64, 128 and 256 mg/L of citral. Mean size (nm), polydispersity (PDI) and surface charge (ζ-potential) were evaluated with Malvern ZetaSizer Nano ZS particle analyser (Malvern, UK). Encapsulation efficiency (%EE) was determined by HPLC-DAD, after separation by ultacentrifugation. Antifungal activities of encapsulated and non-encapsulated phytocompounds (Sigma-Aldrich, USA) was evaluated by microdilution against eight *Candida albicans*, one *Candida auris*, *Candida dubliniensis*, *Candida tropicalis* and *C. albicans* reference strain SC5314. Cytotoxicity was assayed on murine macrophage-like cell line RAW 264.7 by detection of cell metabolic activity by the reduction of MTT to formazan and lactate dehydrogenase activity (LDH) assay.

**Results:** Nanoparticles of 128 mg/L of carvacrol and thymol presented high %EE (70.8 and 69.1%, respectively) without loss of anti-*Candida* activity. Geometric means of MICs of carvacrol and thymol nanoparticles were 101.6 mg/L and 71.8 mg/L, respectively. Nanoparticles of 256 mg/L of citral presented the best %EE (83.3%), but they did not present antifungal activity. Cinnamaldehyde nanoparticles presented the lowest %EE (20.6 to 44.1%) and maintained their antifungal activity. All nanoparticles were stable during four weeks stocked at 4 °C, with PDI values < 0.6 and ζ-potential among + 46.9 to + 55.6 mV. Thymol nanoparticles were the biggest with a particle size of 580.3 ±17.4 nm, followed by cinnamaldehyde, carvacrol and citral nanoparticles (567.9 ± 13.9, 510 ± 9.6 and 497.6 ± 13.8 nm, respectively). Non-encapsulated 32 µg/mL of thymol and carvacrol showed less cytotoxicity levels (76.5% and 74.8% cell viability by MTT assay, respectively; and ≥ 80% by LDH assay), followed by citral 64 µg/mL with 68% by MTT assay and 92.9% by LDH. Thymol and carvacrol nanoparticles were the best tolerated by macrophages with a higher cell viability by MTT assay (83.5-112.8% for thymol nanoparticles and 101-137% for carvacrol nanoparticles) when incubated with 18, 36 and 72 µg/mL of thymol nanoparticles and 18 and 36 µg/mL of carvacrol nanoparticles.

**Conclusion:** Monoolein-based liposomes of DODAB:MO (1:2) loaded with 128 µg/mL of carvacrol or 128 µg/mL of thymol can be a promising alternative of treatment of candidiasis, due to their striking physicochemical properties, appropriate encapsulation efficiencies, stability, reduced cytotoxicity and preserved anti-*Candida* activities. Work supported by Gobierno Vasco-Eusko Jaurlaritza (GIC15/78 IT-990-16) and the Federation of European Microbiological Societies by Research and Training grant (FEMS-GO-2017-020).

P410

In vitro antifungal effect of plant-based compound CIN-102 on filamentous fungi

M. D'Agostino

laboratoire SIMPA, Vandoeuvre-lès-Nancy, France

**Objectives:** Today the increase of invasive fungal infections due to the rise of immunosuppressive therapies is a real problem especially in hospitals. Moreover, the emergence of resistant strains induces a therapeutic failure. Face to this issues, new classes of antifungals are expected. The plant kingdom thus represents an immense potential of natural resources to exploit for these purposes. The SEPTEOS company has recently developed the CIN-102 compound, by using cinnamaldehyde and potentiating compounds from two synergic essential oils of cinnamon. Cinnamaldehyde has already been describe to kill bacteria, yeast and molds growth in both growing and non-growing state (not published). The objective of this study is to determine the spectrum of activity of CIN-102 on the main genus or species of filamentous fungi involved in human pathology.

**Methods:** The activity of CIN-102 product and its main constituent (cinnamaldehyde / CIN) are tested against a representative panel of strains of filamentous fungi (*Aspergillus* (n = 39), *Fusarium* (n = 19) and *Scedosporium* n = 21) of clinical importance. Clinical and reference strains have been studied, comprising isolates characterized by innate or acquired antifungal resistance patterns. A determination of MICs (minimum inhibitory concentrations) was performed using the M38-A2 CLSI reference method. The observed antifungal effect was then characterized by determining an inoculum effect.

**Results:** The MIC of CIN-102 determined for the different strains studied is mainly between 62 (n = 28) and 125 (n = 30) ug / ml, similar to the MIC determined for main active. Strains of the genus *Aspergillus* have on average a slightly higher MIC compared to the genus *Fusarium* and *Scedosporium*:
MIC mainly to 125 µg / ml for *Aspergillus* (n = 23), and 62 µg / ml for *Fusarium* (n = 12) and *Scedosporium* (n = 12). Acquired resistance against azole antifungals (*Aspergillus fumigatus* TR46, TR34 and G54E resistant strains) or innate (naturally antifungals resistant *Scedosporium* strains) do not seem to impact the effect of the compound CIN-102 and cinnamaldehyde. The inoculum as well as the time and dose dependent effect of CIN-102 and cinnamaldehyde will also be characterized.

**Conclusion:** Although the MICs alone do not provide an opinion on the efficacy of CIN-102, these results already show the antifungal effect of the compound. The current results for CIN-102 are very close to those determined for cinnamaldehyde in our study but also in the literature (Homa et al., 2015)(Essid et al., 2017). Further studies will then be conducted, notably on the structural aspect and the mechanism of action, to complete the knowledge on this new molecule. Moreover, an in vivo model of invasive fungal infection is studied to develop our knowledge about this new molecule.

P411

New strategies based on antifungal antibodies for the treatment of aspergillosis

D. Chauvin<fnref n="1" />, M. Hust², M. Schütte², A. Chesnay<fnref n="1" />,³, C. Parent<fnref n="1" />, G.M.S.G. Moreira², J.F. Arroyo⁴, A.B. Sanz⁴, P. Martineau⁵, M. Pugnière⁵, E. Bailly<fnref n="1" />, J. Chandenier<fnref n="1" />,³, N. Heuzé-Vourc'H<fnref n="1" />, G. Desoubeaux<fnref n="1" />,³
¹Cepr Inserm U1100, Université de Tours, Tours, France, ²Institut Für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig, Braunschweig, Germany, ³Parasitology - Mycology - Tropical Medicine, Hôpital Bretonneau, TOURS, France, ⁴Microbiología Y Parasitología, Universidad Complutense, Madrid, Spain, ⁵Institut De Recherche En Cancérologie De Montpellier, Université de Montpellier, Montpellier, France

**Objectives:** *Aspergillus fumigatus* is an airborne opportunistic fungal pathogen responsible for severe infections. For instance, invasive pulmonary aspergillosis is a serious threat for individuals suffering from severe immunosuppression, with mortality rates > 50%. In parallel, allergic bronchopulmonary aspergillosis frequently encountered in cystic fibrosis patients, is also a comorbidity factor. In addition to diagnostic means lacking specificity, current treatments present a high toxicity which prevents their use in weakened subjects, resulting in impaired prognostic. Because of their low toxicity and high specificity, anti-infectious therapeutic antibodies could be a new alternative to conventional therapeutics. In this study, we investigated the potential of *Chitin Ring Formation* cell wall transglycosylases of *A. fumigatus* to be therapeutic targets for therapeutic antibodies.

**Methods:** *In vitro* neutralization test, *in vitro* cultures, fluorescence and confocal microscopy, DNA sequencing, as well as flow cytometry and animal models were used.

**Results:** We demonstrated that the Crf target was highly conserved, regardless of the pathophysiological context; whereas the *CRF1* gene was found to be 100% conserved in 92% of the isolates studied, Crf proteins were expressed in 98% of the strains. In addition, we highlighted the role of Crf proteins in fungal growth, using a deletion mutant for *CRF1* gene, for which a growth decrease of 23.6% was observed after 48 h. It was demonstrated that anti-Crf antibodies neutralized the enzymatic activity of recombinant Crf protein, and delayed fungal growth by 12.3% *in vitro* when added to spores. In a neutropenic rat model of invasive pulmonary aspergillosis, anti-Crf antibodies elicited a significant recruitment of neutrophils, macrophages and T CD4 lymphocytes but it was not correlated with a decrease of fungal burden in lungs and improvement in survival.

**Conclusion:** Overall, our study highlighted the potential relevance of targeting Crf cell wall protein with therapeutic antibodies.

P412

Olorofim EUCAST susceptibility testing of contemporary moulds: *in vitro* activity and improved reproducibility

M.C. Arendrup<fnref n="1" />,²,³, R. Datcu<fnref n="1" />, R. Hare<fnref n="1" />, K.M. Jørgensen<fnref n="1" />

<footnote n="1">¹Unit Of Mycology, Statens Serum Institut, Copenhagen, Denmark, ²Dept. Of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark, ³Dept. Of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark</footnote>
**Objectives:** Olorofim is a novel antifungal orotomide compound for which we previously demonstrated good *in vitro* activity against various moulds. This study sought to (a) investigate whether MIC variation observed for *A. fumigatus* (unimodal range <0.004–0.25 mg/L) was due to inherent MIC variation or true differences in susceptibility and (b) present EUCAST MICs for clinical mould isolates from 2018.

**Methods:** MICs (mg/L) of olorofim were determined by EUCAST E.Def 9.3 using *A. fumigatus* ATCC 204305 for QC. **MIC variability study:** Fifteen *A. fumigatus* strains were selected from the prior study, as follows: five low MIC isolates: MIC = ≤0.002 (n = 2) and MIC = 0.004 (n = 3); five middle MIC isolates: MIC = 0.03 (n = 2) and MIC = 0.06 (n = 3); and five high MIC isolates: MIC = 0.125 (n = 1) and MIC = 0.25 (n = 4). MICs were determined visually by two-three observers blinded to the original MIC. **MICs against 365 clinical mould isolates** were determined during 2018. For *A. fumigatus* olorofim activity was evaluated individually for itraconazole susceptible (MIC≤1) and non-susceptible organisms (MIC>1). The ECOFFinder programme was used for determining statistical wildtype upper limits (highest MIC for organisms without phenotypically detectable acquired resistance mechanisms, WT-ULs) using 95%, 97.5% and 99% subset endpoints. For *Fusarium* isolates the 50%-MIC was also determined spectrophotometrically using 50% growth inhibition.

**Results:** Repetitive testing of low, medium and high MIC *A. fumigatus* isolates resulted in MICs, which for 14/15 isolates fell within two dilutions, whereas 1/10 MICs for isolate #12 and 1/80 MICs for the control strain fell one dilution below the two-dilution range when read by observer 1. The modal MIC for low/middle/high MIC isolates were as follows for repeated testing: Observer 1 and 2: 0.03/0.03/0.016 and for observer 3: 0.03/0.03/0.03. Routine testing during 2018 for species represented by ≥15 isolates generated uniform Gaussian MIC distributions spanning ≤5 dilutions with modal MICs = 0.03-0.06 for all isolates except non-proliferatum *Fusarium* spp. (n = 10) (Table). Modal MIC/WT-UL for *A. fumigatus* was 0.06/0.125 and was unaffected by itraconazole susceptibility and choice of ECOFFinder endpoint. Olorofim displayed MICs ≤0.25 against less common *Aspergillus* spp. except *A. montevidensis* (n = 1) as well as against dermatophytes and other moulds including: *Microsporum gypseum* (n = 1), *T. interdigitale* (n = 1), *T. rubrum* (n = 12), *Rasamsonia aegroticola* (n = 1), *Rasamsonia argillacea* (n = 1), *Scedosporium apiospermum* (n = 3), *Scedosporium boydii* (n = 2). 50%-MICs for *Fusarium* were as follows: *F. dimerum* >8 (n = 2), *F. oxysporum* 0.06 (n = 1), *F. solani* complex MIC range 0.25-1, modal MIC 1 (n = 7), and *F. proliferatum* 0.03 (n = 1).

<table><caption>**Table.** Olorofim *in vitro* activity against 365 clinical mould isolates obtained during 2018.</caption><thead><tr><th rowspan="2"></th><th colspan="10">Olorofim MICs (mg/L)</th><th rowspan="2">Range</th><th rowspan="2">Modal MIC</th><th rowspan="2">MIC<sub>50</sub></th><th rowspan="2">MIC<sub>90</sub></th></tr><tr><th>0.008</th><th>0.016</th><th>0.03</th><th>0.06</th><th>0.125</th><th>0.25</th><th>0.5</th><th>1</th><th>&gt;1</th></tr></thead><tbody><tr><td>A. flavus complex (18)</td><td>7</td><td>8</td><td>3</td><td></td><td></td><td></td><td></td><td></td><td>(0.016-0.06)</td><td>0.03</td><td>0.03</td><td>0.06</td></tr><tr><td>A. fumigatus (220)</td><td>7</td><td>67</td><td><u>113</u></td><td>31</td><td>2</td><td></td><td></td><td></td><td>(0.016-0.25)</td><td>0.06</td><td>0.06</td><td>0.125</td></tr><tr><td>azole wild-type (179)</td><td>6</td><td>55</td><td><u>96</u></td><td>21</td><td>1</td><td></td><td></td><td></td><td>(0.016-0.25)</td><td>0.06</td><td>0.06</td><td>0.125</td></tr><tr><td>azole non-wild-type (41)</td><td>1</td><td>12</td><td><u>17</u></td><td>10</td><td>1</td><td></td><td></td><td></td><td>(0.016-0.25)</td><td>0.06</td><td>0.06</td><td>0.125</td></tr><tr><td>A. nidulans complex (4)</td><td></td><td>1</td><td>2</td><td>1</td><td></td><td></td><td></td><td></td><td>(0.03-0.125)</td><td></td><td></td><td></td></tr><tr><td>A. niger complex (51)</td><td>1</td><td>8</td><td><u>23</u></td><td>17</td><td>2</td><td></td><td></td><td></td><td>(0.016-0.25)</td><td>0.06</td><td>0.06</td><td>0.125</td></tr><tr><td>A. terreus complex (32)</td><td>5</td><td>10</td><td><u>13</u></td><td>4</td><td></td><td></td><td></td><td></td><td>(0.008-0.06)</td><td>0.03</td><td>0.03</td><td>0.06</td></tr><tr><td>Other Aspergillus all (8)</td><td>2</td><td>4</td><td></td><td></td><td></td><td>1</td><td></td><td>1</td><td>(0.008-&gt;1)</td><td></td><td></td><td></td></tr><tr><td>F. proliferatum (1)</td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td>(0.06)</td><td></td><td></td><td></td></tr><tr><td>Other Fusarium (10)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>10</td><td>&gt;1</td><td></td><td></td><td></td><td></td></tr><tr><td>T. rubrum (12)</td><td>1</td><td>1</td><td>1</td><td>8</td><td>1</td><td></td><td></td><td></td><td>(0.008-0.25)</td><td>0.06</td><td>0.06</td><td>0.06</td></tr><tr><td>Other dermatophytes (2)</td><td></td><td></td><td>1</td><td></td><td>1</td><td></td><td></td><td></td><td>(0.03-0.125)</td><td></td><td></td><td></td></tr><tr><td>Other moulds (7)</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>2</td><td></td><td></td><td>(0.008-0.25)</td><td></td><td></td><td></td></tr></tbody></table>

**Conclusion:** Upon repeated testing of *A. fumigatus* isolates with initially low/medium/high MICs, no differences were observed. This suggests that the initial distribution observed can be explained by technical variation and that olorofim has similar and uniform activity against all isolates within the wild-type population. This was confirmed when evaluating results for routine MIC testing during 2018. In conclusion, olorofim displayed promising *in vitro* activity against most moulds included in this study independent of azole susceptibility.
## EUCAST susceptibility testing of manogepix and six comparative agents against contemporary Danish mould isolates

M.C. Arendrup¹,²,³, K.M.T. Astvad¹, K.M. Jørgensen¹

¹Unit Of Mycology, Statens Serum Institut, Copenhagen, Denmark, ²Dept. Of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark, ³Dept. Of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark

**Objectives:** The new antifungal fosmanogepix (APX001) is a first-in-class drug candidate and the methyl phosphate prodrug of the active moiety manogepix (APX001A). Manogepix targets the conserved fungal inositol acyltransferase enzyme Gwt1, thereby preventing GPI-anchored protein maturation and compromising fungal growth. Fosmanogepix is currently in Phase 2 clinical trials. Here we compared the *in vitro* activity of manogepix and six comparators against contemporary clinical mould isolates received for identification and EUCAST susceptibility testing.

**Methods:** A total of 163 clinical mould isolates obtained Aug 2016 to Sep 2017 were included. *Aspergillus* isolates were identified to the species complex level except *A. fumigatus* that was identified sensu stricto using thermotolerance. EUCAST E.Def 9.3.1 reference method using cell-culture treated microtitre plates (Nunc, ThermoFisher Scientific, cat. no. 167008) were prepared using serial two-fold dilutions of manogepix, amphotericin B, isavuconazole, voriconazole, itraconazole and posaconazole. The plates were stored at -80°C for ≥24 h prior to inoculation. Susceptibility endpoints were determined visually as MECs for manogepix (the lowest concentration resulting in aberrant growth) as complete visual or 50% spectrophotometric growth inhibition were not observed (data not shown). For the comparators MICs were read visually according to the EUCAST definition at the lowest concentration giving complete growth inhibition. The ECOFFinder programme was used for determining a statistical single-centre manogepix wild-type upper-limit for *A. fumigatus*.

**Results:** The manogepix MEC and comparator MICs are shown as modes (not done (ND) for isolates represented by five or fewer isolates) and ranges in the Table. Manogepix MECs were ≤0.125 mg/L against all *Aspergillus* and the two *Fusarium* isolates but no activity could be detected against the dermatophyte and three mucorales isolates included at the concentrations tested (MICs >0.5 mg/L). Manogepix *in vitro* activity remained the same against *A. fumigatus* isolates resistant to itraconazole (Table). Using the ECOFFinder programme, a single-centre wild-type upper-limit of 0.125 mg/L was determined for *A. fumigatus* (applying a 97.5% cut-off).

<table><thead><tr><th rowspan="2">Species (n)</th><th colspan="5">EUCAST MEC/MICs (mg/L) presented as mode (range)</th></tr><tr><th>Manogepix</th><th>AMB</th><th>ISA</th><th>VOR</th><th>ITR</th><th>PSC</th></tr></thead><tbody><tr><td>A. flavus (8)</td><td>0.03 (0.016-0.06)</td><td>1 (0.5-4)</td><td>1 (0.5-2)</td><td>1 (0.5-1)</td><td>0.125 (0.125-0.5)</td><td>0.06 (0.06-0.25)</td></tr><tr><td>A. fumigatus (123)</td><td>0.06 (0.016-0.125)</td><td>0.5 (0.25-1)</td><td>1 (0.25-16)</td><td>0.5 (0.25-4)</td><td>0.25 (0.125-&gt;16)</td><td>0.125 (&le;0.004-&gt;4)</td></tr><tr><td>Itraconazole R</td><td>0.06 (0.03-0.125)</td><td>0.5 (0.25-1)</td><td>1 (0.25-16)</td><td>0.5/4 (0.25-4)</td><td>16 (4-16)</td><td>2/&gt;4 (0.5-&gt;4)</td></tr><tr><td>A. nidulans (2)</td><td>ND (0.03)</td><td>ND (0.25-0.5)</td><td>ND (0.25)</td><td>ND (0.25)</td><td>ND (0.125-0.25)</td><td>ND (0.06-0.125)</td></tr><tr><td>A. niger (18)</td><td>0.03 (0.008-0.06)</td><td>0.25 (0.125-0.5)</td><td>8 (1-8)</td><td>2 (0.5-4)</td><td>1 (0.5-&gt;16)</td><td>0.25 (0.125-0.5)</td></tr><tr><td>A. terreus (6)</td><td>0.06 (0.016-0.125)</td><td>4 (1-4)</td><td>2 (0.5-2)</td><td>1 (0.5-2)</td><td>0.125/0.5 (0.06-0.5)</td><td>0.06/0.125 (0.03-0.25)</td></tr><tr><td>F. dimerum (1)</td><td>ND (0.06)</td><td>ND (1)</td><td>ND (&gt;16)</td><td>ND (8)</td><td>ND (&gt;16)</td><td>ND (&gt;4)</td></tr><tr><td>F. solani (1)</td><td>ND (0.03)</td><td>ND (&gt;4)</td><td>ND (&gt;16)</td><td>ND (8)</td><td>ND (4)</td><td>ND (&gt;4)</td></tr><tr><td>T. interdigitale (1)</td><td>ND (&gt;0.5)</td><td>ND (0.5)</td><td>ND (0.25)</td><td>ND (1)</td><td>ND (0.06)</td><td>ND (0.008)</td></tr><tr><td>L. ramosa (1)</td><td>ND (&gt;0.5)</td><td>ND (0.25)</td><td>ND (4)</td><td>ND (&gt;16)</td><td>ND (2)</td><td>ND (1)</td></tr><tr><td>R. pusillus (2)</td><td>ND (&gt;0.5)</td><td>ND (0.06)</td><td>ND (4)</td><td>ND (&gt;16)</td><td>ND (0.25-0.5)</td><td>ND (0.25-0.5)</td></tr></tbody></table>

**Conclusion:** Manogepix displayed uniform and potent *in vitro* activity against the different *Aspergillus* species and *Fusarium* isolates included in the study as determined by MECs. The *in vitro* activity was unaffected by acquired resistance in *A. fumigatus*. Thus, fosmanogepix appears to be a promising therapeutic against these pathogens, many of which are difficult to treat.
Microbiology, Rigshospitalet, Copenhagen, Denmark, ⁴Dept. Of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark

**Objectives:** *Candida auris* is a multidrug-resistant yeast rapidly emerging as a significant cause of nosocomial infections. Ibrexafungerp is a novel oral synthetic enfumafungin derivative inhibiting glucan synthase. It is active *in vitro* and in animal models against *Candida*, *Aspergillus* and *Pneumocystis* and is currently in clinical development for mucocutaneous and invasive fungal infections. Here, we investigate the susceptibility of *C. auris* to ibrexafungerp (formerly SCY-078) compared to that of eight comparators and compared to the activity against *C. albicans* and *C. glabrata*.

**Methods:** EUCAST AFST according to E.Def 7.3.1 was performed for ibrexafungerp (Scynexis, Jersey City, NJ, USA) against 122 clinical *C. auris* isolates (from India (n = 120) and Oman (n = 2)) and three *C. auris* control strains JCM15448, KCTC17809 and KCTC17810. Sixteen Danish clinical *C. albicans* and 16 *C. glabrata* isolates and the control strains *C. albicans* ATCC64548, *C. krusei* ATCC6258 and *C. parapsilosis* ATCC22019 were included as comparators and controls. Cell-culture treated microtiter plates (Nunc, Thermo Fisher Scientific, cat. no. 167008) were prepared using the ISO method. The ibrexafungerp MICs were compared to previously published MICs for anidulafungin, micafungin, amphotericin B, fluconazole, isavuconazole, itraconazole, posaconazole and voriconazole.

**Results:** The *in vitro* activity of ibrexafungerp (IBX) against *C. auris* was uniform with MICs displaying a Gaussian distribution spanning 0.06-2 mg/L suggesting an equal efficacy across the 122 isolates (Table). The modal MIC and MIC₅₀ were 0.5 mg/L. For the *C. auris* reference strains, the MICs were 0.06 mg/L, 0.125 mg/L and 0.5 mg/L, respectively. The MIC ranges for nine repetitive MIC determinations of the control strains MICs were as follows: *C. albicans* ATCC 64548: 0.06 mg/L, *C. krusei* ATCC 6258: 0.5-1 mg/L and *C. parapsilosis* ATCC 22019: 0.125-0.5 mg/L respectively. In contrast, MIC distributions for anidulafungin (ANF), micafungin (MCF), isavuconazole (ISA), voriconazole (VOR), itraconazole (ITR) and posaconazole (PRC) against *C. auris* were wide (spanning 10-13 two-fold dilutions) suggesting differential activity against the isolates. Of note, ibrexafungerp MICs remained low (MICs of 0.25 mg/L and 0.5 mg/L) for the isolates resistant to anidulafungin and micafungin (MICs >32 mg/L). Finally, fluconazole (FLU) MICs for all but one isolate were 16- >256 mg/L suggesting almost universal fluconazole resistance whereas the amphotericin B (AMB) MICs clustered close to the non-species specific breakpoint of 1 mg/L. Next, ibrexafungerp activity was compared for *C. albicans*, *C. glabrata* and *C. auris*. MIC₅₀, (range) were 0.06 mg/L (0.03-0.125 mg/L), 0.25 mg/L (0.25-0.5 mg/L) and 0.5 mg/L (0.06-2 mg/L), respectively.

