**Objectives:** **Introduction:** Neuromeningeal cryptococcosis (NMC) is a serious infection. Frequently described during HIV infection, it can nevertheless occur on any field of cellular immunosuppression and even in the immunocompetent. **Aim:** describe the clinical, para-clinical, therapeutic and evolutionary aspects of NMC cases.

**Methods:** A descriptive retrospective study carried out at the infectious diseases' department of La Rabta Hospital over 25 years, between January 1993 and December 2018, including all patients hospitalized for NMC.

**Results:** We collected 30 cases of (NMC). The median age is 34 years old [18-73] with a sex ratio of 1.7. 76 % of patients were HIV-positive. The average CD4 count was 58.8 cells / µl. Among HIV-negative patients, 4 patients were followed for lymphoma, one patient was diabetic, one had Histiocytosis X and only 1 was immunocompetent.

The median time of treatment was 6 days [1-75]. The main symptoms were fever (73%) and headache (93%). Seizures and signs of localization were present in 1/3 of the cases. A Physical meningeal syndrome was noted in 63% of the cases. Cerebrospinal fluid (CSF) analysis revealed a highly variable cytology [0-3000], hypoglycorrhachia (69%) and hyperproteinorachia (83%). The dosage of cryptococcal antigen in the CSF ranged between 1/64 and 1/25000. The treatment of attack was based primarily on Amphotericin B. The mortality was 56%.

**Conclusion:** NMC is a life-threatening fungal infection. Only a high level of clinical suspicion, early diagnosis and appropriate medical care can improve the prognosis.

P333

**Deep cutaneous mycoses in renal transplant patients: diagnostic and therapeutic challenges**

C. Bertin¹, E. Sitterlé¹, A. Scemla², S. Fraitag³, S. Dellière¹, S. Guegan⁴, S. Leclerc-Mercier⁵, C. Rouzaud⁴, F. Lanternier⁶, M.-E. Bougnoux¹,⁷

¹Parasitology Mycology, APHP Hôpital Necker-Enfants-Malades, Paris, France, ²Transplantation, Hopital Necker, Paris, France, ³Anatomopathology, Hopital Necker, Paris, France, ⁴Infectious Diseases, Hopital Necker, Paris, France, ⁵Hopital Necker, Paris, France, ⁶Infectious Diseases, Necker hospital, Paris, France, ⁷Laboratoire De Parasitologie Mycologie, APHP Hôpital Necker-Enfants-Malades, Paris, France

**Objectives:** Deep cutaneous mycoses are infections extending throughout the dermis and hypodermis, often occurring after inoculation of pathogen fungi. In kidney transplant, deep cutaneous mycoses count for up to 5% of all documented infections. The aim of this study was to describe the clinical features, histopathological findings and mycological characteristics of deep cutaneous fungal infections in renal transplant patients, as well as their management and outcome.

**Methods:** We performed a retrospective review of cases of proven deep cutaneous mycosis (histologically extending to dermis and/or hypodermis with mycological documentation) occurring among kidney transplant recipients in our institution since 2011. We studied the type of transplant, immunosuppression, histopathology, treatment with prospective follow-up of cutaneous lesions

**Results:** Nineteen kidney transplant patients were included; the average age was 41 (range, 28 to 91). The immunosuppressive regimens at the time of diagnosis included corticosteroids (n = 19) and anti calcineurin (tacrolimus (n = 18), cyclosporin (n = 1)). In 10 and 6 patients a treatment by mycophenolate mofetil and azathioprine was associated, respectively. Co-morbidities were frequent: diabetes mellitus (n = 12), cancer (n = 2), HIV (n = 1). In 70% of cases a skin cancer was initially suspected (epidermoid carcinoma n = 5, kaposi sarcoma n = 6). The skin lesions were solitary in ten patients, mainly nodular (84%) and affected the extremities (95%), without fever or fatigue in the majority of cases (63%). Histopathologic examination of skin biopsies showed pseudohyphae and/or irregularly branched hyphae (n = 16/16, 100%), a granulomatous dermatitis (n = 6/16, 37%), and an inflammatory infiltrate (n = 8/16, 50%). Mycological examination of the biopsy showed the presence of hyphae on direct examination in 84% of the cases (n = 16/19). Identification was obtained by culture and/or molecular biology. Eleven different fungal species were observed, including dematiaceous molds (n = 13), hyphomycetes (n = 4), dermatophyte (n = 1), and Mucorale (n = 1). No dissemination to other organs was observed. The detection of the fungal (1-3) beta-D-glucan antigen in serum was
positive in 100% of the cases (n = 17/17). Fifteen patients received antifungal treatment with azoles (posaconazole n = 10, voriconazole n = 3), or liposomal amphotercin B (n = 2) for a mean duration of 3 months, and surgery was associated in 11 patients. Side effects were common (64%) with mainly tacrolimus overdosage (21%) or acute renal failure (50%).

**Conclusion:** This work highlights the great diversity of fungal species responsible for deep cutaneous mycoses in renal transplant patients. The mycological documentation is necessary to adapt the antifungal treatment according to the sensitivity to antifungal agents of the isolated species. Drug interactions between antifungals and immunosuppressive treatment require appropriate monitoring.

P336

Neutrophil profiling in response to the human fungal pathogen, *Candida albicans*, in transplant recipients

M. Mansour

Dept Of Medicine, Division Of Infectious Diseases, Massachusetts General Hospital, Boston, United States of America

**Objectives:** Neutrophils are innate immune cells and the first line of defense against a variety of invading pathogens including yeast and mold species. While low circulating neutrophils numbers (neutropenia) is a clear risk factor for developing invasive fungal infections (IFI), there is lacking evidence if neutrophil dysfunction contributes to infectious risk. In this study, we profiled circulating neutrophil anti-*Candida* fungicidal activity from high-risk patients at our tertiary care hospital, specifically transplant recipients compared to healthy controls.

**Methods:** Solid-organ (SOT) and stem cell transplant (SCT) patients were identified and consented from September 2018 until April 2019. Healthy control patients (HC) were identified at primary care clinics. EDTA-anticoagulated peripheral blood was obtained from healthy and transplant patients 2-4 months post-transplant. Neutrophils were isolated by negative selection. *C. albicans* was co-incubated for 2 hours with and without human neutrophils at a multiplicity of infection (MOI) of 1, 5 and 10. Following neutrophil cell lysis, percent remaining live *Candida* was measured using a viability dye. In addition, growth inhibition of yeast by neutrophil swarming to *C. albicans* spotted onto glass slide arrays was also assessed by live-cell imaging.

**Results:** 22 SOT (15 kidneys, 7 livers), 20 SCT (allograft) and 22 HC were enrolled. Neutrophils from SCT and SOT had lower *C. albicans* killing percentages compared to HC at MOI 10 (HC: 47%, SOT: 29%, SCT 24% p = 0.0041); MOI 5 (HC: 72%, SOT: 35%, SCT 38% p < 0.0001) and MOI 1 (HC: 91%, SOT: 48%, SCT: 45% p < 0.0001). Neutrophil swarming and fungal control of *C. albicans* spots were significantly inhibited by neutrophils from SCT when compared to SOT and controls (p <0.0001). Analysis of medications, including tyrosine kinase inhibitor (TKI) use, did not demonstrate significant differences in a specific drug class when patient groups are compared (SCT versus SOT).

**Conclusion:** This study indicates that neutrophils from patients with SOT and SCT have a decreased ability to kill *C. albicans*, despite normal circulating numbers. There were no medications or laboratory values that predicted a functional neutrophil outcome. These data strongly support the use of functional neutrophil profiling to risk stratify those individuals at higher risk for invasive fungal infections, although additional studies are required to determine if PMN dysfunction results in increased clinical infection. These findings will inform us of potential therapeutic interventions for restoring immune function and preventing invasive fungal infections in high-risk transplant patients.

P340

Invasive candidiasis in neonatal patients in Saint-Petersburg, Russia

E. Shagdileeva<fnref n="1" />, R. Faizullina², O. Belova³, S. Voronovi_H³, T. Kuznetsova⁴, G. Rubin³, S. Vorobeva⁴, T. Bogomolova², I. Vybornova², N. Klimko<fnref n="1" />

<footnote n="1">¹North-West State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ²North-Western State Medical University named after I.I. Mechnikov, St. Petersburg, Russian Federation, ³City Children's Hospital № 17, St. Petersburg, Russian Federation, ⁴City Children's Hospital № 1, St. Petersburg, Russian Federation</footnote>
**Objectives:** To investigate the etiology, risk factors, clinical manifestations, clinical features, and results of treatment of invasive candidiasis (IC) in neonatal patients in St. Petersburg, Russia.

**Methods:** A prospective study was conducted from January 2015 to February 2019. We evaluated neonates with clinical manifestations of infection and a confirmed diagnosis of IC. The EORTC/MSG 2008 criteria were used for IC diagnosis and evaluation of the effectiveness of therapy.

**Results:** "Proven" IC was detected in 29 neonates patients, girls - 62%. Premature neonates were 86%, twins - 21%. Median birth weight was 1280 g (560 - 4000 g), median gestational age at birth - 30 weeks (23 - 40). IC developed on the 5 - 88 days of hospitalization (median - 20). The main underlying conditions: premature neonates - 86%, intraamniotic infections - 72%, HIV - 7%. Broad-spectrum antibacterial drugs were prescribed for 100% patients, mechanical ventilation - 97%, central venous catheter (CVC) use - 93% (median time - 9,5 days, 4 - 25 days), parenteral nutrition - 69 %. Antifungal prophylaxis with fluconazole received 93% patients. The main clinical variants of IC were candidemia - 97%, chorioretinitis - 6%, meningitis - 3%, and hepatitis - 3%. Candida spp. were isolated from CVC in 86% of patients, blood - 41%, cerebrospinal fluid - 3%. The pathogens of IC were C.albicans - 38%, C.parapsilosis - 32%, C.famata - 14%, C.pelliculosa - 7%, C.tropicalis - 3%, C.guiliermondii - 3%, Candida sp. - 3%. In vitro to voriconazole were resistant 3% Candida spp., fluconazole - 3%, dose-dependent susceptibility to fluconazole - 7%. In the first 24 hours after diagnosis antifungal treatment was used in 100% patients: fluconazole (100%), micafungin (38%), amphotericin B deoxycholate (14%), and voriconazole (14%). The median duration of treatment was 20 days (1 - 54). The overall 30-day survival rate was 86%.

**Conclusion:** IC developed in premature neonates (86%), the median gestational age at birth was 30 weeks, the median birth body weight - 1280 g. Risk factors for IC were broad-spectrum antibacterial drugs therapy (100%), mechanical ventilation (97%), and central venous catheter (93%). Main etiology agents were *C. albicans* – 38% and *C. parapsilosis* (32%). The main clinical variant of IC was candidemia (97%). Antifungal treatment was used in 100% patients, fluconazole – 100%, micafungin – 38%. The overall 30-day survival rate was 86%

P341

## AIRE and STAT1 gene mutations in Children with Chronic Mucocutaneous Candidiasis in Saint-Petersburg, Russia

O. Kozlova¹, N. Isaeva¹, E. Frolova¹, T. Bogomolova¹, E. Suspitsin², T. Gabrusskaia³, N. Klimko¹

¹Department Of Clinical Mycology, Allergology And Immunology, North-Western State Medical University n.a. I.I. Mechnikov, Saint-Petersburg, Russian Federation, ²2St. Petersburg State Pediatric Medical University, Saint-Petersburg, Russian Federation, ³Saint-Petersburg State Pediatric Medical University, Saint-Petersburg, Russian Federation

**Objectives:** Publications on AIRE and STAT1 gene mutations in children with chronic mucocutaneous candidiasis (CMC) are limited.

**Methods:** Molecular genetic studies were performed with multigenic targeted sequencing (MiSeq, Illumina, USA).

**Results:** In prospective single center study included 11 patients with CMC. Median age was - 7.5 y, range - 6 to 13 y, males - 92%. Molecular genetic study has shown heterozygous mutation in the STAT1 gene in 4 patients and mutations in autoimmune regulator (AIRE) in 7 patients. CMC started between 2 months - 4 years (median - 2 y). Sites of CMC were oral mucosa - 100 %, skin - 64%, esophagus - 36%, and nails - 36%. Candida albicans was isolated in all patients. Recurring CMC was diagnosed more than 4 times per year in all patients. Endocrine disorders were present in patients with AIRE syndrome: primary adrenal insufficiency - 71%, hypoparathyroidism - 71%, autoimmune thyroiditis with hypothyroidism - 29%; autoimmune disorders: autoimmune gastritis - 71%, alopecia - 29%, and hemolytic anemia - 14%. Patients with impaired STAT1 did not have endocrine disorders and autoimmune gastritis was in one patient (29%).

**Conclusion:** In 100% patients CMC with mutations in autoimmune regulator AIRE endocrine system disorders and autoimmune diseases. STAT1 gene autoimmune diseases diagnosed in 29% patients. This work was supported by Russian Scientific Fund grant 18-15-00256
P342

**Pneumocystis jirovecii pneumonia associated with severe viral infection in children**

O. Kozlova¹, E. Mironuk², V. Kapilov², A. Ustinova², P. Muratov², E. Serikova², M. Pinevskaia², S. Ignatyeva¹, N. Klimko¹

¹Department Of Clinical Mycology, Allergology And Immunology, North-Western State Medical University n.a. I.I. Mechnikov, Saint-Petersburg, Russian Federation, ²Children's City Multidisciplinary Clinical Center for High Medical Technologies. K.A. Rauffusa, Saint-Petersburg, Russian Federation

**Objectives:** As an opportunistic infection, *Pneumocystis jirovecii* pneumonia (PjP) is a severe and life-threatening complication in immunocompromised patients. Publications on PjP associated with severe viral infection in children are limited.

**Methods:** We report the 2 cases of PJP associated with severe viral infection in children.

**Results:** A 13-year-old girl admitted in children's hospital with interstitial pneumonia, sepsis, and respiratory and cardiovascular failure. 7 days before, she presented with the history of fever and dry cough. The patient is diagnosed with influenza. She was in acute respiratory distress and required immediate intubation and transfer to the intensive care unit. On examination, she was hypoxic with SaO2 70-82 % despite 100% flow oxygen. Despite empiric intravenous therapy with ceftriaxone, amikacin, meropenem and vancomycin, she remained hypoxic. Lymphopenia (1,0 × 10⁹/l) was in blood analysis. HIV testing was negative. The chest CT scan showed bilateral interstitial infiltrates. MONOFLUO™ KIT P. jirovecii test and PCR test for P. jirovecii were positive in BAL. Intravenous trimethoprim–sulfamethoxazole (TMP–SMX) (12 mg/kg TMP) and prednisone 0.5 mg/kg twice daily were initiated, which resulted in improvement in respiratory status and weaning of respiratory support over 15 days (SatO2 - 88-92%, PO2 - 46-52%). A 16-year-old boy was diagnosed with chronic bronchiolitis, secondary arterial hypertension, and secondary asymmetric hypertrophic cardiomyopathy. 3 month before, he presented with the history of high fever and maculopapular rashes, spots Koplika and cough. Vaccination details were not available. The patient received several courses of antibiotic therapy and prednisone for a long time at a dose of 2.5-7.5 mg/kg without positive dynamics. Due to the development of severe respiratory failure, the patient was hospitalized. The patient had dyspnea of mixed character (SpO2 - 70-80%). The patient was immunocompetent (CD4 = 2,130*10⁹/l) HIV testing was negative. The serology for measles was positive. The CT showed bilateral interstitial infiltrates. MONOFLUO™ KIT P. jirovecii test was positive in BAL. Intravenous TMP–SMX (12 mg/kg TMP), which resulted in improvement in respiratory status and weaning of respiratory support over 7 days.

**Conclusion:** Severe viral infection associated with several weeks of immune suppression with the consequence may be the cause of secondary infections.

P343

**The first case of successful treatment of invasive mycosis caused by *Candida guiliermondii* and Exophiala dermatitidis* in a neonatal patient**

E. Shagdileeva¹, S. Vorobeva², N. Kotina², T. Bogomolova³, I. Vybornova³, N. Klimko¹

¹North-West State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ²City Children's Hospital № 1, St. Petersburg, Russian Federation, ³Kashkin Research Institute Of Medical Mycology, North-Western State Medical University named after I.I.Mechnikov, St. Petersburg, Russian Federation

**Case Report:** Newborn girl on the 2nd day of life was admitted to the City Children's Hospital № 1 on 25.01.2019. Birth weight was 3050 g, gestational age at birth – 40 weeks. Mother 43 years old: pregnancy - 8, childbirth - 6, HIV since 2014. The underlying condition was early hemorrhagic disease of the newborn, gastric bleeding. Result of HIV-1 DNA PCR test was negative, and antiretroviral prophylaxis (zidovudine) was used. Risk factors for IM were prolonged central venous catheter (CVC) use and broad-spectrum antibacterial therapy. Clinical manifestation of IM was body temperature > 38°C, refractory to broad-spectrum antibacterial therapy. A patient was treated
empirically with fluconazole. Diagnosis of IM was confirmed with positive blood culture: resistant to fluconazole in vitro *C. guiliermondii* and *E.dermatitidis*. Antifungal treatment was changed to micafungin. Clinical condition was improved and results of multiple blood cultures were negative. The total duration of micafungin treatment was 30 days. **Conclusion.** In the treatment of invasive mycosis in neonatal patients, the possibility of several pathogens should be considered.

P344

**Invasive Fungal Infections in a Paediatric Haematological-Oncological centre: a retrospective study of 15 years.**

A. Tragiannidis¹, A. Giantsidi², D. Kotsos², E. Tsotridou², A. Saranti², V. Antari², E. Hatzipantelis², T.-A. Vyzantiadis³

¹Hematology Oncology Unit, 2nd Pediatric Department, Aristotle University of Thessaloniki, AHEPA Hospital, Thessaloniki, Greece, ²Hematology Oncology Unit, 2nd Pediatric Department, Aristotle University of Thessaloniki, AHEPA Hospital, THESSALONIKI, Greece, ³First Department Of Microbiology, MEDICAL SCHOOL, ARISTOTLE UNIVERSITY OF THESSALONIKI, THESSALONIKI, Greece

**Objectives:** Despite the major progress made in the last few years regarding the prevention, diagnosis and treatment of invasive fungal infections (IFIs), they still represent an important cause of morbidity and mortality in paediatric patients with malignancies and those undergoing HSCT. The aim of this retrospective study was to collect data of the incidence and outcome of IFIs in children with haematological malignancies and solid tumors in a Paediatric Hematology Oncology Unit. Additionally, specific data on diagnostics, predisposing factors and treatment was also collected

**Methods:** Data was collected retrospectively from patients with an IFI (proven, probable, possible), who were diagnosed between January 1, 2003 and December 31, 2017 with haematological malignancies and solid tumors in the Haematological-Oncological Unit of the 2nd Department of Paediatrics, AHEPA University Hospital. By reviewing the patients' records, information regarding gender, age at the time of the diagnosis of the IFI, underlying disease, treatment phase, the presence of neutropenia and its duration (≥ or < 10 days) as well as severity (≤ or > 500/mm³) and the site of the fungal infection was obtained. Last follow-up was performed after 12 months after the end of chemotherapy and included the status of disease, cause of death and in case of the occurrence of an IFI, the outcome of the infection.

**Results:** Eleven out of the twenty-six children enrolled in the study were males. The underlying disease was ALL for 19 patients (73%) and AML for 6 (23%). Twenty of them were under parenteral nutrition during the manifestation of the IFIs and 20 of the patients were presented with neutropenia (ANC<500/mm3). The most common location of the IFIs were the lungs and the most common pathogen was Aspergillus spp. According to the revised criteria of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) for the diagnosis of IFSs, 5 of the diagnoses were classified as proven, of which two patients developed zygomycosis, one chronic disseminated candidiasis, one cryptococcosis and one systematic aspergillosis. Out of the rest 21 patients, 12 were classified as being affected by probable and 9 as being affected by possible IFIs. Eleven patients received a combination of antifungal drugs, whereas the rest of them were treated with monotherapy. Twenty received liposomal amphotericin B (LAMB), eleven voriconazole, two caspofungin, two micafungin and one fluconazole. IFI was lethal in 9 out of the 26 patients (34%). Patients with zygomycosis were successfully treated with surgical excision of the lesion combined with high dose of LAMB.

**Conclusion:** The results of this study suggest that, despite the progress regarding the diagnosis and treatment of IFIs with use of new antifungal drugs, mortality remains high. Therefore, it becomes obvious that early suspicion as well as prophylactic and empirical therapy are significant factors contributing in the improvement of the outcome of IFIs.

