patient was referred to tertiary hospital. The patient had no other significant medical history. The patient was admitted to our hospital and arthrocentesis of the left knee was performed. Results of synovial fluid analysis were as follows: leukocyte, 608 cells/µL (neutrophil, 54%); protein, 5.9 g/dL; glucose, 7 mg/dL. Magnetic resonance imaging showed a large abscess around the antero-lateral aspect of the left knee. Fungal culture of synovial fluid reported as
C. pelliculosa susceptible to amphotericin B (minimum inhibitory concentration [MIC], ≤0.25 mg/L), caspofungin (MIC, ≤0.25 mg/L), fluconazole (MIC, 2 mg/L), micafungin (MIC, ≤0.06 mg/L), and voriconazole (MIC, ≤0.12 mg/L) by Vitek 2 yeast identification system. The isolate was confirmed as C. pelliculosa using Internal transcribed spacer 1 and 4 region PCR and sequencing. At 2 weeks of micafungin (100 mg/day) therapy, the patient underwent prosthesis resection with debridement of soft tissue and bone, total synovectomy, and placement of an antibiotic-impregnated spacer. Operative findings showed infected granulation tissue in whole joint space and bony fistula extended to the anterolateral aspect of the lateral tibial component. Postoperatively, the patient was given micafungin for 4 weeks, followed by oral fluconazole (6 mg/kg/day). There were no signs of infection around the left knee and the patient was discharged from the hospital. Oral fluconazole will be given for at least 6 months and implantation of new prosthesis is planned at 3 months after the stage 1 procedure. Rare Candida species could be a causative organism of primary joint infection. Even a single colony of Candida on synovial fluid or bone biopsy should be considered as pathogenic, and the patient should be treated with antifungal agents. A high index of suspicion is required to establish the diagnosis.

P090

**Transdiaphragmatic Mucormycosis**

P. Köhler¹, R. Reimer², R. Whaba³, B. Schömig-Markiefka⁴, O.A. Cornely¹

¹Department I For Internal Medicine, European Excellence Center For Medical Mycology (ecmm), Germany And Cecad Cluster Of Excellence, University of Cologne, Cologne, Germany, ²Department Of Diagnostic And Interventional Radiology, University Hospital Cologne, Cologne, Germany, ³Department Of General, Visceral And Cancer Surgery, University Hospital Of Cologne, University of Cologne, Cologne, Germany, ⁴Institute Of Pathology, University Hospital Cologne, Cologne, Germany

**Objectives:** **Introduction** Mucormycosis is a life-threatening infection in immunocompromised patients and in haematological malignancy patients in particular. **Objectives** To report the phenomenon of transdiaphragmatic mucormycosis and to raise concerns with regard to the extent of imaging studies in patients with mucormycosis.

**Methods:** Retrospective chart review was performed on patients with Mucormycosis treated at the University Hospital of Cologne between January 2011 and January 2019.

**Results:** In a series of three patients we observed a previously unreported contiguous disease pattern, where the pulmonary type of disease contiguously spread through the diaphragm into abdominal organs, mostly liver or spleen. Patient 1, a 53-year-old women with history of B-NHL, ASCT and chronic renal disease developed mucormycosis of the lungs with continuous growth through the diaphragm into the liver. Histology showed invasive growth of fungal hyphae of the Mucorales order in liver and diaphragm. Patient 2, a 35-year-old man with history of AML and no further underlying conditions developed mucormycosis of the lungs with continuous growth through the diaphragm into the spleen. Splenectomy was performed and histology showed invasive growth of fungal hyphae. Fungal cultures of the surgical sample remained negative, but panfungal PCR resulted in *Rhizomucor* spp. Patient 3, a 42-year-old man with history of poorly controlled IDDM and injection drug use developed mucormycosis of the lungs with continuous growth through the diaphragm into the liver. Fungal culture of BAL fluid showed growth and PCR positivity of *Rhizopus microsporus*.

**Conclusion:** This is the first report of the clinical observation of transdiaphragmatic mucormycosis in 3 patients. We observed this phenomenon in 11% (3/27) of patients, raising concerns with regard to the extent of imaging studies in patients with mucormycosis. Clinicians should become aware of this new-described entity.

P091

**Baseline predictors influencing the prognosis of invasive aspergillosis in adults**

P. Köhler¹, J. Salmanton-Garcia², S. Gräfe³, F. Köhler³, S. Mellinghoff⁴, D. Seidel¹, A. Steinbach⁵, O.A. Cornely⁶