<table><caption>**Table.** MICs (mg/L) of ibrexafungerp (IBX) and comparator antifungals against 122 clinical *C. auris* isolates. The MIC₅₀ is highlighted in bold and the modal MIC is underscored. Off-scale MICs are shown in the first concentration outside the tested range.</caption><thead><tr><th></th><th>0.004</th><th>0.008</th><th>0.016</th><th>0.03</th><th>0.06</th><th>0.125</th><th>0.25</th><th>0.5</th><th>1</th><th>2</th><th>4</th><th>8</th><th>16</th><th>32</th><th>&gt;32/64</th><th>128</th><th>256</th><th>&gt;256</th></tr></thead><tbody><tr><th>IBX</th><td></td><td></td><td></td><td></td><td>1</td><td>3</td><td>33</td><td><strong>63</strong></td><td>20</td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>ANF</th><td></td><td></td><td>1</td><td>11</td><td><strong>35</strong></td><td><strong>30</strong></td><td>12</td><td>12</td><td>11</td><td>2</td><td>1</td><td></td><td></td><td></td><td></td><td>7</td><td></td><td></td></tr><tr><th>MCF</th><td></td><td></td><td></td><td>5</td><td>30</td><td><strong>70</strong></td><td>9</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>8</td><td></td><td></td></tr><tr><th>AMB</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>14</td><td><strong>108</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>FLU</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td></td><td></td><td>4</td><td>10</td><td>6</td><td>14</td><td><strong>33</strong></td><td><strong>54</strong></td><td></td></tr><tr><th>ISA</th><td>20</td><td>1</td><td>1</td><td>19</td><td>9</td><td><strong>19</strong></td><td><strong>21</strong></td><td><strong>21</strong></td><td>6</td><td>5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>VOR</th><td>1</td><td></td><td></td><td>1</td><td>1</td><td>16</td><td>13</td><td><strong>34</strong></td><td><strong>38</strong></td><td>13</td><td>5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>ITR</th><td>2</td><td>2</td><td>9</td><td>5</td><td>14</td><td><strong>34</strong></td><td><strong>36</strong></td><td>19</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>PRC</th><td>1</td><td>8</td><td>24</td><td><strong>39</strong></td><td>35</td><td>11</td><td>3</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

**Conclusion:** Ibrexafungerp shows promising *in vitro* activity against *C. auris* suggesting it may be a welcomed therapeutic against this emerging threat with few treatment options. Ibrexafungerp's MICs were in general one step higher against *C. auris* than against *C. glabrata* and, notably, remained unchanged against *C. auris* isolates with antifungal resistance to the comparator drugs, including those highly echinocandin-resistant.
A. *Seyedmousavi*¹, Y.C. *Chang*², D. *Law*³, M. *Birch*³, J. *Rex*³, J. *Kwon-Chung*⁴

¹Department Of Laboratory Medicine, Microbiology Service, National Institutes of Health Clinical Center, Bethesda, United States of America, ²Laboratory Of Clinical Immunology And Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, United States of America, ³F2G Ltd, Manchester, United Kingdom, ⁴Laboratory Of Clinical Immunology And Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, United States of America

**Objectives:** Clinically relevant memebrs of the *Scedosporium/Pseudallescheria* species complex and *Lomentospora prolificans* are generally resistant to currently available systemic antifungal agents and the infection due to these species is difficult to treat. Alternative treatments with new classes of antifungals may improve therapeutic outcomes of the disease caused by these difficult to treat moulds.

**Methods:** We studied the *in vivo* efficacy of olorofim (formerly F901318), a new fungicidal agent that prevents growth of manyascomycetous mold species via inhibition of de novo pyrimidine biosynthesis, against scedosporiosis caused by three species in neutropenic CD-1 mice. Cyclophosphamide immunosuppressed mice infected by *Scedosporium apiospermum*, *Pseudallescheria boydii* and *Lomentospora prolificans* (tail vein infection) were treated by intraperitoneal administration of olorofim (15 mg/kg every 8 h for 9 days). The efficacy of olorofim treatment was assessed by survival rate 10 days post infection, and tissue histopathology 3 days post infection.

**Results:** In the neutropenic CD-1 mice, olorofim therapy significantly improved survival compared to the untreated controls; 80%, 100% and 100% of treated mice survived infection by *Scedosporium apiospermum*, *Pseudallescheria boydii*, *Lomentospora prolificans*, respectively while less than 20% of the control mice (PBS-treated) survived at 10 days post infection. Furthermore, histopathological slides of kidneys revealed no detectable fungal elements in the olorofim-treated mice, whilst numerous fungal hyphae were present in control mice.

**Conclusion:** Overall, treatment with olorofim improved the survival of the mice infected with all three species causing scedosporiosis and showed rapid clearance of fungi from the kidney by histological staining. These results show olorofim to be a promising therapeutic agent for systemic scedosporiosis, a difficult disease to treat with currently available antifungals.

P416

APX001 (Fosmanogepix) is Effective in an Immunosuppressed Mouse Model of Fusariosis

T. *Gebremariam*¹, S. *Alkhazraji*¹, Y. *Gu*¹, A. *Alqarihi*¹, Z. *Mamouei*¹, K.J. *Shaw*², A.S. *Ibrahim*¹

¹Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, United States of America, ²Amplyx Pharmaceuticals, San Diego, United States of America

**Objectives:** Fusariosis has high mortality rates. Owing to its rarity, comparative clinical trials are hard to perform. Animal models are an appropriate complementary avenue for evaluating antifungal therapy. Thus APX001 (fosmanogepix) was evaluated in an immunosuppressed murine model of hematogenously disseminated fusariosis.

**Methods:** The minimum effective concentration (MEC) of APX001A (manogepix, the active moiety of APX001) was determined against a *Fusarium solani* clinical isolate using CLSI M38 method. ICR mice were immunosuppressed with cyclophosphamide and cortisone acetate on days -2, and +3, relative to intravenous infection with 7.5×10⁴ cells of *F. solani*. For survival studies, treatment with placebo (diluent control), APX001 (78 or 104 mg/kg, PO), liposomal amphotericin B (LAmB, 15 mg/kg, IV), or voriconazole (Vori, 40 mg/kg, PO) began 16 h post-infection and continued for 8 days for APX001 or Vori and 4 days for LAmB. To extend the half-life of APX001, mice were administered 50 mg/kg of the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) 2 h prior to APX001 administration. Grapefruit juice in drinking water (50%) was given to Vori-treated mice to enhance the drug's half-life. To assess tissue fungal burden, mice were sacrificed on Day +4 and organs processed for conidial equivalent (CE) by qPCR.

**Results:** The APX001A MEC value for the tested *F. solani* strain was 0.03 µg/mL. Treatment with APX001 or LAmB enhanced median survival time vs. placebo (12, and 10 days for 78, and 104 mg/kg
of APX001, respectively vs. 10 days for LAmB treatment, vs. 8 or 7 days for Vori or placebo, respectively, *P* <0.01). Further, APX001 and LAmB treatments equally enhanced overall survival by day 21 when the experiment was terminated (40% for LAmB or APX001 at 78 mg/kg, and 20% for APX001 at 104 mg/kg, vs. 0% for placebo or Vori treatment). APX001 or LAmB treatments, but not Vori, resulted in ~2-3 log reduction in kidney, and brain CE vs. placebo.

**Conclusion:** APX001A showed significant antifungal activity against *F. solani* *in vitro* which translated to *in vivo* efficacy. APX001 was as effective as LAmB in this mouse model of fusariosis. Continued investigation of APX001 as a novel antifungal agent against this difficult to treat infection is warranted.

P417

Evaluation of the *In vitro* Activity of Olorofim Against *Fusarium* Species

N. Wiederhold¹, H. Patterson¹, M. Birch², D. Law², J. Rex²

¹Fungus Testing Laboratory, The University of Texas Health Science Center at San Antonio, San Antonio, United States of America, ²F2G Ltd, Manchester, United Kingdom

**Objectives:** Invasive infections caused by *Fusarium* species are often associated with significant morbidity and mortality in highly immunocompromised patients, and treatment options are limited. Several species are often resistant to treatment with clinically available antifungals. Olorofim (formerly F901318; F2G Ltd.) is a member of the orotomide class of antifungal agents that inhibits fungal pyrimidine biosynthesis and has potent activity against a variety of filamentous fungi. We evaluated the *in vitro* activity of olorofim against a selection of various *Fusarium* isolates. The *in vitro* activity was also measured for the clinically available antifungals amphotericin B, posaconazole, and voriconazole.

**Methods:** Clinical isolates (n = 111) of *Fusarium* species in the collection of the University of Texas Health Science Center Fungus Testing Laboratory were used. Susceptibility testing was performed by broth microdilution according the CLSI M38 reference standard. MICs for olorofim were determined after 48 hours of incubation using the 50% and 100% inhibition endpoint, while those of the control agents (amphotericin B, posaconazole, and voriconazole) were determined using the 100% inhibition endpoint.

**Results:** Olorofim demonstrated *in vitro* activity against *F. oxysporum* (MIC range 0.125-0.25 mg/L and 0.5-1 mg/L at the 50% and 100% endpoints, respectively), *F. fujikuroi* (≤0.015 mg/L and ≤0.015-0.125 mg/L), *F. verticillioides* (0.06-0.125 mg/L and 0.06-0.25 mg/L), and *F. proliferatum* (≤0.015-≥0.8 mg/L and 0.06-≥0.8 mg/L) (Table). At the 50% inhibition endpoint, olorofim demonstrated *in vitro* activity against some isolates of the *F. solani* species complex (FSSC, now members of the genus *Neocosmospora* 0.5-≥0.8 mg/L) and the *F. incarnatum-equiseti* species complex (FIESC, 0.25-≥0.8 mg/L). In contrast, when the 100% inhibition endpoint was used, limited to no *in vitro* activity was observed against these species complexes (4-≥0.8 mg/L), nor was activity observed against *F. dimerum* using either endpoint (≥0.8 mg/L). Olorofim activity against some of the rarer species of *Fusarium* was mixed, with the growth of some species being inhibited at low MICs (i.e., *F. brachygibbosum*, *F. decemcellulare*, *F. redolens*, and *F. thapsinum*; 0.06-0.25 mg/L at 50% inhibition and 0.25-0.5 mg/L at 100% inhibition). In contrast, limited to no activity was also observed against other rarer species (i.e., *F. delphinoides*, *F. nygamai*, *F. pallidoroseum*, *F. petroliphilum*, and *P. pseudensiforme*; 0.25-0.8 mg/L at 50% inhibition and ≥0.8 mg/L at 100% inhibition). The MIC ranges for the control agents were also wide and differed across *Fusarium*
species. The most consistent activity was observed with amphotericin B. Table. MIC values (mg/L) and geometric mean (GM) MICs for olorofim, amphotericin B, posaconazole, and voriconazole against *Fusarium* species with at least 6 isolates included.

<table><thead><tr><th rowspan="2">Antifungal<br>Species/Species<br>Complex</th><th colspan="2">Olorofim 50%</th><th colspan="2">Olorofim 100%</th><th colspan="2">Amphotericin<br>B</th><th colspan="2">Posaconazole</th><th colspan="2">Voriconazole</th></tr><tr><th>Range</th><th>GM<br>MIC</th><th>Range</th><th>GM<br>MIC</th><th>Range</th><th>GM<br>MIC</th><th>Range</th><th>GM<br>MIC</th><th>Range</th><th>GM<br>MIC</th></tr></thead><tbody><tr><td>FSSC<br>(n = 29)</td><td>0.5 -<br>>8</td><td>1.07</td><td>4 - >8</td><td>>8</td><td>1 - 4</td><td>1.86</td><td>>16</td><td>>16</td><td>4 -<br>>16</td><td>>16</td></tr><tr><td>F. oxysporum<br>(n = 14)</td><td>0.12 -<br>0.25</td><td>0.24</td><td>0.5 - 1</td><td>0.53</td><td>2 - 4</td><td>2.69</td><td>2 -<br>>16</td><td>4.64</td><td>2 - 16</td><td>4.88</td></tr><tr><td>FIESC<br>(n = 16)</td><td>0.25 -<br>>8</td><td>5.19</td><td>>8</td><td>>8</td><td>1 - 4</td><td>2.00</td><td>1 -<br>>16</td><td>2.48</td><td>1 - 8</td><td>4.56</td></tr><tr><td>F. fujikuroi<br>(n = 6)</td><td>≤0.015</td><td>≤0.015</td><td>≤0.015<br>- 0.125</td><td>0.043</td><td>2 - 4</td><td>2.52</td><td>0.5 - 2</td><td>1.26</td><td>0.25 -<br>4</td><td>2.52</td></tr><tr><td>F. proliferatum<br>(n = 21)</td><td>≤0.015<br>- >8</td><td>0.028</td><td>0.03 -<br>>8</td><td>0.113</td><td>2 - 8</td><td>3.87</td><td>1 -<br>>16</td><td>>16</td><td>2 - 16</td><td>9.44</td></tr><tr><td>F. verticillioides<br>(n = 6)</td><td>0.03 -<br>0.125</td><td>0.077</td><td>0.06 -<br>0.5</td><td>0.221</td><td>2 - 16</td><td>3.56</td><td>0.5 - 1</td><td>0.707</td><td>1 - 2</td><td>1.78</td></tr></tbody></table>

**Conclusion:** Olorofim demonstrated *in vitro* activity against some clinical isolates of *Fusarium* species. This activity appeared to be species-dependent, and was also dependent on the endpoint used (50% vs. 100% inhibition of growth). Further work is needed to determine how the *in vitro* activity observed against *Fusarium* species in this study may translate into *in vivo* efficacy, especially for more common species for which there were differences between the activity of olorofim at the different of growth inhibition endpoints.

## P419

### Nanoparticles as antimicrobial agent against *Scytalidium hyalinum* and *Neoscytalidium dimidiatum*

B. De Almeida¹, T. Lemes¹, M. Almeida², D. Volanti¹

¹Unesp, São José do Rio Preto, Brazil, ²FAMERP, São José do Rio Preto, Brazil

**Objectives:** The present study aimed to evaluate the antifungal activity of silver-based nanocomposites (NCs) deposited in hydroxyapatite (HAP), after synthesis by microwave, against clinical strains of *Scytalidium hyalinum* and *Neoscytalidium dimidiatum*.

**Methods:** The analysis was performed with Ag/HAP NCs (4 and 8% Ag) and pure Ag to evaluate the minimum inhibitory concentration to kill *Scytalidium hyalinum* and *Neoscytalidium dimidiatum*, according to CLSI M27-S4 with modifications considering, in triplicate. The fungi strains were maintained in RPMI broth culture media after overnight incubation at 37°C, and each inoculum adjusted according to 0.5 MacFarland standard tube. In 96-well plates, 100 µL of RPMI with fungal inoculum was added and NCs were dispersed into the 10% dimethyl sulfoxide (DMSO) - ranging from 1000 to 7.8 µg/mL. The plates were incubated for 24 hours. After growth, the cell viability was conducted by enzymatic reduction using triphenyl tetrazolium chloride (TTC).

**Results:** The highest antifungal activity was seen for 8% Ag/HAP NCs against *Scytalidium hyalinum* and *Neoscytalidium dimidiatum* with MIC 250 µg/ml. In addition, the pure Ag showed the worst antifungal activity with a MIC 1000 µg/mL for *Scytalidium hyalinum* and MIC 250 µg/mL for *Neoscytalidium dimidiatum*.

**Conclusion:** This study contributes to the knowledge of interactions between nanocomposites and fungal cell, as potential target of control infection caused by *Scytalidium hyalinum* and *Neoscytalidium dimidiatum*. It is clear the occurrence of the synergistic effect of Ag/HAP NCs in relation to pure Ag, event that must be considered, once HAP is biocompatible and could reduce the toxicity of Ag.

## P420

### APX001 (Fosmanogepix) is Effective in an Immunosuppressed Mouse Model of Rhizopus oryzae Infection

T. Gebremariam¹, S. Alkhazraji¹, Y. Gu¹, A. Alqarihi¹, Z. Mamouei¹, K.J. Shaw², A.S. Ibrahim¹

¹Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, United States of America, ²Amplex Pharmaceuticals, San Diego, United States of America
**Objectives:** Mucormycosis is a life-threatening infection that predominantly occurs in immunocompromised hosts. The antifungal APX001A (manogepix) inhibits Gwt1, an enzyme required for the conserved glycosylphosphatidyl inositol (GPI) post-translational modification in eukaryotes. We previously reported the activity of APX001 (fosmanogepix, the prodrug of APX001A) against *Rhizopus delemar* (minimum effective concentration [MEC] = 0.25 µg/mL). Here we assessed the activity against *R. oryzae*, which has an elevated MEC value.

**Methods:** *R. oryzae* 99-892 MIC and MEC values were 0.125 µg/mL and 4.0 µg/mL for isavuconazole (ISAV) and APX001A, respectively. ICR mice were immunosuppressed with cyclophosphamide (200 mg/kg) and cortisone acetate (500 mg/kg) on Days -2, +3, and +8 relative to intratracheal infection with 2.5 x 10⁵ cells of *R. oryzae* 99-892. For survival studies, treatment with 104 mg/kg APX001 was compared to ISAV (110 mg/kg TID). Oral treatment started on Day +1 through Day +7, relative to infection for survival studies, and through Day +4 for tissue fungal burden studies (assessed by conidial equivalent [CE] using qPCR). Placebo mice received vehicle control. To extend the half-life of APX001, mice were administered 50 mg/kg of the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) 2 h prior to APX001 administration.

**Results:** APX001 and ISAV equally prolonged median survival time of mice (n = 20) versus placebo (12 and 14 days for APX001 and ISAV, respectively, vs. 8 days for placebo). Further, APX001 and ISAV treatment both resulted in 30% 21-day survival versus 0% survival of placebo mice (P <0.05 by Log Rank test). Both drug treatments resulted in ~1.5 log₁₀ reduction in lung and brain CE vs. placebo-treated mice (n = 10, P <0.005 by Wilcoxon Rank Sum).

**Conclusion:** Despite a higher MEC value, APX001 showed significant efficacy against *R. oryzae* that was as protective as ISAV in immunosuppressed mice. Given the previously reported activity of APX001 against a strain of *R. delemar* with a lower MEC value, APX001 has now been shown to be efficacious against both species of *Rhizopus*, which together are responsible for ~60-70% of isolates causing lethal mucormycosis. Thus, continued investigation of APX001 against mucormycosis is warranted.

P421

APX001 Protects Immunosuppressed Mice from Scedosporiosis

S. Alkhazraji¹, T. Gebremariam¹, A. Alqarihi¹, Y. Gu¹, K.J. Shaw², A.S. Ibrahim¹

¹Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, United States of America, ²Amplyx Pharmaceuticals, San Diego, United States of America

**Objectives:** Scedosporiosis has high mortality rates. Owing to its rarity, comparative clinical trials are hard to perform. Animal models are an appropriate complementary avenue for evaluating antifungal therapy. Thus, APX001 was evaluated in an immunosuppressed murine model of invasive scedosporiosis.

**Methods:** The minimum effective concentration (MEC) of APX001A (the active moiety of APX001) was determined against 9 clinical isolates of *Scedosporium apiospermum*, *S. boydii* and *Lomentospora prolificans* using CLSI M38 methodology. ICR mice were immunosuppressed with cyclophosphamide and cortisone acetate on days -2, and +3, relative to intratracheal infection with 3.0×10⁷ cells of *S. apiospermum*. For survival studies, treatment with placebo (diluent control), APX001 (26, 52, 104, or 156 mg/kg, po), or liposomal amphotericin B (LAmB, 10 mg/kg, iv) began 16 h postinfection and continued for 7 or 11 days for APX and 4 days for LAmB. To extend the half-life of APX001, mice were administered 50 mg/kg of the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) 2 h prior to APX001 administration. To assess tissue fungal burden, mice were sacrificed on Day +4 and organs processed for conidial equivalent (CE) by qPCR.

**Results:** The APX001A MEC value for all tested isolates was 0.03 µg/mL. Treatment with ABT alone or LAmB, did not enhance survival of mice infected with *S. apiospermum* vs. placebo mice (n = 20 or 30). Treatment with APX001 at 26, 52, 104 or 156 mg/kg for 7 days enhanced median survival time vs. placebo (10, 12, 11, and 15 days for 26, 52, 104, and 156 mg/kg arms, respectively vs. 7 days for placebo, P<0.001). Further, APX001 treatment enhanced overall survival by day 21 when the experiment was terminated (30%, 40%, and 40%, respectively for 26, 52 or 156 mg/kg vs 10% for placebo). Survival
after treatment with APX001 for 7 days or 11 days was not different. All APX001 treatments resulted in ~2 log reduction in lung, brain and kidney CE.

**Conclusion:** APX001A is cidal against *S. apiospermum*, *S. boydii* and *L. prolificans* *in vitro*. APX001 protected immunosuppressed mice from scedosporiosis due to *S. apiospermum*. Continued investigation of APX001 as a novel antifungal agent against scedosporiosis is warranted.

P422

**Evaluation of new therapeutic candidates for the treatment of *Malassezia pachydermatis***

A. Sastoque¹,²,³, K. Ehemann², S. Triana²,³, M. Fernandez-Niño³, A. González³, S. Restrepo², A.M. Celis Ramírez²

¹Biotechnology Institute, Universidad Nacional de Colombia, Bogotá D.C., Colombia, ²Biological Sciences, Universidad de los Andes, Bogotá D.C., Colombia, ³Chemical Engineering, Universidad de los Andes, Bogotá D.C., Colombia

**Objectives:** *Malassezia pachydermatis* is a lipophilic and lipid-dependent yeast, that is part of the microbiota in domestic and wild animals' skin. However, it could be associated with otitis in canines and bloodstream infections in humans. Diseases caused by *M. pachydermatis* frequently exhibit chronic and recurrent clinical course, and the current antifungal therapy based on azoles is associated with increased resistance as well as significant side effects. Genome sequencing, metabolic network reconstruction, and gene essentiality analyses have been performed, allowing to identify fifteen candidates as therapeutic targets such as homoserine dehydrogenase (HSD), homocitrate synthase (HCS) and saccharopine dehydrogenase (SDH). These enzymes participate in the pathways of L-lysine or L-threonine biosynthesis, which have been reported as antibacterials and with antifungal effect. To assess the impact of these amino acids over the growth of *Malassezia pachydermatis* we evaluated the inhibition of HCS and SDH by L-lysine and HSD by L-threonine based on the feedback control, we additionally conducted *in vitro* susceptibility tests.

**Methods:** Recombinant proteins were expressed in *Escherichia coli* and purified to determine enzymatic activity through spectrophotometry detection of corresponding cofactors. Moreover, *in vitro* susceptibility tests using a modified CLSI M27-A3 method and agar diffusion assays were performed. The concentrations tested were (µg/ml) from 1500 to 4000 using Sabouraud dextrose broth (SDB) with tween 40 and tween 60.

**Results:** Therefore, the results of our study showed L-lysine 1 mM and 75 mM were able to inhibit the enzymatic activity of HCS and SDH, respectively while L-threonine 1 mM inhibited the activity of HSD. Lysine and threonine showed to be competitive inhibitors affecting the substrate-enzyme complex (SE) formation. For L-lysine reduction in turbidity was observed at 3100 µg/mL by microdilution method. In addition, agar diffusion showed a diameter of inhibition of 13 mm using 50 mg/mL. On the contrary, L-threonine did not display inhibitory activity in *M. pachydermatis* at 50 mg/mL as the maximum allowed concentration to be evaluated.

**Conclusion:** This study provides evidence that these three enzymes are efficiently regulated by the feedback of the final product of their corresponding metabolic pathway. L-lysine is a potent inhibitor for *M. pachydermatis*, possible for saturation of metabolic pathways and accumulation of high levels of L-lysine that generate toxicity and amino acids starvation in the cell. L-lysine will be considered as a potential antifungal/fungal static candidate. For L-threonine it is necessary to evaluate higher concentrations.