P345
**Candida sepsis successfully treated with micafungin add-on therapy in extremely preterm infants: report of two cases.**

C.S. Kim

Department Of Pediatrics, Keimyung University School of Medicine, Daegu, Korea, Republic of **Case Report: Objectives:** Candida sepsis is a common hospital-acquired infection in the neonatal intensive care unit and occurs usually in extremely preterm infants. Echinocandine is a new drug with a different mechanism of action from existing antifungal agents which induces fungal cell wall damage, and is mainly used in adults, and neonatal applications are extremely rare. **Case 1:** A female infant was born by an emergent Cesarean section with fetal distress at the gestational age 27 weeks. The birth weight was 720 g. After birth, the baby received intensive care for respiratory distress and feeding difficulties, including mechanical ventilation, central venous catheterization, and parenteral nutrition. On the 16th day of admission, the patient was suspected of sepsis and *Candida parapsilosis* was detected in the culture of both blood and central venous catheter specimens. Initially, amphotericin-B (initial dose: 0.5 mg/kg/day, maintenance dose: 1-1.5 mg/kg/day) was administered but fungemia persisted after 10 days of treatment. Thereafter, micafungin (therapeutic dose: 15 mg/kg/day) was added and administered concurrently. After 4 days of the combination therapy, CRP was decreased, and candida was no longer detected in blood samples. The patient was treated with amphotericin-B for 19 days and micafungin for 8 days, and there was no recurrence of fungal infection. There were no adverse effects of antifungal agents in laboratory findings at the end of treatment. **Case 2:** A female infant was born with vaginal delivery at the gestational age 26 weeks and 2 days. The birth weight was 750 g. After birth, the baby received neonatal intensive care, including mechanical ventilation and parenteral nutrition. On the 13th 15th day of hospitalization, C. *parapsilosis* was consecutively detected in blood samples of the patient. Initially, amphotericin-B was administered, but fungemia and elevated titers of CRP were persisted after 10 days of treatment. Thereafter, micafungin was added. Candida was no longer detected in the blood samples after 7 days of the combined therapy. The patient was treated with amphotericin-B for 25 days and micafungin for 14 days, and there was no recurrence of fungal infection. In addition, there were no adverse effects of antifungal agents in laboratory findings at the end of treatment. **Conclusion:** I report two cases of extremely preterm infants with Candida sepsis successfully treated with micafungin add-on therapy that did not resolve with amphotericin-B alone.

P346

**Invasive Aspergillosis in a Paediatric Haematological-Oncological center: a retrospective study of 15 years**

A. Tragiannidis¹, A. Saranti², A. Giantsidi², D. Kotsos², E. Tsotridou², M. Papambougiouki², E. Hatzipantelis², T.-A. Vyzantiadis³

¹Hematology Oncology Unit, 2nd Pediatric Department, Aristotle University of Thessaloniki, AHEPA Hospital, Thessaloniki, Greece, ²Hematology Oncology Unit, 2nd Pediatric Department, Aristotle University of Thessaloniki, AHEPA Hospital, THESSALONIKI, Greece, ³First Department Of Microbiology, MEDICAL SCHOOL, ARISTOTLE UNIVERSITY OF THESSALONIKI, THESSALONIKI, Greece

**Objectives:** Invasive aspergillosis (IA) represents an important cause of morbidity and mortality in patients with primary and secondary immunodeficiencies and them undergoing Hematopoietic Stem Cell Transplantation (HSCT). In particular, Aspergillus spp. constitute the second most frequent cause of fungal infections in immunosuppressed patients, while IA seems to be responsible for higher mortality rates in comparison with candidiasis.

**Methods:** Retrospective case study of IA managed and treated in the Hematological-Oncological Unit between 2001 and 2016. The study included in total 16 children with possible, probable and proven IA according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group criteria.
**Results:** Among the 16 children included in the study, there were 6 boys. The underlying condition was acute lymphoblastic leukaemia (ALL) in 10 patients (62.5%) and acute myeloid leukaemia (AML) in 6 (37.5%). Out of the 16 patients, 13 were neutropenic with an absolute neutrophilic count lower than 500/mm3 at the time of diagnosis. According to the revised criteria of the EORTC/MSG for the diagnosis of IFIs came up that in 1 patient the diagnosis was proven, the diagnosis of 9 patients was classified as probable and finally in 6 as possible. Combined antifungal treatment was administered in 4 patients, while 12 patients received a single antifungal agent. Liposomal amphotericin B (LAMB) was administered in 10/16 patients, voriconazole in 7/16, caspofungin in 1/16 (as combined treatment) and micafungin in 1/16 patients (as combined treatment). Six out of the 16 patients (37.5%) died due to the IFI.

**Conclusion:** According to the above findings there was concluded that despite the progress that was made regarding the diagnosis and treatment of IA with the use of newer formulations of antifungal agents, morbidity is still high. High suspicion and prompt initiation of empiric and prophylactic antifungal treatment in selected cases constitute significant factors for the improvement of the outcome of IA.

P347

Thoracic actinomycosis mimicking tumor in a children

O. Kozlova¹, I. Suslova², Y. Avdeenko², Y. Borzova², N. Klimko³

¹Department Of Clinical Mycology, Allergology And Immunology, North-Western State Medical University n.a. I.I. Mechnikov, Saint-Petersburg, Russian Federation, ²North-Western State Medical University n.a. I.I. Mechnikov, Saint-Petersburg, Russian Federation, ³Department Of Clinical Mycology, Allergy And Immunology, North-Western State Medical University named after I.I.Mechnikov, St. Petersburg, Russian Federation

**Objectives:** Thoracic actinomycosis is rare and publications are limited.

**Methods:** Prospective, single-center study of 186 patients with different clinical forms of actinomycosis (2005–2017 yy.). Pulmonary actinomycosis was in 9% (n = 17) cases of them there were two children. We present two cases successful treatment of thoracic actinomycosis in children without risk factors whose initial diagnosis was malignancy.

**Results:** 14-year-old boy, previously healthy, was admitted with a history of continuous chest pain during the past three weekes, fever, dry cough and hemoptysis. He was referred to the oncology center with the hypothesis of malignant disease. Weight and height of the patient was appropriate the age and sex. Respiratory sounds were decreased in the right lower field. Laboratory findings demonstrated slight anemia (hemoglobin of 110 g/l) and elevated inflammatory markers, such as white blood cell count (leukocyte count of 16 * 10⁹/l) and C-reactive protein (25.5mg/dL). Tuberculin test was negative. A chest CT scan revealed a consolidation (70*40 mm) with spiculated margins and areas of ground-glass attenuation in the right lower lung. A percutaneous thoracoscopic biopsy of right lower lobe was performed. Histological examination revealed the presence filamentous organisms and sulfur granules, without evidence of malignancy. The thoracic actinomycosis was diagnosed. The patient received penicillin 15000000 units/day) during three weeks, followed by 5 months of amoxicillin (40mg/kg/day). During the first month of treatment, he showed clinical and radiograph improvement. He was followed up every 2 months until completing oral antibiotic therapy and remained asymptomatic year after the end of the treatment. Previously healthy 14-year-old girl was admitted with a history of hemoptysis during the past month, continuous chest pain, and fever. Respiratory sounds were decreased in the right lower lobe, the patient's oxygen saturation was 96% while breathing ambient air. CT showed dense consolidation in the right lower lobe. The disease was initially interpreted as the malignant disease. A lobectomy was performed. Histological evaluation of surgical specimens with hematoxylin and eosin staining shows classic sulfur granules consistent with actinomycosis. She was started on intravenous sodium penicillin 8 000 000 units/day during two weeks, followed by 3 months of amoxicillin (40mg/kg/day), resulting in a rapid clinical response with the disappearance of all symptoms. General state of health was satisfactory, indicators of clinical and diagnostic studies within the normal range. CT imaging was normal.
**Conclusion:** Actinomycosis is an uncommon disease in children. Thoracic actinomycosis should be considered in the differential diagnosis of neoplasia.

P348

Coccidioidomycosis disseminated disease in pediatric patient

V. Lora-Téllez, M.F.D.C. Lara-Hernández, A. Bonifaz-Trujillo, G. Rodríguez-Coello

Mycology Laboratory, Hospital para el Niño Poblano, Puebla, Mexico

Case Report: Objective. To describe the clinical manifestations of the disease and laboratory diagnosis. Methods. Clinical characteristics, symptoms, direct examination of the pathological material, culture and biology molecular methods. Results. 11 years old female patient with malnutrition, who lives in semiarid region in the south-east of Mexico. 3 months of clinical evolution. Fever 38°C. Multiple lesions such as subcutaneous nodes tissue with localization in back, ilium region, knee, and feet. Without pleuritic, aching chest pain and cough. Subcutaneous abscesses at computer assisted tomography suggests with sarcoma diagnosis. Pathological material biopsy for laboratory diagnosis by Hematoxylin Eosin, Periodic Acid-Schiff stains, KOH 10% solution, culture on fungal media. Direct microscopic observation of the histopathological sections demonstrated the inflammatory response contained granulomatous elements, collections of neutrophils, areas of caseous necrosis and mature, ruptured spherules. The mature spherules measure 30 to 60 µm, with double walls up to 2 µm in thickness. Inside of the mature spherules multiple endospores measure 2 to 5 µm containing. The growth of colonies white and floccose at first 3 days was evident by culture methods. Coccidioidomycosis for diagnosis of the disease by the direct examination of the pathological material was enough. The fungal therapy with amphotericin B was successful. The molecular biology method by polymerase chain reaction in real time technique (PCR-rt) match with Coccidioides posadasii by the examination of the in vitro fungal growth. Conclusion. Coccidioidomycosis is usually acquired through inhalation of arthroconidia and the disease is known to be endemic to the United States, Mexico restricted to the semiarid and desert regions. It is also found in a lesser degree in Central and South America. At present, the etiological agents of coccidioidomycosis in man are Coccidioides immitis and Coccidioides posadasii. Although the both etiological agents have equal phenotypic characteristics and produced the same disease, there are differences between them, such as the geographic distribution and genetic characteristics. Coccidioides immitis is located in the north region of Mexico, whereas Coccidioides posadasii is located in the south region of the country. It is important to know the specific etiological agent for epidemiological reasons. Lesions of skin and subcutaneous tissue are among the most common manifestations of hematogenous dissemination. The usual dissemination as a complication of primary infection it was because malnutrition and the endemic region habitat.

P349

Zygomycosis in pediatric patients and laboratory diagnosis.

V. Lora-Téllez, M.L. Pérez-Ricardez, M.F.D.C. Lara-Hernández, G. López-Hernández

Mycology Laboratory, Hospital para el Niño Poblano, Puebla, Mexico

Case Report: Objective. To describe the distribution of the clinical manifestations and the etiological agents of zygomycosis identification in pediatric patients. Methods. Various clinical forms description in four pediatric patients with underlying condition. Etiological agent identification by direct examination and culture on Sabouraud Dextrose Agar of the pathological material. Results. Female 7 years old, acute lymphocytic leukemia as underlying condition. Sites of the infection were paranasal sinuses, eye's orbital section, palate and face. Paranasal sinuses biopsy was selected as pathological material for direct examination and fungal culture. Aseptate hyphae branching at angle of 90 degrees approximately was observed by hematoxylin eosin and periodic acid-Schiff stains by direct examination. The etiological agent was Lichthiemia spp by fungal culture. Female 7 years old, acute lymphocytic leukemia as underlying condition. Site of the infection was paranasal sinuses. In the paranasal sinuses biopsy was observed aseptate hyphae 10 µm measure. Fungal culture on Sabouraud Dextrose Agar at 25°C, after 4 days a woolly colony growth was observed. Microscopic
morphology of the colony was identified Mucor spp as etiological agent. Male 8 years old, burn trauma as underlying condition. Sites of the infection were legs, arms and hands. Tissue debridement was observed by direct examination with potassium hydroxide KOH 40% solution, and cultured on fugal media. A white-gray woolly colony growth was observed after 2 days on Sabouraud Dextrose Agar at 25°C, after 4 days incubation, the surface of the colony was covered with dark spots like a "salt - and- pepper" appearance. At microscope examination, the differential characteristics of the Rhizopus were observed, such as a single umbrached sporangiophore, a globose sporangia, sporangiospores and rhizoids. Microscopic morphology of the colony was identified Rhizopus spp. Female 1 month old, gastric perforation as underlying condition. At computer assisted tomography examination a mass like a tumor was observed in the gastric cavity. Gastric mucosa biopsy by direct examination multiple aseptate and very broad hyphae were observed. Histopathological examination of the gastric mucosa biopsy demonstrated the vascular invasion by hyphae, leading to thrombosis and infarction of tissue. Rhizopus spp as etiological agent by fungal culture examination. Conclusion. Zygomycosis is characterized by vascular invasion with hyphae, infarction, and necrosis of tissue and by an acute or subacute course. Zygomycosis caused by Rhizopus, Lichtheimia, Rhizomucor and Mucor has a wide geographic distribution. The distribution of the clinical manifestations was usual sites of infection because underlying condition such as acute lymphocytic leukemia with neutrocytopenia, and burn trauma. But gastric cavity was uncommon as site of infection due was introduced by oral-gastric catheter. Because the hallmark of Zygomycosis is vascular invasion by hyphae, leading to thrombosis and infarction of tissue, the antifungal therapy with Amphotericin B was successful while is delimited the original site of the infection, such as paranasal sinuses, before it presents in contiguous structures of the orbit, palate, face, nose, or brain. The early diagnosis as much as the site of infection contributed for a good clinical evolution.

P350

**Spectrum of invasive yeast infections in children in Pakistan over the last five years: 2015-2019**

J. Farooqi¹, S. Memon², S. Laiq¹, F. Naqvi¹, K. Iqbal¹, S. Tariq¹, A. Zafar³, K. Jabeen⁴

¹Pathology And Laboratory Medicine, Aga Khan University, Karachi-KHI, Pakistan, ²Microbiology, Karachi University, Karachi, Pakistan, ³Pathology And Laboratory Medicine, Aga Khan University, Karachi, Pakistan, ⁴Pathology And Laboratory Medicine, Aga Khan University, Karachi, Pakistan, Pakistan

**Objectives:** We aim to describe here the changing spectrum of invasive yeast infections in children in the last 5 years in Pakistan, using retrospective laboratory data from 2015-May 2019

**Methods:** Records of archived yeast isolates from Jan 2015-May 2019 kept at the Aga Khan University Clinical Laboratories, section of Microbiology in Karachi, were retrieved for analysis. A total of 1119 non-duplicate isolates were identified, of which 992 were invasive specimens from blood (833), CSF (51), wounds (47), intraabdominal collections (36), Central venous lines (17) and other sterile fluids (8). The patients were divided into neonates (259), paediatric age group (253) and adults (477). Frequencies of different *Candida* and non-*Candida* species isolated over the last 5 years were computed and compared for age groups using chi-square test. Hypothesis of a change in the spectrum over the years among each age group was also tested.

**Results:** *Candida* species made up 87.4% and 86.6% of all invasive yeasts in neonates and children, respectively. The most common group in neonates was a diverse group of rare *Candida* species (including *C. lusitaniae*, *C. guilliermondii* and *C. pelliculosa* to name a few) at 45.4%, followed by *C. tropicalis* (24%) and *C. parapsilosis* (15.7%); *Ustilago* species were the most common non-*Candida* yeasts at 9.2%. In children, the most common species was *C. tropicalis* (32%), followed by rare *Candida* species as described above (22.8%) and *C. albicans* (15.1%). *Ustilago* was again the most common non-*Candida* yeast at 11.1%. There was a statistically significant difference in the distribution of invasive yeasts between neonatal and paediatric age groups (p<0.001). The change in spectrum of invasive yeast over the years was significant for infants and children (p = 0.035) and adults (p = 0.029); however, we could not demonstrate a significant difference in neonatal (p = 0.096) and elderly (p = 0.270) spectrum of
invasive yeast infections over the years. Fluconazole resistance in yeasts isolated from neonates was found to be 8.2% while it was 22.8% in infants and children, possibly due to an emergence of *C. auris* infections in children (12.8%) versus neonates (1.3%).

**Conclusion:** We conclude that it is essential to monitor the spectrum of yeast infections in neonates and children as they are the population where there is greatest diversity of invasive species, not limited to *Candida* species. Emerging pathogens are most likely to arise from this population, including resistant strains, hence, it is also important to monitor resistance trends.

P351

**Changing epidemiology of *candida* infection in NICU and PICU – multicenter study**

S. Pleško¹, M. Novak², V. Benjak², D. Hadžić³, D. Kardum⁴, K. Lah Tomulić⁵, B. Polić⁶, M. Bucat⁷, V. Mišanović⁸, K. Kondža⁹, M. Mikšić¹⁰, M. Bakoš², T. Ćaleta²

¹Department For Clinical And Molecular Microbiology, University Hospital Centre Zagreb, Zagreb, Croatia, ²Department Of Pediatrics, University Hospital Center Zagreb, Zagreb, Croatia, ³Department Of Intensive Therapy And Care Pediatrics Clinic, University Clinical Center Tuzla, Tuzla, Bosnia and Herzegovina, ⁴Department Of Pediatrics, Neonatal Intensive Care Unit, University Hospital Osijek, Osijek, Croatia, ⁵Department Of Pediatric, University Hospital centre Rijeka, Rijeka, Croatia, ⁶Department Of Pediatrics, University Hospital Centre Split, Split, Croatia, ⁷Department Of Pediatrics, Nicu, University Hospital Centre Split, Split, Croatia, ⁸Department Of Pediatrics, University clinical Center Sarajevo, Sarajevo, Bosnia and Herzegovina, ⁹Department Of Pediatrics, Childrens Hospital Zagreb, Zagreb, Croatia, ¹⁰Department Of Pediatrics, university Hospital Centre Maribor, Maribor, Slovenia

**Objectives:** Invasive *Candida* infections are considered as very important cause of morbidity and mortality in neonatal intensive care unit (NICU) and pediatric intensive care unit (PICU). Although *C.albicans* is the most commonly isolated species from invasive candida infections the frequency of non-albicans candida has increased. Geographical differences in epidemiology, and even more important changes in susceptibility patterns have been described. It is very important to know the epidemiology in order to recognize patients in risk and to start the appropriate empirical therapy and improve patient outcome. The aim of this study was to determine changes in *Candida* infections epidemiology in NICU and PICU from three countries.

**Methods:** We included the data from three neighboring countries: Croatia, Bosnia and Herzegovina and Slovenia (10 centers altogether), during the two year period. We collected data about species of *Candida* causing infection, site of infection and antifungal susceptibility of strains. In first year, overall 65 patients with invasive fungal infection (IFI) (25 from NICU and 40 from PICU), and in second year 107 patients with IFI (59 from NICU and 48 from PICU) were enrolled in the study.

**Results:** During the first year of surveillance in NICU, *Candida albicans* was the most frequent isolate in IFI patients in 4 centers and *Candida parapsilosis* in one center. According to collected data, *Candida tropicalis*, *Candida krusei* and *Saccharomyces cerevisiae* were observed as rare and sporadic causes of IFI. During the second year of surveillance we observed the shift of species as leading cause of IFI between centers: *C. parapsilosis* was most frequent isolate and *C. albicans* was less frequent. In some centers *C. famata*, *C. glabrata*, *C. krusei*, *C. guillermondii* was reported, sporadically. In PICUs, during first year *C. albicans* was most common in four centers followed by *C. parapsilosis* except in one center where *C. lusitaniae* and *C. krusei* were recorded. *C. glabrata* and *C. guillermondii* were recorded, sporadically. In second year, we also noted the shift of species as leading cause of IFI: the number of *C. albicans* decreased and number of *C. parapsilosis* increased in 4 centers. It is important to emphasize that 60% of *C. parapsilosis* in second year were susceptible only to amphotericin B (resistant to fluconazole ad intermediate to echinocandins according to the EUCAST interpretation breakpoints). *C. glabrata*, *C. tropicalis*, *C. famata*, *Crytococcus laurentii* and *C. lusitaniae* were reported, sporadically.

**Conclusion:** The results of this observational study indicate the changing epidemiology of IFI pointing to differences in fungal isolates as leading causes of IFI between centers and in the same centers, during observational period of two years. In addition, this study noted the increase of MICs
for some antifungals and shift in susceptibility of fungi, addressing the importance of monitoring and optimization of therapeutic guidelines of IFI in NICU and PICU.

P355

Liposomal Vaccine Containing Aspergillus Proteins Protects Immunosuppressed Male and Female Mice Against Pulmonary Aspergillosis Caused by Azole Resistant or Sensitive Strains of *Aspergillus fumigatus*

M. Slarve¹, N. Holznecht², J. Adler-Moore², S. Ho³, G. Fuji³

¹Biology, Cal Poly Pomona, Riverside, United States of America, ²Biology, Cal Poly Pomona, Pomona, United States of America, ³Molecular Express, Inc, San Dominguez, United States of America

**Objectives:** This study tested the efficacy of an immunotherapeutic liposomal *Aspergillus* vaccine (VesiVax® LAsV), with or without additional antifungal drug treatment. The vaccine contained *Aspergillus* proteins Aspf3 and Aspf9, and the adjuvant lipidated tucaresol (LT1). LAsV was tested in female mice challenged with two azole resistant strains of *Aspergillus fumigatus* (V29 and V80), and in male mice with an azole sensitive strain of *A. fumigatus* (ATCC#13073).

**Methods:** Six week old Swiss Webster mice were vaccinated with LAsV (Molecular Express, Inc.) at 7.5 µg Aspf3/dose, 7.5 µg Aspf9/dose and 5 µg LT1/dose, subcutaneously on d0, with intranasal boosts on d21 and d42. Control mice were given phosphate buffered saline (PBS). Prior to challenge, spleens and blood were collected (n = 5/group) for ELISpot cytokine analysis and anti-spore antibody agglutination assays. On d53, d55, and d57, vaccinated mice were immunosuppressed intraperitoneally with 28 mg/kg triamcinolone acetonide. On d56, mice were intranasally challenged with *A. fumigatus* conidia (2-2.8 X 10⁷/mouse depending on fungal strain). Some vaccinated groups were also given a short course of 7.5 mg/kg liposomal amphotericin B (AmBisome®, AmBi, Gilead Sciences Inc.) intravenously at 12h, 36h, and 60h post-challenge. Lungs were collected (n = 7/group) 3 days post challenge for fungal burden, and remaining mice (n = 9/group) were monitored for survival to d77.