P423

**The effect of the orotomide antifungal olorofim on the growth and viability of *Scedosporium and Lomentospora* species.**

S. Du Pré¹, D. Law¹, G. Sibley¹, N. Read², M. Bromley², J. Oliver¹, M. Birch¹

¹F2G Limited, Manchester, United Kingdom, ²Manchester Fungal Infection Group, University of Manchester, Manchester, United Kingdom

**Objectives:** Olorofim is the first member of the new orotomide class of systemic antifungals acting through inhibition of fungal DHODH. Olorofim is active against all species of Aspergillus tested to
date including cryptic species. In addition, there is activity against a wide range of filamentous fungi including *Scedosporium*, *Paecilomyces* and endemic moulds. *Scedosporium* and *Lomentospora* species represent some of the most clinically challenging fungal infections often showing resistance to currently available antifungal agents. Previously olorofim has been shown to be highly active in vitro against a range of *Scedosporium* species and the closely related multidrug-resistant species *Lomentospora prolificans* (Wiederhold *et al* 2017, Biwas *et al* 2018). In this study we examined the effects of olorofim on isolates from the *Scedosporium* complex at a cellular level using microscopy and viability dyes.

**Methods:** *In vitro* MICs were undertaken using standard CLSI methodology. Images of the bottom of each well were obtained using a Leica SP8X confocal microscope. Studies on viability were carried out by allowing conidia to germinate for 8h and then exposing to olorofim at the MIC concentration for up to 120h. The viability dye DiBAC was used to determine cell death following exposure to olorofim over time.

**Results:** Olorofim MICs for *S. apiospermum* 451 and *L. prolificans* 206 were both determined to be 0.25 µg/mL. Imaging of microtitre wells above and below this MIC value showed that olorofim dramatically reduced growth of both *S. apiospermum* 451 and *L. prolificans* 206 at concentrations several dilutions below the MIC value. DiBAC staining was conducted with one of the study strains (*L. prolificans* 206) and showed that hyphae began to take up the dye after 24h and that the uptake increased steadily over time. When observations were stopped at 120h, >90% of hyphae had taken up DiBAC and were non-viable.

**Conclusion:** Olorofim demonstrated time-dependent killing of *L. prolificans* hyphae, consistent with previous observations with *Aspergillus* spp. Microscopic analysis of olorofim treated *S. apiospermum* and *L. prolificans* cultures, demonstrated profound effects on growth even at concentrations below the MIC level.

P425

*In Vitro* Activity of APX001A (Manogepix) and Comparator Agents against 1,706 Fungal Isolates
Collected During an International Surveillance Program (2017)

M.D. Huband¹, M.A. Pfaller¹, R.K. Flamm¹, P. Bien², M. Hodges², M. Castanheira¹

¹JMI Laboratories, North Liberty, United States of America, ²Amplyx Pharmaceuticals, San Diego, United States of America

**Objectives:** Current antifungal agents cover a majority of opportunistic fungal pathogens; however, breakthrough invasive fungal infections continue to occur and increasingly involve relatively uncommon yeasts and/or moulds that tend to exhibit decreased susceptibility to current agents. APX001A (manogepix) is a first-in-class small molecule inhibitor of the fungal Gwt1 enzyme that is required for acylation of inositol during glycosylphosphatidylinositol (GPI) anchor biosynthesis. It is active against the major fungal pathogens: *Candida* (except *C. krusei*), *Aspergillus*, and hard-to-treat moulds, including *Fusarium* and *Scedosporium*. In this study, we tested APX001A, anidulafungin (ANF), micafungin (MCF), fluconazole (FLU), and others against 1,706 contemporary clinical fungal isolates collected worldwide during 2017.

**Methods:** A total of 1,706 non-duplicate fungal isolates were collected from 68 medical centers in North America (37.3%), Europe (43.4%), Asia-Pacific (12.7%), and Latin America (6.6%). Among the isolates tested, 78.5% were *Candida* spp, 3.9% were non-*Candida* yeasts, including 30 *Cryptococcus neoformans* var. *grubii* (1.8%), 14.7% were *Aspergillus*, and 2.9% were other moulds. All isolates were tested by CLSI reference broth microdilution methods.

**Results:** APX001A (MIC₅₀/₉₀, 0.008/0.06 µg/mL) was the most active agent tested against *Candida* isolates (Figure); corresponding ANF, MCF, and FLU MIC₉₀ values were 16- to 64-fold higher. Similarly, APX001A (MIC₅₀/₉₀, 0.25/0.5 µg/mL) was ≥8-fold more active than ANF, MCF, and FLU against *C. neoformans* var. *grubii*. Against *Aspergillus*, APX001A (MIC₅₀/₉₀, 0.015/0.03 µg/mL) was comparable in activity to ANF and MCF. APX001A (MIC₉₀, 0.06 µg/mL) was ≥128-fold more active than ANF and MCF against *Scedosporium* isolates.
**Conclusion:** APX001A demonstrated potent *in vitro* activity against 1,706 fungal isolates, including echinocandin- and fluconazole-resistant strains. The extended spectrum of APX001A was also notable for its potency against many less common, yet antifungal-resistant, strains such as *Candida auris*, *A. lentulus*, *A. ustus*, *Fusarium solani* species complex, and *Scedosporium*. Further studies are needed to demonstrate the utility of APX001A in difficult-to-treat resistant fungal infections.

<table><thead><tr><th rowspan="2">Organism (no. tested)</th><th colspan="4">MIC<sub>50/90</sub> (µg/mL)</th></tr><tr><th>APX001A</th><th>Anidulafungin</th><th>Micafungin</th><th>Fluconazole</th></tr></thead><tbody><tr><td>Candida spp. (1.340)</td><td>0.008/0.06</td><td>0.06/2</td><td>0.015/1</td><td>0.25/4</td></tr><tr><td>C. albicans (414)</td><td>0.008/0.008</td><td>0.015/0.03</td><td>0.015/0.015</td><td>≤0.12/0.25</td></tr><tr><td>C. glabrata (321)</td><td>0.06/0.12</td><td>0.06/0.12</td><td>0.015/0.03</td><td>4/32</td></tr><tr><td>C. parapsilosis (270)</td><td>0.008/0.015</td><td>2/2</td><td>1/1</td><td>0.25/2</td></tr><tr><td>C. tropicalis (151)</td><td>0.015/0.03</td><td>0.03/0.06</td><td>0.03/0.06</td><td>0.25/0.5</td></tr><tr><td>C. krusei (43)</td><td>>2/≥2</td><td>0.06/0.12</td><td>0.06/0.12</td><td>32/32</td></tr><tr><td>C. dubliniensis (49)</td><td>0.004/0.008</td><td>0.03/0.12</td><td>0.015/0.03</td><td>≤0.12/0.25</td></tr><tr><td>C. lusitaniae (39)</td><td>0.03/0.12</td><td>0.25/0.5</td><td>0.12/0.25</td><td>0.25/2</td></tr><tr><td>Other Candida spp. (40)<sup>a</sup></td><td>0.008/0.03</td><td>0.5/2</td><td>0.25/0.5</td><td>1/16</td></tr><tr><td>Cryptococcus neoformans var. grubii (30)</td><td>0.25/0.5</td><td>>4/≥4</td><td>>4/≥4</td><td>2/4</td></tr><tr><td>Aspergillus spp. (251)</td><td>0.015/0.03</td><td>0.015/0.03</td><td>0.015/0.015</td><td>- / -</td></tr><tr><td>A. fumigatus (182)</td><td>0.015/0.03</td><td>0.015/0.03</td><td>≤0.008/0.015</td><td>- / -</td></tr><tr><td>Other Aspergillus spp. (18)<sup>b</sup></td><td>0.015/0.03</td><td>0.03/0.12</td><td>0.03/0.12</td><td>- / -</td></tr><tr><td>Scedosporium spp. (13)<sup>c</sup></td><td>0.03/0.06</td><td>4/8</td><td>>8/≥8</td><td>- / -</td></tr></tbody></table>

## Rezafungin *In Vitro* Activity against Phase 2 STRIVE Part A and Contemporary Nordic Clinical Candida Isolates Determined by the EUCAST Reference Method

M. Helleberg¹, K.M. Jørgensen², R. Datcu², R. Hare², M.C. Arendrup²,³,⁴

¹Dept. Of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark, ²Unit Of Mycology, Statens Serum Institut, Copenhagen, Denmark, ³Rigshospitalet, Dept. of Clinical microbiology, Copenhagen, Denmark, ⁴University Of Copenhagen, Dept. of Clinical Medicine, Copenhagen, Denmark

**Objectives:** Rezafungin is a novel echinocandin with favourable safety and pharmacokinetic characteristics, which allows for high drug exposures and once-weekly dosing. Epidemiological cut-off values (ECOFFs) of rezafungin have not yet been established. We determined the *in vitro* activity of rezafungin and four comparators against contemporary Nordic clinical isolates of *Candida* and other yeasts and established single centre wildtype upper limits (WT-UL). Subsequently, these WT-UL were used to evaluate rezafungin susceptibility for clinical isolates from the completed part A of STRIVE, the phase 2 trial of rezafungin for treatment of candidaemia and invasive candidiasis (NCT02734862).

**Methods:** 1,249 clinical isolates (19 *Candida*, 13 other yeast species) from Denmark, Norway and Sweden received at the Danish mycology reference laboratory in 2017-18 were identified using CHROMagar, matrix-assisted laser desorption ionization-time of flight mass spectrometry and, when needed, internal transcribed spacer sequencing. EUCAST E.Def 7.3.1 susceptibility testing included rezafungin, anidulafungin, micafungin, amphotericin B and fluconazole. WT-UL were established following EUCAST principles for visual and statistical ECOFF value setting for rezafungin and for comparators against species without EUCAST ECOFFs, allowing classification as wildtype or non-wildtype. The FKS target genes were sequenced for echinocandin non-wildtype isolates.

**Results:** Rezafungin MIC distributions for the most common *Candida* species and modal MICs for rezafungin and comparators are shown in Table 1. Rezafungin had species-specific *in vitro* activity similar to that of anidulafungin and micafungin. At a mg/L basis rezafungin was overall less active (modal MICs ≥ 2 two-fold dilutions higher) than anidulafungin and micafungin, but equally or more active than fluconazole and amphotericin B, except against *C. parapsilosis*, other *Candida* (amphotericin B only) and other yeasts. Rates of rezafungin non-wildtype isolates among the most common *Candida* species were <3%, only marginally higher than for amphotericin B and lower than for fluconazole, anidulafungin and micafungin (Table 2). We identified 27 (2.0%) echinocandin non-wildtype isolates: *C. albicans* (n = 11, 1.9%), *C. glabrata* (n = 11, 3.3%), *C. tropicalis* (n = 2, 2.7%), *C.*

<footnote><sup>a</sup> Candida auris (1), C. fabianii (2), C. fermentati (9), C. guilliermondii (6), C. haemulonii (1), C. intermedia (1), C. metapsilosis (6), C. nivaniensis (1), C. orthopsilosis (8), C. pararugosa (1), C. pelliculosa (2), C. rugosa (1), C. utilis (1)</footnote>

<footnote><sup>b</sup> Aspergillus lentulus (3), A. nidulans (7), A. nidulans species complex (2), A. sclerotiorum (1), A. thermomutatus (1), A. ustus (2), A. ustus species complex (1), A. versicolor (1)</footnote>

<footnote><sup>c</sup> S. apiospermum (2), S. apiospermum/boydii (5), S. boydii (2), S. dehoogii (2), S. prolificans (2)</footnote>
*dubliniensis* (n = 2, 2.9%) and *C. krusei* (n = 1, 1.2%). Alterations in Fks1 and/or Fks2 hot-spots (or within ±3 amino acids) were found in 20 of 25 sequenced non-wildtype isolates: Fks1: *C. albicans*: D648Y, P1354P/S (2), P1354S (3), R1361R/S, R1361G; *C. tropicalis*: F650S and S654P; *C. dubliniensis*: S645P (2); *C. krusei*: S659F. Fks2: *C. glabrata*: S663P (2), S663F (3). Fks1+Fks2: *C. glabrata*: S663F/L630Q and L664Q+Y658N+premature stop-codon. The rezafungin MICs for non-wildtype isolates were 0.25–2 mg/L. Rezafungin susceptibility testing of 95 *Candida* blood isolates from the STRIVE trial yielded MIC distributions comparable to those for the Nordic clinical isolates (Table 1). One *C. rugosa* isolate had an MIC of >8 mg/L, suggesting intrinsic resistance, and one *C. glabrata* isolate had an MIC of 0.5 mg/L, but no mutations in *FKS* target genes.

**Conclusion:** Rezafungin displayed broad *in vitro* activity with a species-specific susceptibility pattern similar to that of other echinocandins. WT-UL were suggested for the most common species. Few non-wildtype strains with alterations in *FKS* target genes or reduced susceptibility to rezafungin were identified among clinical *Candida* isolates from Nordic surveillance and the STRIVE trial.

<table><caption>**Table 1:** MIC ranges (mg/L) for rezafungin and modal MICs (mg/L) for rezafungin and comparators against clinical isolates of *Candida* and other yeasts</caption><thead><tr><th rowspan="3">Species</th><th colspan="7">Nordic Isolates</th><th colspan="3">STRIVE 2 isolates</th></tr><tr><th rowspan="2">n</th><th colspan="2">RZF</th><th rowspan="2">ANF<br>MIC</th><th rowspan="2">MFG<br>MIC</th><th rowspan="2">AMB<br>MIC</th><th rowspan="2">FLC<br>MIC</th><th rowspan="2">n</th><th colspan="2">RZF</th></tr><tr><th>MIC range</th><th>Modal<br>MIC</th><th>MIC range</th><th>Modal<br>MIC</th></tr></thead><tbody><tr><td>C. albicans</td><td>569</td><td>0.016 - 1</td><td>0.06</td><td>0.004</td><td>0.016</td><td>0.25</td><td>0.125</td><td>43</td><td>0.016 - 0.03</td><td>0.03</td></tr><tr><td>C. dubliniensis</td><td>68</td><td>0.03 - 2</td><td>0.125</td><td>0.008</td><td>0.03</td><td>0.06</td><td>0.125</td><td>3</td><td>0.03 - 0.06</td><td>ND</td></tr><tr><td>C. glabrata</td><td>329</td><td>0.03 - 2</td><td>0.125</td><td>0.03</td><td>0.016</td><td>0.25</td><td>4</td><td>17</td><td>0.06 - 0.5</td><td>0.06</td></tr><tr><td>C. krusei</td><td>82</td><td>0.03 - 1</td><td>0.125</td><td>0.03</td><td>0.125</td><td>0.5</td><td>16</td><td>1</td><td>1</td><td>ND</td></tr><tr><td>C. lusitaniae</td><td>20</td><td>0.125 - 0.5</td><td>0.125</td><td>0.03</td><td>0.06</td><td>0.125</td><td>0.5</td><td>0</td><td>ND</td><td>ND</td></tr><tr><td>C. parapsilosis</td><td>61</td><td>1 - 4</td><td>2</td><td>1</td><td>2</td><td>0.5</td><td>0.5</td><td>13</td><td>2 - 4</td><td>4</td></tr><tr><td>C. tropicalis</td><td>73</td><td>0.03 - 2</td><td>0.125</td><td>0.03</td><td>0.03</td><td>0.25</td><td>0.5</td><td>15</td><td>0.03 - 0.125</td><td>0.06</td></tr><tr><td>Saccharomyces<br>cerevisiae</td><td>15</td><td>0.125 - 0.5</td><td>0.25</td><td>0.06</td><td>0.125</td><td>0.25</td><td>8</td><td>0</td><td>ND</td><td>ND</td></tr><tr><td>Other Candida</td><td>41</td><td>0.03 - >4</td><td>1</td><td>0.25</td><td>0.25</td><td>0.25</td><td>4</td><td>3</td><td>0.125 - >4</td><td>ND</td></tr><tr><td>Other yeast</td><td>36</td><td>0.03 - >4</td><td>>4</td><td>>4</td><td>>4</td><td>0.5</td><td>0.25</td><td>0</td><td>ND</td><td>ND</td></tr></tbody><tfoot><tr><td>Total</td><td>1,249</td><td>0.016 - >4</td><td>0.06</td><td>0.004</td><td>0.016</td><td>0.25</td><td>0.125</td><td>95</td><td>0.03 - >4</td><td>0.06</td></tr></tfoot></table>

AMB: amphotericin B, ANF: anidulafungin, FLC: fluconazole, MFG: micafungin, RZF: rezafungin, ND: Not determined

<table><caption>**Table 2:** Rates of non-wildtype isolates for rezafungin and comparators</caption><thead><tr><th rowspan="2">Species</th><th colspan="5">Nordic isolates</th><th colspan="2">STRIVE 2 isolates</th></tr><tr><th>RZF<br>% >WT-UL</th><th>ANF<br>% >ECOFF</th><th>MFG<br>% >ECOFF</th><th>AMB<br>% >ECOFF</th><th>FLC<br>% >ECOFF</th><th>RZF<br>% >WT-UL</th><th></th></tr></thead><tbody><tr><td>C. albicans</td><td>0.5</td><td>1.1</td><td>6.2</td><td>0</td><td>5.6</td><td>0</td><td></td></tr><tr><td>C. dubliniensis</td><td>2.9</td><td>2.9<sup>1</sup></td><td>2.9<sup>1</sup></td><td>0<sup>1</sup></td><td>6.3<sup>1</sup></td><td>5.9</td><td></td></tr><tr><td>C. glabrata</td><td>1.5</td><td>3.3</td><td>4.9</td><td>0</td><td>8.2</td><td>0</td><td></td></tr><tr><td>C. krusei</td><td>1.2</td><td>2.4</td><td>3.7</td><td>0</td><td>ND</td><td>0</td><td></td></tr><tr><td>C. lusitaniae</td><td>0</td><td>0<sup>1</sup></td><td>5.0<sup>1</sup></td><td>5.0<sup>1</sup></td><td>10.0<sup>1</sup></td><td>ND</td><td></td></tr><tr><td>C. parapsilosis</td><td>0</td><td>0</td><td>0</td><td>0</td><td>4.9</td><td>0</td><td></td></tr><tr><td>C. tropicalis</td><td>2.7</td><td>2.7</td><td>2.7</td><td>0</td><td>8.2</td><td>0</td><td></td></tr><tr><td>Saccharomyces cerevisiae</td><td>0</td><td>0<sup>1</sup></td><td>0<sup>1</sup></td><td>0<sup>1</sup></td><td>0<sup>1</sup></td><td>ND</td><td></td></tr></tbody></table>

1: % >WT-UL based on 1,249 contemporary Nordic clinical strains. AMB: amphotericin B, ANF: anidulafungin, FLC: fluconazole, MFG: micafungin, RZF: rezafungin, ND: not determined
**Methods:** This was a retrospective service evaluation of 177 patients with various forms of pulmonary aspergillosis attending the National Aspergillosis Centre in Manchester. Due to risk of intolerance/bronchospasm, patients first received a challenge test with nebulised Fungizone in the hospital setting with spirometry pre/ post Fungizone. To improve tolerability, nebulised short acting beta agonist was given prior to administration of Fungizone (10mg in 4ml water for injection).

**Results:** The baseline FEV1 and FVC of the patients ranged from 0.94-2.97L and 13.3-5.15L respectively. 66% (117) were able to tolerate their first test dose, of whom 26 (21%) stopped using the nebulised therapy before their next clinic visit. Reasons cited for Fungizone intolerance included increased breathlessness (8), wheeze (7), bronchospasm (6), sore throat/voice (4), coughing (4), chest tightness (3), light-headedness/dizziness (2), diarrhoea (2), fatigue (2), mouth blisters/ulcers (2), headaches (2), nausea/vomiting (2) in addition to other symptoms. Eighteen continued nebulisation of Fungizone 10mg twice a day for >3 months. Eleven had allergic bronchopulmonary aspergillosis (ABPA), 4 had severe asthma with fugal sensitization (SAFS), 2 had Aspergillus bronchitis and 1 had Aspergillus sensitisation with cavitating nodules. Treatment courses ranged from 4months to 4years. At their first clinic follow-up, there were reported benefits with sputum production (7), sinus symptoms (2), dyspnoea (2), exercise tolerance (2), reduction in prednisolone maintenance dose (1) and one demonstrated radiologic improvement. Three had interruptions to their Fungizone treatment which had to be restarted following clinical deterioration off therapy (2 of whom had dose change to once daily 12.5mg). Five patients have remained on treatment with sustained benefit (e.g. clinical stability, marked reduction in frequency of chest infections, reduction in Aspergillus specific IgE and increased exercise tolerance). Four patients discontinued therapy as there was no perceived clinical benefit. Aspergillus PCR on sputum at first repeated measure following initiation of Fungizone treatment changed from positive to negative in 7 and stayed negative in 7. No pre-treatment PCR tests were done in 5 patients and no follow-up PCR tests were performed in 6 patients. Fungal cultures of sputum went from positive to negative after >3 months treatment in 6 patients, remained positive in 3 patients, remained negative in 4 patients and became positive in 2 patients.

<table><thead><tr><th>Aspergillus IgE</th><th>Aspergillus IgG</th><th>Total IgE</th></tr></thead><tbody><tr><td>-1.5</td><td></td><td>3</td><td>30</td></tr><tr><td>-19.3</td><td></td><td>-26</td><td>-400</td></tr><tr><td></td><td></td><td>7</td><td>1</td></tr><tr><td>-4.2</td><td></td><td>0</td><td>-66.7</td></tr><tr><td>0.1</td><td></td><td></td><td>3.6</td></tr><tr><td>-5.5</td><td></td><td>8</td><td>-357</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>-10.8</td><td></td><td>-9</td><td>-326</td></tr><tr><td>-2.19</td><td></td><td>-25</td><td>-252</td></tr><tr><td>-40.9</td><td></td><td>11</td><td>-440</td></tr><tr><td>0</td><td></td><td></td><td>-3.2</td></tr><tr><td></td><td></td><td></td><td>140</td></tr><tr><td>0.4</td><td></td><td></td><td>100</td></tr><tr><td>-0.31</td><td></td><td></td><td>-210</td></tr><tr><td>0.04</td><td></td><td>21</td><td>4.7</td></tr><tr><td>-1</td><td></td><td>-149</td><td>-239</td></tr><tr><td>-2.4</td><td></td><td>-6</td><td>-884</td></tr><tr><td>3.8</td><td></td><td></td><td>-7925</td></tr></tbody></table>

**Conclusion:** Nebulised Fungizone is a poorly tolerated treatment for pulmonary Aspergillosis with high dropout rates. There appears to be both clinical and serologic benefits following sustained treatment with nebulised Fungizone in some patients
# In-Silico and In-Vitro Evaluation of Anticandidal Activity of Cinnamaldehyde

G. Uttam<fnref n="1" />, K. Singh<fnref n="1" />, D. Katiyar<fnref n="2" />

**Objectives:** *Candida albicans* is a common commensal which resides in human body and causes invasive fungal infections especially in immunocompromised hosts. This study aims to evaluate the anticandidal activity of cinnamaldehyde through *in-silico* approach and *in-vitro* methods.

**Methods:** Interactions between cinnamaldehyde and potential drug targets of *C. albicans* was elucidated using *in-silico* approach which was further validated by determination of minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of cinnamaldehyde using broth microdilution method recommended by CLSI 2009 for yeast (M27-A2). The effect of cinnamaldehyde on micromorphology of *C. albicans* was studied. Sorbitol Assay (effect on cell wall) and Ergosterol Binding Assay of *C. albicans* were also performed. Time-kill curve was conducted using cell viability testing.

**Results:** *In-silico* studies revealed that cinnamaldehyde showed good binding affinity with potential targets of *C. albicans* such as ergosterol, N-myristoyl transferase and Secreted aspartic proteinase 5. MIC and MFC of cinnamaldehyde were found to be 8.2 µg/ml and 16.4 µg/ml respectively. In the morphological interference assays, it was observed that the cinnamaldehyde inhibited pseudohyphae, blastospores and chlamydospores formation. The time-kill curve of cinnamaldehyde showed that it required only few hours of exposure to effectively kill >90% of the inoculum.

**Conclusion:** Cinnamaldehyde showed good *in-vitro* antifungal potential against *C. albicans*. However, evaluation of pharmacodynamics and pharmacokinetics studies is desirous for complete elucidation of its action as a therapeutic which will be helpful for mankind.

# Antifungal Activity of Cinnamaldehyde against Some Human Pathogenic Fungi

K. Singh, A. Kumari, G. Uttam, N. Srivastava

Animal Mycology Laboratory, Department Of Zoology, Mmv, Banaras Hindu University, Varanasi, India

**Objectives:** Despite the availability of effective antifungals, the morbidity and mortality of fungal infections has remained high especially in individuals with impaired immunity. The limited targets of existing antifungals may be one of the reasons for the emergence of drug-resistant strains. In this regard, the anti-fungal potential of cinnamaldehyde, a plant product and active constituent of cinnamon was tested through *in-silico* approach which was further validated by *in-vitro* methods.

**Methods:** Antifungal activity of cinnamaldehyde was tested against *Cryptococcus neoformans* (ATCC 6352), *Candida albicans* (MTCC 3017), *Aureobasidium pullulans* var. *pullulans* (CBS 577.93), *Aspergillus terreus*, *Alternaria alternata* and *Fusarium oxysporum* (MTCC 2773) using broth micro-dilution methods recommended by CLSI (2009) for yeasts and moulds. Further, PDB structures of drug targets of said fungus species were downloaded or molleded (if not available) and their interactions with cinnamaldehyde were elucidated *in-silico* using RCSB Protein databank, PHYRE2 and PatchDock servers.

**Results:** Cinnamaldehyde showed potent anti-fungal activity against all the fungal pathogens screened. However, *Cryptococcus neoformans* (ATCC 6352) was found highly susceptible against it with the MIC value 1.367±0.260 mg/ml. *In-silico* interaction between targets of *Cryptococcus neoformans* (lanosterol 14-alpha demethylase), *Candida albicans* (ergosterol, N-myristoyl transferase and Secreted aspartic proteinase 5), *Aureobasidium pullulans* (chitinase), *Aspergillus terreus* (chitinase), *Alternaria alternata* (ornithine descarboxylase) and *Fusarium oxysporum* (Thiamine thiazole synthase) with cinnamaldehyde exhibited high binding energies and hydrogen bonds.

**Conclusion:** Cinnamaldehyde possesses broad spectrum and potential anti-fungal activity. Being a plant product, it also has advantage for structural diversity and therefore, could be a potential drug candidate for the treatment of fungal diseases. However, its drug likeness properties should be

<footnote n="1">¹Animal Mycology Laboratory, zoology, Mmv, Banaras Hindu University, Varanasi, India,</footnote>

<footnote n="2">²Chemistry, Mmv, Banaras Hindu University, Varanasi, India</footnote>
evaluated by ex-vivo and in-vivo studies.

*Aureobasidium pullulans*

<figure><img src="image_95.png" /><figcaption>*Aspergillus terreus*</figcaption></figure>

<figure><img src="image_96.png" /></figure>

<figure><img src="image_97.png" /><figcaption>*Fusarium oxysporum*</figcaption></figure>

<figure><img src="image_98.png" /><figcaption>*Cryptococcus neoformans*</figcaption></figure>

<figure><img src="image_99.png" /></figure>

<figure><img src="image_100.png" /><figcaption>*Alternaria alternata*</figcaption></figure>

<figure><img src="image_101.png" /></figure>

<figure><img src="image_102.png" /><figcaption>*Candida albicans*</figcaption></figure>

P430

Assessment of anti-*Candida* activity of Benzo(a)phenoxazines

J.I. Carvalho-Pereira¹,², M.S. Gonçalves³, M.J. Sousa¹,², P. Sampaio¹,²

¹Centre Of Molecular And Environmental Biology (cbma), Department Of Biology, University of Minho, Braga, Portugal, ²Institute Of Science And Innovation For Bio-sustainability (ib-s), University of Minho, Braga, Portugal, ³Centre Of Chemistry, University of Minho, Braga, Portugal

**Objectives:** Fungal infections represent a major health problem due to their prevalence, increasing number of cases and high complexity of the etiological agents involved. *Candida* species are the most common and clinically important being able to cause from less-severe superficial lesions to life-threatening disseminated mycosis. *C. albicans* remains the most common species isolated, however, infections caused by non-*albicans* species, such as *C. auris*, are increasing. Current treatments for fungal infections are not 100% effective due to the drug toxicity, pathogen resistance, low drug bioavailability and late diagnosis. At the moment, few groups of antimycotics are available to deal with these infections. The loss of clinical efficacy of these drugs creates a potential risk of spreading of resistant strains through the emergence of clones with high pathogenic potential. This illustrates that the development of new antimicrobials is crucial. Phenoxazine derivatives, namely benzo(a)phenoxazines have been shown to present good antifungal properties. Thus, in this work we evaluated the antifungal activity of five benzo(a)phenoxazin derivatives, against distinct *Candida* species and selected the most promising to develop an effective antifungal nanoformulation.

**Methods:** The revised *European Committee on Antimicrobial Susceptibility Testing* (EUCAST) protocol was used to determine the minimum inhibitory concentrations (MICs) of five benzo(a)phenoxazines against seven *Candida* species, namely *C.albicans*, *C.glabrata*, *C.parapsilosis*, *C.tropicalis*, *C.krusei*, *C.bracarensis* and *C.auris*. The most effective compounds were selected for the development of formulations based on DODAB:MO liposomes. All formulations were evaluated regarding the mean size, polydispersity index, surface charge, liposome stability and encapsulation efficiency. The most stable formulations were selected for antifungal activity re-evaluation against all seven *Candida*
species and for cytotoxicity assessment with different cell lines and using both the MTT and the
lactate dehydrogenase (LDH) release assays.

**Results:** demonstrated that all five benzo(α)phenoxazin derivatives tested presented antifungal activity against all seven *Candida* species, with MICs between 3.75 to 60 µM. Two of these benzo(α)phenoxazin derivatives presented MICs of 3.75-15 µM against all species tested, being selected to develop DODAB:MO formulations. The liposomal formulations were positively charged, presenting mean sizes between 300-500nm and polydispersity indexes of 0.4-0.5. The encapsulation efficiency was between 80-95% for both benzo(α)phenoxazin derivatives tested. The activity of formulation against all seven *Candida* species were evaluate with the liposomal formulations. Results demonstrated that empty liposomes did not affect *Candida* growth and that the encapsulation of benzo(α)phenoxazins increased their activity against *Candida* species. The cytotoxicity of the promising formulations was tested using different cell lines and the non-toxic formulations were selected.

**Conclusion:** The results obtained demonstrated that benzo(α)phenoxazins derivatives could be potential antifungal drugs since present activity against all *Candida* species tested, including the multidrug resistant *C. auris*. Moreover, the encapsulation of the benzo(α)phenoxazin derivatives increase the antifungal activity and reduce the cytotoxicity of compounds.

P431

Inhibitory effect of a curcumin-based compound against *Epidermophyton floccosum* clinical isolates

V. Camilo Pattini¹, C.R. Polaquini¹, T. Lemes¹, N. Brizzotti², J.P. Siqueira², M. Almeida², L.O. Regasini³
¹São Paulo State University - Institute of Biosciences, Humanities and Exact Sciences (UNESP - IBILCE), São José do Rio Preto, Brazil, ²FAMERP, São José do Rio Preto, Brazil, ³Chemistry And Environmental Sciences, UNESP (Sao Paulo State University), São José do Rio Preto, Brazil

**Objectives:** The present study aimed to evaluate the antifungal activity of a curcumin-based compound against clinical strains of *Epidermophyton floccosum* isolated from dermatomycoses.

**Methods:** For this study, the curcumin-based compound was obtained from the Laboratory of Antibiotics and Chemotherapeutics – LAQ, of the Institute of Biosciences, Letters and Exact Sciences (IBILCE, Unesp São José do Rio Preto). This new compound is a hybrid curcumin compound with substitutions to make the substance more stable and active. The clinical strains were from the culture collection of the Laboratory of Microbiology of the Medical School in São José do Rio Preto (FAMERP), Brazil. Susceptibility tests were performed for two *E.floccosum* strains (6069 and 6113), using the Clinical and Laboratory Standards Institute M38-A2 guidelines as reference. Percentage of inhibition was calculated by spectrophotometry using the : I = 1 - (AbsT-AbsCT / AbsCC) × 100; where: I = percentage of inhibition; AbsT = absorbance of the inoculum plus the compound solution; AbsCT absorbance of sterility control; AbsCC = absorbance of growth control. The curcumin (Merck®) was used as a control.

**Results:** The minimum inhibitory concentration (MIC) obtained for the curcumin-based compound against *E.floccosum* 6069 and 6113 strains was 15.62µg/mL, inhibiting 80 and 87% the growth of the strains, respectively. On the other hand, pure curcumin(Merck®) exhibited no activity at the concentrations tested.

**Conclusion:** The curcuminoid showed potent antifungal activity against *E. floccosum* strains, higher than pure curcumin (Merck®), showing a potential alternative against *E. floccosum*. In the future, toxicity tests will be performed allowing new therapeutic approaches in the treatment of dermatomycoses.

P432

Antifungal activity of Kombucha *Hibiscus sabdariffa* infusion extract against *Trichophyton mentagrophytes* and *Trichophyton rubrum*.

V. Camilo Pattini¹, N. Brizzotti², T. Lemes¹, J.P. Siqueira², M. Marques², M. Caetano³, T. Maschio-Lima³, B. De Almeida³, L. Bianco¹, C. Silva², C.R. Polaquini¹, L.O. Regasini⁴, E. Castilho², M. Almeida²
São Paulo State University - Institute of Biosciences, Humanities and Exact Sciences (UNESP - IBILCE), São José do Rio Preto, Brazil, FAMERP, São José do Rio Preto, Brazil, São Paulo State University - Institute of Biosciences, Humanities and Exact Sciences (UNESP - IBILCE), São José do Rio Preto, Brazil, Chemistry And Environmental Sciences, UNESP (São Paulo State University), São José do Rio Preto, Brazil

**Objectives:** The present study aimed to explore the antifungal potential of Kombucha infusion extracts on planktonic cells of potentially pathogenic fungal isolates, as an alternative to synthetic drugs.

**Methods:** Firstly, a SCOBY (Symbiotic Culture of Bacteria and Yeast), obtained from a local natural products store, was placed in 1 liter of distilled water with 20g of sucrose weekly for 30 days to remove any previous residues. The *Hibiscus sabdariffa* infusion (HI) was prepared with four grams of leaves/flowers boiled in 450 mL of sterile mineral water for 5 minutes and filtered on filter paper. The initial fermentation was obtained by mixing 20 g of SCOBY and 80 g of sucrose to the infusion, and the final volume was adjusted to 800 mL with sterile mineral water. The culture was then incubated at 25 °C for 20 days. The experiment was carried out in triplicate. After incubation, the solution was filtered in the MILLIPORE® System with 0.2 µm membrane. The chemical compounds of the filtrate were extracted using ethyl acetate. To obtain the dry and purified extract of the chemical compounds, the acetate phase was placed on a rotary evaporator. Minimal inhibitory concentrations (MIC) and minimal fungicide concentrations (MFC) of Kombucha HI extract against *Trichophyton rubrum* and *Trichophyton mentagrophytes* clinical isolates were investigated by the microdilution method, using as reference the document M38-A2 of the *Clinical and Laboratory Standards Institute*.

**Results:** The results obtained for the MIC of Kombucha HI extract against the *T. mentagrophytes* and *T. rubrum* strain were 125 µg/mL and 62.5 µg/mL, respectively. For the MFC, values observed were 250 µg/mL for *T. mentagrophytes* and 125 Oµg/mL for *T. rubrum*.

**Conclusion:** Kombucha HI extract showed antifungal activity against *T. mentagrophytes* and *T. rubrum*. In the future, isolation and identification of the extract compounds may allow new therapeutic approaches in the control of fungal infections.

P433

**Characterization of the specificity of the Ca37 monoclonal antibody and its protective role against Candida albicans infection**

A. Antoran¹, L. Aparicio-Fernandez¹, I. Buldain¹, L. Martin-Souto¹, M. Areitio¹, A. Rementeria¹, M.A. Ghannoum², B.B. Fuchs³, E. Mylonakis³, F.L. Hernando¹, A. Ramirez-Garcia¹

¹Immunology, Microbiology And Parasitology, University of the Basque Country (UPV/EHU), Leioa, Spain, ²Dermatology, Case Western Reserve University, Cleveland, United States of America, ³Division Of Infectious Diseases, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, United States of America

**Objectives:** *Candida albicans* is a commensal yeast of mucosal surfaces of human body. However, when the host is immunocompromised, this yeast can cause invasive infections, that present an unacceptably high mortality rate (up to 50%). Therefore, looking for alternative treatments, we have focused on monoclonal antibodies. Alcohol dehydrogenase (Adh) protein was selected for antibody development, due to its role in carcinogenic acetaldehyde production, its surface location and its immunogenicity potential. The objective of this work was to characterize the specificity of this Ca37 monoclonal antibody against *Candida albicans* and its ability to interfere with yeast infection.

**Methods:** Adh1 protein was cloned in *Escherichia coli*. This recombinant protein was used for mice immunization and Ca37 monoclonal antibody development. Proteomic analysis by two dimensional (2-DE) western blotting and LC-MS/MS identification, in yeast and hyphal fractions, were performed in order to identify the proteins detected by Ca37 and to determine its specificity against Adh1 protein. Indirect immunofluorescence was carried out to study surface location of the protein. In this case, sodium metaperiodate treatment was used for surface carbohydrate removal. Effect of Ca37 on yeast germination, growth and interaction with antifungals was studied. *Galleria mellonella in vivo* model infection, using an ΔADH1 mutant and parental strains was used to study the effect of ADH1
gene loss on virulence, and for testing Ca37 antibody role as therapeutic agent against C. albicans infection.

**Results:** The 2-DE western blot performed on cell wall-associated protein fraction showed that Ca37 detected Adh1 protein in yeast and Adh2 in hyphal cells. Immunofluorescence results indicated that the epitope recognized by the Ca37 was partially covered by carbohydrates, as antibody only bound to fungal cells after sodium metaperiodate treatment, or after long incubation times. Yeast germination was slightly inhibited, but growth inhibition reached up to 90%. The antibody also showed an additive effect with amphotericin B and fluconazole antifungals. Finally, in *Galleria mellonella* infection, we detected a higher survival when using the ΔADH1 strain, and the same level of survival using fluconazole or Ca37 treatment.

**Conclusion:** The Ca37 monoclonal antibody is specific for Adh proteins, which are located at fungal cell surface, partially hidden by carbohydrates, and seem relevant for virulence *in vivo*. Moreover, Ca37 is effective against yeast growth, provides an additive effect with antifungal drugs, and confers protection *in vivo*. Altogether, Adh proteins seem interesting targets against *Candida albicans* infections. *Financial support: This study was founded by a grant of UPV/EHU (PPG17/41). LA-F is recipient of a PhD grant of UPV/EHU and LM-S of the Basque Government.*

P436

**Rezafungin Clinical Safety and Efficacy in the Treatment of Candidemia and/or Invasive Candidiasis: Combined Results from the STRIVE Phase 2 Trial Parts A and B**

G.R. Thompson¹, P.M. Honore², J.P. Horcajada³, J. Fortun⁴, M. Hites⁵, M. Bassetti⁶, K. Mena⁷, L. Navalta⁷, R. Viani⁷, T. Sandison⁷, P. Pappas⁸

¹University of Davis Medical Center, Davis, United States of America, ²Brugmann University Hospital, Brussels, Belgium, ³Hospital del Mar, Barcelona, Spain, ⁴University Hospital Ramon y Cajal, Madrid, Spain, ⁵Erasme Hospital, Brussels, Belgium, ⁶Dept Of Medicine, University of Udine, Udine, Italy, ⁷Cidara Therapeutics, San Diego, United States of America, ⁸University of Alabama at Birmingham, Birmingham, United States of America

**Objectives:** Rezafungin (RZF) is a novel echinocandin designed to have next-generation properties, such as increased stability and safety and novel pharmacokinetics/pharmacodynamics (PK/PD), distinct from other systemic antifungals. The PK profile of RZF includes a prolonged half-life and front-loaded plasma exposure that allow for once-weekly therapy, as well as maximized pharmacometrics of antifungal efficacy. STRIVE is a global, double-blind, randomized, clinical trial that compared the safety and efficacy of RZF once-weekly to caspofungin once-daily in patients with candidemia and/or invasive candidiasis (IC). After successful completion of STRIVE Part A (Thompson et al, 2018), a second randomization (Part B) was conducted. Combined results from Parts A and B are presented herein.

**Methods:** Adults (≥18 y) with systemic signs and mycological confirmation of candidemia and/or IC were randomized to RZF once-weekly or standard of care (SOC) with caspofungin for ≥14 days (up to 4 weeks) (Figure 1). Efficacy was assessed by overall response (resolution of clinical signs of infection + mycological eradication) at day 14 (primary endpoint) and PI assessment of clinical response at day 14 and all-cause mortality at day 30 (secondary endpoints). Safety was evaluated by
adverse events (AEs) and mortality.

<figure><img src="image_103.png" /><figcaption>**Figure 1.** Phase 2 STRIVE Trial of Rezafungin Treatment of Candidemia and/or Invasive Candidiasis*: Part A and Part B (ITT)</figcaption></figure>

**Results:** Of 207 patients enrolled, 183 patients were included in the microbiological intent-to-treat population. Treatment groups were well balanced and matched in demographics and baseline characteristics. Patients with IC comprised ~21% of the combined population. RZF 400 mg/200 mg demonstrated greater efficacy than SOC, with the highest rates of overall success and clinical cure and the lowest rate of 30-day all-cause mortality of all 3 groups (Table 1). The rate of all-cause mortality through follow-up in the pooled RZF group was 15.6% (19/122) and 19.7% (12/61) for SOC. No concerning safety trends were observed. The incidence of ≥1 treatment-emergent adverse event (TEAE) related to study drug was 13/134 (9.7%) among RZF-treated patients (pooled) and 9/68 (13.2%) with SOC. Between the pooled RZF and SOC groups, rates of study drug-related TEAEs leading to study discontinuation (2.2% and 1.5%, respectively) and serious AEs (1.5% and 2.9%, respectively) were comparable.

<table><caption>**Table 1.** Topline Results from STRIVE – Part B and Combined (Part A + Part B), mITT</caption><thead><tr><th rowspan="3">Endpoint</th><th colspan="2">RZF once weekly<br>400 mg</th><th colspan="2">RZF once weekly<br>400 mg Wk 1/200 mg</th><th colspan="2">CSF once daily<br>70 mg D1/50 mg</th></tr><tr><th>Part B</th><th>Combined</th><th>Part B</th><th>Combined</th><th>Part B</th><th>Combined</th></tr><tr><th>N=43</th><th>N=76</th><th>N=15</th><th>N=46</th><th>N=33</th><th>N=61</th></tr><tr><th colspan="7">n (%)</th></tr></thead><tbody><tr><td>Overall Response<br>Success<sup>a,b</sup> (Day 14)</td><td>27 (62.8)</td><td>46 (60.5)</td><td>13 (86.7)</td><td>35 (76.1)</td><td>23 (69.7)</td><td>41 (67.2)</td></tr><tr><td>PI Assessment of<br>Clinical Response<br>Cure<sup>c,d</sup> (Day 14)</td><td>28 (65.1)</td><td>53 (69.7)</td><td>13 (86.7)</td><td>37 (80.4)</td><td>23 (69.7)</td><td>43 (70.5)</td></tr><tr><td>Mortality<br>All-cause (Day 30)<sup>d</sup></td><td>7 (16.3)</td><td>12 (15.8)</td><td>1 (6.7)</td><td>2 (4.4)</td><td>5 (15.2)</td><td>8 (13.1)</td></tr></tbody></table>

CSF=caspofungin; mITT=microbiological intent-to-treat population (all who received any study drug and had documented Candida infection); PI=Principal Investigator; RZF=rezafungin.

aExcludes indeterminate responses, defined as inability to assess outcome due to missing data point(s).

bOverall Success defined as resolution of attributable systemic signs of candidemia/IC present at baseline plus mycological eradication.

cOutcome that most closely approximates the primary outcome in prior candidemia/IC clinical trials.

dPhase 3 ReSTORE trial primary endpoint for the FDA.

**Conclusion:** RZF was safe and efficacious in the Phase 2 trial of RZF treatment in patients with candidemia and/or IC. RZF 400 mg/200 mg, the dosing regimen used in the ongoing Ph 3 study, demonstrated greater efficacy than SOC and the most favorable efficacy of all groups. No concerning trends in TEAEs were observed and, again, the RZF 400 mg/200 mg once weekly group demonstrated the most favorable safety outcomes in terms of TEAEs related to study drug. The recently completed Part B of STRIVE, combined with the previous success of STRIVE Part A, demonstrates the safety and efficacy of RZF treatment and supports its Phase 3 development.

P440

Invasive aspergillosis in adult non-hematologic patients

O. Shadrivova<fnref n="1" />, M. Tonkoshkur<fnref n="1" />, E. Desyatik², A. Volkova³, M. Popova³, S. Ermolova⁴, T. Bogomolova², S. Ignatyeva², L. Zubarovskaya³, B. Afanasyev³, N. Klimko<fnref n="1" />

<footnote n="1">¹Department Of Clinical Mycology, Allergy And Immunology, North-Western State Medical University named after I.I.Mechnikov, St. Petersburg, Russian Federation, ²Kashkin Research Institute Of Medical Mycology, North-Western State Medical University named after I.I.Mechnikov,</footnote>
St. Petersburg, Russian Federation, ³I.Pavlov First Saint Petersburg State Medical University, St. Petersburg, Russian Federation, ⁴Department Of Pulmonology, ³Leningrad Regional Clinical Hospital, St. Petersburg, Russian Federation

**Objectives:** To investigate the features of invasive aspergillosis (IA) in non-hematologic patients.

**Methods:** Retrospective analysis of clinical data of adult non-hematologic patients with IA. The EORTS/MSG 2008 criteria were used for IA diagnosis and assessment of response of therapy.

**Results:** In the study were included 46 non-hematologic patients with IA, median age – 48 years (19 - 99), females – 57%. The background conditions were autoimmune diseases – 24%, kidney diseases with acute or chronic renal failure – 20%, oncology – 15%, heart and vascular diseases – 11%, severe viral-bacterial pneumonia – 8%, HIV infection – 7%, lung pathology – 7%, severe influenza H3N2 – 4%, other – 4%. The risk factors of IA were glucocorticosteroid therapy – 50%, prolonged lymphocytopenia (<1,0 x10⁹/l, median – 18 days) – 48%, immunosuppressive therapy – 35%, ICU stay – 30%, precede surgical treatment – 28%, organs transplantation – 20%, severe neutropenia (median – 10 days) – 13%, diabetes – 11%, allogeneic hematopoetic stem cells transplantation (HSCT) – 2%, autologous HSCT – 2%. The main sites of infection were lungs – 85%, central nervous system (CNS) – 11%, and paranasal sinuses – 9%. Disseminated (≥2 organs) IA was in 17% patients. Clinical signs of IA were fever (≥ 38,5°C) – 79%, cough – 76%, respiratory failure – 59%, chest pain – 26%, and hemoptysis – 22%. CT scan signs were bilateral lesions – 56%, air crescent sign – 12%. The main etiological agents were *A.fumigatus* (62%), *A.niger* (14%), *A.flavus* (14%), *A.ustus* (5%) and *A.ochraceus* (5%). «Proven» IA was diagnosed in 28% patients, «probable» – 72%. Antifungal therapy was used in 93% patients, surgical treatment – 7%. Overall 12-weeks survival of patients was 76%.

**Conclusion:** Invasive aspergillosis developed in non-hematologic patients usually with autoimmune (24%), renal (20%) or oncology diseases (15%). The main risk factors were glucocorticosteroid therapy (50%), lymphocytopenia (48%), immunosuppressive therapy (35%), organs and hematopoetic stem cells transplantation (24%). The main etiological agents were *A.fumigatus* (62%), *A.niger* (14%), and *A.flavus* (14%). The main sites of infection were lungs (85%), disseminated (≥2 organs) infection was in 17% patients. Overall 12-weeks survival of patients was 76%.

D. Stubbe¹, M. Hendrickx², P. Becker²

¹Mycology & Aerobiology, Bccm/ihem Collection, Sciensano, Brussels, Belgium, ²Mycology, Sciensano, BCCM/IHEM, Brussels, Belgium

**Objectives:** Open science aims at sharing scientific outputs in order to maximize the impact of research. This allows follow-on studies, facilitates new discoveries, improves reproducibility of experiments and favours transparency of results. Although open data is becoming a well-known concept, less attention is given to the availability of research materials. In medical sciences, public culture collections represent a historical example of open science, thanks to their longstanding experience in the preservation of living microorganisms and their distribution for further scientific investigations or development. These microbial resource centres provide well-characterized, quality-controlled and authenticated strains and associated data. In medical mycology, the diversity of fungal pathogens is important and needs to be secured following (inter)national legislations for future basic and applied health questions. We wish to promote the deposit of isolates in public collections. However, the responsibility to make fungal strains available is shared by researchers, funding agencies and publishers.

**Methods:** We made a random selection of articles in the field of medical mycology to check whether or not studied isolates were deposited and made available in public collections.

**Results:** A random selection was made of 90 articles issued between 2017 and 2019 in seven journals specialized in medical mycology. Out of them, only 13 (less than 15%) mentioned the deposit of the strains.