**Results:** LAsV vaccinated male and female mice had reduced lung fungal burdens compared to PBS control mice regardless of the strain of *A. fumigatus* challenge. Female mice vaccinated with LAsV, challenged with strain V29 or V80, and then given subsequent AmBi treatment, had higher survival than PBS control mice (V29 = 79% vs 21%; V80 = 57% vs 25%) and the vaccine alone also yielded some protection when mice were challenged with V29 (55% survival). Non-vaccinated female mice given AmBi alone had only 37% survival following V29 challenge, and 22% survival with V80 challenge. Results in male mice vaccinated with LAsV and given AmBi after challenge, were similar to the female mice, yielding 55% survival following *A. fumigatus* challenge (#13073) versus 22% survival for mice given AmBi alone or PBS. ELISpot cytokine analysis showed that LAsV vaccinated female and male mice had a significantly higher number of IL-4 than IFN-γ secreting splenocytes following incubation with Aspf3 and Aspf9 (p = 0.0178 for males, p = 0.0290 for females), which is characteristic of a Th2 response. Anti-spore antibody agglutination titers demonstrated that LAsV vaccinated mice also had increased anti-*A. fumigatus* spore antibodies in the serum than PBS control mice (p = 0.0009 for males, p<0.0001 for females).

**Conclusion:** Prophylactic LAsV vaccination of immunocompetent female or male mice, which were then immunosuppressed prior to Aspergillus challenge, followed by a short course of AmBi treatment, provided more protection against pulmonary aspergillosis than AmBi treatment alone whether the infection was caused by azole resistant or sensitive strains of *A. fumigatus*. Elevated levels of anti-*A. fumigatus* spore antibody titers correlated with protection, serving as a possible biomarker to indicate protective immune responsiveness to the vaccine. The LAsV has the potential to be used as an immunomodulatory treatment to further enhance the efficacy of antifungal drugs.

P356

Anti-interferon-γ autoantibodies underlie disseminated *Talaromyces marneffei* infection

C. Cao

Dermatology, The first affiliated hospital of Guangx medical university, Nanning, China
**Objectives:** Anti-interferon-γ autoantibodies underlie disseminated *Talaromyces marneffei* infection
Obsjects: *T. marneffei* causes life-threatening opportunistic infections, mostly in Southeast Asia and
South China. Most *T. marneffei* infections occur in patients infected with human immunodeficiency
virus (HIV), but this infection can, nevertheless, occur in HIV-negative individuals in the absence of
immunosuppression. We investigated the role of anti-interferon (IFN)-γ autoantibodies in severe *T.
marneffei* infection in HIV-negative patients.

**Methods:** Anti-interferon-γ autoantibodies underlie disseminated *Talaromyces marneffei* infection
Obsjects: *T. marneffei* causes life-threatening opportunistic infections, mostly in Southeast Asia and
South China. Most *T. marneffei* infections occur in patients infected with human immunodeficiency
virus (HIV), but this infection can, nevertheless, occur in HIV-negative individuals in the absence of
immunosuppression. We investigated the role of anti-interferon (IFN)-γ autoantibodies in severe *T.
marneffei* infection in HIV-negative patients. Method: We enrolled 58 HIV-negative adults who
developed *T. marneffei* severe infections but were otherwise healthy in this study. We evaluated the
presence of anti-IFN-γ autoantibodies and their neutralizing activity.

**Results:** The prevalence of neutralizing anti-IFN-γ autoantibodies was high (94.8%) in these previously healthy adults suffering from severe *T. marneffei* infections. The presence of anti-IFN-γ autoantibodies was strongly associated with HLA-DRB1*16:02 and DQB1*05:02 in these patients.

**Conclusion:** Adult-onset acquired immunodeficiency due to autoantibodies against IFN-γ is the major cause of severe *T. marneffei* infection in HIV-negative patients in the regions in which this fungus is endemic. The high prevalence of pathogenic HLA class II DRB1*16:02 and DQB1*05:02 alleles, which are strongly associated with anti-IFN-γ autoantibodies, may account for the restriction of severe *T.marneffei* infection to Southeast Asia. Our findings clarify the pathogenesis of *T. marneffei* infection and pave the way for the development of novel treatments.

P358

Phagocytic activity and cytokines production by murine RAW-264.7 macrophages in response to
Microsporum canis

J. Neves¹, F. Konno¹, M. Lallo¹, A. Alvares-Saraiva², E. Hurtado¹, J. Alvarez¹, S.D.A. Coutinho¹

¹Post-graduation Program In Environmental And Experimental Pathology, Paulista University, São Paulo, Brazil, ²Institute Of Physical Activity And Sports Sciences, Cruzeiro do Sul University, São Paulo, Brazil

**Objectives:** Dermatophytes are important fungi for public health, as they are transmitted between animals and humans causing zoonoses. The innate immune system plays an important role in the establishment of dermatophytic infection; however, there are few studies on the relationship between innate immune response and *Microsporum canis*. Thus, the aim of this work was to evaluate the response in vitro of macrophages challenged with *Microsporum canis* microconidia. We evaluated the ability of macrophages stimulated or not with LPS, to phagocytize, to produce nitric oxide and cytokines, and the cell viability.

**Methods:** Murine Raw-264.7 macrophages were cultivated overnight on glass coverslips in a 24 well plates. Macrophages were infected with *M. canis* (ATCC-36299) in the ratio of 0.25:1 (microconidia/macrophages) and incubated at 37°C with 5% CO₂ for 30 min, 1 h, 3 h and 6 h. After incubation intervals, glass coverslips were collected and submitted to Giemsa stain. Two hundred cells were counted and calculated the percentage of macrophages that internalized at least one spore. LPS (1mcg/mL) was added to the macrophages 24 h prior *M. canis* infection in one group. Supernatants were collected for detection of nitric oxide and cytokine levels, and to check the cell viability.

**Results:** The phagocytic index showed an increase in phagocytosis over time (Fig. 1 and 2), reaching the maximum at 6 h. The percentage of non-LPS stimulated macrophages that phagocytosed *M. canis* microconidia was 28.0%, 33.5%, 62.5% and 71.5%, respectively in 30 min, 1 h, 3 h and 6 h (Fig. 1). In macrophages stimulated with LPS the phagocytic index was 34.5%, 47.5%, 67.0%, and 74.0%, respectively in 30 min, 1 h, 3 h and 6 h (Fig. 1). It was observed similar nitric oxide production in both, macrophages challenged and not with *M. canis*. Levels of TNF-α have increased over time in
macrophages challenged with *M. canis* stimulated or not with LPS when compared with macrophages non-infected (statistically significant). Higher levels of IL-6 were produced by macrophages stimulated with LPS and infected with *M. canis* at 1 h (statistical difference). Cell viability decreased over time, with the peak of macrophages killing at 6 h. Microbicity test showed that, even after phagocytosis, spores of *M. canis* remained viable inside macrophages.

<figure><img src="image_82.png" /><figcaption>**Figure 1.** Phagocytic index of RAW-264.7 macrophages challenged with *Microsporum canis* in the ratio of 0.25:1 (microconidia:macrophages), stimulated and non-stimulated with LPS, at 30 min, 1 h, 3 h, and 6 h.</figcaption></figure>

<figure><img src="image_83.png" /><figcaption>**Figure 2.** The uptake of *Microsporum canis* by RAW-264.7 macrophages at 3 h. The presence of phagocytosed microconidia and intracellular multiplication with germ tubes formed inside the macrophages (arrows) (Giemsa stain 1,000x).</figcaption></figure>

**Conclusion:** Raw-264.7 macrophages showed high phagocytic activity against *M. canis*, which increased when macrophages were previously stimulated with LPS. LPS stimulation also influenced the production of cytokines, which suggests greater modulation in the response to infection. Increased production of the pro-inflammatory cytokines TNF-α and IL-6, which are important in the recruitment of new phagocytic cells and in the resolution of the disease, have been found in macrophages challenged with *M. canis*. The results suggest that, although macrophages present phagocytic activity and cytokine production when challenged with *M. canis*, their viability and microbicidal activity are impaired.

**Objectives:** *Candida albicans* is a clinically important opportunistic yeast and has evolved different immune evasion molecules on its cell surface. In the study on the role of one of these, the transmembrane protein "High affinity glucose transporter 1" (Hgt1p), involved in glucose metabolism, but also high-jacking the complement regulator factor H (FH) from host serum, we have used different media, one of them the commonly used YPD medium (yeast extract 1%, peptone 2% and dextrose 2%) to study the role of this evasion molecule in immune defence.
**Methods:** *C. albicans* strains (SC5314 und others) were opsonized with human serum and incubated with anti-FH and anti-complement C3b/iC3b antibodies; their deposition on the cell wall surface was assessed by fluorescence-activated cell sorter (FACS). In parallel, *C. albicans* strains were co-cultured with fresh human polymorphonuclear neutrophils (PMNs). All these experiments were done at different glucose concentrations, mainly because a glucose transporter was involved.

**Results:** *Candida* wild type strain SC5314 showed a clear FH deposition on its cell wall at the physiological glucose concentration of 0.1%, but this was completely higher and thus overestimated when using the two high glucose (0.3%, 2%) containing media. Consequently, the resulting low C3b/iC3b deposition at 0.1% was even lower at the unphysiologically high glucose levels. Also the thus impaired phagocytosis, observable at 0.1%, appeared much more dramatic at glucose levels which are not achievable in vivo.

**Conclusion:** The main events in immune defence, such as opsonisation by C3b/iC3b and phagocytosis by PMNs and the counteracting evasion strategy by *Candida* to high-jack FH are all highly dependent on the glucose concentration and all experimental data are thus completely misleading, if media with unphysiological glucose concentration are employed, such as the commonly used YPD.

P360

Immune response in C57BL mice in *Encephalitozoon cuniculi* infection

A. Miyuki Hidifira¹,², A. Pereira¹,², A. Alvares-Saraiva¹, E. Hurtado¹, F. Konno¹, J. Alvarez¹, M. Lallo¹,²

¹Post-graduation Program In Environmental And Experimental Pathology, Paulista University, São Paulo, Brazil, ²Biomedicina, Centro Universitário São Camilo, São Paulo, Brazil

**Objectives:** *Encephalitozoon cuniculi* is a microorganism belonging to the phylum Microsporidia and to the kingdom Fungi, considered an opportunistic and obligatory intracellular pathogen. The first line of defense against *E. cuniculi* is the innate immune response through the action of phagocytic cells and production of cytokines that are responsible for the activation of T lymphocytes. The immune response against *E. cuniculi* has not been fully elucidated and further studies are needed to understand the involvement of cell types such as macrophages in the role of microsporidiosis. This study evaluated the populations of macrophages and lymphocytic cells in *E. cuniculi* infection in C57BL/6 mice.

**Methods:** SPF C57BL/6 mice were used and divided in 4 groups: uninfected, infected, uninfected-immunosuppressed and infected-immunosuppressed. Immunosuppression was made by intraperitoneal injection of cyclophosphamide and euthanasia was performed by anesthetic deepening to collect the peritoneal lavage and remove the spleen. Lymphocytes and macrophages (F4/80) were identified by cell surface markers using flow cytometry.

**Results:** The results of the evaluation of spleen and peritoneal cells indicate that in the peritoneum there was an increase in CD8⁺ T cells in infected group in relation to the uninfected and infected-immunosuppressed groups and a decrease in CD4⁺ T population in infected-immunosuppressed group. The B cells populations were decreased in both groups infected (immunosuppressed and non-immunosuppressed). In the spleen, there was an increase of CD8⁺ T cells and B cells in infected-immunosuppressed mice and a decrease in CD4⁺ T in the same groups evaluated. Macrophage markers analysis revealed expression of F4/80⁺ CD80/86⁺high MCHIIhigh in both infected animals (immunosuppressed and non-immunosuppressed) in the peritoneal lavage and in the spleen these cells were increased only in infected-immunosuppressed group.

**Conclusion:** The results of this study help to understand the involvement of cells as macrophages in the *E. cuniculi* infection in C57BL/6 mice.

P361

Level of Mannose binding Lectin protein in dermatophytic patients and its correlation with MIC values

V. Hemanth¹, A.J. Kindo², A. Subramanian³, A. Krishnan³

¹Microbiology, Sri Ramachandra Medical college and Research Institute, chennai, India,
²Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, India,
³Dermatology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, India

**Objectives:** 1.Speciation of dermatophytes from the clinical isolates 2.To study the antimicrobial pattern of frequently used antifungal agents 3.To determine the serum MBL levels in patients with dermatophytosis 4.To compare MIC levels with serum MBL levels

**Methods: METHODS:** Clinically diagnosed dermatophytic patients were sampled for microscopy and culture, grown dermatophytes was subjected to phenotypic identification by conventional method. Antifungal susceptibility testing was done by broth microdilution method according to CLSI M38-A2 document guidelines. *Trichophyton mentagrophytes* ATCC 4439 was included in each run of susceptibility testing as a quality control. Final drug concentrations in the microdilution plates ranged from 64 to 0.25 µg/ml for fluconazole and AmphotericinB and from 16 to 0.25 µg/ml for Itraconazole, Voriconazole, Terbinafine and Griesofulvin. This is an ELISA based assay performed in microwells coated with a monoclonal antibody against the MBL carbohydrate- binding domain. Bound MBL is detected with the same antibody that has been labeled with biotin, followed by development with horseradish peroxidase (HRP)-conjugated streptavidin and incubation with a chromogenic substrate. Reading was taken at 620nm.

**Results:** Out of the 100 clinical isolates tested, 58 of them were identified as *Trichophyton mentagrophytes*, 40 were *Trichophyton rubrum*, and the remaining two isolates were identified as *Epidermophyton floccosum* and *Microsoprum nanum*. Antifungal susceptibility testing was carried out for 100 isolates of *Trichophyton* species against 6

<figure><img src="image_84.png" /></figure>

<table><thead><tr><th colspan="5">MIC RANGE FOR THE CONVENTIONAL AND NEWER ANTIFUNGAL AGENTS</th></tr><tr><th>Species identified</th><th>Trichophyton mentagrophyte</th><th>Trichophyton rubrum</th><th>Epidermophyton floccosum</th><th>Microsoprum nanum</th></tr><tr><td></td><td>n = 58</td><td>n=40</td><td>n =1</td><td>n =1</td></tr></thead><tbody><tr><td>AmpB (64-0.25µg/ml)</td><td>4-&gt;64 µg/ml</td><td>4-&gt;64 µg/ml</td><td>4 µg/ml</td><td>4 µg/ml</td></tr><tr><td>Fluconazole (64-0.25µg/ml)</td><td>2-64 µg/ml</td><td>2-64 µg/ml</td><td>2 µg/ml</td><td>&gt;64 µg/ml</td></tr><tr><td>Itraconazole (16-0.06µg/ml)</td><td>0.06 -1 µg/ml-</td><td>0.06-16 µg/ml-</td><td>8 µg/ml</td><td>0.06 µg/ml</td></tr><tr><td>Voriconazole (32-0.125µg/ml)</td><td>0.125 -4 µg/ml</td><td>0.06-4 µg/ml-</td><td>0.125 µg/ml</td><td>0.5 µg/ml</td></tr><tr><td>Griesofulvin (16-0.06µg/ml)</td><td>0.06-4 µg/ml</td><td>0.125 - 16µg/ml</td><td>0.25 µg/ml</td><td>0.5 µg/ml</td></tr><tr><td>Terbinafine (16-0.06µg/ml)</td><td>0.06-8µg/ml</td><td>0.06-2µg/ml</td><td>0.06 µg/ml</td><td>0.06 µg/ml</td></tr></tbody></table>

<figure><img src="image_85.png" /></figure>

antifungal agents. All isolates showed detectable growth after 4-5days of incubation. MICS for *Trichophyton mentagrophytes* ATCC 4439 were within the established range. Mannose Binding Lectin ELISA: Results awaited for ELISA Assay.

**Conclusion:** In this study *Trichophyton mentagrophytes* was the most common cause of dermatophytosis. The findings of this study showed that the patients with dermatophytosis have higher MBL serum levels, the acute phase protein plays a role in dermatophyte infections. It is well known that mutations in MBL genes are associated with reduced serum levels, which is a risk factor for infection. In this study, the high level of MBL levels could be used as prognostic marker for dermatophytic infections. Further studies with larger samples have to be under taken to come to definite conclusion.
**Aspergillus infection in STAT3-deficient patients is associated with defective interferon-gamma and Th17 responses**

F. Danion¹, V. Amanianda², J. Bayry³, A. Duréault¹, M.-E. Bougnoux⁴, C. Tcherakian⁵, M.-A. Alyanakian⁶, H. Guegan⁷, A. Puel⁸, C. Picard⁹, O. Lortholary¹, F. Lanternier¹, J.-P. Latgé²

¹Infectious Diseases, Necker hospital, Paris, France, ²Institut Pasteur, Paris, France, ³Equipe-immunopathologie Et Immunointervention Thérapeutique, INSERM, Paris, France, ⁴Parasitology Mycology, APHP Hôpital Necker-Enfants-Malades, Paris, France, ⁵Pneumology, Hopital Foch, Paris, France, ⁶Immunology, AP-HP Necker Hospital, Paris, France, ⁷Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France, ⁸Inserm U1163, Institut Imagine, Paris, France, ⁹Centre D'étude Des Déficits Immunitaires (cedi), AP-HP Necker Hospital, Paris, France

**Objectives:** Signal transducer and activator of transcription 3 (STAT3)-deficient patients are highly susceptible to many bacterial and fungal infections including aspergillosis, although its pathogenesis remains largely unknown. **Objective:** To investigate the immune responses of STAT3-deficient patients against *Aspergillus fumigatus* infection

**Methods:** Phagocytic and killing efficiencies as well as cytokine responses of mononuclear cells and neutrophils of STAT3-deficient patients were investigated against *A. fumigatus*.

**Results:** We compared results among three groups, STAT3 deficient patients with or without aspergillosis and healthy controls. STAT3-deficient patients with aspergillosis showed elevated titers of *A. fumigatus* specific IgE and IgG compared to STAT3-deficient patients without aspergillosis as well as healthy controls. *A. fumigatus* conidial phagocytic and killing efficiencies of monocytes and neutrophils from STAT3-deficient patients with or without aspergillosis were similar to those from healthy controls. When stimulated with *A. fumigatus* conidia, peripheral blood mononuclear cells from patients with STAT3-mutation showed reduced levels of secreted (pg/mL) and intracellular (%) cytokines compared to healthy controls (IFN-γ: 1836 vs 3248 pg/mL and 3.7% vs 11.4%; IL-17: 16 vs 80 pg/mL and 2.6 vs 7.0%; IL-22: 64 vs 170 pg/mL). In addition, there were significantly lower levels of IFN-γ and TNF-α in STAT3-deficient patients with aspergillosis compared to those without aspergillosis (IFN-γ: 683 vs 3420 pg/mL; TNF-α: 779 vs 4743 pg/mL). In the STAT3-deficient cohort, three patients with aspergillosis were treated with IFN-γ in addition to antifungals; two of them showed favorable outcome.

**Conclusion:** STAT3-deficiency leads to defects in adaptive immune responses against *A. fumigatus*, particularly with a low IFN-γ response in those with aspergillosis, suggesting potential therapeutic benefit of recombinant IFN-γ in these patients.

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**Functionality of neutrophils against Aspergillus fumigatus in patients with alcoholic cirrhosis**

M. Blaize¹, D. Blez¹, P. Lebray², A. Meghraoui-Keddar¹, C. Combadière¹, A. Boissonnas¹, M. Rudler², A. Fekkar¹

¹Sorbonne Université, Inserm, CNRS, Centre d'Immunologie et des Maladies Infectieuses, Cimi-Paris, F-75013, PARIS, France, ²AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Service d'Hépato-Gastro-Entérologie, F-75013, Paris, France

**Objectives:** Patients with liver cirrhosis are at risk of developing bacterial or fungal infections. The incidence and severity of these infections depend on the stage of the disease. Severe alcoholic hepatitis, the most serious stage of liver failure in patients with alcohol abuse and chronic liver disease, has been associated with the development of many cases of invasive aspergillosis and is now considered a risk factor for it. A dysfunction of the immune system, namely systemic inflammation and immunodeficiency, is described in cirrhosis with, in particular, an alteration of the phagocytic and oxidative capacities of neutrophils. The latter is known to be the main immune effector against *Aspergillus*. Therefore, we aim to study neutrophil's responses from patients with alcoholic cirrhosis at different stages of their disease against *Aspergillus fumigatus*.

**Methods:** To achieve this objective, we conducted a prospective monocentric study involving 23 patients and 12 healthy controls. Patients were divided into 4 groups: stable cirrhosis (n = 5), decompensated cirrhosis without severe alcoholic hepatitis (n = 3), decompensated cirrhosis with
severe alcoholic hepatitis (*n* = 11) and decompensated cirrhosis with severe alcoholic hepatitis after 7
days of steroid treatment (*n* = 4). Interactions between neutrophils and *Aspergillus* hyphae were
analyzed by video microscopy, while the oxidative burst of neutrophils before and after fungal
stimulation and cytokine production was determined by flow cytometry and enzyme immunoassay
(EIA).