**Conclusion:** Medical mycologists need to be more aware towards strain conservation. Governmental funding policies should request the deposit of strains isolated during financed projects. Regarding
publishers, most journals encourage authors to make biological material available but few specifically require deposit and most isolates investigated in publications are not preserved. Editors should therefore implement mechanisms for active agreement by authors to deposit strains when submitting an article. Such mechanisms could follow Transparency and Openness Promotion guidelines¹ for journals that include a standard for research materials. Two level of stringency are proposed for this standard: (1) the article states whether strains are available and, if so, where to access them and (2) the mandatory deposit of the strains in a trusted repository.

P442

**Fungicidal and Yeasticidal activity of chemical disinfectant and antiseptic products: European standards & Real hospital life - How to be successful?**

S. Loeffert-Frémiot

Microbiology Laboratory, Member Of Wg 1 Of The Cen, Laboratoires ANIOS, LILLE, France

**Objectives:** Chemical disinfectants and antiseptic products are a crucial part of the infection control strategy in hospital and are upregulated by European standards. If infection control fails, the consequences could be severe especially for immunocompromised. This is why it is important that testing procedures of chemical disinfectants are sufficient and well understand to ensure that disinfectants for the medical area are reliable. The aim of this work is to help users to understand where, when and how to use these products and to make and update of the conception and evolution of these standards following what is happening in real work.

**Methods:** Explain the European Standards to which products have to conform in order to support the claims for Fungicidal and yeasticidal activity. This include the description of EN 14885 "Chemical disinfectants and antiseptics – Application of European Standards for chemical disinfectants and antiseptics" (2018) requirements and the tests conditions of the EN 13624 "Chemical disinfectants and antiseptics – Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity in the medical area -Test method and requirements (phase 2, step 1)" (2013). In order to adapt the European standards to what happen in medical area, standards are systematically revised by group of expert members of the Working Group 1 of the European Comission of standardization (Commission Europeenne de Normalisation :CEN).

**Results:** It is known that some strains could be challenging for healthcare workers by their emerging character or by the apparition/ evolution of their resistance in different part of European country. Therefore, European standards need to continuously follow and adapt to the field situation. The choice of test organisms and conditions of EN 13624 is under discussion to see their relevance. It is a critical point that need to be raised and share with expert people of the field.

**Conclusion:** European Standards requirements must be continuously revised to be suitable for effective infection strategy in medical area and particularly in healthcare settings. These standards are more often not really known by healthcare people working on the field, fighting everyday against resistant pathogens. They need to have all the keys to ensure a reliable startegy against these pathogens.

P443

**Comparing the efficacy of three adsorbents, bentonite, activated charcoal and fuller's earth on the detoxification of aflatoxin-contaminated feeds.**

A. Mgbeahuruike

Department Of Veterinary Pathology And Microbiology, Faculty Of Veterinary Medicine,, University of Nigeria, Nsukka, Nsukka, Enugu, Nigeria

**Objectives:** To test the ability of three low-cost and locally available adsorbents (activated charcoal, bentonite and fuller's earth) to detoxify poultry feeds contaminated with aflatoxin.

**Methods:** Mould growth was induced on poultry feeds by sprinkling water on the feeds until the feed was fully wet, the feed was packed in dark nylon bags and covered tightly. The bags were stored in dark environments for 3 weeks. The feeds were divided into 4 groups, Contaminated but untreated, Contaminated but treated with bentonite, Contaminated but treated with Activated
charcoal and contaminated but treated with Fuller's earth. Aflatoxin concentration in the feeds was measured using reverse phase HPLC. Fungal contaminants in the feeds were isolated and molecularly identified. Phylogentic analysis was reconstructed using MEGA 5.0. Measurement of performance indices of the birds like haematological and serum biochemical parameters were carried out and analysed statistically using Minitab.

**Results:** Bentonite was the most effective adsorbent and lowered the total aflatoxin concentration from 120 ± 38 µg/kg to 15 ± 5.0 µg/kg, while the concentration was reduced to 20 ± 6.5 µg/kg in feed treated with fuller's earth. The three adsorbent treatments resulted in 63-100% weight increase of the birds, compared with birds fed the untreated, aflatoxin-containing feed. Based on sequencing of the internal transcribed spacer and phylogenetic analysis, 86% of the identified fungal contaminants were *Aspergillus* species, with *Aspergillus tamarii* and *A. nominus* being the most prevalent with a frequency of occurrence of 21% each. The number of white blood cells in blood samples was increased by 13-17% in birds that consumed adsorbent-treated feed, compared with the untreated, aflatoxin-contaminated feed. Serum biochemical analysis further showed that aspartate amino transferase activity was 2.3 to 2.6 fold higher in birds that consumed the contaminated but untreated feed, compared with feed treated with adsorbents. Hepatic lesions were also prominent in the liver of the birds fed contaminated but untreated feed but were reduced in the group that were fed the adsorbent-treated feeds, especially in the bentonite-treated feed.

**Conclusion:** Conclusively, the adsorbents were able to reduce the concentration of aflatoxin in the feed and this reflected positively on the general performance of the birds.

P444

Evaluation of the growth of *Sporothrix schenckii* and *Sporothrix brasiliensis* when exposed to the iron element

M.L. Scroferneker¹, A.H. Da Silva Hellwig², H. Klein De Abreu², A. Carvalho Ribeiro¹, H. Tedesco De Oliveira¹, E. Heidrich Pezzi¹, R. Adriel Zanette³

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, ³Postgraduate Program In Biological Sciences: Pharmacology And Therapeutics, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

**Objectives:** The aim is to evaluate the viability of *Sporothrix* spp. when exposed to iron, using 11 isolates of *S. schenckii* and 10 isolates from *S. brasiliensis*.

**Methods:** All the samples used were previously analyzed by molecular methods. Conidia were collected from cultures with seven days of growth in potato dextrose agar medium and suspended in 0.85% saline solution. Each suspension was standardized in a Neubauer chamber, adjusted to the concentration of 10⁶ conidia / ml, and inoculated at the center of petri plates containing potato dextrose agar plus the following iron concentrations (FeCl₃): 0.0625%; 0.125%; 0.25%. The isolates were also inoculated into plates containing potato dextrose agar pure or added with 1 mM ascorbic acid and 1 mM ferrozine to eliminate traces of iron present in the medium. Plates were incubated in triplicates at 35 °C for 28 days to measure the mean diameter of colonies (mm) and perform analysis of variance followed by the Tukey post-hoc test in the GraphPad Prism statistical program, with significance p < 0.05.

**Results:** It has been found that high iron concentrations are toxic to *Sporothrix* spp., as seen in the concentration of 0.25% FeCl₃, where there was no growth of the isolates. In addition, medium with absence of iron also had to lower growth of *Sporothrix* spp. when compared to the potato dextrose agar medium and the medium plus 0.0625% and 0.125% FeCl₃.

**Conclusion:** It is possible to conclude that the viability of *Sporothrix schenckii* and *Sporothrix brasiliensis* is affected by iron, which is an important element for its biological activity.

P445

The interaction between *Sporothrix schenckii* sensu sctricto and *Sporothrix brasiliensis* with *Acanthamoeba castellanii*
M.L. Scroferneker¹, P. Lemos Tavares², F. Kercher Berte¹, A. Carvalho Ribeiro¹, M. Brittes Rott¹

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

**Objectives:** The aim of our study was to verify the profile of interaction between *S. schenckii sensu stricto* and *S. brasiliensis* and *A. castellanii* by an *in vitro* model of coculture as a modulator factor of both microorganisms intrinsic characteristics.

**Methods:** For this purpose the phagocytosis index of *S. schenckii sensu stricto* and *S. brasiliensis* by *A. castellanii* was evaluated; the *S. schenckii sensu stricto* and *S. brasiliensis* viability after contact with *A. castellanii*; the amoeba viability after contact with the fungus; and the influence of *S. schenckii sensu stricto* and *S. brasiliensis* in encystment process of *A. castellanii* were performed. The analyses indicated that *S. schenckii* and *S. brasiliensis* have suffered phagocytic events by *A. castellanii* and this index is significantly higher for *S. schenckii* in the first two hours compared to *S. brasiliensis*.

**Results:** Our results showed a significant increase in conidia and hyphae count after 72h of coculture of *S. brasiliensis* and lysis of amoebae occurred after fungal internalization.

**Conclusion:** *S. schenckii sensu stricto* and *S. brasiliensis* probably used amoebae as nutritional source. Our results were obtained *in vitro* and may not demonstrate the same behavior *in vivo*, being required *in vivo* studies with co-infection in order to have a thorough understanding of this relationship.

P446

**Estimation of fungal risk in cystic fibrosis: lessons from the MucoFong prospective project**

L. Delhaes¹, F. Francis², R. Enaud³, P. Soret⁴, M. Consortia⁵, S. Bui³, M. Fayon³, R. Thiebaut²

¹Parasitology Mycology, Univeristy of Bordeaux, Centre de Recherche Cardio-Thoracique de Bordeaux, U1045, F-33000, Bordeaux, France, ²CHU Bordeaux, Department of Public Health, Bordeaux, France, ³University of Bordeaux, Centre de Recherche Cardio-Thoracique de Bordeaux, U1045, CHU de Bordeaux, CRCM Pédiatrique, CIC 1401, Bordeaux, France, ⁴University of Bordeaux, Inserm, Bordeaux Population Health Research Center, UMR 1219, INRIA SISTM Team, Bordeaux, France, ⁵The Mucofong Investigation Group, Bordeaux, France

**Objectives:** Beside the main objectives of the multicentric project "MucoFong" (PHRC-19021906) that evaluated the respiratory fungal composition in CF and provided the first French guidelines for mycological analysis of CF sputum, data collected during this two-year follow-up of 299 CF patients (3 visits per patient including biological, radiological, and clinical data) were analyzed to assess associations between the microorganisms identified in the sputum and the clinical evolution.

**Methods:** Relations between microorganisms identified in the sputum and clinical course of patients were longitudinally analyzed taking as output variable FEV1 (Forced Expiratory Volume in 1s expressed as percent of predicted value). As data were collected several times throughout the follow-up, a mixed-model analysis was performed to evaluate the effect of a sporadic or chronic colonization with bacteria and/or fungi at the inclusion on the evolution of lung function over two years. Univariate plus multivariate analysis were achieved. A sensitivity analysis was performed to identify interactions between microbes.

**Results:** *P. aeruginosa*, *A. xylosoxidans*, *S. maltophilia*, and *C. albicans* were associated to a more significant decrease in FEV1 during the two-year follow-up, adjusted on age, gender, hospital centre, BMI, and gastrooesophageal reflux disease. Unexpectedly, FEV1 evolution was lower of 11.26% in patients presenting an intermittent colonisation with non-pneumoniae *Streptococcus* than others.

**Conclusion:** To conclude, these results were in agreement with data recently published, and provided new insights into the bacterial but also the fungal colonization as a key factor in assessing and predicting lung function evolution in CF patients. **The Mucofong Investigation Group:** Marc Pihet, Emilie Fréalle, Yolande Lemeille, Claudine Pinel, Hervé Pelloux, Gilles Gargala, Loic Favennec, Isabelle Accoceberry, Isabelle Durand-Joly, Frédéric Dalle, Frédéric Huet, Annlyse Fanton, Amale Boldron, Guy-André Loeuille, Philippe Domblides, Bérengère Coltey, Isabelle Pin, Catherine Llerena, Françoise Troussier, Christine Person, Christophe Marguet, Nathalie Wizla, Caroline Thumerelle, Dominique Turck, Anne Prévotat, Benoit Wallaert, Sylvie Leroy, Jean-Philippe Bouchara Grant:
Project MucoFng 2.0 (RF20160501626/1/1/275) from Vaincre la Mucoviscidose (VLM)

P447

Evaluation of metabolic adaptations of *Fonsecaea pedrosoi* using tricyclazole and FTIR

M.L. Scroferneker¹, A. Koehler², V. Corbellini³

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, ³Department Of Chemistry And Physics, Universidade de Santa Cruz do Sul, Santa Cruz do Sul, Brazil

**Objectives:** Evaluating the metabolic adaptations of *Fonsecaea pedrosoi* when exposed to tricyclazole using Fourier Transform Infrared Spectroscopy (FTIR).

**Methods:** Twelve isolates of *F. pedrosoi* from the collection of the Pathogenic Fungi Laboratory, Department of Microbiology, ICBS, UFRGS, were cultivated for 14 days at 30°C in Petri dishes with Sabouraud agar. For each isolate were used two plates with 4 µg/mL or 16 µg/mL of tricyclazole. The spectra were acquired through attenuated total reflection (ATR) using fragments of the cultures on dehydrated agar. Five spectra were recorded from 4000 to 650 cm⁻¹ for each isolate. The data set was preprocessed and normalized by amplitude. The Pirouette® software was used to perform the Principal Component Analysis (PCA) and the Partial Least Squares Discriminant Analysis (PLS-DA) using the first derivative and an orthogonal signal correction. These analyzes were perfomed to verify if it was possible to separate the classes (growth with 4 or 16 µg/mL) through the spectra, showing if there are metabolic adaptations of *F. pedrosoi* when exposed to tricyclazole.

**Results:** The PCA didn't allowed class differentiation due to irrelevante background information. With PLS-DA, using only one component, the minimum error of class prediction was about 0.001, with Bayes limit between classes close to 0.5 and 100% of accuracy in the identification of the cultures in 4 or 16 µg/mL of tricyclazole. The results show that the metabolic adpatations are reproducible, but discrete, and the regions of the spectra that most contributed to the differentiation were the bands of 989 cm⁻¹ and 1086 cm⁻¹ (sugars); 1508 cm⁻¹ (proteins) and 1617 cm⁻¹ (unsaturated carbon bonds, evidence of the formation of the 1,8-dihydroxynaphtalene (DHN) melanin precursors).

**Conclusion:** There are metabolic adaptations of *F. pedrosoi* with the exposition to tricyclazole. These adaptations can be evaluated through FTIR, which is relevant for the development of techniques to make correlations with models of virulence and pathogenicity of these fungi.

P449

Bridging the knowledge gap on mycoses in Africa; setting up a Pan-African Mycology Working Group

R. Oladele¹, I. Akase², A. Fahal³, N. Govender⁴, M. Hoenig¹, J.-P. Gangneux⁶, T. Chiller⁷, O.A. Cornely⁸, D. Denning⁹, A. Chakrabarti¹⁰

¹Medical Microbiology & Parasitology, College of Medicine of University of Lagos, Lagos, Nigeria,
²Internal Medicine, Lagos University Teaching Hospital, Lagos, Nigeria, ³Mycetoma Research Centre, Kartoum, Sudan, ⁴National Institute for Communicable Diseases, Johannesburg, South Africa,
⁵Division of Infectious Diseases, Department of Medicine, UCSD, San Diego, United States of America, Austria, United States of America, ⁶Universite de Rennes, Rennes, France, ⁷Mycotic Division, Centre for Disease Control, Atlanta, United States of America, ⁸Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany, ⁹Global Action Fund for Fungal Infections, Geneva, Switzerland, ¹⁰Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

**Objectives:** The African continent has an estimated population of 1.3 billion people accounting for roughly about a quarter of the world's population and has an estimated 700,000 fungal related deaths in the setting of HIV infections. From the data generated by GAFFI; an estimated 47,643,919 Africans suffer from fungal diseases, of which 1,728,657 suffer from a serious fungal infection annually (GAFFI Factsheet). This is a course for concern considering the fact that this is data from only 15 (out of 57) African countries (Table 1). Almost all African countries lack a surveillance system for fungal
infections; the only exception is South Africa. Interestingly, only South Africa has a reference mycology laboratory. Also, there is a pervasive picture of inadequate/poor diagnostic capacity, low level of awareness/index of suspicion among health care workers and policymakers, unavailability, and non-accessibility to antifungal medications. Most African countries have poorly funded and overburdened health systems. Additionally, a high prevalence of HIV in Sub-Saharan Africa contributes to a high burden of opportunistic fungal infections. Recently, the African sub-region developed a network of mycology experts whose goal is to provide a common platform for a compressive discussion and collaboration on fungal infections.

**Methods:** Recent outreach efforts of ISHAM and also the European Confederation of Medical Mycology (ECMM) aimed to increase the involvement of African countries and experts in e.g. the "One World One Guideline" initiative and also the ECMM Academy. The Medical Mycology Society of Nigeria with support from ISHAM and ECMM held its first international conference on March 2019, which cumulated with the establishment of the PanAfrican Mycology Working Group (PAMWG).

**Results:** PAMWG will organize and engage African leaders in the field of mycology in the African sub-region linked to ISHAM. The aim is to provide better interaction&synergy among regional leaders in order to develop educational programs for capacity building to aid in the diagnosis and care of patients with fungal infections. It will also encourage countries' initiatives to develop clinical guidelines for the clinical management of fungal infections and support the need for the establishment of reference mycology laboratories. Objectives The ISHAM-PAMWG aims to provide a platform for all the leaders in the field on mycology from different African countries in order to initiate epidemiological studies, regional guidelines, and educational programs to increase the capacity/ expertise of African specialists in the early detection and treatment of fungal infections. These activities will be achieved via the following: Education and training programs for health care workers involved in the care and evaluation of patients with fungal infections. South-south, and north-south collaborations and partnership. Organization of regional collaborative studies. Participation in the ECMM One World One Guideline program. Encourage all PAWG members to affiliate with ISHAM Organize local meetings in African countries in collaboration with ISHAM and ECMM Participation of leading centers in Africa in the ECMM Excellence Center program To advocate the recognition of neglected fungal diseases in Africa.

**Conclusion:** Meeting the objectives listed above will generate the needed data for advocacy needed to drive government commitment to allocate the resources required.
<table><caption>Table 1: Distribution of burden of fungal infections reported from Africa</caption><thead><tr><th>Country</th><th>2019 Population [million]</th><th>Total burden of serious fungal infection</th><th>Oesophageal candidiasis</th><th>Oesophageal candidiasis</th><th>RVVC</th><th>Fungal keratitis</th><th>Tinea capitis</th><th>Serious fungal infections</th></tr></thead><tbody><tr><td>Algeria</td><td>42.63</td><td>588,942</td><td>832</td><td></td><td>485,188</td><td></td><td>4,265</td><td>78657</td></tr><tr><td>Burkina Faso</td><td>20.28</td><td>1,360,280</td><td>7,450</td><td>24,300</td><td>179,002</td><td></td><td>1,132,781</td><td>16747</td></tr><tr><td>Cameroon</td><td>25.45</td><td>1,126,332</td><td>43,300</td><td></td><td>316,555</td><td></td><td>721,000</td><td>45477</td></tr><tr><td>Egypt</td><td>101.05</td><td>1,649,686</td><td>700</td><td>2,250</td><td>1,307,765</td><td>11,550</td><td></td><td>327420</td></tr><tr><td>Ethiopia</td><td>109.97</td><td>8,820,437</td><td>57,344</td><td>166,050</td><td>1,426,988</td><td></td><td>7,051,736</td><td>118319</td></tr><tr><td>Ghana</td><td>30.06</td><td>1030563</td><td>18,292</td><td></td><td>442,621</td><td>810</td><td>598,840</td><td>30,000</td></tr><tr><td>Kenya</td><td>51.13</td><td>3,186,523</td><td>114,000</td><td>306,000</td><td>594,660</td><td></td><td>1,712,676</td><td>459187</td></tr><tr><td>Malawi</td><td>19.68</td><td>1,338,523</td><td>73,000</td><td>216,000</td><td>326,960</td><td>1,826</td><td>670,900</td><td>49837</td></tr><tr><td>Mozambique</td><td>31.35</td><td>1,836,374</td><td>75,718</td><td>184,307</td><td>348,179</td><td></td><td>1,181,686</td><td>46484</td></tr><tr><td>Namibia</td><td>2.63</td><td>60,456</td><td>9,861</td><td>6,660</td><td>37,390</td><td></td><td></td><td>6545</td></tr><tr><td>Nigeria</td><td>200.01</td><td>17,983,517</td><td>144,195</td><td>253,062</td><td>1,512,520</td><td></td><td>15,581,400</td><td>492340</td></tr><tr><td>Senegal</td><td>16.71</td><td>1,743,507</td><td>1,946</td><td></td><td>191,228</td><td></td><td>1,523,700</td><td>26633</td></tr><tr><td>S/ Africa</td><td>58.01</td><td>4,047,138</td><td>623,598</td><td>1,150,560</td><td>1,002,499</td><td></td><td>1,003,490</td><td>266991</td></tr><tr><td>Tanzania</td><td>60.79</td><td>1,422,204</td><td>88,509</td><td>81,051</td><td>759,500</td><td></td><td>420,000</td><td>73144</td></tr><tr><td>Uganda</td><td>45.62</td><td>2,500,000</td><td>12,062</td><td>30,959</td><td>375,540</td><td></td><td>1,300,000</td><td>781439</td></tr><tr><td>Total</td><td>815.37</td><td>47,643,919</td><td>1270807</td><td>2421199</td><td>9,306,596</td><td>14,186</td><td>32,902,474</td><td>1728657</td></tr></tbody></table>

<table><caption>Table 2: Description of some fungal infections which are unique to Africa</caption><thead><tr><th>Disease</th><th>Agent</th><th>Presentation</th></tr></thead><tbody><tr><td>Entomophthoromyces</td><td>Conidiobolus coronatus, C. necongruentis, Basidiobolus ranarum</td><td>Chronic subcutaneous zygomy costs of the face and limbs</td></tr><tr><td>Emergomycosis</td><td>Emergomyces africana</td><td>...</td></tr><tr><td>African histoplasmosis</td><td>Histoplasma duboi</td><td>Skin and subcutaneous nodules/ abscesses, and osteomyelitis</td></tr><tr><td>Favus</td><td>Trichosporon schoenlinii</td><td>Crusts of the scalp (scutulum) in children and few adolescents</td></tr></tbody></table>
<figure><img src="image_104.png" /><figcaption>Figure 1: Reports of burden estimate done so far in African</figcaption></figure>

P450

**Importance of Sphingolipid Biosynthetic Pathway for Growth, Biofilm Formation and Membrane Integrity of the Pathogenic Fungus *Scedosporium boydii***

R. Rollin Pinheiro¹, V. Pereira Rochetti¹, M.I.D.S. Xisto¹, B. Bastos², A. Rella³, S. Rozental², M. Del Poeta³, E. Barreto-Bergter¹

¹Instituto De Microbiologia Paulo De Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, ²Instituto De Biofísica Carlos Chagas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, ³Department Of Molecular Genetics And Microbiology, Stony Brook University, Stony Brook/New York, United States of America

**Objectives:** *Scedosporium boydii* is a worldwide emerging pathogen able to cause a wide-spectrum infection ranging from mycetoma to invasive infections in immunocompromised patients. It is also considered the second more frequent pathogenic fungus associated to cystic fibrosis patients. Sphingolipids are abundant components of membranes in fungal cells, playing a variety of roles such as heat stress response, signal transduction, hyphal growth, endocytosis and apoptosis. Particularly, glucosylceramides (GlcCer) have been studying by our group during the last decades in *Scedosporium/Lomentospora* complex, being associated to fungal growth and pathogenesis. This study aims to evaluate the sphingolipid synthesis during the germination process, as well as the importance of different sphingolipids for fungal growth, membrane integrity using some inhibitors of the biosynthetic pathway.

**Methods:** S. *boydii* conidia were treated with Myriocin, a serine palmitoyltransferase inhibitor, or PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol), a glucosylceramide synthase inhibitor, and the results were compared to the non-treated control. Sphingolipid synthesis was evaluated by mass spectrometry of lipids extracted by fungal cells treated or not with the inhibitors. The effect of the inhibitors on fungal growth was analyzed by germination assay, colony forming units (CFU) quantification and biofilm formation. Filipin staining, checked by fluorescence microscopy, was performed to evaluate the accumulation of membrane microdomains on hyphal tips. Membrane integrity was analyzed by transmission electron microscopy (TEM) and fungal susceptibility to membrane stressors, such as SDS and NaCl. Susceptibility to antifungal agents in the presence of Myriocin and PPMP was evaluated through the checkerboard method.

**Results:** S. *boydii* treated with Myriocin and PPMP displayed decreased sphingolipid production and showed a reduced growth, forming less CFU and biofilms compared to the control. Additionally, the germination process was impaired, since treated cells formed short germ tubes compared to mature hyphae in the control. It was confirmed with filipin staining, in which lipid raft accumulation in hyphal tips, essential for hyphae elongation, was not observed in treated cells. Analysis by TEM revealed the presence of regions with loss of membrane integrity, what was confirmed through the increase in membrane susceptibility to SDS and NaCl in treated cells. Myriocin-treated cells became
more susceptible to different classes of antifungal drugs, suggesting that sphingolipids on the membrane could interfere in drug susceptibility.

**Conclusion:** Sphingolipids have been considering a potent target for the develop of new antifungal drugs. Thus, the study of these molecules in pathogenic fungal becomes a useful approach in medical mycology. The present data highlight the participation of sphingolipids in different fungal cell events that are crucial for fungal growth, virulence and drug resistance. Hyphae elongation and biofilm formation are important processes for mold proliferation and host tissue invasion. In addition, membrane integrity, especially regarding lipid raft areas, is essential for signal transduction, stress resistance and a variety of other physiological processes in fungal cells. For these reasons, the use of sphingolipid inhibitors helped to show how these molecules are promising targets for new drugs against *Scedosporium/Lomentospora* infections.

P451

**Studying the unknown: implementation of two recognized protocols to characterize lipid droplets in *Malassezia pachydermatis***

M. Mantilla¹, G. Ariza¹, C. Cabrera², S. Restrepo¹, A.M. Celis Ramírez¹

¹Biological Sciences, Universidad de los Andes, Bogotá D.C., Colombia, ²Chemistry, Universidad de los Andes, Bogotá D.C., Colombia

**Objectives:** *Malassezia pachydermatis* is a lipid-dependent yeast that is part of animal skin mycobiota and is associated with otitis externa and dermatitis in animals, as well as fungemia in humans. The lipid metabolism of this yeast is of high relevance because it may play an essential role during pathogenesis; however, these processes are yet to be well understood. Lipid droplets (LD) have become a subject of study in *Malassezia*, as they are known to store lipids and proteins in other biological models such as *Mycobacteria*, Hepatitis B virus, *Saccharomyces cerevisiae* and, mammal eukaryotic cells. Therefore, this study aims to standardize for the first time two protocols for the analysis of LD in *Malassezia pachydermatis*: The first one to characterize their structure by observation under a laser scanning confocal fluorescence microscopy; and the second one for the extraction and purification of LD for future lipidomic analysis.

**Methods:** Here we standardized two protocols to describe LD: the first one to characterize the structure is based on co-staining yeast cells and LDs with different fluorochromes: Nile Red (Sigma-Aldrich), LipidTox red neutral (Invitrogen) and BODIPY 493/503 (Invitrogen), in combination with calcofluor white (Sigma-Aldrich). The first three fluorochromes bind to neutral lipids present in the LD and calcofluor binds to the cellulose and chitin in the fungal cell wall. The second protocol to extract and purify LD induces yeast cell lysis by a combination of chemical and physical techniques, followed by the modification of the density of the solution with different buffers to allow the extraction and purification of LD.

**Results:** We standardized a protocol to optimize LD fluorescent staining to characterize their structure under confocal microscopy. We found that the optimal steps are as follows: 1) Preparation of the staining solution with a mixture of 4% formaldehyde and the three neutral lipid fluorophores.

2) Incubation at room temperature of LipidTox and Nile Red for 2 hours, and BODIPY for 30 minutes.

3) Double washing with PBS, resuspension in 4% formaldehyde and mounting on microscope slides.

4) Addition of calcofluor white, and 0.01% and 10%(v/v) KOH to the slide. We also optimized the protocol to get the of rupture *M. pachydermatis* cells for the extraction and purification of LDs. We found that the optimal conditions are: 1) Enzymatic digestion with *Trichoderma harzianum* enzymes (Sigma-Aldrich) for 2 hours at 47°C. 2) Mechanical disruption by adding 0.5 mm zirconium pearls to the sample and ten cycles of 1 minute of a vortex and 30 seconds on ice. In addition, cell wall removal by stroking the sample 10 times with a Dounce homogenizer. 3) Finally, several ultracentrifugation cycles in buffers containing Ficoll 400 (Sigma-Aldrich) to separate the LDs in the supernatant from the organelles that stay in the pellet.

**Conclusion:** Improving and standardizing these protocols can contribute to the study of the morphology, structure and, in the future, the lipid composition of LD in *Malassezia pachydermatis*. This study also could disclose clues about the lipid metabolism field in this yeast.
P452

**Characterization of the local inflammatory reaction after inhalative infection of immunocompromised mice with the mucormycete *Rhizopus arrhizus***

C. Speth<sup>1,2</sup>, J. Marcabruni<sup>1</sup>, N. Parth<sup>1</sup>, F. Koci<sup>1</sup>, C. Lass-Flörl<sup>2</sup>, H. Maier<sup>3</sup>, G. Rambach<sup>4</sup>

<sup>1</sup>Div Hygiene & Med. Microbiology, Med. Univ. Innsbruck, Innsbruck, Austria, <sup>2</sup>Christian Doppler Laboratory for Invasive Fungal Infections, Innsbruck, Austria, <sup>3</sup>Institute Of Pathology, INNPATH, Innsbruck, Austria, <sup>4</sup>Div Hygiene & Med. Microbiology, Med. Univ. Innsbruck, Medical University of Innsbruck, Austria

**Objectives:** The mucormycete species *Rhizopus arrhizus* mainly enters the body of immunosuppressed patients via inhalation of the spores. The local pathogenesis of inflammatory processes in the lung is poorly understood. For that reason we investigated the inflammatory reaction and the antifungal immune reaction in the lung of *Rhizopus*-infected mice.

**Methods:** Mice treated with cortisone or cyclophosphamide were intravenously infected with a high dose of *Rhizopus arrhizus* spores. For comparison mice with selective deficiency in neutrophils, alveolar macrophages or complement were used. Lung parameters were investigated by histology and by isolation of bronchoalveolar lavage (BAL). Immune processes were quantified in blood samples taken from the animals.

**Results:** Lack of neutrophils as well as treatment with cyclophosphamide were the most prominent risk factors for a lethal outcome after *Rhizopus* infection; in contrast, depletion of alveolar macrophages or cortisone treatment represented only moderate risk factors. Interestingly, complement deficiency even ameliorated the outcome, presumably due to a reduced inflammatory process. Lack of macroscopic organ abnormalities beside the lung indicated that fungal infection was mainly limited to the lung. BAL analyses revealed clear signs of excessive inflammation such as presence of erythrocytes, massive infiltration of granulocytes and high levels of the marker haptoglobin. Furthermore, macrophages in the lung were significantly augmented and these immune cells were highly activated. Most spores found in BAL were bound to or phagocytosed by neutrophils and macrophages and only few fungal hyphae were visible.

**Conclusion:** Inflammatory processes in the lung after inhalation of *Rhizopus arrhizus* are the main reason for fatal outcome; fungal growth and/or dissemination plays a minor role.

P453

**Pathogenesis and amphotericin B treatment of *Lichtheimia corymbifera* infection in a murine model**

G. Rambach<sup>1,2</sup>, J. Marcabruni<sup>1</sup>, N. Parth<sup>1</sup>, C. Lass-Flörl<sup>1,2</sup>, C. Speth<sup>1,2</sup>

<sup>1</sup>Div Hygiene & Med. Microbiology, Med. Univ. Innsbruck, Innsbruck, Austria, <sup>2</sup>Christian Doppler Laboratory for Invasive Fungal Infections, Innsbruck, Austria

**Objectives:** Immunocompromised individuals are the main risk group for invasive mucormycosis, but the relevance of the precise nature of immunodeficiency is insufficiently investigated. Furthermore, only limited information exists about differences in the efficiency of amphotericin B (AmB) therapy in various patient groups. For that purpose we aimed to study pathogenesis and AmB treatment of inhalative *Lichtheimia corymbifera* infection and to compare the susceptibility of pancytopenic mice versus mice with selective defects in innate immunity.

**Methods:** Mice treated with cyclophosphamide or cortisone or selectively deficient in neutrophils, alveolar macrophages or complement, were inhalatively infected with *Lichtheimia corymbifera*. Half of the animals received therapy with liposomal AmB. Survival, clinical status and immunological processes were monitored.

**Results:** Immunosuppression of the animals with cyclophosphamide represented the most relevant risk factor for fatal outcome of *Lichtheimia* infection, followed by treatment with cortisone. Although neutropenia is assumed to be the correlate for susceptibility against fungal infection, selective depletion of neutrophils predisposed only moderately for severe disease and mortality. Interestingly, neither complement deficiency nor depletion of alveolar macrophages represented major risk factors
for the animals. Various clinical parameters such as body temperature and weight loss as well as immunological parameters confirmed these results. For those animals dying from invasive *Lichtheimia* infection, neurological symptoms were the second most reason for scarifying the mice. Treatment with liposomal AmB successfully improved the outcome in the animal groups with neutropenia, cortisone acetate and cyclophosphamide with the most prominent effect in the cortisone group.

**Conclusion:** Affectation of several immune cell types is necessary to make the animals susceptible for invasive mucormycosis by *Lichtheimia corymbifera*, whereas selective loss of neutrophils or alveolar macrophages have only minor effects.

P454

**The role of Aspergillus fumigatus fumagillin in fungal virulence.**

X. Guruceaga¹, U. Perez-Cuesta¹, A. Pellon², B. Uribe³, O. Gonzalez³, N. P. Keller⁴, F.L. Hernando¹, J. Anguita², R.M. Alonso³, A. Ramirez-Garcia¹, A. Rementeria¹

¹Immunology, Microbiology And Parasitology, University of the Basque Country (UPV/EHU), Leioa, Spain, ²Inflammation And Macrophage Plasticity Laboratory, CIC bioGUNE, Derio, Spain, ³Department Of Analytical Chemistry, University of the Basque Country (UPV/EHU), Leioa, Spain, ⁴Department Of Medical Microbiology And Immunology, University of Wisconsin, Madison, United States of America

**Objectives:** *Aspergillus fumigatus* is a saprophytic fungus capable to cause pulmonary infections in immunosuppressed patients. We have previously demonstrated an overexpression of 67% of the genes involved in the fumagillin metabolic pathway during experimental infection of immunosuppressed mice. The aim of this work was to demonstrate the implication of this molecule in fungal pathogenesis.

**Methods:** We performed a set of *in vitro* experiments using commercial toxin preparations to first standardize the UHPLC-PDA analytical method and then identify its toxic effects on macrophages (RAW 264.7), melanome (B16-F10), and the lung epithelial (A549) cell lines. Moreover, we carried out fungus-host cell co-incubation assays using murine primary bone marrow derived macrophages (BMMs). Finally, *in vivo* assays were performed by intranasal infection of immunosuppressed Swiss mice treated with 100 mg/kg of cyclophosphamide intraperitoneally every 72 h, starting four days before the infection. The mice, in group on 10, were infected with, the non-fumagillin producer mutant strain, *ΔfumR*, as well as the wild type (WT), *ΔakuB*<sup>ku80</sup>, and the complemented strains. All the experiments were repeated at least thrice.

**Results:** The UHPLC-PDA method assay showed that *A. fumigatus* WT strain is able to produce the toxin at a 1.31 µg/ml growing in RPMI during 24 h at 37°C. Furthermore, when we incubated the cell lines in the presence of 1 µg/ml of commercial toxin for 24 h, eighty percent of the toxin disappeared from the media of A549 cultures, 58% in the case of B16-F10 melanome and only 4% in cultures with RAW 264.7 cells. However, the viability of the A549 cells was not significantly affected, while B16-F10 and RAW264.7 cells showed a significant reduction in their viability compared to. untreated cells. Assays *in vitro* showed that the null strain, *ΔfumR*, was significantly more susceptible to phagocytosis by the BMMs than the complemented or WT control strains. On the other hand, no differences in germination were observed during the co-incubation with BMMs, but *ΔfumR* showed a significant increase in the percentage of double branches conidia compared to the other two strains. Indeed around 25% of the germinated *ΔfumR* conidia showed two branches after 8 hours of co-incubation. The *in vivo* study did not show any significant differences in mice survival between the groups infected with the *ΔfumR* and the WT strains. In contrast, mice infected with the complemented strain showed a higher mortality rate, maybe due to the increase secretion of fumagillin by complemented strain, as we showed in the *in vitro* assays.

**Conclusion:** This study shows that *A. fumigatus* is able to secrete the mycotoxin fumagillin, a toxin that penetrates inside the cells causing cell death. Furthermore, this toxin could be a fungal defense factor against phagocytosis and may play an important role during *A. fumigatus* pulmonary infection.
**Acknowledgement.** This study was funded by a grant of UPV/EHU (PPG17/41). Pre-doctoral grants of the Basque Government and UPV/EHU have supported XG, and BU and UPC, respectively.

P456

Establishment of an experimental protocol of chronic tobacco exposure to study its effects on a
mice model of pulmonary paracoccidioidomycosis

R. Buccheri¹,², A. Nunes Duarte Neto¹, F.L. Batista Da Silva², G.O. Marques Haddad Carvalho², L. Buffoni Roque Da Silva³, R. Soares De Azevedo Neto¹, C.P. Taborda³, G. Benard⁴

¹Department Of Pathology, Medical School, University of São Paulo, São Paulo, Brazil, ²Laboratório De Investigação Médica Lim53, Insituto de Medicina Tropical, São Paulo, Brazil, ³Laboratório De Fungos Dimórficos Patogêncios, Biomedical Sciences Institute, University of São Paulo, São Paulo, Brazil, ⁴Laboratório De Investigação Médica Lim53, Instituto de Medicina Tropical/Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil

**Objectives:** Paracoccidioidomycosis (PCM) is caused by *Paracoccidioides spp*, and the most important systemic mycosis in non-immunosuppressed patents in Latin America. It has two major clinical presentations, the acute/subacute, more rare (≤5% of the cases) form (AF), and the more prevalent, chronic form (CF). In the AF, the phagocytic-mononuclear system is predominantly affected, while the lung is the target organ in the CF. The AF in general develops some time (weeks to months) after exposure to *Paracoccidioides* spp. and the CF develops from reactivated quiescent foci many years or decades after the initial exposure. However, we still do not know the factors that trigger the reactivation of the fungus and the development of the CF. Of note is the observation that most (>90%) CF patients refer tobacco smoking. It is well known that chronic tobacco exposure leads to imbalance of the pulmonary immune responses, and has a role in the development of other chronic pulmonary infections such as tuberculosis. We therefore aimed to develop a protocol of chronic tobacco exposure to study its effects on an experimental mice model of pulmonary PCM. We present here the preliminary results of this protocol.

**Methods:** C57BL/6 animals were exposed to cigarette smoke for 120 minutes (equivalent to ~16 cigarettes) twice/day, 5 days/week, for a total of 20 weeks, using a previously described device [Biselli PJC et al. Braz J Med Biol Res. 2011;44:460-468]. Three groups of animals were studied: an infected/exposed group, inoculated intra-tracheally with 10² *P. brasiliensis* yeast cells 2 weeks prior to initiation of tobacco exposure, an infected only group, and an exposed only group. The tobacco exposure protocol was adjusted to result in carboxyhemoglobin (COHb) levels of 10-15% after the daily first exposure, and 15-20% after the second exposure, to simulate the levels observed in chronic moderate smokers [Stewart RD et al. JAMA. 1974;26:1187-95]. Animals were sacrificed at weeks 4, 12 and 20 and their lungs examined for colony forming units, area involved by the granulomatous inflammation and histological assessment of the effects of tobacco exposure.

**Results:** The tobacco exposure protocol was successful in mimicking the effect of chronic tobacco smoking in humans: a) the mean level of COHb after the first tobacco exposure (measured monthly) was 15,5% (range 10,9-16,1%), and of 20,5% (range 15,9-21,1%) after the second exposure, while in the no-exposed infected group it was 0,1% (range 0-0,3%); b) By week 20 there were moderate dysplasia of the columnar epithelium, increased mucous production, alveolar septum destruction and focal areas of bronchiolitis. Preliminary data showed that tobacco exposure increased both the CFU and
the area involved by the granulomatous inflammation as shown below.

<figure><img src="image_105.png" /></figure>

**Conclusion:** These preliminary results indicate that: 1) We were able to establish a protocol of tobacco exposure mimicking that observed in patients with chronic form paracoccidioidomycosis. 2) Tobacco exposure impacts the pulmonary disease in a mouse model of PCM. Financial support: Fapesp #2016/08730-6 and #2017/12374-3. CPT and GB receive a senior researcher fellowship from CNPq

P457

**Phenotypic characterization of *Aspergillus fumigatus* *maiA* deficient strain.**

X. Guruceaga¹, U. Perez-Cuesta¹, A. Martin-Vicente², A.C.O. Souza², A.V. Nywening², Q. Al Abdallah², W. Ge², F.L. Hernando¹, J. Fortwendel², A. Ramirez-Garcia¹, A. Rementeria¹

¹Immunology, Microbiology And Parasitology, University of the Basque Country (UPV/EHU), Leioa, Spain, ²Department Of Clinical Pharmacy And Translational Science, University of Tennessee Health Science Center (UTHSC), Memphis, United States of America

**Objectives:** Invasive aspergillosis is the most dangerous disease produced by *Aspergillus fumigatus*. To identify genes important for host-pathogen interactions, we analyzed fungal gene expression in contact with macrophages and lung epithelium cell lines, as well as during an *in vivo* infection in immunosuppressed mice and detected multiple genes overexpressed in all the situations. One of these genes, *maiA*, is involved in the Tyrosine metabolic pathway and could be an important gene for energy production during host-pathogen interactions. For that, the aim of this work was to characterize a mutant strain deficient in *maiA* gene.

**Methods:** The loss of function mutant strain was built by CRISPR-Cas9 technology using the *A. fumigatus* genetic background Af-293. The transformation method was carried out using a standard protocol. For the phenotypic spot characterization assays, we used Glucose minimal medium (GMM) agar plates supplemented with Congo red (80 µg/ml), Calcofluor white (80 µg/ml), Voriconazole (0,5 µg/ml), Caspofungin (4 µg/ml) or Anidulafungin (4 µg/ml) and minimal medium supplemented with L-tyrosine (10 mM) or Phenylalanine (10 mM) as the sole carbon source. Antifungal susceptibility tests using Voriconazole, Itraconazole, Isavuconazole, Posaconazole, Caspofungin and Anidulafungin were carried out following the EUCAST method. Finally, glucan quantification, following the Aniline blue assay, and pyomelanin quantification, using GMM broth and GMM broth supplemented with L-Tyrosine (10mM) or Phenylalanine (10mM), were also performed. All the assays were performed in triplicate.

**Results:** The CRISPR-Cas9 technique provided a gene-targeting efficiency of 60% (3 out of 5 colonies) using the *A. fumigatus* genetic background Af-293. After spot dilution assays analysis, differences in the fungal growth between the wild type and the mutant strain were not found in presence of Calcofluor white, Voriconazole, Caspofungin or Anidulafungin. In contrast, Δ*maiA* was hypersusceptible to Congo red despite a lack of significantly different amounts of glucan per milligram of mycelia. Furthermore, no significant differences were observed in the MIC or MEC for the triazoles or the echinocandins, respectively. However, Δ*maiA* was unable to use either L-tyrosine or Phenylalanine as the sole carbon source and pyomelanin production by the mutant strain was
significantly higher than the wild type when GMM broth was supplemented with L-tyrosine or Phenylalanine. In GMM broth non-supplemented, both the wild type and Δ*maiA* showed the same pyomelanin production pattern.

**Conclusion:** CRISPR-Cas9 is an accurate technique that allowed us to generate the mutant strain Δ*maiA* using the *A. fumigatus* genetic background Af-293 with a great efficiency. This mutant was not able to grow in presence of Congo red or to use L-Tyrosine or Phenylalanine as a sole carbon source. Pyomelanin production was higher for Δ*maiA* in the presence of both amino acids, which is consistent with results published using other mutants of the Tyrosine degradation pathway. Future studies will explore in deep the role of *maiA* in animal models of IA. **Acknowledgement.** This study was funded by a grant of UPV/EHU (PPG17/41). EMBO Short-Term Fellowships and pre-doctoral grants of the UPV/EHU have supported XG and UPC respectively.

P458

*Scedosporium apiospermum* *sidD* gene is responsible for the biosynthesis of the hydroxamate-type siderophore Nα-methylcoprogen B, and is essential for fungal growth

Y. Le Govic¹,², V. Havlicek³, N. Papon¹, S. Le Gal¹, J.-P. Bouchara¹,⁴, P. Vandeputte¹,⁴

¹Host-Pathogen Interaction Study Group, SFR ICAT 4208, Univ Angers, Univ Brest, Institute of Biology in Health, IRIS, University Hospital Center, Angers, France, Angers Cedex, France,

²Laboratory of Parasitology-Mycology, University Hospital Center, Fort-de-France, Martinique, Fort-de-France, Martinique, ³Institute Of Microbiology, Academy of Science, Prague, Czech Republic,

⁴Laboratory of Parasitology-Mycology, University Hospital Center, Angers, France, Angers, France

**Objectives:** *Scedosporium* species are filamentous fungi which commonly colonize the airways of patients with cystic fibrosis (CF). Nevertheless, there is only scarce information on their virulence mechanisms. Iron acquisition is crucial for the survival of nearly all microorganisms and is implicated in virulence of numerous pathogens that chronically inhabit the CF lung. Several genes relevant for siderophore-mediated iron uptake were previously identified in *Scedosporium apiospermum* genome, notably an orthologue of *sidD*, a non-ribosomal peptide synthetase (NRPS) gene that was demonstrated to drive the production of the extracellular hydroxamate-type siderophore fusarinine C (FsC) in *Aspergillus fumigatus*. Here, we aimed to determine the chemical structure of the siderophore produced by *S. apiospermum* *sidD* gene and to assess its role in fungal growth.

**Methods:** First, *sidD* gene was disrupted in a wild-type (WT) strain after previous disruption of the gene *ku70* encoding one of the components of the DNA repair system. Next, siderophore analysis was performed on lyophilized filtrates from WT and Δ*sidD* strains grown in minimal Yeast Nitrogen Base (YNB) medium for 48 h at 37°C. WT strain was cultured in YNB supplemented or not with 100 µM bathophenантroline disulfonate (BPS), while the mutants (n = 3) were grown in YNB without BPS only. The impact of *sidD* deletion was then studied by cultivating the fungus on yeast extract-peptone-dextrose-agar (YPDA) plates containing BPS and supplemented with culture filtrates from either WT or mutant strains. Finally, the ability of *S. apiospermum* to assimilate iron from xenosiderophores was assessed by using iron-saturated ferrichrome, ferrioxamine or pyoverdine (20 µM each) as sole source of iron.

**Results:** Analysis of the extracellular siderophores by high-resolution mass spectrometry performed on culture filtrate from WT strain yielded a molecular mass of *m/z* (M-2H + Fe)⁺ = 794.314 for the iron-saturated siderophore, perfectly matching Nα-methylcoprogen B. This compound was absent in all Δ*sidD* mutant strains, indicating that *sidD* gene is essential for its synthesis. Other siderophores like FsC or TAFC were not found in any sample. Moreover, the disruption of *sidD* resulted in the lack of growth under iron-restricted conditions, and growth could be restored by supplementation of the culture medium (YPDA+BPS) with a culture filtrate from the WT strain. Conversely, incorporation of lyophilized filtrates from mutant strains in the culture medium did not rescue growth, corroborating the biochemical analyses. Interestingly, the use of iron-saturated ferrichrome or ferrioxamine as sole source of iron permitted a normal growth of all strains, indicating their direct assimilation by the fungus independently of siderophore production. Conversely, pyoverdine supported growth of the
WT strain only, suggesting that N-methylcoprogen B is important for iron acquisition from pyoverdine.

**Conclusion:** Our results revealed that the *S. apiospermum* *sidD* gene drives the synthesis of a unique extracellular siderophore, N<sup>α</sup>-methylcoprogen B, that is essential for fungal growth. This compound seems important for iron acquisition from pyoverdine, which might explain why *S. apiospermum* and *Pseudomonas aeruginosa* are rarely found concomitantly in the CF lungs. Works are in progress to determine the contribution of *sidD* in the virulence of *S. apiospermum*.

P459

Effect of *Candida albicans* on the malignant characteristic of tumor cells

L. Aparicio-Fernandez, A. Antoran, L. Martin-Souto, I. Buldain, M. Areitio, A. Rementeria, F.L. Hernando, A. Ramirez-Garcia

Immunology, Microbiology And Parasitology, University of the Basque Country (UPV/EHU), Leioa,
Spain

**Objectives:** *Candida albicans* is an opportunistic pathogenic fungus usually present in different areas of the human body such as mouth and gut. Under certain circumstances, this yeast is able to reach the bloodstream causing disseminated candidiasis. Therefore, *C. albicans* is in close relationship with human cells, being in constant contact with them. This microorganism has been related to cancer and metastasis through different mechanisms such as carcinogenic product synthesis and induction of pro-metastatic microenvironment. Among the types of cancer studied in relation to *C. albicans* are oral malignancies and melanoma metastasis. Hence, this work aims to determine whether the exposure to *C. albicans* is able to change tumor cells phenotype, increasing their aggressiveness.