**Results:** Compared to healthy controls, patients with severe alcoholic hepatitis had an increase in the production of reactive oxygen species by their neutrophils (1727 *versus* 2545, expressed as mean fluorescence intensity, p = 0.041). An increase in plasma IL-8 levels (95 *versus* 378 ng/mL respectively, p = 0.006) was also observed between these groups. Also among patients with severe alcoholic hepatitis, the production of reactive oxygen species decreased after 7 days of corticosteroids compared to other patients, both under basal conditions (937 *versus* 2545; expressed as mean fluorescence intensity, p = 0.011) or after stimulation with germinating conidia (2031 *versus* 9272; expressed as mean fluorescence intensity, p = 0.014). In addition, video microscopy experiments (Figure 1) showed that an alteration in the ability of neutrophils to kill *A. fumigatus* hyphae appears in the severe alcoholic hepatitis group after 7 days of corticosteroid therapy compared to patients sampled before corticosteroid therapy began (12% of mortality *versus* 46% respectively, p = 0.03) (Figure 2).
<figure><img src="image_86.png" /><figcaption>Figure 1. The time sequence shows one germinating conidia before engulfment by a neutrophil sampled from a cirrhotic patient (time: 24 minutes).
The neutrophil kills the *Aspergillus* conidia (as indicated by the fungus stained with Sytox green) after approximately one hour of close interactions.</figcaption></figure>

<figure><img src="image_87.png" /><figcaption>Figure 2. Killing of *A. fumigatus* germinating conidia by neutrophils is impaired in severe alcoholic hepatitis patients receiving corticosteroids. (sAH: severe alcoholic hepatitis; sAH 7d: severe alcoholic hepatitis after 7 days of corticosteroids therapy)</figcaption></figure>

**Conclusion:** In conclusion, a pro-inflammatory state is present in patients with severe alcoholic cirrhosis as well as in patients with stable cirrhosis. The introduction of corticosteroids as a treatment for severe alcoholic hepatitis quickly leads to a dramatic decrease in the inflammatory environment and the neutrophil's ability to kill *Aspergillus fumigatus*.
¹Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR8204 - CIIL - Center for Infection and Immunity of Lille, Lille, France, ²Parasitology-mycology, CHU Lille, Lille, France, ³Clinique Des Maladies Respiratoires, CHU Lille, Lille, France, ⁴Mycology - Umr6249 Cnrs Chrono-environnement, University Hospital Besançon, Besançon, France, ⁵Pôle De Réanimation, CHU Lille, Lille, France

**Objectives:** Chronic obstructive pulmonary disease (COPD) is a complex chronic respiratory disease which affects 8% of subjects over 45 years-old in France. It is mainly caused by cigarette smoke, which induces chronic lung inflammation and is considered a key etiological factor in the development and pathogenesis of COPD. Other environmental factors, such atmospheric pollution, can also be involved in the evolution of the disease, modulating inflammation and promoting exacerbations. Here, we assessed the contribution of domestic mould exposure to lung and systemic inflammation in COPD patients, during exacerbation or at stable state.

**Methods:** Sixty-two COPD patients were recruited during severe acute exacerbation requiring hospitalization between August 2011 and November 2015 in Lille University Hospital. Mycological analysis of sputa and anti-*Aspergillus* antibodies detection was performed in order to assess *Aspergillus* colonization and sensitization status. CXCL8, TNFα (recruiting/activation of monocytes and neutrophils), IL-1β, IL-6, IL-17, IL-22 (Th17 response) and CXCL4, CD62P, CD40L (platelet activation) levels were measured in the sputum and in the serum. The inflammatory response was compared at inclusion (exacerbation) and after an 18-months follow-up (at stable state) between (i) *Aspergillus fumigatus* colonized or non-colonized patients, (ii) *Aspergillus* sensitized or non-sensitized patients, and (iii) patient with or without mould exposure, which was assessed using electrostatic dust collectors (EDCs) that were exposed during 10 weeks in the patient's home.

**Results:** *A. fumigatus* colonization was detected in 16.9% of patients at inclusion (during exacerbation) and 4% of patients at stable state. Anti-*Aspergillus* antibodies detection was positive in 32.2% of patients at inclusion and 36.4% after the 18-months follow-up. No significant differences were found between the cytokine levels during exacerbation or at stable state when *A. fumigatus* colonized or non-colonized patients, and *Aspergillus* sensitized or non-sensitized patients were compared. However, during exacerbation, patients with mould exposure showed significantly higher levels of serum CXCL8 (19.6±41.2 pg/mL vs <7 pg/mL for non-exposed patients, P = 0.001). Furthermore, at stable state, mould exposure was associated with significantly higher levels of serum IL-6 (28.9±87.9 pg/mL vs <2 pg/mL, P = 0.016), and significantly lower levels of serum IL-22 (86.8±31.6 pg/mL vs 285.8±363.3 pg/mL, P = 0.0008).

**Conclusion:** These data suggest that domestic mould exposure could contribute to the alteration of the inflammatory response in the course of COPD. *Funding: Conseil Régional Hauts-de-France, Gilead, MSD, Pfizer*

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Effects of ibrutinib treatment on neutrophil response against *A. fumigatus*

M. Blaize¹, D. Blez¹, C. Soussain², A. Boissonnas¹, A. Meghraoui-Keddar¹, N. Menezes³, A. Portalier⁴, C. Combadière¹, V. Leblond⁴, D. Ghez⁵, A. Fekkar¹

¹Sorbonne Université, Inserm, CNRS, Centre d'Immunologie et des Maladies Infectieuses, Cimi-Paris, F-75013, PARIS, France, ²Hématologie, Institut Curie - Site de Saint-Cloud, 35 rue Dailly, F-92210, Saint-Cloud, France, ³AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Service de Parasitologie-Mycologie, F-75013, Paris, France, ⁴AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Service d'Hématologie, F-75013, Paris, France, ⁵Département d'Hématologie, Gustave Roussy, 114 rue Edouard Vaillant, F-94805, Villejuif, France

**Objectives:** Ibrutinib, a small irreversible Bruton's tyrosine kinase (Btk) inhibitor has become a leading therapy against chronic lymphoid leukemia and has significantly improved patient care. Recently, ibrutinib has been associated with the occurrence of invasive fungal infections, in particular invasive aspergillosis. This observation was surprising because patients with lymphoid malignancies are at low-risk of invasive aspergillosis. The mechanisms underlying the increased susceptibility to fungal infections associated with ibrutinib exposure are currently unknown. Polymorphonuclear
neutrophil is the cornerstone of anti-*Aspergillus* immunity. However, since the potential effect of ibrutinib on the antifungal capacities of neutrophils has been studied to a very limited extent, we decided to do so in more detail.

**Methods:** To answer this question, we studied the consequences of *in vivo* and *in vitro* ibrutinib exposure on the neutrophil's anti-*Aspergillus* responses. Thirty-three patients receiving ibrutinib for lymphoid malignancies were included. A total of 63 blood samples were collected from patients at different time points: just before ibrutinib treatment initiation (*n* = 23) then approximately 1 month (*n* = 22) and 3 months later (*n* = 18). Both flow cytometry and video-microscopy approaches were used to analyze neutrophils' cell surface molecule expression, cytokine production, oxidative burst and killing activity against *Aspergillus*.

**Results:** We observed different alterations between the patient group at 1 month of treatment and the untreated group, as a decrease in the CD11b (major receptor for beta-glucan) expression or IL8 production. Above all, we observed a decrease in the production of reactive oxygen species (ROS) by neutrophils, both in the basal state (2333 *versus* 1131, expressed as mean fluorescence intensity, p<0,05) or after stimulation by *Aspergillus* germinating conidia (9330 *versus* 4569; p<0,05). Among the interactions observed between germinating conidia and neutrophils in 16-hours video microscopy co-cultures experiments, we focus on the engulfment and destruction of fungus by cells. A decrease in the ability of neutrophils to spread over the fungus was observed between treated and untreated patient groups (Figure1). Regarding the destruction of *Aspergillus* by neutrophils, we observed a dramatic decrease with 50% of germinating conidia killed by neutrophils in the untreated group compared to only 1% in the group after one month of treatment and 0.8% in the group after 3 months of treatment (p<0.01) (Figure2). The results were confirmed by the analysis of healthy neutrophils (from blood donors) exposed (or not) *in vitro* to ibrutinib.
<figure><img src="image_88.png" /><figcaption>Figure 1: Comparison of two different time sequences of a sample from a single patient with chronic lymphoid leukemia before ibrutinib therapy (upper) and after 34 days of ibrutinib therapy (lower). In the ibrutinib treatment sample, the inability of the neutrophil to kill the *A. fumigatus* germinating conidia led to uncontrolled fungal growth.</figcaption></figure>

<figure><img src="image_89.png" /><figcaption>Figure 2: Rate of killing of *Aspergillus fumigatus* germinating conidia by neutrophils sampled from patients before ibrutinib therapy and after one or three months after the start of ibrutinib therapy.</figcaption></figure>

Kruskal-Wallis with Dunn's multiple comparison post-test was applied; **: p<0.005

**Conclusion:** Ibrutinib is associated, both *in vitro* and in patients receiving this treatment, with several functional defects in neutrophils, including a decrease in the production of reactive oxygen species, a decrease in their ability to engulf the fungus and an inability to effectively kill *Aspergillus* hyphae. Our results provide a plausible explanation for the emergence of invasive aspergillosis in patients receiving ibrutinib. Further work is needed to understand the exact mechanisms underlying this effect, particularly if it is caused by inhibition of the Btk itself in neutrophils or by an off-target effect. On May 2019, part of the data has been provisionally accepted for publication in Haematologica journal.
**Objectives:** *Candida glabrata* (*C. glabrata*) is a commensal yeast of the mucosal surfaces in humans. Incidence of invasive candidiasis due to this species has recently increased. Thus the high level of resistance that this species can present to different classes of antifungals, including echinocandins is of major concern. Nowadays, the impact of resistance acquisition in this opportunistic pathogen to the immune response of the host remains poorly understood. Knowing the mechanisms by which *Candida*'s intrinsic and adaptive resistance to antifungals can modulate (i.e. 'escape to' or 'recognition by') the immune system of the host is crucial. Indeed, most of the antifungal resistance mechanisms causes a direct or indirect modification of the fungal cell-wall. In addition, by establishing the first contact with the host tissues, the fungal cell-wall is involved in the recognition of *C. glabrata* by the host cells through interactions with Pattern Recognition Receptors (PRRs). We hypothesize that changes in the fungal cell-wall, as a result of resistance, could alter the recognition of *C. glabrata* by PRRs and thus trigger a different immune response, from a qualitative (i.e. cytokines production and immune cell recruitment) or quantitative (i.e. amplitude and duration of the immune response) point of view. The purpose of this study was (i) to characterize the cell wall modifications associated with resistance to echinocandins in *C. glabrata* and then (ii) to investigate the impact of such modifications on the virulence of the fungus and the host immune response.

**Methods:** Clinical isolates of *C. glabrata* included in the study displayed echinocandins' resistance for which the mechanisms of resistance were molecularly characterized. The virulence of these clinical isolates was performed using two *in vitro* models of interaction of the fungus with the host compared to the ATCC reference strain: (i) the Yeast-to-Enterocyte interaction model and (ii) the Yeast-to-Macrophages interaction model. Cell wall characterization of the clinical isolates of *C. glabrata* was performed using microscopy and flow cytometry.

**Results:** Data support a change in the virulence and recognition by the host of echinocandins-resistant *C. glabrata* clinical isolates compared to the ATCC reference strain.

**Conclusion:** This study will allow (i) to better understand the prevalence of echinocandins-resistant *C. glabrata* isolates in high-risk patients, (ii) to specify whether high levels of *in vitro* resistance correlate or not to *in vivo* therapeutic failure and (iii) to establish effective immunotherapeutic strategies to combat *Candida* resistance to antifungals.

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## Gene variants of key TH-1 adaptive immune response proteins as predictors of invasive aspergillosis in oncohematological patients

A. Taraskina¹, O. Shadrivova¹,², I. Boyko¹, E. Frolova¹, A. Uchevatkina¹, L. Filippova¹, N. Vasilyeva¹

¹Kashkin Research Institute Of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ²Department Of Clinical Mycology, Allergy And Immunology, North-Western State Medical University named after I.I.Mechnikov, St. Petersburg, Russian Federation

**Objectives:** Fungi of the genus *Aspergillus* are widely distributed in nature and are relevant opportunistic pathogens for immunocompromised patients, causing a life-threatening infectious fungal complication — invasive pulmonary aspergillosis (IPA). Disruption of immune barriers associated with suppressive therapy, such as cytostatic polychemotherapy, can affect both the mechanisms of the innate immune response and acquired, in particular, the differentiation of T-helper cells (Th). The regulation of T-cell effector functions occurs due to cytokine-induced intracellular signaling and transactivation of transcription factors. Single nucleotide polymorphisms (SNPs) of cytokine genes that direct T cell differentiation can lead to their different expression and / or affinity for complementary receptors on immune cells, increasing the risk of infectious processes *The aim of this study* was to investigate gene variants of key protein-inductor of Th1 adaptive immune response as biomarkers of IPA and effect on expression of IP-10 in serum in oncohematological patients.

**Methods:** 178 oncohematological patients on the background of cytostatic polychemotherapy with symptoms of lung injury were recruited. 86 oncohematological patients (44,5%) either developed proven or probable IPA as defined by criteria of EORTC/MSG 2008 (median age 43y ±14, range [22-
77y], 57% males) whereas controls (92 oncohematological patients (55,5%) without IPA comparable in age and sex) did not fulfill these criteria. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the SNPs of rs458917, rs56061981 *CXCL10* gene, rs1800629 *TNFα* gene and rs2430561 *INFγ* gene. Chemokine IP-10 amount was determined by the enzyme-linked immunosorbent assay with the use of commercial ELISA kit sets (Cloud-Clone Corp, USA) and is presented in ng/ml serum. Statistical analysis was performed using SPSS21 (IBM, USA).

**Results:** The genotype and allele distribution of the study SNPs were no significant differences between oncohematological patients with probable IPA and without IPA. In the study we found that allele variants of G-135A (rs56061981) do not appear to affect serum IP-10 levels although the presence of allele A is associated with a slight increase in serum chemokine, while the presence of minor allele-1447G (rs4508917) is associated with significantly higher concentrations of IP-10 in serum.

**Conclusion:** The presence of the *CXCL10*-1447G allele in serum of oncohematological patients was associated with increased IP-10 protein expression. The expansion of the studied groups of patients will allow making conclusions about the prospect of typing SNPs as a predictive marker of the risk of pulmonary mycosis development.

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In vitro phagocytosis of *Aspergillus fumigatus* conidia by chicken thrombocytes and heterophils

P. Bigeard, C. Filleul, T. Vahsen, L. Zapata, F. Botterel, J. Guillot, P. Arné, V. Risco

Ea Dynamyc Upec, Université de Créteil, Créteil, France

**Objectives:** Avian respiratory diseases can be responsible for substantial economic losses to the poultry industry. This is particularly true for aspergillosis, which is pervasive in both domestic and wild avian communities, and is a common cause of respiratory distress and morbidity. Mammal immune response against pulmonary aspergillosis is characterized by an early interaction of *Aspergillus* conidia with phagocytic cells such as macrophages and neutrophils, but knowledge remains limited regarding the avian immune response after inhalation of *Aspergillus* conidia. Avian thrombocytes and heterophils (homologues to mammal neutrophils) are known for their phagocytic activity, but nothing has been described so far regarding their potential role during *Aspergillus* infection in birds. This study aimed to investigate the phagocytic activity of chicken thrombocytes and heterophils against *Aspergillus* conidia.

**Methods:** We used filtered GFP- or DsRed-expressing *A. fumigatus* conidia to inoculate primary cultures of heterophils or thrombocytes previously purified by Percoll's discontinuous gradient density from whole blood of chicken. Cultures and conidia inoculation were performed on poly-L lysine treated Labtek® slides or in suspension. When slides were used, cultures were fixed at different time points to measure internalization rates by immunolabeling with goat anti-chicken CD45 (leucocytes) and anti-chicken CD41 (thrombocytes) antibodies. When cells were in suspension, they were inoculated with *Aspergillus* conidia for 30 min, fixed and analyzed by flow cytometry (BD Accuri® C6) using the antibodies above described. Finally, activation of phagolysosomal acidification was evaluated by lysosome staining using the Lysotracker® method.

**Results:** We obtained cultures with up to 95.3% pure thrombocytes while heterophils purification was less efficient (<50%) because of heterophil layer contamination with erythrocytes and thrombocytes during the purification and heterophil aggregation during culture. Nevertheless, identification of avian heterophils by morphology and CD45+ labeling demonstrated conidial internalization as soon as 15 min after culture inoculation. Conidial internalization rate reached its peak 30 min after inoculation, with 22-28% of thrombocytes containing at least one conidia, while heterophils showed up to 93% of internalization. Eight hours after inoculation, conidial swelling and germination was observed in almost all thrombocytes while internalized conidia in heterophils remained quiescent. Lysotracker® staining confirmed phagolysosome fusion in heterophils but not in thrombocytes containing conidia.

**Conclusion:** The results of the present study confirm for the first time the phagocytic capacity of both chicken heterophils and thrombocytes to *A. fumigatus* conidia, and suggest differences in
internalization efficacy between these phagocytic cells. In thrombocytes, phagocytic activity was lower than that detected in heterophils, which allowed conidia swelling and germination inside the cells. Further studies shall determine whether survival inside thrombocytes might be used by *Aspergillus* as a strategy to ensure dissemination in birds.

## Evaluation of the liposome properties in the development of immune response during an immunization protocol with *C. albicans* proteins loaded on DODAB:MO liposomes

I. Pacheco¹,², A. Costa-Barbosa¹,², A.M. Barbosa³,⁴, M. Dias¹,², J.I. Carvalho-Pereira¹,², M.E.C.D. Real Oliveira⁵, C. Pais¹,², A. Correia⁶,⁷,⁸, E. Torrado³,⁹, P. Sampaio¹,²

¹Institute Of Science And Innovation For Bio-sustainability (ib-s), University of Minho, Braga, Portugal, ²Centre Of Molecular And Environmental Biology (cbma), Department Of Biology, University of Minho, Braga, Portugal, ³Life And Health Sciences Research Institute (icvs), School Of Health Sciences, University of Minho, Braga, Portugal, ⁴I3s, Instituto De Investigação E Inovação Em Saúde, University of Porto, Porto, Portugal, ⁵Centre Of Physics (cfum), University of Minho, Braga, Portugal, ⁶Ibmc, Instituto De Biologia Molecular E Celular, University of Porto, Porto, Portugal, ⁷I3s, Instituto De Investigação E Inovação Em Saúde, University of Porto, Braga, Portugal, ⁸G Dgaot, Faculdade De Ciências, University of Porto, Porto, Portugal, ⁹Icvs/3b's Pt Associate Laboratory, University of Minho, Guimarães, Portugal

**Objectives:** *Candida albicans* is a normal colonizer of the human body, persisting in a harmless state most of the time. However, given the opportunity, this species becomes one of the most important opportunistic pathogens, which increases the risk of developing systemic candidiasis. A promising antifungal strategy is the use of liposomal vaccine delivery systems. Previous studies reported that cationic liposomes prepared with DODAB:MO (1:2) and loaded with *C. albicans* cell wall surface proteins (CWSPs) were stable, non-cytotoxic and avidly phagocytized by macrophages. However, standardizing these liposomal formulations is challenging and complicated, since their wide range of characteristics must be taken into consideration according to the application we desire and the performance we expect. The liposome preparation method greatly influences these characteristics. Our main objective was to analyze the implications of the liposome preparation method on the biophysical properties of the DODAB:MO liposomes loaded with *C. albicans* proteins and evaluate the consequences at the immune response level, in order to develop a formulation that could be used in vaccination strategies against systemic *C. albicans* infections.

**Methods:** CWSPs from *Candida albicans* SC5314 were encapsulated in DODAB:MO (1:2) liposomes. The DODAB:MO liposomes were prepared using the thin lipid-film hydration method. The lipid film was hydrated with ultrapure water (1ª experiment) or with HEPES-buffer and sonicated to reduce the vesicles' size (2ª experiment). For both, the final concentration of CWSPs was 50 µg/mL in 25mM of HEPES-buffer and the final total lipid concentration was 888 µg/mL. The mean size (nm), polydispersity (PDI) and surface charge (ζ-potential) were evaluated with the Malvern ZetaSizer Nano ZS (Malvern, UK) particle analyzer and the immunization procedure was carried as previously reported (Carneiro *et al.*, 2015). The production of IFN-γ, IL-17, IL-4 and IL-10 in splenic CD4⁺ T cells was evaluated through flow cytometry. "*ex vivo*" cytokine production was evaluated through re-stimulation of the splenocytes with CWSPs and CWSP-specific antibodies were quantified by ELISA.

**Results:** Liposomes from the first experiment had a size around 666.23 ± 91,31 nm, with a PDI of 0.36 ± 0.08, and a ζ-potential of -2.30 ± 0.31 mV. Changing the liposome preparation method, we obtained smaller liposomes (249.69 ± 29.34 nm), with a PDI of 0.20 ± 0.06 and a ζ-potential of -8.53 ± 0.66. While IL-4 was the prevailing cytokine when taking into consideration the liposomes from the first experiment, the liposomes from the second experiment led to much higher production of IFN-γ and IL-17, which clearly shifted the immune response from a Th2 to a Th1/Th17 response. The liposomes from the second experiment also demonstrated a significant increase (10x) in CWSP-specific antibodies.

**Conclusion:** Our results indicate that the biophysical properties of DODAB:MO liposomes are important in the type of adaptative cellular response, and that in reducing significantly these
liposomes sizes a shift from a Th2 to a Th1/Th17 response was observed, which is known to be more protective against systemic candidiasis. Therefore, with this liposome system, the liposome size has a huge impact in the immune response.

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# The Impact of NFAT Inhibition on Neutrophil Effector Functions, Antifungal Defense, and Myelopoiesis in Cyclosporine A-Treated and NFATc1LysM Mice and in Patients after Allogeneic Hematopoietic Stem Cell Transplantation

D. Teschner¹, K. Plein¹, C. Michel¹, S. Pruefer², M. Bros³, H. Beckert⁴, E.M. Wagner-Drouet¹, M. Theobald¹, H. Schild², M.P. Radsak¹

¹Department Of Hematology, Medical Oncology, & Pneumology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany, ²Institute Of Immunology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany, ³Department Of Dermatology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany, ⁴Clinic For Pneumology, Ruhrland Clinic, Faculty of Medicine of the University of Duisburg-Essen, Essen, Germany

**Objectives:** Calcineurin inhibitors substantially contribute to the risk for opportunistic fungal infections in patients after allogeneic transplantation (HSCT). It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin especially in T cells. However, NFAT also seems to play a role in innate antifungal immune responses by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation.

**Methods:** Firstly, isolated PMN from CsA-treated patients after allogeneic HSCT (n = 17) and healthy donors (n = 8) were analyzed *in vitro* and *ex vivo* regarding their different effector functions. Secondly, we used a murine IPA model (C57BL/6) and treated CsA-treated or LysM-specific NFATc1 knockout (NFATc1LysM) mice with *Aspergillus fumigatus* intratracheally. PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) using flow cytometry and cytometric bead array and murine lungs by fungal culture assays and histopathologic examination. Furthermore, survival was studied with neutropenic animals serving as positive controls. In addition, we investigated myelopoiesis and myeloid differentiation by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysM mice using flow cytometry and simultaneously counting PMN in PB under steady state conditions.

**Results:** CsA significantly enhanced phagocytosis of PMN *in vitro* and *ex vivo* in patients' blood samples. Moreover, PMNs migratory capabilities were reduced *in vitro*, whereas other effector functions or release of IL-8 were rather unaffected. PMNs of CsA-treated patients showed increased activation, degranulation and production of inflammatory mediators, but production of ROS was slightly decreased. In our *in vivo* model, IPA was lethal in neutropenic mice whereas solely CsA or vehicle treated mice survived the infection. CsA treatment resulted in enhanced PMN recruitment in BAL by trend, while pulmonary inflammation and PMN counts in PB remained stable. Indeed, fungal clearance was clearly constrained in CsA treated animals. In our murine knockout model, NFATc1LysM mice infected with *Aspergillus fumigatus* showed unimpaired survival without displaying detectable differences in PMN recruitment or fungal clearance. However, pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice. Interestingly, BALs of CsA treated mice showed increased levels of IL-6 by trend but decreased levels of MCP-1 and TNF-α. In contrast, MCP-1, RANTES and TNF-α were enhanced by trend in BALs of NFATc1LysM mice, while IL-6 was reduced compared to wild type controls. PMN counts in PB were unaffected in NFATc1LysM mice but distribution of bone marrow derived murine myeloid progenitor cells was significantly impaired especially in megakaryocyte-erythroid progenitor cells, whereas solely CsA treatment had no influence.

**Conclusion:** Results from our *in vitro* and *ex vivo* studies on patients' blood samples as well as from our murine *in vivo* IPA model indicate that NFAT regulates not only myelopoiesis, but also PMN
functionalities in mice and humans. Nevertheless, these interactions are obviously multidimensional
and potentially derive from involvement of different pathways. The underlying molecular
mechanisms and clinical relevance of our findings in HSCT remain to be determined.

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Novel assay for sterol 24-C-methyltransferase enzymatic activity, a primary antifungal target of Abafungin

H. Naruse, T. Ito, E. Noshi, Y. Tanaka, H. Maki

SHIONOGI & CO., LTD., Osaka, Japan

**Objectives:** Abafungin is a broad-spectrum antifungal agent. It has been presumed that abafungin inhibits sterol 24-C-methyltransferase (SMT) from the report that abafungin decreased L-[Methyl-14C]methionine incorporated intracellular sterol. SMT is one of the enzymes involved in ergosterol biosynthesis pathway which is well-known antifungal target (e.g. azoles). As *erg6* gene encoding SMT is conserved in many fungal species, SMT could be a promising drug target. However, it is not known whether abafungin actually inhibits SMT enzymatic activity, and there is no high throughput SMT enzyme assay for drug discovery. Therefore, we developed a novel SMT enzymatic assay by homogeneous time-resolved FRET (HTRF). We also evaluated the effects of SMT inhibition on antifungal activity.

**Methods:** HTRF is based on a competitive immunoassay between SAH, byproduct of SMT reaction, and d2 labelled SAH (SAH-d2) towards Lumi4-Tb cryptate labelled anti-SAH antibody. *A. fumigatus* SMT and its substrates, lanosterol and SAM, were mixed and incubated at room temperature, then SAH-d2 and anti-SAH antibody were added. After incubation at room temperature, HTRF signal was measured. The HTRF ratio was calculated as a two-wavelength signal ratio (665 nm/620 nm). SMT inhibitory activity of abafungin and its derivatives were calculated as IC₅₀ values. MICs of these compounds against *A. fumigatus* and *C. albicans* were measured according to the method of the CLSI.

**Results:** SAH was detected by 0.001 µM to 0.1 µM in the optimized condition and SMT produced SAH in a dose dependent-manner. SMT of microsomal fraction indicated 3-fold higher enzymatic activity than that of cytoplasmic fraction. In this HTRF assay abafungin inhibited SAH production and exhibited IC₅₀ of 0.21 µM. The MICs of abafungin against *A. fumigatus* and *C. albicans* were 2 µg/ml and 0.0625 µg/ml, respectively. The derivatives without SMT inhibitory activity didn't exhibit antifungal activity against *A. fumigatus* and *C. albicans*.

**Conclusion:** We established a novel assay to evaluate *A. fumigatus* SMT activity. Abafungin was revealed to inhibit SMT enzymatic activity, which led to antifungal activity. This result would help us develop new antifungals targeting SMT.

P376

Metagenomic analysis and culture-dependent diagnosis of cystic fibrosis patient airways

M.R. Von Zeska Kress¹, L. Tonani¹, L.M. Fonseca¹, O.G.G. Almeida¹, C.P.D.C. Capizzani¹, E.C.P. De Martinis¹, L.A.G.M.M. Torres²

¹Faculdade De Ciencias Farmacéuticas De Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto-SP, Brazil, ²Faculdade De Medicina De Ribeirão Preto, Universidade de São Paulo, Ribeirao Preto-SP, Brazil

**Case Report:** Cystic fibrosis (CF) is a debilitating autosomal recessive disease in which the patient carries a mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes a chloride channel involved in electronic exchanges through the plasma membrane of epithelial cell types. Consequently, a defective lung mucociliary clearance occurs, producing thick and sticky bronchial mucus, suitable for entrap airborne bacteria and fungi. The culture-dependent diagnostics of CF patients airways show the frequent colonization by *Haemophilus influenzae*, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Burkholderia cepacia* complex and atypical mycobacteria. Additionally, prolonged antibiotic treatments contribute to the colonization by multidrug-resistant pathogens such as *Achromobacter xylosoxidans* and *Stenotrophomonas maltophilia*. On the other hand, culture-independent technologies, as the metagenomic approach, revealed that the CF patients
airways are not inhabited by these few pathogens, but by complex polymicrobial communities. Fungal species are usually found colonizing the CF patients airways (e.g. *Aspergillus fumigatus* and *Candida albicans*), however, few metagenomics studies are reported in the literature. Here, we show the culture-dependent identification and metagenomic approach (16S ribosomal DNA and the internal transcribed spacers (ITS) of the nuclear ribosomal DNA) of CF patient sputum. The detailed description of the CF lung microbiome is presented and compared with culture-dependent identification. The isolation of microorganisms on media culture from the sputum of the CF patient revealed the bacteria *Stenotrophomonas maltophilia*, *Pseudomonas aeruginosa*, *Achromobacter xylosoxidans* and *Staphylococcus aureus*, and the fungi *Aspergillus fumigatus* and *Candida albicans*. The identification was performed by classical and molecular methods, respectively. As the culture-based identification, the 16S and ITS metagenome revealed the bacteria *Stenotrophomonasp*, *Pseudomonas putida*, *Achromobacter sp*., and *Staphylococcus aureus*, and the fungi *Aspergillus fumigatus* and *Candida albicans*. But metagenome additionally identified *Streptococcussp*, *Neisseriasp*., and *Saccharomyces cerevisiae* in high relative abundance, and *Tectona grandis*, *Actinobacillus sp*, *Schizophyllum commune*, *Agaricales* order and *Halosphaeriaceae* family in intermediate and low relative abundance. Thus, the culture-based analyses correlated qualitatively with the relative abundance of data on obtained bacterial and fungal taxa. However, additional identifications were obtained by metagenome data. Here we observed that the taxonomic identification by Regarding 16S and ITS metagenome to the species level were not achieved for the fungal kingdom and the estimation of the relative abundance of species in the community is imprecise. This is justified by the primer-based amplicons sequencing method which has a limited ability to resolve this taxonomic identification. Thus, although we have identified several species colonizing the CF patients airways, the metagenome shotgun approach is indicated to provide information about the composition of the community up to the level of clonal complexes.

P377

Mechanisms of resistance to elevated copper in the pathogenic mold *Aspergillus fumigatus*

Z. Meir¹, H. Sandovsky¹, Y. Shadkchan¹, G. Masrati², P. Wiemann³, N. Ben-Tal², N. P. Keller³, N. Osherov¹

¹Clinical Microbiology And Immunology, Tel Aviv University, Tel Aviv, Israel, ²Biochemistry & Molecular Biology, Tel Aviv University, Tel Aviv, Israel, ³Medical Microbiology And Immunology, University of Wisconsin, Madison, United States of America

**Objectives:** *Aspergillus fumigatus* is the most common opportunistic mold pathogen in the growing immunocompromised population. Patients with weak immune systems are at risk to develop Invasive Aspergillosis (IA), a deadly type of infection with high mortality rates caused by *A. fumigatus*. Fungi use unique pathways to control the levels of essential metals inside the cell. Copper is a transition metal essential for all living systems and its homeostasis in *A. fumigatus* under copper excess is dependent on the copper sensing transcription factor AceA and its downstream target, the copper transporter CrpA. Studies suggest that genes participating in copper homeostasis can be promising drug targets. Our goals were to reveal the detailed function of the copper transporter CrpA and to identify and characterize additional participants acting in the Cu-resistance pathway of *A. fumigatus* and assess their relevance to virulence.

**Methods:** To achieve these goals we constructed a model structure of *A. fumigatus* CrpA, in collaboration with Prof. Nir Ben-Tal, and identified evolutionarily conserved fungal-specific domains in the protein. We generated GFP-tagged CrpA and studied its subcellular localization. We performed RNAseq analysis of *A. fumigatus* exposed to high copper levels and deleted selected upregulated genes. Finally, we performed a screen to identify genes that provide Cu-resistance when overexpressed.

**Results:** Significant progress was made in the localization of CrpA protein. We located the protein mostly in the ER but also in the membrane. We also created an inducible overexpression strain of CrpA using the xylose-inducible promoter *xylP* and showed that CrpA overexpression confers increased copper resistance when exposed to copper *in vitro* but not *in vivo*. We generated deletion strains for six genes that were up-regulated in WT vs AceA-null strains following exposure to copper
as identified by RNA-seq. These genes are chaperones and metal-binding proteins that may
participate in *A. fumigatus* copper resistance. The phenotypes of these deletion strains will be
described. We performed a genetic screen using an overexpression library in which *A. nidulans* was
transformed with the AMA-1-based overexpression genomic library and plated on excess Cu. We
found four new genes that confer resistance to *A. nidulans*

**Conclusion:** Our results provide a more detailed picture of copper resistance in *A. fumigatus*, indicating that it is dependent on the combined activities of the CrpA copper exporter, Cu-chaperones and accessory stabilizing proteins.

P378

Genotyping of *Cryptococcus* spp. isolated from HIV-negative patients with Neurocryptococcosis

E. Nascimento¹, P.H.G. Barião², M. Von Zeska Kress², R. Martinez¹

¹Department Of Internal Medicine, Medical School /University Of Sao Paulo/Ribeirao Preto, Ribeirao Preto, Brazil, ²school Of Pharmaceutical Sciences Of Ribeirao Preto, University Of Sao Paulo, Ribeirao Preto, Brazil

**Objectives:** The aim of this study was to determine the species and molecular types of *Cryptococcus* spp. isolates from patients not co-infected with HIV from the Medical Hospital of Ribeirao Preto - Medical School of Ribeirao Preto - University of Sao Paulo - Brazil, from 2000 to 2017, and correlate with the comorbidity of the patients.

**Methods:** A total of 43 strains of *Cryptococcus* spp. isolated from 43 patients not co-infected with HIV were genotyped. The species were determined by PCR with the primers CN70 and CN49. The molecular types *C. neoformans* VNI, VNII, VNIII and VNIV, and *C. gattii* VGI, VGII, VGIII and VGIV were determined by PCR-RFLP with the amplification of the *URA5* gene and subsequent enzymatic restriction with *HhaI* and *Cfr131*. The identification of *C. gattii* and *C. laurentii* was confirmed by the sequencing of the rDNA ITS regions.

**Results:** From a total of 43 clinical isolates, 28 were identified as *C. neoformans*, 13 as *C. gattii* and 2 as *C. laurentii*. Among *C. neoformans* clinical isolates, 27 were identified with the molecular type VNI and 1 VNII. Additionally, 2 *C. gattii* VGI and 11 *C. gattii* VGII were identified. The correlation between species and patient comorbidity revealed 28 clinical cases (Group 1) of patients not coinfected with HIV but with other underlying diseases (e. g. diabetes, adenocarcinoma, chronic lung disease, transplants, systemic lupus erythematosus and others ) and 15 clinical cases (Group 2) of immunocompetent patients. Most of the isolates identified in Groups 1 were *C. neoformans* (75%) and in Group 2 were *C. gattii* (47%). *C. laurentii* were isolated from the Group 2.

**Conclusion:** Most of the clinical isolates were identified as *C. neoformans* and were more present in individuals not co-infected with HIV but who had another underlying disease. On the other hand, *C. gattii* is more frequent in the group of immunocompetent patients. The molecular types VNI and VGII were the most identified in our study, which corroborate with other studies carried out in Southeast Brazil.

P379

Classification of dermatophytes by a multilocus phylogenetic approach based on Tef-1α, beta tubulin and ITS genes.

R. Sacheli¹, C. Tomé¹, C. Adjetey¹, R. Darfouf¹, L. Palmeira², M.-P. Hayette¹

¹Clinical Microbiology- Nrc For Mycoses, University Hospital of Liège, Liège, Belgium, ²Human Genetics, University Hospital of Liège, Liège, Belgium

**Objectives:** Identification of dermatophytes to the species level is epidemiologically, ecologically and therapeutically significant. The use of phylogenetic species concepts based on rDNA internal transcribed spacer (ITS) regions have improved the taxonomy of dermatophytes. However, it has been shown that for some species, confirmation and refinement using other genes are needed. Indeed, there are problems with accurate definition and characterization of dermatophytes especially among the *Trichophyton mentagrophytes* series. Intra- and interspecies variations of the translation elongation factor 1-α (*Tef-1α*) gene were evaluated as a new identification marker in a wide range of
dermatophytes. The aim of this study was to evaluate the discriminatory power of a phylogenetic tool based on concatenated sequences of three genomic regions including *Tef-1α*, *ITS* (ITS1 to ITS2) and *beta-tubulin* to differentiate: 1/ the anthropophilic *Trichophyton interdigitale* species from the zoophilic *Trichophyton mentagrophytes* 2/ the two closely related African anthropophilic species *Trichophyton violaceum* from *Trichophyton soudanense*.

**Methods:** 26 well characterized strains of *T. interdigitale/mentagrophytes* have been selected including 3 IHEM reference strains; 30 well characterized strains of *T. soudanense/T.violaceum* including 2 IHEM reference strains were also included. All strains were submitted to *ITS*, *beta-tubulin* and *Tef-1α* PCR/sequencing. Moreover, identification was completed by real time PCR *DermaGenius®* (PathoNostics). Alignments were performed using *MUSCLE* in Seaview. The alignments for each of the three genes were then concatenated together based on the name of the sample and gaps were added between genes. Phylogenies were then inferred on the concatenated alignment using *PhyML*.

**Results:** After generation of the phylogenetic tree by concatenation of the three sequence genes, the differentiation between *T. interdigitale* and *T. mentagrophytes* was clear. The 16 strains of *T. interdigitale* were well classified into one distinct group on the dendrogram. The 10 strains of zoophilic *T. mentagrophytes* were well distinct of the anthropophilic group and defined by another clade on the dendrogram. Reference strains were correctly classified into the two previously defined groups. Regarding the differentiation between *T. soudanense* and *T. violaceum*, the concatenation permitted to define two well distinct clades on the dendrogram. One clade containing the 10 *T. violaceum* tested and the other containing the 20 *T. soudanense* included in the study. Reference strains were correctly classified into each corresponding clade.

**Conclusion:** The concatenation of *ITS*, *beta-tubulin* and *Tef-1α* genes sequences allows the generation of a discriminating dendrogram between the zoophilic *T. mentagrophytes* and the anthropophilic *T. interdigitale* and between the two closely related African anthropophilic species *T. violaceum* and *T. soudanense*. These species are not clearly well defined by *ITS* analysis only. This multilocus phylogenetic approach allows to better define the species boundaries between these dermatophytes species and facilitates the molecular characterization of these species in routine diagnostic.

P380

**Pulmonary mycobiome of patients with suspicion of respiratory fungal infection – an exploratory study**

M. Oliveira¹,², M. Pinto³, C. Verissimo⁴, R. Sabino²

¹Animal Biology Department, Faculty of Sciences of the University of Lisbon, Lisbon, Portugal,
²Reference Unit For Parasitic And Fungal Infections, Infectious Diseases, National Health Institute
Dr. Ricardo Jorge, Lisbon, Portugal, ³Bioinformatics Unit, Infectious Diseases, National Health
Institute Dr. Ricardo Jorge, Lisbon, Portugal, ⁴Infectious Diseases, Reference Unit for Parasitic and
Fungal Infections, National Health Institute Dr. Ricardo Jorge, Lisbon, Portugal

**Objectives:** This pilot study aimed to characterize the pulmonary mycobiome of patients with suspicion of fungal infection of the respiratory tract as well as to identify potentially pathogenic fungi colonizing/infecting their lungs.

**Methods:** A cohort of 10 patients was analyzed, including HIV+ patients and patients with active infection caused by *Mycobacterium* species. Their respiratory samples (bronchoalveolar lavage fluid/ bronchial secretions) were pre-treated with lyticase and proteinase K; DNA was extracted using the High Pure PCR Template Preparation kit following the manufacturer's instructions. The internal transcribed spacer region 1 (ITS1) and calmodulin gene were amplified by PCR and the resulting amplicons were sequenced using the Illumina MiSeq platform with pair-end reads of 150 bp. The obtained results were analyzed using the PIPITS pipeline as described by Gweon *et al.* [1]. Operational taxonomic units (OTU) to which less than 0.1% of the total reads attributed were disregarded.

**Results:** Thirty-seven different OTU were identified from which two belonged to the *Plantae* kingdom, 11 had less than the 0.1% threshold of the total reads and were therefore disregarded. The remaining 24 different OTU (grouped in 17 phylotypes), were considered as part of the pulmonary
mycobiome of patients. Two phyla were identified: *Basidiomycota* (33.3%) and *Ascomycota* (54.2%). Regarding the *Basidiomycota* phylum, reads were classified in three classes (Agaricomycetes, Tremellomycetes and *Walleomycetes*), while for the *Ascomycota* phylum four different taxonomical classes were identified: Pneumocystidomycetes, Dothideomycetes, Eurotiomycetes and Saccharomycetes, with the latter being the most frequent class. Twelve fungal genera were identified, being *Candida* the most frequently detected. The median number of fungal genera detected in patients' pulmonary mycobiome was six (ranging from two up to nine). The genus *Papilotrema* and the potentially pathogenic genera *Cryptococcus* and *Pneumocystis* were exclusively found in the pulmonary mycobiome of HIV+ patients. Other potentially pathogenic fungi such as *Aspergillus* spp., *Trichosporon* spp., *Saccharomyces* spp. and *Schizophyllum* spp. were also detected.

**Conclusion:** This pilot study illustrates how the pulmonary mycobiome is rich and highly variable in patients with fungal infections. The obtained results suggest that the described metagenomic analysis may possess a great ability to quickly and effectively detect potentially pathogenic fungi in the mycobiome of patients, making it a promising future diagnostic tool. Thus, further optimization, standardization and clinical validation of these NGS methodologies should be warranted in the future. Reference: Gweon HS, Oliver A, Taylor J, Booth T, Gibbs M, Read DS, Griffiths RI, Schonrogge K. PIPITS: an automated pipeline for analyses of fungal internal transcribed spacer sequences from the Illumina sequencing platform. Methods Ecol Evol. 2015 Aug;6(8):973-980.