**Methods:** Three tumor cell lines were used for this study: murine melanoma cells (B16M), human oral squamous cell carcinoma (H357) and murine colon carcinoma cells (C26). First, an adhesion assay was performed to test the effect of *Candida albicans* on tumor cells. Then, cancer cell lines were stimulated with antibiotic-inactivated fungus and conditioned media of the fungus, and wound healing assay was performed to measure migration activity of tumor cells. Afterwards, RNA was extracted from tumor cells to examine the expression of cancer-related genes by RT-qPCR and the supernatant was collected to measure the level of pro-inflammatory and cancer-related cytokines by ELISA.

**Results:** First, we observed that *C. albicans* was able to adhere to the three tumor cell lines. Then, wound healing assays with both antibiotic-inactivated *C. albicans* and conditioned media of *C. albicans* showed an increase in migration capacity of the different cell lines in comparison with the control, which indicates that the exposure to this fungus can induce a phenotypical change in tumor cells. The pro-tumor microenvironment was also altered, being the expression of different studied cytokines and cancer-related genes increased in presence of the fungus.

**Conclusion:** These results suggest that exposure to *C. albicans* induce alteration or changes in tumor cells and the related microenvironment that can lead to an increased malignancy. Acknowledgement: This study was funded by a grant of UPV/EHU (PPG17/41). Pre-doctoral Grants of Basque Government and UPV/EHU have supported LMS and LAF respectively.

P465

Refining the therapeutic range of posaconazole and isavuconazole for efficient therapeutic drug monitoring using a bioassay approach

T. Willeman<fnref n="1" />, J. Tonini², G. Cécile³, S. Bailly⁴, P. Gandia⁵, F. Stanke-Labesque⁴, D. Maubon⁶, E. Gautier-Veyret⁴

<footnote n="1">¹Laboratoire de Pharmacologie, Pharmacogénétique et Toxicologie, CHU Grenoble Alpes, France, GRENOBLE Cedex, France, ²Laboratoire de Pharmacologie, Pharmacogénétique et Toxicologie, CHU Grenoble Alpes, France, GRENOBLE Cedex, France, ³Univ. Grenoble Alpes, CNRS, CHU Grenoble Alpes, Grenoble INP*, TIMC-IMAG, 38000 Grenoble, France * Institute of Engineering Univ. Grenoble Alpes, GRENOBLE, France, ⁴Univ. Grenoble Alpes, Inserm, CHU Grenoble Alpes, HP2, 38000 Grenoble, France, GRENOBLE, France, ⁵UMR1436-INTHERES, 31076 Toulouse, France,</footnote>
Toulouse, France, ⁶Univ. Grenoble Alpes, CNRS, CHU Grenoble Alpes, Grenoble INP*, TIMC-IMAG, 38000 Grenoble, France * Institute of Engineering Univ. Grenoble Alpes, GRENOBLE Cedex, France

**Objectives:** Therapeutic drug monitoring for posaconazole (POS) tablet and isavuconazole (ISA) is still under debate and target trough concentrations (Cmin) need to be specified. We aimed to investigate the pharmacokinetic-pharmacodynamic relationship of POS and ISA, using a bioassay approach as surrogate marker of antifungal activity, in order to refine the therapeutic Cmin of both antifungals.

**Methods:** A bioassay using a cellulose disk diffusion method was performed to determine the growth inhibition zone (GIZ) of POS and ISA on *A. fumigatus* (POS and ISA) and *C. parapsilosis* (ISA only). GIZs of plasma from patients undergoing routine TDM for POS (n = 136) or ISA (n = 40) were determined.

**Results:** GIZs of plasma patients and antifungal Cmin were highly correlated both for ISA (*A. fumigatus*: rho = 0.942, p<0.0001; *C. parapsilosis*: rho = 0.949, p<0.0001) and POS (rho = 0.922, p<0.0001), and these relationships were represented with a Michaelis-Menten model. Based on this modeling, the recommended thresholds of 0.7, 1, and 1.25 mg/L for the POS Cmin corresponded to 50.1, 55.2, and 59.1% of the maximal GIZ, respectively. We propose an upper threshold of 4.8 mg/L for the POS Cmin and a lower threshold of 2.0 mg/L for the Cmin of ISA, as they respectively corresponded to concentrations leading to 90% and 50% of the maximal GIZ on *A. fumigatus*.

**Conclusion:** The determination of antifungal activity using this bioassay allowed refining target Cmin for both POS and ISA, especially the upper threshold of POS (4.8 mg/L) and the lower threshold of ISA (2.0 mg/L).

P466

**Prospective evaluation of a combined genetic score for the individualization of voriconazole dose**

E. Gautier-Veyret¹, A. Thiebaut-Bertrand², M. Roustit¹, X. Fonrose³, L. Bolcato⁴, J. Depeisses², C. Lefebvre⁵, D. Masson⁶, G. Schummer⁷, J.-Y. Cahn⁸, F. Stanke-Labesque¹

¹Univ. Grenoble Alpes, Inserm, CHU Grenoble Alpes, HP2, 38000 Grenoble, France, GRENOBLE, France, ²Hématologie Clinique CHU Grenoble Alpes, Grenoble, France, ³Chu Grenoble Alpes, Laboratoire de Pharmacologie, Pharmacogénétique et Toxicologie, GRENOBLE, France, ⁴Laboratoire de Pharmacologie, Pharmacogénétique et Toxicologie, GRENOBLE, France, ⁵Laboratoire d'Hématologie, CHU Grenoble Alpes, Grenoble, France, ⁶Etablissement Français du Sang, La Tronche, France, ⁷Centre de ressources biologiques, CHU Grenoble Alpes, GRENOBLE Cedex, France, ⁸Univ. Grenoble Alpes, CHU Grenoble Alpes, TIMC-Therex, Grenoble, France

**Objectives:** Voriconazole (VRC) exhibits a highly variable pharmacokinetics which is partly related to genetic polymorphisms affecting activities of cytochromes (CYP) 2C19 and 3A. This prospective observational study aimed to evaluate the impact of a previously described genetic score combining CYP2C19 and CYP3A genotypes (Gautier-Veyret, AAC 2015) to predict infratherapeutic plasma VRC trough concentration (Cmin) (defined as VRC Cmin < 1mg/L) in order to individualize VRC dose.

**Methods:** CYP2C19, CYP3A4 and CYP3A5 genotypes were determined in hematological patients treated by VRC and who benefited from longitudinal therapeutic drug monitoring in Grenoble University Hospital between January 2016 and December 2018. The genetic score was determined for each patient and patients were classified according to their genetic score ≤ or > 2. The higher the genetic score, the faster the metabolism of the patient. Statistical analyzes were performed on VRC Cmin adjusted on dose (VRC Cmin/D), considering either the initial VRC Cmin/D or all VRC Cmin.

**Results:** Forty-three patients (43 % of female, median age [Q1-Q3]: 57 [45-67] years) were included and 127 VRC Cmin analyzed. Ninety five percent of patients (41/43) received VRC for curative treatment of invasive aspergillosis (22 possible, 17 probable and 2 proven). Median initial VRC Cmin was 2.2 [1.1-4.3] mg/L and 21 % of patients (n = 9/43) had infratherapeutic initial VRC Cmin. Sixty-three percent of patients had at least one genetic variant for CYP2C19, 3A4 or 3A5, with 8 patients with a genetic score <2. Among 9 patients with infratherapeutic initial VRC Cmin, 8 (89%) had a genetic score ≥ 2. In univariate analysis, initial VRC Cmin/D, as well as all VRC Cmin/D was not influenced by genetic score, but significantly associated with advanced age, increased CRP and
bilirubin levels. Multivariate analysis performed in all VRC Cmin/D identified CRP (p<0.0001), bilirubin (p = 0.01) and age (p = 0.007) as independent determinants of VRC Cmin/D, while genetic score had no impact. Genetic score was significantly associated with VRC Cmin/D only in the subgroup of VRC Cmin (n = 51) determined in absence of severe inflammation (defined by CRP level < median CRP of 65 mg/L).

**Conclusion:** Our genetic score that combined genetic variants of CYP2C19, 3A4 and 3A5 seems interesting to predict subtherapeutic VRC Cmin, but its impact is suppressed by severe inflammation. Strategies for individualization of VRC dose must integrate pharmacogenetics variants, but also the inflammatory status of patient.

P467

**Pharmacokinetics of posaconazole in critically ill patients undergoing extracorporeal membrane oxygenation (ECMO)**

R. Van Daele¹,², R.J.M. Brüggemann³,⁴, M. Holtappels⁵, P. Depuydt⁶, B. Rijnders⁷, F. Cotton⁸, D. Fage⁸, E. Dreesen², J. Wauters⁹,¹⁰, I. Spriet¹,²

¹Pharmacy Department, University Hospitals Leuven, Leuven, Belgium, ²Department Of Pharmaceutical And Pharmacological Sciences, KU Leuven, Leuven, Belgium, ³Radboud University Medical Center, Department of Pharmacy and Radboud Institute for Health Sciences, Nijmegen, Netherlands, ⁴Center Of Expertise In Mycology Radboudumc /cwz, Radboud University Medical Center, Nijmegen, Netherlands, ⁵Laboratory For Clinical Infectious And Inflammatory Disorders, KU Leuven, Leuven, Belgium, ⁶Department Of Intensive Care, Ghent University Hospital, Ghent, Belgium, ⁷Department Of Infectious Diseases, Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands, ⁸Department Of Clinical Chemistry, LHUB-ULB, Erasme Hospital and Université Libre de Bruxelles, Bruxelles, Belgium, ⁹Department Of Microbiology And Immunology, KU Leuven, Leuven, Belgium, ¹⁰Medical Intensive Care Unit, University Hospitals Leuven, Leuven, Belgium

**Objectives:** The use of posaconazole as prophylaxis for influenza-associated aspergillosis is currently investigated in critically ill patients. An important subset of patients admitted to the intensive care unit (ICU) for influenza is undergoing extracorporeal membrane oxygenation (ECMO) for severe acute hypoxemic respiratory failure. Little is known on the exposure to posaconazole in this population. This study presents the pharmacokinetics (PK) of seven ICU patients treated with intravenous posaconazole while on ICU undergoing ECMO.

**Methods:** All but one of the included patients were participating in the posaconazole-arm of the ongoing multicenter Posa-Flu study (NCT03378479), assessing the efficacy of a 7-day prophylactic intravenous treatment with posaconazole in ICU patients admitted for severe influenza. The additional patient was a lung transplant recipient treated with posaconazole for invasive aspergillosis. Blood samples were collected at an early (day 2-3) and late (day 5-7) time point, using a full (predose, 1.5,2,3,4,6,8,10,12,18,24h) or limited (predose, 1.5-3, 4-8, 8-12, 12-24h) sampling scheme. Trough concentrations were collected on all other days. All patients received an intravenous loading dose of 300 mg BID followed by a maintenance dose of 300 mg QD. Written informed consent was obtained from the patients or their relatives. PK parameters were estimated in Excel®.

**Results:** All patients received veno-venous ECMO and two patients were supported by continuous veno-venous hemodialysis (CVVH). The median area under the curve (AUC₀-24), clearance (CL) and volume of distribution (Vd) were 35.69 mg.h/L, 8.42 L/h and 421.0 L, respectively. Exposure documented in the ECMO patients is shown in Table 1, along with the results previously documented in healthy volunteers, patients with hematological disease and critically ill patients. As shown in Figure 1, all trough concentrations (Cmin) were above 0.7 mg/L, which is the suggested threshold for prophylaxis in patients with hematological disease, and 71% of the Cmin were also ≥ 1 mg/L, which is the suggested hematological threshold for treatment of invasive aspergillosis (ECIL-6 and ESCMID-ECMM-ERS 2017). The median average concentration (Cavg) of both exposure days, i.e. 1.49 mg/L, was within the European Medicines Agency (EMA) target range (0.5-2.5 mg/L). The targeted posaconazole AUC₀-24 of 20.87-22.5 mg.h/L for a MIC value of 0.125 mg/L (DRU 2014), was also
achieved.

<table><caption>**Table 1.** PK parameters of posaconazole documented in patients undergoing ECMO vs. previously reported results from literature</caption><thead><tr><th rowspan="2"></th><th colspan="3">Study population<br>(median [IQR])</th><th rowspan="2">Kersemaekers et al.,<br>AAC 2015<br>(mean (CV%))</th><th rowspan="2">Cornely et al.,<br>JAC 2017<br>(mean (CV%))</th><th rowspan="2">Sime et al.,<br>AAC 2018<br>(median (IQR))</th></tr><tr><th>Early + late day<br>(n= 8 days)</th><th>Early day<br>(n= 4 days)</th><th>Late day<br>(n= 4 days)</th><th>Healthy volunteers</th><th>Patients with<br>hematological disease</th><th>Critically ill patients</th></tr></thead><tbody><tr><td>AUC<sub>0-24</sub> (mg·h/L)</td><td>35.69 [30.32-38.84]</td><td>34.03 [30.13-38.64]</td><td>35.83 [32.80-40.65]</td><td>46.40 (26) (AUC<sub>0-∞</sub>)</td><td>36.10 (35)</td><td>11.61 [9.90-18.25]</td></tr><tr><td>CL (L/h)</td><td>8.42 [7.76-9.91]</td><td>8.89 [7.80-9.98]</td><td>8.39 [7.51-9.21]</td><td>6.90 (27)</td><td>.</td><td>16.8 [11.1-21.7]</td></tr><tr><td>Vd (L)</td><td>421.0 [334.7-755.6]</td><td>771.4 [608.6-822.8]</td><td>370.9 [334.7-405.2]</td><td>236 (17)</td><td>.</td><td>529.1 [352.2-720.6]</td></tr><tr><td>T<sub>1/2</sub> (h)</td><td>34.73 [30.40-53.76]</td><td>54.53 [44.34-62.16]</td><td>33.22 [30.40-34.15]</td><td>24.6 (20)</td><td>.</td><td>23.0 [19.1-31.6]</td></tr><tr><td>C<sub>min</sub> (mg/L)</td><td>1.07 [0.89-1.27]</td><td>0.96 [0.87-1.13]</td><td>1.17 [1.05-1.41]</td><td>.</td><td>1.09 (44)</td><td>0.22 [0.20-0.56]</td></tr><tr><td>C<sub>max</sub> (mg/L)</td><td>2.78 [1.91-3.47]</td><td>3.64 [2.67-3.80]</td><td>2.35 [1.91-2.91]</td><td>2.84 (30)</td><td>3.28 (74)</td><td>1.70 [1.35-2.14]</td></tr><tr><td>C<sub>over</sub> (mg/L)</td><td>1.49 [1.26-1.62]</td><td>1.42 [1.26-1.61]</td><td>1.49 [1.37-1.69]</td><td>.</td><td>1.50 (35)</td><td>0.75 [0.58-1.16] (estimated)</td></tr></tbody></table>

<figure><img src="image_106.png" /><figcaption>**Figure 1.** Posaconazole trough levels documented from day 2-7. Boxplots representing the median + IQR posaconazole trough concentrations.</figcaption></figure>

**Conclusion:** Posaconazole exposure was similar compared to that documented in hematology patients but higher than in ICU patients without ECMO. The lower limit for prophylaxis, as recommended in patients with hematological malignancies, is attained in all patients. This should be interpreted with caution as the optimal exposure to ensure prophylactic efficacy might be different in critically ill patients admitted for severe influenza. This optimal exposure is currently being investigated in the Posa-Flu trial. Based on this limited dataset, ECMO does not seem to influence posaconazole exposure. An *a priori* dose adjustment does not seem to be necessary but therapeutic drug monitoring is recommended to confirm the exposure. Despite the fact that this observation should be established in a larger number of patients, these results suggest that posaconazole exposure is maintained in critically ill patients undergoing ECMO. Hence we recommend the standard dosing regimen.

<footnote n="1"><sup>1</sup>Pharmacy, Radboudumc, Nijmegen, Netherlands, <sup>2</sup>Center of Expertise in Mycology Radboudumc / CWZ, Nijmegen, Netherlands, <sup>3</sup>Clinical Pharmacy, St. Antonius Hospital, Nieuwegein, Netherlands,</footnote>

<footnote n="4"><sup>4</sup>Division Of Systems Biomedicine And Pharmacology, Leiden Academic Centre For Drug Research, Leiden University, Leiden, Netherlands, <sup>5</sup>Department Of Anesthesiology, Intensive Care And Pain</footnote>
Management, St. Antonius Hospital, Nieuwegein, Netherlands, ^6Department Of Surgery, St. Antonius Hospital, Nieuwegein, Netherlands

**Objectives:** One in five individuals will be obese in 2025. Obesity is associated with underdosing of antimicrobial drugs for prophylaxis and treatment of (life-threatening) infections. Posaconazole is a broad-spectrum triazole antifungal drug frequently used for prophylaxis and treatment of mold infections. There is anecdotal evidence that posaconazole exposure is impacted by weight resulting in suboptimal target attainment. We performed a prospective clinical trial to investigate the effect of body size in obese subjects and determined the probability of reaching the target trough concentration for prophylaxis (>0.7mg/L) and therapy (>1.0mg/L).

**Methods:** Obese subjects (BMI above 35 kg/m2) undergoing bariatric surgery and normal-weight subjects (BMI 18.5-25 kg/m2) were included in an open-label, single-dose, multicenter, pharmacokinetic study. Obese subjects were randomized to receive either 300 or 400 mg posaconazole IV while normal-weight subjects all received 300 mg posaconazole IV. Blood samples were collected at nine time points up to 24 hours after start of infusion. Statistical analysis and population pharmacokinetic modeling was performed using R and NONMEM 7.3.

**Results:** 8 obese subjects on 300 mg, 8 obese subjects on 400 mg and 8 normal-weight subjects on 300 mg were included. Weight [range] was 129 kg [109-190]), 144 kg [107-175]), 72.3 kg [61.4-85.4] respectively. The observed geometric mean [range] AUC~0-24h~ in normal-weight versus obese subjects receiving 300 mg posaconazole was 21.4 mg*h/L [15.6-29.1] versus 13.1 mg*h/L [9.1-18.5] (p<0.05). Obese subjects receiving 400 mg posaconazole had an AUC~0-24h~ of 16.8 mg*h/L [12.2-25.6]. A two-compartment model with first-order elimination, a proportional residual error model and inter-individual variability on the central compartment best described the data. Total body weight was the best predictor of variability of clearance and the central and peripheral compartment. Simulations demonstrated that a 300 mg dose results in a >90% PTA in patients up to 190 kg for prophylaxis. For treatment, a 300 mg dose is sufficient up to 120 kg after which a dose increase to 400 mg daily should result in a >90% PTA up to 170 kg.

**Conclusion:** We found that obese individuals have a significant lower exposure to posaconazole compared to normal-weight individuals even when using a 400 mg dose. Nevertheless, PTA (at 0.7 mg/L) was high using the IV formulation. In the treatment setting higher dosages are warranted.

P469

**The effects of Cytochrome P450 2C19 profile on voriconazole serum trough concentrations and hepatotoxicity in Haematology patients**

L.L. Chong^1, L.M. Ling^2

^1Haematology, Tan Tock Seng Hospital, Singapore, Singapore, ^2Infectious Diseases, Tan Tock Seng Hospital, Singapore, Singapore

**Objectives:** Cytochrome P450 (CYP) 2C19 genetic polymorphism contributes to variability of voriconazole pharmacokinetics. Supra-therapeutic voriconazole trough concentrations have been correlated with hepatotoxicity. This study aims to establish the effects of CYP2C19 profile on voriconazole troughs and hepatotoxicity.

**Methods:** A prospective, observational study was done to determine voriconazole trough concentrations at pre-steady state (day 2 to 4 of therapy) and steady state (day 6 onwards). Blood sampling was repeated weekly until week 12 of recruitment or death. The CYP2C19 genotype was also identified for patients on voriconazole.

**Results:** 11 patients were analyzed for CYP2C19 polymorphism: 1*/1* normal metabolizers (n = 5, 45%), 1*/2* intermediate metabolizers (n = 3, 27%) and 2*/2* poor metabolizers (n = 3, 27%), respectively. 4 out of 6 patients were intermediate or poor metabolizers whom had supra-therapeutic voriconazole concentrations (trough > 5.5 mg/L). All poor metabolizers developed abnormal liver function tests (range of ALP: 230 to 630).

**Conclusion:** Knowledge of CYP2C19 profile would allow pre-emptive dose adjustments during the first week of voriconazole therapy and thus reduce the incidence of hepatotoxicity.
**Obese patients require a fixed dose of liposomal amphotericin B**

R.E. Wasmann¹,², C. Smit³,⁴, E. Van Dongen⁵, R. Wiezer⁶, J. Adler-Moore⁷, Y. De Beer⁸, D. Burger¹, C. Knibbe³,⁴, R.J.M. Brüggemann⁹,¹⁰

¹Pharmacy, Radboudumc, Nijmegen, Netherlands, ²Center of Expertise in Mycology Radboudumc / CWZ, Nijmegen, Netherlands, ³Clinical Pharmacy, St. Antonius Hospital, Nieuwegein, Netherlands, ⁴Division Of Systems Biomedicine And Pharmacology, Leiden Academic Centre For Drug Research, Leiden University, Leiden, Netherlands, ⁵Department Of Anesthesiology, Intensive Care And Pain Management, St. Antonius Hospital, Nieuwegein, Netherlands, ⁶Department Of Surgery, St. Antonius Hospital, Nieuwegein, Netherlands, ⁷Biology, Cal Poly Pomona, Pomona, United States of America, ⁸Department Of Clinical Pharmacy & Toxicology, Maastricht University Medical Center+, Maastricht, Netherlands, ⁹Pharmacy, RadboudUMC, Nijmegen, Netherlands, ¹⁰Center Of Expertise In Mycology Radboudumc /cwz, Radboud University Medical Center, Nijmegen, Netherlands

**Objectives:** In 2014, approximately 11% of adult men and 15% of adult women were obese worldwide, a number that is increasing. Like other patients, obese patients may be confronted with invasive fungal disease and require treatment with antifungal agents such as liposomal amphotericin B (L-Amb, Ambisome®). We performed a pharmacokinetic study in morbidly obese individuals to define the optimal dose of L-Amb by quantifying the impact of obesity on the pharmacokinetics of L-Amb.

**Methods:** In this open-label study, morbidly obese but otherwise healthy subjects (BMI > 40 kg/m²) received L-Amb received a single dose of 1 or 2 mg/kg (n = 8+8) the day before undergoing laparoscopic bariatric surgery. Each subject was intensively sampled (n = 9) up to 48 hours to determine amphotericin B plasma concentrations. The following information was collected: body size descriptors (e.g. total body weight [TBW], lean body weight, BMI, ideal body weight calculated according to Janmahasatian *et al.*, body surface area), age and sex. Population pharmacokinetic analyses was performed in NONMEM 7.3 with PsN 4.7. For the covariate analyses the relationships between empirical Bayes estimates and covariates were investigated in scatter plots. The final model was used to simulated AUC₀-24h and Cmax in steady state conditions in 10.000 subjects with body weights uniformly distributed between 60 and 180 kg. Each virtual subject < 100 kg received a daily 3 mg/kg L-Amb infusion in one hour, subjects ≥100 kg received either 3 mg/kg or a fixed 300 mg dose as a one hour infusion.

**Results:** 16 morbidly obese subjects with median [range] BMI of 45.9 [40.2-52.1] kg/m² and a total body weight of 137 [104-177] kg were included. A two-compartment model best explained the data. There was no relationship between clearance of L-Amb and any of the body size descriptors. A linear relationship was found between the central volume of distribution (Vc) and TBW. Simulations show that the AUC₀-24 increases with weight when patients are dosed on a mg per kg basis. In parallel, Cmax also increases with body weight when L-Amb is dosed on a per kg basis. This phenomenon is primarily driven by the absolute increase in the dose with a clearance that does not change with weight. When using a dose cap of 300 mg for patients > 100 kg the AUC would remain similar across weight while a similar Cmax (13% lower) in a 140 kg patient compared to a 70 kg patient will be obtained.