P382

**Comparative genomic analysis of *Scedosporium* species and identification of pathogenicity determinants.**

L. Duvaux<sup>1,2</sup>, J. Shiller<sup>3</sup>, P. Vandeputte<sup>2</sup>, M. Bahut<sup>4</sup>, C. Lemaire<sup>1</sup>, W. Meyer<sup>5</sup>, L. Hibrand Saint Oyant<sup>4</sup>, J.P. Renou<sup>3</sup>, B. Le Cam<sup>1</sup>, J.-P. Bouchara<sup>2</sup>, <u>A. Gastebois</u><sup>2</sup>

<sup>1</sup>Irhs-ecofun, INRA, Beaucouzé, France, <sup>2</sup>Geihp, Université d'Angers, Angers cedex, France, <sup>3</sup>Irhs-bio-info, INRA, Beaucouzé, France, <sup>4</sup>Sfr-quasav-anan, Université Angers, Beaucouzé, France, <sup>5</sup>Molecular Mycology Research Laboratory, University of sydney, Sydney, Australia

**Objectives:** **Objectives** *Scedosporium* species are soil saprophytes that may cause a large variety of infections in human. Especially, some of them are capable to chronically colonize the lungs of patients suffering from cystic fibrosis (CF) which constitutes a major risk of disseminated infection in case of lung transplantation. Molecular and biochemical analysis revealed that the genus *Scedosporium* comprises at least ten distinct species with differences in their susceptibility to antifungals and in their pathogenicity. Among them, three species, *S. boydii*, *S. apiospermum* and *S. aurantiacum* are predominant in CF patients, while other species like *S. minutisporum* are occasionally encountered in this clinical context. Conversely, *S. dehoogii* has never been reported from respiratory secretions of CF patients whereas some environmental studies revealed its common occurrence in soils where it accounts for up to 40% of all *Scedosporium* isolates. The aim of our project was to identify by a comparative genomic approach, regions of the genome or genes that might be responsible for the ability of *Scedosporium* species to establish within the respiratory tract of CF patients and to cause chronic colonization of the airways.

**Methods:** **Methods** Five clinical or environmental isolates from each of the five species *S. boydii*, *S. apiospermum*, *S. aurantiacum*, *S. minutisporum*, and *S. dehoogii* were sequenced. To identify candidate proteins that may enable *Scedosporium* species to establish within the respiratory tract of CF patients, we first searched clusters of orthologous proteins conserved in all isolates of the pathogenic species, but absent in *S. dehoogii* isolates. Genomic data were also analyzed searching for genes present in all pathogenic species, as well as in *S. dehoogii*, but exhibiting in this last species point mutations potentially leading to a loss of function.

**Results:** **Results** At the beginning of the project, the whole genome of a first strain of *S. apiospermum sensu stricto* (Vandeputte et al., 2014) and of *S. aurantiacum* (Pérez-Bercoff et al., 2015) were already available. The whole-genome sequencing project allowed us to obtain a total of 2.4-8.9 mega-reads per isolate which permitted good quality draft genome assemblies. For 3 species, we did *de novo* sequencing, genome assembly and performed comparative genomic analysis with the 25 strains.
Finally, comparative genomics allowed us to identify a series of 134 genes lost or with a potential loss of function in *S. dehoogii* but still functional in all other species.

**Conclusion:** **Conclusion** This comparative genomic analysis of the main *Scedosporium* species allowed us to identify a set of candidate genes potentially involved in the adaptive mechanisms developed by pathogenic *Scedosporium* species to establish within the respiratory tract of patients suffering from CF. Functional genomic approaches focused on the genes here identified will now be developed to elucidate their role in pathogenesis. Acknowledgment This project was financed by Angers University with the AAP-CS program.

P383

**Molecular phylogenetic study of strains morphologically identified as *Exophiala dermatidis* from clinical and environmental specimens in Japan**

Y. Alimu, R. Tanaka, S. Ban, T. Yaguchi

Division Of Bio-resource, Medical Mycology Research Center, Chiba University, Chiba, Japan

**Objectives:** With support from the National Bio-Resource Project, at the Medical Mycology Research Center (MMRC), Chiba University, the collection, storage and distribution of pathogenic fungi are carried out mainly on clinical isolates. The molecular phylogenetic position of the isolates, the mycological characterization, the genomic and the clinical information are managed and added to the database to increase the value of the stock and lead to an increase in the number of aliquots. Since old isolates are morphologically identified and conserved, their identifications are reviewed as needed based on the base sequence of the ITS region of rDNA. We report here on *Exophiala dermatitidis*, which is an important pathogen of deep-skin dermatomycosis, it has been reconfirmed with identification and fitted with the latest classification. *E. dermatitidis* has been reported to have a molecular phylogenetically polymorphism in strains from South America. However, the investigation of the distribution in Japan has not been reported. The aims of this study, is investigate the phylogenetic distribution of *E. dermatitidis* in Japan.

**Methods:** In this investigation, we examined 66 clinical and environmental isolates morphologically identified and conserved as *E. dermatidis* previously, which were stocked in Bioresource Management Office in MMRC. The identification based on the ITS region. The sequences were aligned and compared through neighbor-joining phylogenetic tree analyses on the ITS1 region, using the Molecular Evolutionary Genetics Analysis (MEGA) version 7.0.26 software package. Furthermore, the strains of different gene types will test for mycological characterization such as the growth temperature, the growth rate, and the drug sensitivity.

**Results:** The examined 66 clinical and environmental isolates were composed of 47 ones from Japan, 4 ones from USA, 5 ones from Brazil, 6 ones from Venezuela and 4 ones from China. According to establish a phylogenetic tree for the ITS1 region, we knew that 45 of 47 isolates from Japan, 2 of 4 ones from China, 2 of 6 ones from Venezuela and all of 4 ones from the USA were classified to the gene type A1. Only 2 of Japan and China's isolates belonged to the gene type A2, respectively. Interestingly, all of the isolates from Brazil were part of the gene type B and most of the Venezuela samples were close to the gene type C. The relationship between the types and their mycological characterizations was examined.

**Conclusion:** In our study, it is confirmed that, in South American strains, polymorphisms were found molecularly within species, and molecular phylogenetic diversity of the isolates in Japan was relatively uniform.

P384

**Investigation of the impact of amino acid changes in the substrate binding site of fungal sterol 14α-demethylase on protein structure and function using *Saccharomyces cerevisiae* as an expression model.**

M. Lackner<fnref n="1" />, M.V. Keniya², B. Monk²

<footnote n="1">¹Division Of Hygiene And Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria, ²Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand</footnote>
**Objectives:** Mucormycetes are intrinsically short-tailed azoles resistant. We used the high resolution X-ray crystal structure of *S. cerevisiae* lanosterol 14α-demethylase (LDM) in complex with voriconazole (VCZ) to postulate that the amino acid substitution Y129F in *Rhizopus arrhizus* LDM (RaLDM) F5 is associated with their intrinsic resistance. Our aim was to experimentally test this hypothesis.

**Methods:** Full-length recombinant C-terminal hexahistidine-tagged LDM (F1 and F5) of *R. arrhizus* were overexpressed from the *PDR5* locus in a *S. cerevisiae* host lacking 7 drug efflux pumps. Their cognate NADPH-cytochromeP450 reductase (RaNCP) was overexpressed from the *PDR15*. Phenotypic and biochemical analysis were used to determine structural and functional features of RaLDMF1, RaLDMF5, without and with co-expression of RaNCP.

**Results:** RaLDMF1 and F5 were functionally overexpressed in *S. cerevisiae*. RaLDMF5 appeared to be the major contributor to azole resistance, with its expression resulting in a 32x higher VCZ minimal inhibitory concentration (MIC) than the recipient strain. Co-expression of RaLDMF5 with NCP resulted in additional 4-fold increase of VCZ MIC when compared with RaLDMF5 single expression. MICs for long-tailed azoles were unaffected by the LDM supplementation. *S. cerevisiae* co-expressing RaLDMF1 with NCP had similar MIC values as the recipient strain. Azole-susceptibility patterns of *S. cerevisiae* co-expressing RaLDMF5 and RaNCP closely matched those of *R. arrhizus*. Phenotypic analysis of recombinant LDMs expressed in *S. cerevisiae* provides insights on the relevance of mutations located in the substrate binding pocket. This system allows assessment of effects on the drug target without interference from drug efflux pathways.

**Conclusion:** LDMF5 was found to be the dominant isoform in *R. arrhizus* that confers short-tailed azole resistance. Biochemical and structural analysis of the purified protein are expected to provide deeper understanding of the observed phenotypes.

P385

**A metagenomic and whole genome sequencing comparative approach to study the impact of azole therapy on chronic colonisation with *Aspergillus fumigatus* during cystic fibrosis.**

J.P. **Gangneux**¹, L.-E. **Vandenborght**², B. **Bidon**³, C. **Belleguic**⁴, E. **De Neuville**⁴, H. **Guegan**¹, S. **Leroy**⁵, B. **Laviolle**⁴, A. **Horsley**⁶, D. **Denning**⁷, C. **Cuomo**⁸, J.-P. **Bouchara**³, L. **Delhaes**⁹, N. **Papon**¹⁰

¹Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France, ²Genoscreen, Lille, France, ³CHU d'Angers, Angers, France, ⁴CHU de Rennes, Rennes, France, ⁵CHU de Nice, Nice, France, ⁶University Hospital of South Manchester, Manchester, United Kingdom, ⁷University of Manchester, Manchester, United Kingdom, ⁸Broad Institute of MIT and Harvard, Cambridge, United States of America, ⁹Parasitology Mycology, Univeristy of Bordeaux, Centre de Recherche Cardio-Thoracique de Bordeaux, U1045, F-33000, Bordeaux, France, ⁱ⁰Ea Geihp, UFR Pharmacie d'Angers, Angers, France

**Objectives:** The impact of early azole therapy during cystic fibrosis (CF) remains debated. The ATCF (Antifungal Therapy during Cystic Fibrosis) is a longitudinal phase III study with the following objectives: - to assess the microbiological and clinical effects of azole therapy: negativation of sputum cultures for *Aspergillus*, course of FEV1, clinical signs and quality of life, and the impact on co-prescription of antibiotic courses and/or steroid therapies. - to study the genomic impacts of azole treatment on the evolution of micro- and mycobiota and on genomic evolution of *Aspergillus* isolates.

**Methods:** The ATCF study followed CF patients with a chronic colonisation with *A. fumigatus* and randomized to receive an early oral azole therapy with itraconazole (ITRA) or voriconazole (VORI) for 4 to 6 months in a phase III study. A long-term follow-up was conducted until 2 months after end of treatment (EOT). All sputa were submitted to DNA extraction for further metagenomic approach to determine the fungal (ITS2) and bacterial (16S) microbiota. Besides, twenty isolates of *Aspergillus fumigatus* recovered before and after treatment were sequenced with a shotgun strategy.

**Results:** Eleven patients were included and followed until 2 months after EOT. All assessable patients had a successful outcome, with a negativation of sputa, an increase in FEV1 and a decrease in the number of antibiotic/steroids co-prescriptions. However, 2 months after EOT, most of clinical assessment criteria were similar to the baseline values. Fungal diversity increased under treatment
with a large decrease of the more abundant fungal taxa, particularly *Aspergillus*. However, 2 months after EOT the fungal diversity returned to baseline values and *Aspergillus* reappeared, mostly in a larger proportion. Besides, *Streptococcus*, *Pseudomonas* and *Staphylococcus* were the main genera detected and their proportion increased under azole therapy. The bacterial communities were similar after EOT compared to before. The analysis of micro – and mycobiota must be further documented. All SNPs and InDels variants were determined for 20 isolates. Isolates from a single sputum and more generally from a single patient during treatment were closely related. By contrast, isolates obtained 2 months after EOT were far different suggesting reinfections with new genotypes. In particular, one azole resistant *A. fumigatus* was recovered with a TR34/L98H mutation of the *CYP51A* gene, suggesting an environmental origin rather than an acquired resistance under azole drug pressure.

**Conclusion:** When a chronic colonisation with *A. fumigatus* is associated with clinical deterioration, EOT can temporarily reduce airway infection and inflammation, and extend periods of stable disease status. While only a limited number of patients were included, the longitudinal phase III design of ATCF study combined to a genomic approach underlines the need for azole therapy rationalization during CF. Indeed, the reversible effects 2 months after the EOT and the limited tolerance of both ITRA and VORI encourages short courses of therapy driven by the clinical improvement. The longitudinal changes of the mycobiota were restored 2 months after EOT and genetic characterization of isolates showed that reinfection with new genotypes is more frequent than *Aspergillus* in host evolutions under azole treatment.

P386

**Role of *Candida glabrata* candidate genes in echinocandin tolerance and resistance development**

R. Garcia-Rubio¹, A. Clear¹, K.R. Healey², E. Shor¹, D.S. Perlin¹

¹Center For Discovery And Innovation, Hackensack Meridian Health, Nutley, United States of America, ²Department Of Biology, William Paterson University, Wayne, United States of America

**Objectives:** *Candida glabrata* is the second most prevalent bloodstream fungal pathogen in North America and Europe, and its prevalence is rising. Echinocandin class compounds are first-line agents for the treatment of these infections, but therapeutic failures are increasingly encountered. However, the reasons why *C. glabrata* shows elevated potential to develop echinocandin resistance, relative to other *Candida* sp., are currently not well understood. Although echinocandins are fungicidal drugs, multiple cellular mechanisms can stabilize *C. glabrata* during drug exposure and promote drug tolerance, allowing cells to survive long enough to acquire mutations leading to stable clinical drug resistance. Interestingly, genomic analyses of different *C. glabrata* clinical isolates have revealed a high level of genetic diversity both within the same MLST sequence type (ST) clade and between them. Depending on the population subset, genomic plasticity may be responsible for differences in tolerance phenotype. The objective of our research is to elucidate the factors involved in promoting echinocandin drug tolerance in *C. glabrata*.

**Methods:** Because echinocandins target the fungal cell wall, we have focused on interrogating the role of cell wall integrity pathway components in promoting echinocandin tolerance. We have used an *in vitro* killing assay developed in our group to measure tolerance over a range of micafungin concentrations (0.016-32 µg/ml). Multiple knock-out mutants (Δslt2, Δsnf1, Δrlm1, Δsbe2, Δmid2, Δcka1, Δcbk1, Δypk2, Δslg1, Δyps1, Δada2, Δmot3, Δgas1, Δrta1, Δchs5, Δcrz1, Δpdr1, Δssd1, Δsbe22, Δchs3) were tested in this assay looking for significant changes in tolerance phenotypes. Additionally, to identify genetic polymorphisms that may be influencing echinocandin tolerance, we have sequenced some of the most relevant cell wall integrity pathway genes, particularly focusing on those whose deletion was found to affect drug tolerance in the killing assay.

**Results:** Most of the knock-out mutants showed no or minor changes in echinocandin tolerance. However, three mutants – Δyps1, Δypk2 and Δslg1 – resulted in significantly reduced tolerance to micafungin. These genes were sequenced in a number of *C. glabrata* echinocandin sensitive clinical isolates. Different polymorphism combinations were found in these strains, with specific polymorphisms being characteristic of particular STs. Several of these genetic polymorphisms resulted in amino acid changes.
**Conclusion:** Changes in micafungin tolerance phenotype were found in mutants lacking certain cell wall integrity pathway genes, implicating them in promoting drug tolerance in *C. glabrata* and representing potential targets to limit development of antifungal drug resistance. Further studies will evaluate whether polymorphisms in these genes are responsible for variation in drug tolerance among clinical strains.

P387

**Genetic modification of opportunistic pathogenic Mucoromycotina species using a plasmid free CRISPR/Cas9 system**

G. Nagy¹,², C. Szebenyi¹,², A. Vaz¹,², O. Jáger¹,², S. Ibragimova¹, Y. Gu³, A.S. Ibrahim³, C. Vágvölgyi¹, T. Papp¹,²

¹Deapartment Of Microbiology, University of Szeged, Szeged, Hungary, ²MTA-SZTE “Lendület” Fungal Pathogenicity Mechanisms Research Group, Szeged, Hungary, ³Los Angeles Biomedical Research Institute at Harbor-UCLA Med Center, Torrance, United States of America

**Objectives:** Most fungal species, which can cause human infections belong to the genera *Candida* and *Aspergillus*, but in the recent years, the number of infections caused by Mucoromycotina species (e.g., *Rhizopus oryzae*, *Lichtheimia corymbifera* and *Mucor circinelloides*) has significantly increased. The properties and potential virulence factors that make the Mucoromycotina species enable to cause disease (mucormycosis) in patients, are not known. Potential virulence factor genes may play role in fungal dimorphism, proteolytic enzyme production, iron uptake, and production of cell surface proteins.

**Methods:** Genetic manipulation of Mucoromycotina species, based on homologous recombination still can be a great challenge. In this study, a plasmid free CRISPR/Cas9 system was used for the genetic manipulation of *Mucor*, *Lichtheimia* and *Rhizopus* species. PEG-mediated protoplast transformation was used to introduce the Cas9 enzyme and the synthesized gene-specific gRNA with or without a deletion cassette into the fungus.

**Results:** In case of *Lichtheimia* and *Rhizopus*, the plasmid free CRISPR/Cas9 system was successfully used to disrupt the *pyrG* gene by NHEJ. Our results suggested that the disruption of the *pyrG* gene has no effect on the growth of fungal strains. These strains and the developed methods will be suitable for further transformation procedure in the future.

**Conclusion:** Based on the potential virulence factor genes (*cotH*, *svf*, *cyp51*, *hsbA*), a mutant library has been constructed from *M. circinelloides*. We have started to analyse the function of these genes in the pathogenesis and azole resistance of *Mucor*. This study was supported by the “Lendület” Grant of the Hungarian Academy of Sciences (LP2016-8/2016) and the Hungary grant 20391-3/2018/FEKUSTRAT of the Ministry of Human Capacities.

P388

**Mycobiome and microbiome in chronic fungal rhinosinusitis**

S. Dellière¹, C. Angebault¹,², M. Fieux³, P. Bonfils³, I. Podglagen⁴, P.-L. Woerther²,⁵, E. Dannaoui⁴, F. Botterel¹,²

¹Mycology And Parasitology, Henri-Mondor, Créteil, France, ²Ea Dynamyc Upec, Université de Créteil, Créteil, France, ³Ent, HEGP, Paris, France, ⁴Microbiology, HEGP, Paris, France, ⁵Microbiology, Hôpital Henri-Mondor, Créteil, France

**Objectives:** Fungal ball rhinosinusitis is the main type of non-invasive fungal rhinosinusitis, presenting as a dense mass of mycelium obstructing the sinus. Despite fungal hyphae being observed on direct examination of biopsy samples, culture remains negative in more than 60% of cases.¹ The aim of this study was to evaluate the performances/efficacy of targeted metagenomics to describe and analyze bacterial and fungal microbiota in fungal balls compared to other types of sinusitis and find possible microbial co-occurrence/associations intra or extra kingdom.

**Methods:** A total of 46 sinus biopsies were studied from 46 patients who underwent surgery for chronic rhinosinusitis from 2015 to 2017 in Paris, France. Direct examination of biopsies was performed by calcofluor and culture was performed on Sabouraud media. DNA was extracted and
fungal ITS1 and ITS2 loci and bacterial V3-V4 16S ribosomal DNA locus were amplified and sequenced (MiSeq™ Illumina, V2 kit, 500 cycles). To analyze the microbial diversity, operational taxonomic units were defined via QIIME and assigned to SILVA and UNITE databases for 16S and ITS, respectively. Statistical analyses were performed using SHAMAN. These data were compared to microbiological and clinical data.

**Results:** Among these 46 patients, 38 had fungal balls and 8 had non-fungal chronic rhinosinusitis and were considered as controls. Among the 38 fungal balls, direct examination and culture were positive for fungi in 95% and 30% of biopsies, respectively. By contrast, targeted metagenomic analysis of the 38 fungal balls yielded one or more fungal genera in 100% of cases, with *Aspergillus* spp. alone or in combination with other fungi in 92.1% (35/38) of patients. Control samples (n = 8) generated only a limited number of fungal reads, mainly from the genus *Malassezia* (4/8). Overall, the main bacterial genera found by targeted metagenomics were *Haemophilus* (20.1%), *Staphylococcus* (14.6%), *Pseudomonas* (7.66%) and *Enterobacter* (5.84%). Using principal coordinate analysis (Bray-Curtis distance; permanova test), we found significantly different bacterial and fungal metagenomic profiles in patients with fungal balls compared to controls (p = 0.047 and p<0.001, respectively) (Figure 1). One bacterial taxon, *Haemophilus*, was significantly over-represented in patients with fungal balls compared to controls (p<0.001).

<figure><img src="image_90.png" /></figure>

**Conclusion:** Targeted metagenomics allowed identification of fungal genera responsible for chronic rhinosinusitis when the culture remains negative. *Aspergillus* was the most commonly fungus identified. The genus *Haemophilus* appeared statistically associated with fungal balls. Fungal balls pathogenesis could result from microbial interactions between fungi and bacteria in mixed biofilm-like structure. 1. Jiang, R.-S., Huang, W.-C. & Liang, K.-L. Characteristics of Sinus Fungus Ball: A Unique Form of Rhinosinusitis. Clin Med Insights Ear Nose Throat (2018).

**Objectives:** The germination is the first adaptive process that *Aspergillus fumigatus* must overcome to colonize the environment or infect animals. The *A. fumigatus* germination process involves a first step known as swollen and the subsequent hyphae elongation. Morphologically, both are well-characterized processes but molecularly there are only a few studies describing the transition from swollen to germination in the literature. This study, therefore, focuses on the transcriptomic analysis of the genes differently expressed between swelling conidia and early hyphae output to understand the mechanisms involved in these steps.
**Methods:** The *A. fumigatus* reference strain Af293 was incubated at 37°C without agitation in RPMI supplemented with 5% fetal bovine serum for 4.5 and 6 hours to obtain swelling conidia or early hyphae respectively. Total RNA was extracted and maintained in RNA later until use. Finally, RNA samples were studied using the AWAFUGE microarray (Agilent Whole *A. fumigatus* Genome Expression 44K v.1) designed by our research group.

**Results:** The transcriptomic analysis detected 158 and 256 genes overexpressed in the swelling and early hyphal stages, respectively. GO enrichment analysis showed that transport (13.2%), organelle organization (11.9%) and regulation (10.7%) were the most common categories in the swelling stage, and transport (10.2%), regulation (10.2%) and filamentous growth (7.4%) in early hyphal stage. Unfortunately, only 36% and 53.5% of the overexpressed genes had known molecular function in swelling or hyphal stages, respectively. Among them, the oxidoreductase (17.6%), hydrolase (8.8%) and transferase (8.8%) were the most usual activities detected in swelling stage, while hydrolase (14.1%), oxidoreductase (10.2%) and transferase (6.3%) activities were the most abundant in early hyphal stage. According to component ontologies, the most overexpressed genes in the swelling conidia codified proteins located in mitochondrion (24.5%), membrane (23.3%) and nucleus (13.8%), while the ones in the early hyphae stage codified proteins of the membrane (13.7%), extracellular region (5.9%) and plasma membrane (4.7%). On the one hand, in the swelling step highlights the primary metabolism being the production of energy the principal priority. In addition, the regulation of glutamate homeostasis as well as the iron metabolism may also play a crucial role. On the other hand, in hyphal output, an overexpression of genes implicated in vitamins and cofactors production, calcium regulation and cell wall were detected.

**Conclusion:** This work might enable a better understanding of the germination process of *Aspergillus fumigatus* from a transcriptomic point of view. This project could involve important advances in the knowledge of the ecology and the mechanisms of adaptation of the fungus that could be exploited by both the biotechnological and the biosanitary sector.

P390

Genotyping of azole resistance in *Candida auris*

J. Li¹, A.T. Coste¹, D. Bachmann¹, D. Sanglard¹, F. Lamoth²

¹Microbiology Institute, University Hospital Lausanne, University of Lausanne, Lausanne, Switzerland, ²Infectious Diseases Service, CHUV, Lausanne, Switzerland

**Objectives:** Since 2009, *Candida auris* has emerged as a multidrug-resistant yeast pathogen in all continents. Its ability to cause nosocomial outbreaks with high mortality rate has attracted worldwide attention. Four distinct geographic and genetic clades have been described: I: South Asia (India, Pakistan), II: East Asia (Japan, South Korea); III: South Africa; IV: South America (Venezuela, Colombia). Remarkably, rapid development of fluconazole resistance is a hallmark of most *C. auris* isolates. However, mechanisms of azole resistance and type of genetic mutations still remain unclear. Hotspot mutations in the target gene (*ERG11*) are classical cause of azole resistance in *Candida spp*, but other mechanisms exist, such as overexpression of drug transporters induced by gain-of-function mutations in transcription factors like *TAC1* and *MRR1*, both of which are important regulators of efflux systems in *Candida albicans*. Nevertheless, *TAC1* and *MRR1* have not been studied in *C. auris* yet. The objective of the study was to analyze the genotype of these genes in *C. auris* isolates from different clades and with different levels of azole resistance.

**Methods:** Twenty-two *C. auris* strains from India, Japan, Israel, Colombia and Switzerland were collected. Susceptibility to fluconazole (FLC) and voriconazole (VOR) was tested according to CLSI protocol. Minimal inhibitory concentration (MIC) thresholds for resistance were defined according to arbitrary cut-offs previously reported by Lockhart *et al.*: MIC ≥32 µg/mL for FLC and ≥2 µg/mL for VOR. Entire sequencing of *ERG11*, *TAC1* and *MRR1* was performed for all 22 isolates.

**Results:** By antifungal susceptibility testing, 2 isolates were classified as azole susceptible (MIC FLC 4 µg/mL and VOR ≤1 µg/mL), 13 as resistant to FLC only and 7 as resistant to both FLC and VOR. *ERG11* sequencing revealed three hotspot mutations that have already been described in *C. auris* (F126T, Y132F and K143R). These isolates (n = 8) exhibited high level resistance to FLC and variable
VOR susceptibility. In addition, we identified two new hotspot mutations: V125A from Israeli and Swiss strains (n = 3) with high resistance to both FLC and VOR, and F444L from Colombian strains (n = 4) with high resistance to FLC and variable VOR susceptibility. Putative *TAC1*(QG37_07465) sequencing uncovered twenty mutations possibly associated with azole resistance (i.e. present in azole-resistant isolates, but not in susceptible ones): S89Y, E200K, F214L/S, R215K, K225N, K247E, I268V, Q298K, T346I, Q503R, F580L, P595H, P607S, S611P, A640V, Y647C, I772-, N773- and L774M. Compared to *ERG11* mutants exhibiting high FLC MIC (≥64 µg/mL) and relatively high VOR MIC (≥1 µg/mL), *ERG11* wild-type isolates harboring *TAC1* mutations exhibited intermediate level of FLC resistance (MIC: <64 µg/mL) and low VOR MIC (<1 µg/mL). Putative *MRR1*(QG37_07783) sequencing unveiled three mutations: N167K, G280R and N647T. *MRR1* mutations were always associated with *ERG11* mutations and their role in azole resistance thus remains unclear.

**Conclusion:** Overall, we identified two new mutations in *ERG11* (V125A and F444L) within conserved hotspot regions. Moreover, we identified twenty mutations in *TAC1-like* that were associated with intermediate level of fluconazole resistance in the absence of *ERG11* mutations. Their putative role in azole resistance deserves further investigations.

P391

Comparison of phenotypic and genotypic methods of speciation and gene sequencing of
Malassezia from the skin samples in a tertiary care hospital

N. Romald¹, A.J. Kindo², M. Veeraragavan³

¹Department Of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, India, ²Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, India, ³Dermatology, Sri Ramachandra Medical College and Research Institute, Chennai, India

**Objectives:** Speciate *Malassezia* phenotypically. Confirm the phenotypic identification through molecular method of speciating *Malassezia* by PCR and Gene Sequencing. Comparison between phenotypic and genotypic result.

**Methods:** *Malassezia* species was isolated from the skin scrappings of patients with hypo/hyper pigmented lesions. Microscopic Examination was done with 10%KOH. Samples showing meat ball and spaghetti appearance were cultured onto Sabourauds Dextrose Agar (SDA), SDA with olive oil overlay (SDA-O) and Modified Dixon's agar (MDA) containing chloramphenicol and incubated at 32° C upto 3 weeks. *Phenotypic Identification* was done based on the colony growth characteristics, gram staining, urease test, catalase test, bile esculin with tween 60 hydrolysis and tween assimilation. *Molecular Identification* The isolates which were cultured and identified by phenotypic methods were confirmed by Molecular methods DNA was extracted by Phenol: Chloroform method. Extracted DNA was amplified by Polymerase chain reaction (PCR) using thermocycler *Primers used Pan-fungal PCR primers*:- PCR amplification of ITS1 and ITS4 regions ITS1 5' TCC GTA GGT GAA CCT GCG G 3' and ITS4 5' TCC TCC GCT TAT TGA TAT GC 3' *Malassezia* specific nested primers Pan fungal PCR positive samples were subjected to species specific nested PCR using *Malassezia* specific primer ITSIF-N 5'GGATCATTAGTGATTGCCTTTATA 3' and ITS4-R 5' TCCTCCGCTTATTGATATG 3' *Agarose Gel Electrophoresis* was done and band obtained PCR products were sequenced by *Sanger's sequencing* and *NCBI blast* was done and species identified.

**Results:** *Phenotypic method of speciation of Malassezia* Out of 31 samples 7 isolates were *Malassezia furfur* 12 isolates were *Malassezia sympodialis* 9 samples grew Malassezia globosa 3 samples grew *Malassezia obtusa* MOLECULAR IDENTIFICATION *Pan-fungal PCR* PCR by Pan fungal primers ITS 1 and ITS 4 shows band between 600-1000bp (fig). All samples showing positive band was subjected to *Malassezia* specific nested PCR. *Malassezia* specific nested PCR *Malassezia* specific nested PCR using *Malassezia* specific primers showed band at 200-300bp.(fig) **Sequence result(table)**
<figure><img src="image_91.png" /></figure>

<table><thead><tr><th></th><th>PHENOTYPIC IDENTIFICATION</th><th>GENOTYPIC IDENTIFICATION</th></tr></thead><tbody><tr><td>Malassezia furfur</td><td>7</td><td>15</td></tr><tr><td>Malassezia sympodialis</td><td>12</td><td>4</td></tr><tr><td>Malassezia globosa</td><td>9</td><td>12</td></tr><tr><td>Malassezia obtusa</td><td>3</td><td></td></tr></tbody></table>

<figure><img src="image_92.png" /></figure>

<figure><img src="image_93.png" /></figure>

**Conclusion:** Phenotypic speciation showed *Malassezia sympodialis* as the predominant species. Genotypic speciation showed *Malassezia furfur* as the predominant species and the least was *Malassezia sympodialis*. Genotypic speciation is more confirmatory than phenotypic method. Speciation of *Malassezia* is important because of variation in resistant pattern to anti-fungals among different species.
**phylogenetic analysis.** MLST analysis was performed by the individual application of the six housekeeping genes CAP59, GPD1, LAC1, PLB1, SOD1 and URA5 along with the IGS1 region according as previously described (Meyer 2009). The allele types and the sequence type (ST) were identified via sequence comparisons with the *Cryptococcus* MLST database. The allelic profile of the strains and the global dataset were applied to the goeBURST algorithm in PHILOVIZ software. Clona complex (CC) was defined when the strain had no more than one difference in housekeeping gene allele. **Statistical analysis** X2 and Fischer's exact test were performed for categorical variables and student's T test for continuous variable.

**Results:** **Molecular types** Of a total of 55 clinical isolates, molecular types were identified in 50, in the 5 remaining the pattern of bands is reproducible but not concordant with the patterns of the references strains. Three of them corresponded to *C.gattii* complex. 28 (56,0%) isolates were *C.neoformans* var *grubii*, VNI (now *C. neoformans*), 16 (32,0%) were *C.neoformans* var *neoformans*, VNIV (now *C. deneoformans*), 6 (12%) were VNIII AD hybrids. The strains that were not identified by PCR fingerprinting were studied by MLST. **MLST (Mutilocus sequence type) and phylogenetic analysis.** 20 strains selected by their genetic variability and phenotypic characterization were analyzed by MLST. 7 different allele were identified for CAP59, 4 for GPD1,11 for LAC1, 7 for PLB1, 5 for SOD1, 7 for URA5 and 8 for IGS1 region. A total of 16 ST were observed, 13 *C. neoformans* complex isolates and 3 STs for 3 *C. gattii* complex isolates (Table 1). Of the 13 STs corresponding to *C. neoformans* complex, 8 were not reported until now and were included in the database (ST605, ST606, ST607, ST608, ST609, ST610, ST611 and ST612). The more frequent ST were ST32 (n = 3), ST23 (n = 2), ST63 (n = 2).

<table><caption>Table 1. Characterization with MLST</caption><thead><tr><th>Strain Identification</th><th>CAP59 Allele</th><th>GPD1 Allele</th><th>IGS1 Allele</th><th>LAC1 Allele</th><th>PLB1 Allele</th><th>SOD1 Allele</th><th>URA5 Allele</th><th>ST</th><th>Molecular type</th></tr></thead><tbody><tr><td>UYIH9*</td><td>5</td><td>11</td><td>24</td><td>16</td><td>10</td><td>1</td><td>28</td><td>605</td><td>VNIV</td></tr><tr><td>UYIH10*</td><td>38</td><td>11</td><td>34</td><td>20</td><td>10</td><td>1</td><td>18</td><td>606</td><td>VNIV</td></tr><tr><td>UYIH12</td><td>7</td><td>11</td><td>1</td><td>3</td><td>1</td><td>1</td><td>2</td><td>311</td><td>VNI</td></tr><tr><td>UYIH14</td><td>7</td><td>1</td><td>1</td><td>18</td><td>1</td><td>1</td><td>1</td><td>63</td><td>VNI</td></tr><tr><td>UYIH16</td><td>7</td><td>1</td><td>1</td><td>2</td><td>1</td><td>1</td><td>2</td><td>23</td><td>VNI</td></tr><tr><td>UYIH17*</td><td>1</td><td>11</td><td>10</td><td>3</td><td>4</td><td>1</td><td>1</td><td>607</td><td>VNI</td></tr><tr><td>UYIH21*</td><td>7</td><td>11</td><td>46</td><td>5</td><td>1</td><td>1</td><td>2</td><td>608</td><td>VNI</td></tr><tr><td>UYIH23</td><td>1</td><td>1</td><td>10</td><td>3</td><td>4</td><td>1</td><td>1</td><td>32</td><td>VNI</td></tr><tr><td>UYIH26</td><td>1</td><td>1</td><td>4</td><td>4</td><td>1</td><td>14</td><td>7</td><td>20</td><td>VGII</td></tr><tr><td>UYIH28</td><td>1</td><td>1</td><td>10</td><td>3</td><td>4</td><td>1</td><td>1</td><td>32</td><td>VNI</td></tr><tr><td>UYIH29*</td><td>38</td><td>11</td><td>34</td><td>22</td><td>10</td><td>1</td><td>18</td><td>609</td><td>VNIV</td></tr><tr><td>UYIH30*</td><td>5</td><td>11</td><td>34</td><td>19</td><td>10</td><td>1</td><td>18</td><td>610</td><td>VNI</td></tr><tr><td>UYIH37</td><td>7</td><td>1</td><td>1</td><td>18</td><td>1</td><td>1</td><td>1</td><td>63</td><td>VNI</td></tr><tr><td>UYIH38</td><td>1</td><td>1</td><td>10</td><td>3</td><td>4</td><td>1</td><td>1</td><td>32</td><td>VNI</td></tr><tr><td>UYIH43*</td><td>7</td><td>11</td><td>1</td><td>1</td><td>1</td><td>1</td><td>2</td><td>612</td><td>VNI</td></tr><tr><td>UYIH46</td><td>7</td><td>1</td><td>1</td><td>2</td><td>1</td><td>1</td><td>2</td><td>23</td><td>VNI</td></tr><tr><td>UYIH47*</td><td>5</td><td>11</td><td>34</td><td>16</td><td>14</td><td>1</td><td>1</td><td>611</td><td>VNI</td></tr><tr><td>UYIH50</td><td>1</td><td>3</td><td>10</td><td>3</td><td>4</td><td>1</td><td>1</td><td>338</td><td>VNI</td></tr><tr><td>UYIH52</td><td>44</td><td>11</td><td>59</td><td>5</td><td>13</td><td>63</td><td>14</td><td>157</td><td>VGI</td></tr><tr><td>UYIH72</td><td>2</td><td>16</td><td>55</td><td>4</td><td>26</td><td>15</td><td>2</td><td>132</td><td>VGII</td></tr></tbody></table>

(*) Strains with news ST reported.

**Conclusion:** This is the first molecular characterization of Uruguayan isolates of *Cryptococcus spp* from cases of meningoencephalitis. We demonstrated the great genetic variability of circulating *Cryptococcus spp* strains in our environment that cause meningoencephalitis. The new STs reported are closely related with each other and with the rest of the STs, although they did not belong to the same CC.
P393

Identification of genetic mediator of fluconazole tolerance in Candida albicans

E. Delarze1, E. Trachsel1, L. Brandt1, C. D'Enfert2, D. Sanglard1

1Institute of Microbiology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland, 2Pasteur Institute, Paris, France

Objectives: Fluconazole (FLC) resistance in *Candida albicans* has long been reported and is involving several well described molecular resistance mechanisms. FLC resistance is based on measurement of MIC values and threshold values have been provided to distinguish between wild-type susceptible isolates and resistant isolates. Another phenomenon, called FLC tolerance (known also as "trailing effect" is susceptibility assays) is characterized by the ability of *C. albicans* to grow persistently at supra-MIC drug concentrations without acquisition of drug resistance. Little is known about molecular mechanisms behind FLC tolerance and we aimed here to undertake identification of genetic mediators of FLC tolerance.

Methods: We used in this study a collection of 579 *C. albicans* strains, each of which could overexpress a specific *C. albicans* gene in a tetracycline-dependent manner and was tagged by a distinct sequence barcode. The whole collection was pooled in RPMI and, after repeated subcultures under FLC pressure at 32 µg/ml for 5 consecutive days in RPMI with 2% glucose, the relative population of each barcode was determined by barcode sequencing. In addition, each barcoded strain was subjected to single tolerance assays yielding tolerance indexes reflecting relative growth of strains at a single drug concentration (8 µg/ml FLC) after 24 h growth in RPMI in 2% glucose (growth conditions similar to EUCAST susceptibility assays).

Results: Combining this genetic selection with single isolates testing, two transcription factors (*CRZ1* and *GZF3*) and a protein kinase (*YCK2*) were confirmed to be positive mediators of FLC tolerance when overexpressed. *CRZ1* is part of the calcineurin pathway that is known critical for *C. albicans* in response to antifungal stress, while *GZF3* and *YCK2* are less well characterized. Using overexpression of these genes in deletion mutants, we showed that *GZF3* acts probably upstream of *CRZ1*. *CRZ1* and *GZF3* were deleted in azole-susceptible clinical strains but exhibiting different levels FLC tolerance. Our results demonstrate that *CRZ1* plays a critical role in FLC tolerance in these strains while *GZF3* deletion has minor effects. Consistent with these data, transcriptional profiles of clinical strains in the presence of FLC at supra-MIC drug concentrations was reminiscent of a calcineurin-like transcriptional response.

Conclusion: A genetic screen permitted the identification of mediators of FLC tolerance, one of which (*CRZ1*) is known to be part of the calcineurin pathway. FLC tolerance of clinical strains is dependent on the presence of at least *CRZ1*. The precise mechanisms of FLC tolerance mediated by *CRZ1* and the other identified genes still need further investigations.

P394

Genotypic characterization of *Trichosporon* sp. from a Tertiary care centre in South India

T. Premamalini<fnref n="1" />, S.V. Rajyoganandh<fnref n="2" />, H. Veena<fnref n="2" />, R. Vijayakumar<fnref n="2" />, A.J. Kindo<fnref n="2" />, S.K.M. Rungmei<fnref n="3" />, K.S. Sridharan<fnref n="2" />, B. Vijayalakshmi⁴

**Objectives:** To determine the species distribution of the *Trichosporon* sp. based on genotypic characterization. To assess the genetic relatedness of the most commonly isolated *Trichosporon asahii* strains using RAPD primers GAC1 and M13.

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

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

<footnote n="3">³Microbiology, SG-PGI Lucknow, Lucknow, India, ⁴Infecious Disease Consultant, SIMS, Chennai, India</footnote>
**Methods:** Seventy two *Trichosporon* clinical isolates recovered from patients of SRMC & RI, a few hospitals in Chennai, and a hospital in Lucknow, were considered for the study. PCR was performed initially with *Trichosporon* genus specific primers to double check for accurate identification of this genus. Genotypic characterization was done by PCR amplification of rRNA gene sequences by *T. asahii* specific primers which identified only *T. asahii* isolates. Species confirmation was done by IGS1 sequencing and phylogenetic analysis. Maximum parsimony clustering was used for construction of phylogenetic tree. Its stability was assessed by parsimony bootstrapping with 1,000 pseudo replications. Phylogenetic analysis was done by comparing the obtained nucleotide sequences with the nucleotide sequences of *Trichosporon* reference strains obtained from the GenBank database. DNA fingerprinting of the *T. asahii* isolates was carried out by RAPD analysis, using primers GAC-1 and M13.

**Results:** Distribution of sample source of the 72 isolates were as follows: 43 (59.7%) urine, 12 (16.7%) blood, 7 (9.7%) Percutaneous nephrostomy (PCN) tube, 5 (6.9%) respiratory isolates, 4 (5.6%) pus, and 1 (1.4%) peritoneal dialysate fluid. *Trichosporon* specific PCR confirmed all the 72 isolates as belonging to this genus. *Trichosporon asahii* specific PCR identified 65 isolates as *T. asahii* (Fig. 1). The remaining 7 isolates were identified further by phylogenetic analysis of the IGS1 sequences. In phylogenetic analysis, six representative *T. asahii* clinical isolates clustered together with *T. asahii* KR265116.1 and *T. asahii* KR872659.1 (bootstrap ≥95), confirming the molecular identification results. One isolate identified as *Trichosporon* species clustered in the same clade (bootstrap ≥79) was found to be closely related to *T. ovoides*. All these isolates belonged to the Ovoides clade. Two isolates clustered along with *T. loubieri* AB066428 & *T. loubieri* KT936593.1 (bootstrap ≥99). They belonged to the Gracile clade. The remaining 4 isolates clustered together in Cutaneum clade along with *T. cutaneum* AB 066415.1 (bootstrap ≥47) (Fig. 2). Finally, the 72 *Trichosporon* clinical isolates were identified as 65 *Trichosporon asahii*, 4 *Trichosporon cutaneum*, 2 *Trichosporon louberi* and one *Trichosporon* sp. closely related to *T. ovoides*. DNA fingerprinting of the mostly commonly isolated *T. asahii* (64/72) isolates carried out by RAPD analysis, identified more heterogeneity among the *T. asahii* isolates by GAC1 than M13 primer.
<figure><img src="image_94.png" /></figure>

**Conclusion:** *Trichosporon asahii* is the most common species causing human trichosporonosis, though other members of the genus may also be involved. The members of the genus *Trichosporon* are most frequently under-reported due to inconsistency of the phenotypic methods used routinely in most laboratories. *Trichosporon* infections when treated with fluconazole or caspofungin, which is commonly used to treat yeast infections, results in poor clinical outcome. Hence, correct identification of the *Trichosporon* species using molecular methods, helps the clinician in providing appropriate treatment.