**Conclusion:** Based on our results we see limitations to using a mg per kg dose regime for adult patients as clearance and therefore exposure to Amb is not affected by body weight. In addition, body weight derived dosing might lead to an increased risk of toxicity in heavy patients since toxicity is associated with AUC. A fixed dose regimen would not only be more appropriate but also more practical for clinicians. In obese patients specifically, we recommend using the licensed 3 mg/kg dose and cap the dose at a maximum weight of 100 kg resulting in a 300 mg fixed dose.

**Evaluation of the in vitro antifungal activities of combinations of amphotericin B with anidulafungin or caspofungin against *Candida auris* by time-kill curves**
U. Caballero¹, S. Nuñez-Marcos¹,², E. Eraso², J. Pemán³, G. Quindós², N. Jauregizar¹

¹Pharmacology, University of the Basque Country, Leioa, Spain, ²Inmunology, Microbiology, Parasitology, University of the Basque Country (UVP/EHU), Bilbao, Spain, ³Servicio De Microbiología, Hospital Universitario y Politécnico La Fe, Valencia, Spain

**Objectives:** *Candida auris* has recently emerged as a cause of nosocomial outbreaks. The health alert caused by this pathogen is related to the difficulty of a proper identification, its persistence in the hospital environment and multidrug resistance. Antifungal combination therapy may allow to overcome this multiresistance. Despite their relevance, research about the activity of antifungal drug combinations against *C. auris* is still scarce. The objective of this study was to evaluate the activity of the combinations of amphotericin B with anidulafungin or caspofungin against *C. auris* clinical isolates by time-kill curves.

**Methods:** *In vitro* static time-kill curves experiments were performed in duplicate in micro titre plates in RPMI-1640 medium with six *C. auris* clinical blood isolates (Hospital La Fe, Valencia, Spain). Inoculum size ranged from 1 to 5 x 10⁵ CFU/mL and concentrations assayed in combination experiments were 0.5 and 1 mg/L for amphotericin B and 0.25, 0.5, 1 and 2 mg/L for anidulafungin and caspofungin. Samples for viable counts were taken at 0, 2, 4, 6, 8, 24 and 48 h. Plots of averaged colony counts (log CFU/mL) versus time were constructed for data analysis. The first-order killing rate constant K (h⁻¹) obtained after fitting an exponential equation to the data and time to achieve the fungicidal endpoint t₉₀.₉% (h) were calculated to assess antifungal activity and to compare different combinations between them. Fungistatic or fungicidal activities, and synergistic, antagonist or indifferent interactions between these drugs were considered according to published definitions [1].

**Results:** Combinations of 0.5 mg/mL of amphotericin B with 0.5, 1 and 2 mg/mL of anidulafungin or caspofungin were synergistic (>2 log reduction compared to the drug with greater activity) and fungistatic. However, combinations of 1 mg/mL of amphotericin B with 0.25 and 0.5 mg/mL of anidulafungin or caspofungin were additive (<2 log reduction) and fungicidal. There were not significant differences between the use of anidulafungin or caspofungin in combinations with amphotericin B.

**Conclusion:** Combination of amphotericin B with anidulafungin and caspofungin showed promising results. Combinations of 0.5 mg/L of amphotericin B with low doses of echinocandins were synergistic, whereas the interaction with 1 mg/mL of amphotericin B was additive but exerted fungicidal activity. This is a remarkable finding, specially if we take into account that echinocandins do not show fungicidal behaviour against *C. auris*. The results could support the use of these antifungal combinations for treating *C. auris* deep infections. **Funding:** This study was partially financed by GIC 15/78 IT-990-16 (Gobierno Vasco-Eusko Jaurlaritza), PI17/01538 (FIS, Spain) and SAF2017-86188-P (MINECO, Spain). Unai Caballero has received a grant by the University of the Basque Country (UPV/EHU) [1] Serena C et al., *In vitro* interactions of micafungin with amphotericin B against clinical isolates of *Candida* spp. Antimicrob Agents Chemother. 2008; 52(4):1529-32

S. Kurland¹, M. Furebring¹, A. Shams¹, E. Chryssanthou², J. Sjölin¹, E. Löwdin¹

¹Section Of Infectious Diseases, Department Of Medical Sciences, Uppsala University, Uppsala, Sweden, ²Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden

**Objectives:** The echinocandin caspofungin is used as first-line therapy in candidemia, the most common invasive candida infection. The protein bound fraction of caspofungin is 96.5% (in healthy volunteers). In our recent study on critically ill patients treated with caspofungin, a median AUC in plasma of 58 (range 44-113) mg*h/L* was reported, corresponding to an average plasma concentration of 2.4 (1.8-4.7) mg/L. This would result in a free unbound concentration of 0.08 (0.06-0.16) mg/L, concentrations equivalent to the MIC distribution for wild type *Candida glabrata*. For fungicidal effect higher free concentrations would be needed. In critically ill patients the plasma protein level is habitually reduced. The objectives of this study were to determine the killing effect of caspofungin
on *C. glabrata* with clinically relevant concentrations of caspofungin in an *ex vivo* model using human plasma and furthermore to study the efficacy in relation to different plasma protein levels.

**Methods:** The efficacy of caspofungin on four blood isolates of *C. glabrata* was determined by time-kill studies performed in plasma from healthy blood donors. In order to investigate the significance of the protein level on the killing effect of caspofungin, *C. glabrata* with an inoculum of 5x10⁴ CFU/ml was added to plasma, plasma diluted with 50% Ringer's Acetate (Ri-Ac) and plasma diluted with 75% Ri-Ac. The MIC values for the isolates were determined by Sensititre Yeast One method and were 0.06, 0.12 and 1 mg/L, respectively. The caspofungin concentrations investigated were 1, 2, 5 and 9 mg/L. CFU/ml were determined at 0, 2, 6 and 24 hours.

**Results:** The results showed a higher reduction in CFU/ml than expected based on MIC and the calculated unbound caspofungin concentration. There was a ≥ 2 log reduction in log CFU/ml with a concentration of 1 mg/L (corresponding to 0.035 mg/L free drug/unbound concentration) for the two strains with MIC 0.06 mg/L. For the strain with MIC 0.12 mg/L the corresponding reduction was approximately 1 log CFU/ml. Furthermore, there was a limited but significant decrease in CFU/ml in the time-kill studies performed in plasma with reduced levels of protein in comparison with plasma with normal protein level. For the strain with MIC 1 mg/L there was no reduction in CFU/ml in plasma. Although, there was a killing with the concentrations 5 and 9 mg/L in the experiments in plasma diluted with 75% Ri-Ac.

**Conclusion:** The killing of *C. glabrata* with caspofungin in plasma was higher than expected based on MIC and the reported amount of unbound caspofungin in normal plasma. At lower protein levels this killing was augmented which is consistent with an increased concentration of free caspofungin.

P475

**Subcutaneous nodule caused by Phaeoacremonium fuscum in a non-immunocompromised patient.**

S. Colman, L. Cattoir, A. Boel

Microbiology, OLV Hospital, Aalst, Belgium

**Case Report:** Introduction Phaeohyphomycosis (PHM) is a chronic infection caused by dematiaceous fungi which usually involves the skin and subcutaneous tissue. As a result of the use of molecular diagnostic techniques, previously unrecognized causative agents of phaeohyphomycosis have been discovered. Here we report the first case of phaeohyphomycosis caused by *Phaeoacremonium fuscum* in an immunocompetent patient. Case presentation A 60 year old Filipino male presented with rotator cuff pathology to the orthopedics consultation. On physical examination a painless, soft, subcutaneous mass above the right patella was noted, which didn't cause the patient any complaints. The suprapatellar nodule had been growing slowly for 3 years. The patient denied any apparent antecedent trauma, arthropod bite, family history or contact with a person with similar lesions. The man still visits the Philippines on an infrequent basis. He had no fever, weight loss or other symptoms. Detailed physical examination further revealed a 4x3 cm, immobile, non-pulsatile light red mass. There were no other signs of inflammation of the skin. A serous effusion was aspirated from the mass. Fungal culture of the aspirated fluid showed growth of a filamentous fungus on chocolate blood agar and Sabouraud's dextrose agar. Molecular identification was performed by sequence analysis of the internal transcribed spacer region 2 (ITS2). BLAST search revealed a similarity of 100% with *Phaeoacremonium fuscum*. The antifungal susceptibility profile was established by EUCAST broth microdilution: itraconazole >16 mg/L, posaconazole 0.25 mg/L, amphotericin B 0.50 mg/L, voriconazole 0.50 mg/L. This susceptibility profile is similar to those previously published. A wide local excision of the lesion was performed a couple of weeks later. The patient had an uneventful postoperative recovery. No antifungal therapy was started postoperatively since the patient was not immunocompromised and there was lack of reimbursement. Histopathological examination of the specimen showed fibrous tissue with a mixed cellular infiltrate including lymphocytes, plasma cells, macrophages and neutrophils. Periodic acid-schiff stain and Grocott stain showed evidence of branching septate hyphae. Discussion PHM is caused by brown pigmented fungi containing melanin in their cell wall. Phaeoacremonium species are known as vascular plant pathogens, responsible for wilting and dieback of woody plants. They are also opportunistic
pathogens in humans, mainly causing cutaneous or subcutaneous PHM. However, sporadic cases of respiratory tract infection, septic arthritis and disseminated infection are described. The true prevalence of Phaeoacremonium species is probably underestimated due to the asymptomatic nature of most subcutaneous lesions and the difficult identification. There is no standard antifungal regimen or evidence regarding the necessity for antifungal treatment as concomitant with surgery for localized infections. Long courses of antifungals, such as voriconazole treatment, are often employed to reduce the risk of recurrence. Case reports suggest that surgery can be curative if the infection is localized and adequately removed, as was the case in our patient. Regardless of the immune status of the patient, clinicians should consider the possibility of a fungal infection when they encounter chronic subcutaneous nodules without major symptoms.

P476

**Belgian national survey on tinea capitis: epidemiology and molecular investigations.**

R. Sacheli¹, X. Menatong¹, C. Labarbe¹, C. Crutzen¹, S. Harag², J. André², S. Evrard³, K. Lagrou⁴, K. Laffineur⁵, D. Rousseaux⁶, L. Detollenaere¹, C. Adjetey¹, L. Seidel⁷, M.-P. Hayette¹

¹Clinical Microbiology- Nrc For Mycoses, University Hospital of Liège, Liège, Belgium,
²Dermatology, CHU St Pierre, Brussels, Belgium, ³Clinical Microbiology, CHR Citadelle, Liège, Belgium,
⁴Clinical Bacteriology And Mycology, Katholieke Universiteit Leuven, Leuven, Belgium,
⁵Clinical Biology, Clinique St Luc Bouge, Bouge, Belgium, ⁶Clinical Biology, Centre Hospitalier Chretien Heusy, Verviers, Belgium, ⁷Statistics, University Hospital of Liège, Liège, Belgium

**Objectives:** Tinea capitis (TC) is a superficial infection of the scalp caused by dermatophyte fungi which affects mainly prepubescent children. This last decade, a huge increase of African anthropophilic strains causing tinea capitis, has been observed in Europe, probably due to immigration waves from African countries. The Belgian National Reference Center for Mycosis (NRC) has conducted a surveillance study about TC in 2018. This work presents final results of the study for the epidemiological part and preliminary results for the molecular part.

**Methods:** Belgian laboratories were invited to send all dermatophytes strains isolated from the scalp from January to December 2018. Dermatologists were involved and were asked to fill a form containing several epidemiological information about the patient. Strains identification was confirmed by ITS sequencing. A multiplex pan-dermatophyte real time PCR assay (DermaGenius®, PathoNostics) was applied if necessary. Typing of the *M.audouinii* strains was done using rep-PCR method (Diversilab, BioMérieux).

**Results:** A total of 337 strains have been collected from 337 patients. The main population concerned by TC was children from 5-9 years (165/337, 49,01%). Males (214/337, 63,5%) were more affected than females (123/337, 36,5%), the sex ratio M/F was of 1,74. The majority of the strains was collected in Brussels area (181/337, 53,8%), followed by Liege area (73/337; 21,7%). Other Belgian cities were less concerned by TC. Among known ethnical origins (n = 119), African people (114/119, 96,2%) were more concerned by TC than European people (5/119, 3,8%), (p<0,0001). The majority of patients were from Guinea (26/119, 21,8%), followed by Cameroun (14/119, 11,8%) and RDC (14/119, 11,8%), many other African nationalities were represented (12 different countries, all over Africa). The main transmission mode of TC was the familial way (83,3% among known cases n = 126, 105/126). The major etiological agent was *Microsporum audouinii* (118/337, 35%) followed by *Trichophyton soudanense* (83/337, 24,6%), *T. tonsurans* (27/337, 17%), *M. canis* (36/337, 10,7%), *T. violaceum* (28/337, 8,3%), *T. benhamiae* (7/337, 2,1%), *T. mentagrophytes* (5/337, 1,48%) and *M. incurvatum* (1/337, 0,3%). This last rare dermatophyte has never been reported as responsible for TC in Belgium before. *M. audouinii* strains have been genotyped by rep-PCR and three genotypic variants have been characterized, one of them circulating mainly in Brussels area. No link with a particular ethnical origin could be found among genotypic groups.

**Conclusion:** African anthropophilic dermatophytes such as *M.audouinii* and *T. soudanense* are mainly responsible for tinea capitis in Belgium. Large cosmopolitan cities like Brussels and Liege are the most concerned. People from African origin are mostly affected by TC. Among the *M. audouinii* strains circulating in Belgium, a genotypic diversity has been characterized.
# Identification of dermatophytes isolated from *tinea capitis* infections using a liquid media MALDI-TOF MS protocol

P. Lecerf¹, Y. Jazaeri¹, M. Hendrickx², B. Richert¹, A. Packeu³

¹Faculty Of Medecine, Université Libre de Bruxelles, Brussels, Belgium, ²Mycology, Sciensano, BCCM/IHEM, Brussels, Belgium, ³Mycology & Aerobiology, Sciensano, Brussels, Belgium

**Objectives:** *Tinea capitis* is a common childhood superficial infection of the scalp caused by dermatophytes. Conventional methods based on morphological characteristics to identify theses fungi are time-consuming and complex, requiring expert mycological knowledge. Recently, MALDI-TOF MS has become a powerful tool in clinical microbiology for rapid identification of these microorganisms. In the present study, a liquid media MALDI-TOF MS protocol was developed and validated for the fast and accurate identification of dermatophytes species responsible for *tinea capitis* infection, using both dermatophytes from reference strains and clinical isolates. The main purpose was to work with primary isolates in order to avoid the culture step and lead to a faster diagnosis. Identification techniques (conventional identification and MALDI-TOF MS based on liquid and solid culture) were evaluated and compared for their rate of correct identification and turnaround time. for the fast and accurate identification of dermatophytes species responsible for *tinea capitis* infection, using both dermatophytes from reference strains and clinical isolates. The main purpose was to work with primary isolates in order to avoid the culture step and lead to a faster diagnosis. Identification techniques (conventional identification and MALDI-TOF MS based on liquid and solid culture) were evaluated and compared for their rate of correct identification and turnaround time.

**Methods:** The in-house BCCM/IHEM database made from references strains on solid culture was used. First, the liquid media MALDI-TOF MS protocol was validated using references strains. Secondly, the same protocol was applied on clinical isolates collected from Saint-Pierre University Hospital and paralleled with solid media MALDI-TOF MS protocol.

**Results:** The use of the liquid media MALDI-TOF MS protocol resulted in a rate of 100% of correct identification at the species level for reference strains and 78,8% for clinical isolates. The protocol was statistically significantly faster than the conventional method. The results of the different methods disagreed for 17 isolates. The complete extraction from the solid media MALDI-TOF MS gave the highest correct species identification with the highest mean of log-scores.

**Conclusion:** The liquid media MALDI-TOF MS technique is an accurate method for the correct identification of dermatophytes at the species level and is much faster than the conventional technique. The main advantage lies in not needing a culture step for primary isolates, thus leading to a faster diagnosis. For the identification of closely related species, the complete extraction from solid media MALDI-TOF MS protocol remains the most reliable technique.

# Dermatophytes' identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) - the experience of a clinical laboratory

C. Verissimo, H. Simões, R. Sabino, D. Simões

Infectious Disease, National Health Institute, Lisbon, Portugal

**Objectives:** Dermatophytes are a challenging group of fungi that infect the keratinized tissues. The taxonomy of these fungi has changed recently with the reclassification of some species and description of new ones. However, many clinical laboratories still base the identification of dermatophytes on their phenotype. Since dermatophytes are very pleomorphic, macro and micromorphology are often insufficient to reach a correct classification and may lead to misidentifications. The identification based on MALDI-TOF relies on the protein profile of the microorganism. Thus, this study aims to summarize our current laboratorial experience of dermatophyte identification using MALDI-TOF MS.

**Methods:** From january to april 2018, 95 dermatophytes isolates, collected from human keratinized samples and also from quality control programs were characterized by phenotypic analysis, and by
VITEK MS V3.2 bioMerieux. Before identification procedure, isolates were inoculated on Sabouraud Dextrose agar plates and incubated at 27°C during 5 to 10 days. Species were identified taking into account clinical features, as well as cultural, microscopic and physiological characteristics. Prior to MALDI-TOF MS analysis, the samples were pre-treated according to the manufacturer's protocol for filamentous fungi. Molecular identification by sequencing of the internal transcribed spacer 1 (ITS1) was performed in 34 of those isolates

**Results:** Through phenotypic analysis eight different species were identified (54 Trichophyton rubrum; 4 T.soudanense; 22 T.interdigitale; 1 T.mentagrophytes; 3 T.tonsurans; 7 Microsporum canis; 3 M.audouinii; 1 Microsporum spp.- (non canis or audouinii). MALDI-TOF analysis showed an identification agreement in 80 cases (84,2%) with a confidence level of 99,9%. Eight isolates showed divergent identification results: three T.rubrum were identified as T.violaceum, three T.soudanense were identified as T.rubrum, one T.mentagrophytes was identified as T.interdigitale and one T.tonsurans was identified as T.rubrum. In four cases MALDI-TOF analysis did not get a profile. The ITS sequencing analysis of discrepant results corroborated the MALDI-TOF identification in five of them. On the other hand, T.soudanense was only identified by phenotypic analysis since MALDI-TOF and ITS sequencing result was T.rubrum. MALDITOF identification of T.violaceum was not confirmed by ITS sequencing that identified T. rubrum instead, in accordance with the phenotypic identification.

**Conclusion:** Correct identification of dermatophytes to species level requires sequencing of the ITS, LSU, and/or beta-tubulin regions. The implementation of this methodology in a clinical laboratory is expensive and time consuming. MALDI-TOF identification is a good option for dermatophytes' identification performed in laboratory routine, since costs of consumables as well as time of sample preparation are lower than for PCR analysis and doesn't require long training period as phenotypic identification does. In this study, however, both methods failed to identify some species variants like Trichophyton soudanense or T. violaceum. The combined use of both MALDI-TOF and phenotypic methods seems to be the better approach for dermatophytes' identification since some species show significant phenotypic and clinical differences.

A retrospective study of dermatophyte infections caused by *Tricophyton rubrum* and *T. interdigitale* in the County of Stockholm, Sweden.

L. Vargas, E. Alvarado, C. Stenström, S. Forsblom, B. Girestam, G. Loko, E. Chryssanthou
Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden

**Objectives:** Dermatophytosis is a common fungal infection caused by keratinophilic fungi that are capable of invading nail, hair and superficial layers of the skin of humans and animals. The aim of this study was to determine the epidemiologic profile of the most frequent dermatophytosis such as *Tinea unguium* and *T. pedis* caused by *Tricophyton rubrum* or *T. interdigitale* in Stockholm, Sweden.

**Methods:** In 2015 we developed and validated a rapid and sensitive real-time PCR method for detection of the two most prevalent dermatophytes in Northern Europe. Fungal DNA was extracted directly from clinical samples (toe or finger nail and skin scrapings from the feet) by using proteinase K and heat as pre-lysis step, followed by automated DNA extraction on the PSH/MagNA Pure 96 Compact. Real-time PCR was performed using pan-dermatophyte primers for detection of all dermatophytes and specific primers for identification of *T. rubrum* and *T. interdigitale*. Specimens from finger nails were also cultured on Chrom agar plates for detection of onychomycosis caused by yeasts.

**Results:** Laboratory records comprising PCR results of 13,683 specimens (3,304 skin respective 10,379 nail fragments) collected from May 2015 through March 2019 were retrospectively analyzed. *Tricophyton rubrum* (42%) was the predominant pathogen identified from these cases, followed by *T. interdigitale* (4,9%). In contrast, 2% of the samples were positive for pan-Derm indicating the prevalence of infections caused by other pathogens distinct to *T. rubrum* or *T. interdigitale*. Sex distribution analysis of the patients showed that males (40%) were more susceptible to suffer dermatophyte infections compared to females (21,9%). (Furthermore, the prevalence of
onychomycosis in finger nails caused by yeasts were also evaluated in this study.) Culture analysis from total 941 finger nails showed that 25% were positive for yeast infection. *Candida parapsilosis* (52%), followed by *C. albicans* (35%) and *C. guilliermondii* (6%) were the predominant species isolated in such cultures.

**Conclusion:** The findings presented in our study are in concordance with previous studies where *T. rubrum* is the most frequent dermatophyte species isolated in the general population of industrialized countries. Detection of dermatophytes using real-time PCR represents a faster and sensitive method to evaluate large volume of clinical samples.

P480

**Evaluation of Diafactory tinea unguium®, a new rapid immunochromatographic test for the diagnostic of onychomycosis**

E. Rupin, H. Pelloux, H. Fricker-Hidalgo, M.-T. Leccia, O. Cognet

Parasitology-mycology, Grenoble Alps University Hospital, GRENOBLE, France

**Objectives:** Suspected nail onychomycosis remains a frequent and still increasing problem despite therapeutic advances. Based on clinical features the diagnosis of onychomycosis is confirmed by direct examination and culture. Direct examination can be done on the same day but cultures required several weeks what delays diagnosis and therapy. Furthermore, direct examination and culture have low sensitivity. Diafactory tinea unguium®, a new rapid immunochromatography test was recently developed. The aim of this study was to compare this new test to conventional methods (direct examination and culture) in suspected tinea unguium.

**Methods:** Diafactory tinea unguium® (Biosynex, France) detects dermatophytes by using monoclonal antibodies that react specifically with polysaccharide present in the cell wall of dermatophytes. Direct microscopic examination was performed using KOH enhanced with chlorazol black E and dimethyl sulfoxide. Cultures were carried out on Sabouraud medium with or without cycloheximide (bioMérieux, France), incubated at 25°C. The three methods were evaluated on nails samples (n = 70) collected from patients (n = 50) with suspected nail mycosis infection in the Grenoble Alps University Hospital.

**Results:** Among the 70 samples, cultures were positive in 53 nails. Dermatophytes were isolated in 40 samples (*Trichophyton rubrum* n = 35, *Trichophyton interdigitale* n = 3, *Trichophyton tonsurans* n = 2), yeasts in 8 samples (*Candida parapsilosis* n = 6, *Candida albicans* n = 2), and moulds in 5 samples (*Scopulariopsis* spp. n = 4, *Fusarium* spp. n = 1). The results were positive with Diafactory tinea unguium® in 48 samples, correlated with direct microscopy (i.e. presence of filamentous fungal elements) in 36 samples and with cultures in 39 samples. In one case, the result of Diafactory tinea unguium® was negative and the culture was positive (*T. rubrum*). In three cases, the result of Diafactory tinea unguium® was negative and the direct examination was positive. In the 13 cases of moulds or yeasts isolation, the immunochromatographic test was negative. The positive and negative predictive value of Diafactory tinea unguium® were respectively 75% and 86%.

**Conclusion:** In order to reinforce the importance of confirmation in the diagnosis of mycological onychomycosis, Diafactory tinea unguium® provides a reliable, convenient and quick method (5 to 30 minutes). Because of its good detection capacity, this device improved the accuracy of diagnosis of tinea inguium in complement of conventional fungus testing methods.

P481

**Molecular identification of Dermatophytes from clinical samples**

Z. Gazit<fnref n="1" />, S. Gefen-Halevi², N. Belausov<fnref n="1" />, N. Keller<fnref n="1" />, G. Smollan<fnref n="1" />

**Objectives:** Dermatophytes are fungi that can invade and infect the keratinized layers of skin, hair, and nails. Three genera of fungi, Trichophyton (T), Microsporum (M), and Epidermophyton (E), account for most Dermatophytic infections and cause a pathology called tinea (ringworm fungi). The

<footnote n="1">¹Clinical Microbiology, Sheba medical center, Ramat Gan, Israel, ²Clinical Microbiology, Sheba medical center, ramat gan, Israel</footnote>