**Objectives:** The goal of the study was to assess the relationship between the fungal mycobiome detected in samples from the Lower Respiratory Tract (LRT) of non-infectious patients, and the mycobiota detected in the air and dust of their dwellings.
**Methods:** The mycobioma of the LRT was analysed by the study of Bronchoalveolar Lavage (BAL) samples taken from patients with indication of bronchoscopy. After informing and getting their consent for the study, they were asked about the possibility of a domestic sampling that was scheduled. Negative controls were included for every laboratory procedure and samples from the bronchoscopes were also taken as controls. All samples were processed for DNA extraction and for cultures. The fungal Internal Transcribed Spacer (ITS2) of the extracted DNA was amplified and sequenced by the illumina system. Results of the massive sequencing were analyzed by QIIME 1.8.0 and compared with the ones in the UNITE Database. NCBI nucleotide database was also used for identification. Cultures of samples were incubated at room temperature and 37°C and the fungal isolates were studied for identification.

**Results:** Forty-six BAL, 4 samples from bronchoscope and 19 domestic environmental (MA) samples, together with a negative control from the PCR were submitted to massive sequencing analysis (Solexa/Illumina system). The first results yielded over 7000000 sequences (5060431 from BAL, 108178 from bronchoscopes and 10861275 from MA samples). In order to filter contaminants, we used a negative control from PCR and three negative controls from each sequencing run. Samples were clustered in different fungal Operational Taxonomic Units (OTUs) at 99% identity and sequences clustered in OTUs present in control samples were removed from the analysis. After contaminant filtering, we obtained 2840808 clean sequences belonging to 122 different fungal Operational Taxonomic Units (OTUs) at 97% identity. The similarity among different microbial communities was assessed using phylogenetic information. Among the fungal species detected by massive sequencing, 6 OTUs were detectable in more than 75% of the BAL and 75% of the MA samples. These sequences corresponded to *Cladosporium angustisporum*; *Candida orthopsilopsis*; *Rhodotorula mucilaginosa*; *Cryptococcus neoformans* sensu stricto; *Naganishia liquefaciens* and *Malassezia restricta*. Most of these species could be detected also by culture in the domestic environment, but almost none in BAL samples cultures. When comparing the mycobiome of the LRT of patients with one of their domestic environment, important coincidences can be seen for specific species of *Candida*, *Cryptococcus* and *Malassezia*.

**Conclusion:** The results show an important similarity between the mycobiota of the environment and the mycobiome of the LRT samples of the patients. Most of the fungal species detected in both environments correspond to fungi which are important opportunistic pathogens like *Cryptococcus neoformans* and *Candida orthopsilosis*.

P396

**Identification of Mycotoxigenic Fungi from Grains in a Nigerian Region Using the Modern Polyphasic Methodology.**

O.F.I. Okeke¹, S.O. Fapohunda², C. Soares³, N. Lima⁴, G.M. Ayanbimpe¹

¹Medical Microbiology, University of Jos, Plateau State, Nigeria, ²Bioscience And Biotechnology, Babcock University, Ogun State, Nigeria, ³Centre Of Biological Engineering, Micoteca da Universidade do Minho, University of Minho, Campus of Gualtar,, Braga, Portugal, ⁴Centre Of Biological Engineering,, Micoteca da Universidade do Minho, University of Minho, Campus of Gualtar, Braga, Portugal

**Objectives:** Mycotoxins are poisonous substances produced by mycotoxigenic fungi which contaminate agricultural commodities. Many foods and feeds can become contaminated with mycotoxins since they can form in commodities before harvest, during the time between harvesting and drying, and in storage. Mycotoxins may also be carried over to animal products due to consumption of contaminated feed. Maize (*Zea mays*) and guinea corn (*Sorghum bicolor*) form a major staple of the study area and are high risk commodities for contamination with mycotoxigenic fungi. This study was carried out to identify mycotoxigenic fungi from maize and guinea corn in Plateau state, north-central of Nigeria.

**Methods:** In a multistage sampling technique, markets for the study were selected. Using simple random sampling method, 270 samples were collected from various sampling points within selected
markets where these grains are sold. Isolation and identification of the mycotoxigenic fungi employed the modern polyphasic methodology for filamentous fungi identification.

**Results:** The mycotoxigenic fungi *Aspergillus*, *Fusarium*, and *Penicillium* were isolated. Further work on these mycotoxigenic fungal genera identified them as follows: *A. niger*, *A. aculeatus*, *A. tamari*, *A. flavus*, *F. verticillioides* and *F. equiseti*. The *Penicillium* isolate was identified to belong to the section Sclerotiora and closely related to *P. malochii*. *Aspergillus* accounted for 75% of all mycotoxigenic fungi isolated while *Fusarium* and *Penicillium* accounted for 23% and 2% respectively.

**Conclusion:** Further work is also ongoing to determine if our strain of *Penicillium* is a *P. malochii* or an entirely novel species. Our findings indicate contamination of maize and guinea corn in the study area with mycotoxigenic fungi which may pose serious health risks for the human and animal population and also have implications for food safety and public health in Nigeria. In view of this, further investigation is necessary to assess the health risks linked with the consumption of these grains. Key words: Mycotoxigenic fungi, grains, Nigeria

**Regulation of DNA repair machinery and Ras pathway in *Paracoccidioides brasiliensis* during oxidative stress**

W. Batista¹, M. Navarro², D. Castilho², J. Calado², P. Conceição¹, B. Castro¹, R. Silva², A. Chaves²

¹Pharmaceutical Sciences, Universidade Federal de São Paulo, São Paulo, Brazil, ²Microbiologia E Imunologia, UNIFESP, São Paulo, Brazil

**Objectives:** Paracoccidioidomycosis is a granulomatous disease caused by fungi of the genus *Paracoccidioides* and widely distributed in Latin America. The disease has high disabling potential and yet is still absolutely neglected. These fungi undergo cell differentiation induced by changes in temperature and this event is critical to the ability to cause disease. The knowledge of which molecular patterns of the fungus may be true virulence factors is still limited by the difficulties inherent to genetic manipulation of these fungi. Studies that have tried to understand these molecular aspects rely on the method of transformation mediated by *Agrobacterium tumefaciens*. To overcome these barriers, we need to understand the mechanisms of DNA repair used by the fungus. This is because the establishment of a knockout strain depends on the occurrence of homologous recombination events. The aim of the present communication was to evaluate the role of the DNA repair machinery of *P. brasiliensis* in response to oxidative stress.

**Methods:** *P. brasiliensis* yeast cells were treated with different concentrations of H₂O₂, in the presence or absence of pharmacological inhibitors (Ras, Hog-1 or DNA ligase 4 pathways). We used the RBD-GST (Ras Binding Domain fused with GST) probe to detect the Ras activation and western blot to evaluate the Hog-1 phosphorilation levels. Gene expression was analyzed by qRT-PCR. Cell viability were assessed by MTT assay or cell counting.

**Results:** The double-strand breaks repair pathways communicate with the Ras GTPase and Hog1 Map kinase pathways. The genes *PbRAD51* and *PbRAD52* are active elements during DNA repair and participate in the response to oxidative stress in *P. brasiliensis*. In addition, inhibition of the non-homologous end joining (NHEJ) mediated repair pathway has been observed to provide a protective response to *P. brasiliensis* yeasts during a normally lethal oxidative stress condition for the fungus. Perhaps, this event is related to the greater efficiency of the repair by homologous recombination induced by the inhibition of NHEJ. Finally, it was observed that the combination of the damping of the NHEJ pathway, through the inhibition of DNL4 or GSK3, together with the induced oxidative stress do increase the efficiency of transformation processes in yeasts of *P. brasiliensis*. Indeed, inhibitors of NHEJ pathway caused a protective effect on yeast cells challenged with high concentration of H₂O₂. This effect correlates with an increase in expression of scavenger genes like catalase and superoxide dismutase.

**Conclusion:** Taken together, our results indicate that there is a significant crosstalk between two major cellular damage response pathways, ROS signaling and DNA repair, for *P. brasiliensis* survival.
**Matched-paired analysis of patients treated for invasive mucormycosis - standard treatment vs. posaconazole new formulations (MoveOn)**

J. Salmanton-Garcia¹, D. Seidel¹, P. Köhler¹, S. Mellinghoff¹, H. Wisplinghoff², J.-J. Vehreschild¹, O.A. Cornely¹, M.J.G.T. Vehreschild¹

¹Department I Of Internal Medicine, University Hospital of Cologne, Cologne, Germany, ²Antibiotic Resistance, Molecular Epidemiology, Labor Wisplinghoff, Cologne, Germany

**Objectives:** Current first-line (1st) antifungal treatment for invasive mucormycosis (IM) consists of liposomal amphotericin B (AMB). Salvage (SAL) treatment options are limited and often based on posaconazole oral suspension (POSsusp). However, with the approval of posaconazole new formulations (POSnew), patients could benefit from improved pharmacokinetics, safety and tolerability. Therefore, our aim is to assess the effectiveness of POSnew as first-line and SAL treatments for IM.

**Methods:** We performed a case-matched analysis with proven or probable IM patients from the FungiScope® Registry. 1st-POSnew and 1st-AMB+POSnew cases were matched with 1st-AMB-based treatment controls, and SAL-POSnew cases were matched with SAL-POSsusp controls. Each case was matched with up to three controls based on severity, haematological/oncological malignancy, surgery and/or renal dysfunction.

**Results:** Five patients receiving first-line POSnew alone, 18 receiving first-line POSnew combined with AMB, and 22 receiving salvage POSnew were identified. By day 42, favourable response was reported for 80.0% (n = 4/5) of patients receiving first-line POSnew, for 27.8% (n = 5/18) receiving first-line POSnew plus AMB, and for 50.0% (n = 11/22) receiving salvage POSnew. Day-42 all-cause mortality of patients receiving POSnew was lower compared to mortality in their respective controls (20.0% (n = 1/5) in 1st-POSnew vs. 53.3% (n = 8/15) in 1st-AMB; 33.3% (n = 6/18) in 1st-AMB+POSnew vs. 52.0% (n = 26/50) in 1st-AMB; 0.0% (n = 0/22) in SAL-POSnew vs. 4.4% (n = 2/45) in SAL-POSsusp).

**Conclusion:** In the observed patients, POSnew was effective in terms of treatment response and associated mortality of IM. POSnew may be an alternative for treatment of IM.

P401

**Efficacy of Celecoxib solely or in combination with Itraconazole in the treatment of murine paracoccidioidomycosis**

E. Burger¹, L.A. Santos¹, J.C. Grisolia¹, N.A. Dias¹, F.B.D.A. Paula², A.M. De Oliveira³, A.M. Rodrigues⁴, Z.P.D. Camargo⁴

¹Department Of Microbiology And Immunology, Federal University of Alfenas, Alfenas, Brazil,
²Department Of Clinical And Toxicological Analysis - Pharmaceutical Sciences Faculty, Federal University of Alfenas, Alfenas, Brazil,
³Laboratory Of Pathology - José Do Rosário Vellano University,
UNIFENAS, Alfenas, Brazil,
⁴Department Of Microbiology, Immunology And Parasitology, Federal University of Sao Paulo, Sao Paulo, Brazil

**Objectives:** Our objective is to evaluate the effectiveness of Celecoxib, of Itraconazole and both, as a combined treatment for paracoccidioidomycosis, a highly prevalent granulomatous mycosis in Latin America using a murine model.

**Methods:** Mice were intraperitoneally infected with a virulent *P. brasiliensis* isolate and three days later, daily administration by gavage of either 3mg/mL Itraconazole (I), or 6mg/mL Celecoxib (C) or both 3mg/mL Itraconazole and 6mg/mL Celecoxib (I+C) was initiated and kept for 15 days. Controls included infected, non-treated mice (Pb) and non-infected mice. Body weight and survival rate were monitored daily. At 7 and 15 days of infection, delayed type hypersensitivity (DTH) was evaluated and serum was obtained for specific anti-*P. brasiliensis* IgG antibody determination. At the last day of treatment, the mice were sacrificed and spleen, lungs, liver and epiploo/pancreas were collected and their macroscopic characteristics were analyzed. Following, the organs were either processed and stained with H/E for general histology, or macerated, for quantification of viable fungi.

**Results:** There was no difference in the body weight of infected, treated or uninfected controls and there were no deaths registered during this early phase of the infection in mice from any group. In all experimental groups, DTH decreased from day 7 to day 15 and, in contrast, specific IgG titers
increased from day 7 to day 15. But At 15 days, both, DTH reactivity and IgG titers were the highest in the Pb group, lower in the I group and in the C group, and the lowest in the I+C group. This decrease in both cellular and humoral specific immunity in the groups most treatment-covered may be explained by the elimination of viable *P. brasiliensis* by the drugs, rendering a full immune response less necessary. The histological aspects demonstrated that treatment with I+C was the one that reduced the most the number of lesions and the severity of the granulomatous response in all organs. When analyzing the control of fungal growth in the tissues, both mice from the I and from the C groups showed less fungi scattered in the tissue than mice from the Pb group; however the lowest numbers of fungi were found in mice from the I+C group. In the epiploo, which is the organ of shock for this model of *P. brasiliensis* infection, the animals of the I+C group had the fewest fungi with preserved morphology. The same was found in terms of viable fungi, as the number of CFUs was lower in all organs of treated mice, and more markedly in those receiving the combined I+C therapy.

**Conclusion:** We showed the effect of the antifungal drug Itraconazole and also of the anti-inflammatory drug Celecoxib at the tissue level. This is the first report of the direct effect of Celecoxib on *P. brasiliensis* as well as in combination with Itraconazole. Our results suggest that this combined treatment employing an antifungal and an anti-inflammatory drug may constitute a new efficient therapeutic strategy. Grants: CNPq 305216/2016-3 and FAPEMIG APQ 012941-16. L.A. Santos and J.C. Grisolia are recipient of CAPES scholarships.

P402

**Successful Treatment of a Patient with Retroperitoneal Abscess caused by *Candida krusei* with the Investigational Agent, Ibrexafungerp (formerly SCY-078): A Case Report from the FURI study**

G. Thompson¹, D. Angulo²

¹Infectious Disease, University of California at Davis, Davis, United States of America, ²SCYNEXIS, Inc., Jersey City, United States of America

**Case Report: Objectives:** Intraabdominal candidiasis is the second most common *Candida* infection after candidemia. *Candida albicans* is the predominant cause of these infections, with non-*albicans* *Candida* becoming more frequently observed in this clinical setting. Echinocandins are the recommended treatment for intraabdominal *Candida* infections but have varied clinical response. This is postulated to be based on poor drug levels in tissue and abscesses. Ibrexafungerp (formerly SCY-078) is a novel IV/oral glucan synthase inhibitor (triterpenoid) antifungal with activity against *Candida*, *Aspergillus* and *Pneumocystis*. A Phase 3 open-label, single-arm study of oral ibrexafungerp (FURI; NCT03059992) is ongoing for the treatment of patients intolerant of or with fungal disease refractory to standard antifungal therapy. We present a patient case of retroperitoneal abscess caused by *Candida krusei* from the FURI study. **Methods:** A 71 year old male patient with ischemic stroke, pulmonary edema, and tracheostomy was being treated for a retroperitoneal abscess, a complication of a perforated duodenal ulcer. *Candida krusei* was isolated in peri-duodenal drain cultures and the patient was initiated on micafungin therapy for 21 days. During micafungin therapy, cultures from the peri-duodenal drain on three occasions remained positive for *Candida krusei*. The patient underwent abscess drainage and was enrolled into the FURI study due to micafungin treatment failure and was treated with oral ibrexafungerp 750mg BID for two days followed by 750mg daily.

**Results:** The patient was treated with oral ibrexafungerp therapy for 21 days. Clinical improvement was observed during therapy. At End of Treatment visit, the clinical signs and symptoms of fungal disease were considered by the investigator to be resolved and the physical exam noted minimal discharge from the patient's retroperitoneal drain. As of the 6-week follow-up visit, clinical signs and symptoms of infection were still resolved. No drug-related adverse events were reported.

**Conclusion:** This case report shows the potential of oral ibrexafungerp to treat intraabdominal infections caused by *Candida krusei* in patients who fail echinocandin therapy. Continued enrollment in the FURI study is warranted.

P403
In vitro study of Antifungal Biopharmaceutical Dectin1-Fc

L. Lim

Bioprocessing Technology Institute/National University of Singapore, Singapore, Singapore

**Objectives:** Invasive fungal infections are a neglected category of disease that has a high mortality and implicated in immunocompromised patients. Dectin-1 (CLEC7A) is a type II transmembrane C-type lectin pattern recognition receptor expressed on cells of the innate immune system like macrophages, monocytes and neutrophils that recognizes β-glucans through its single C-type lectin-like domain at its extracellular region. It functions by binding to cell walls of different fungal species, such as *Saccharomyces cerevisiae*, *Candida albicans*, *Pneumocystis carinii*, *Coccidioides posadasii*, and *Aspergillus fumigatus*; triggering immune cell responses like phagocytosis and cytokine production. The absence of β-glucans in human cells presents the opportunity to exploit Dectin-1 as therapeutic vehicle to enhance immune response during a fungal infection. This project aims to develop recombinant Dectin1-Immunoglobulin G1 Fc fusion protein as a potential biopharmaceutical for passive immunization to aid the innate immune system against Candida albicans fungal infection. The in vitro properties of the recombinantly expressed fusion protein are examined in this study.

**Methods:** The fusion protein was expressed in DG44 Chinese Hamster Ovarian (CHO) cells and amplified with methotrexate. The fusion protein was extracted from culture supernatant via GE Histrap histag affinity and purified with GE Superdex 200 size exclusion chromatography on a GE Akta Purifier FPLC. Immunofluorescence imaging with anti-Fc Alexa Fluor 568 conjugated antibody from ThermoFisher Scientific was used to assay binding of the fusion protein to unicellular or hyphae Candida albicans. Binding affinity of the Fc fusion protein versus a human IgG1 to Fcγ receptors from R&D Systems were screen by surface plasmon resonance on a GE Biacore T200.

**Results:** Immunofluorescence imaging shows the fusion protein capable of binding to Candida albicans both in the unicellular and hyphae morphology. Surface plasmon resonance study of the Fc-fusion protein versus a IgG1 control reveal that the fusion protein binds to FcγRI, FcγRIIIa and FcRn. However, the binding constants for the Fc-fusion is generally weaker than a full fledge IgG1 antibody. Activity testing of the fusion protein using THP-1 monocytes against unicellular Candida albicans in a 1 hr phagocytosis assay shows a dose response. For the highest dose of 100µg/ml of fusion protein, a reduction of greater than 50% of surviving Candida albicans cells compared to the untreated control was observed.

**Conclusion:** The in vitro results reveal that the respective domains of the Fc-fusion protein function as intended though binding to the Fc receptors are not as strong as an antibody. This in turn may affect the efficacy of the drug. Further testing with primary immune cells would be appropriate to study the full therapeutic potential of this fusion protein. In addition, in vivo studies with an appropriate mouse model will give a better picture of the performance of the Fc-fusion protein drug.

The *in vitro* effect of farnesol on the activity of voriconazol and amphotericin B against
*Aspergillus* isolates

Y. Oz¹, S. Onder², G. Durmaz²

¹Department Of Microbiology, Division Of Mycology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, Turkey, ²Department Of Microbiology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, Turkey

**Objectives:** Although new antifungal agents are being developed, there is an increasing resistance to standard antifungal therapy, and no new classes of antifungal agents have been approved for a long time. Currently, three antifungal drug classes including triazoles, polyenes and echinocandins are available to use in treatment of IFIs. However, treatment is often complicated due to their high toxicity, low tolerability, drug interactions and limited spectrums of activities. Therefore, the requirement of new drug or treatment alternatives especially those with a wider spectrum, lower toxicity and cheaper are increasing day by day. Farnesol is an extracellular quorum-sensing molecule producing by *C.albicans* and inhibits the yeast-to-hypha transition in *C.albicans* and blocks biofilm formation. FAR is also a sesquiterpene alcohol existing in many herbal products and exogenously
FAR inhibits the conidiation in *Aspergillus niger* and the germination of macroconidia in *Fusarium graminearum*. However, the number of studies assessing the antifungal efficacy of FAR with standardised methods is limited. In this study, we investigated the contribution of farnesol on the activity of antifungals such as voriconazole and amphotericin B against clinical *Aspergillus* isolates.

**Methods:** Clinical *Aspergillus* isolates, *A.fumigatus* (n = 30) and *A.flavus* (n = 15), were included in this study. The MIC values of antifungal drugs and farnesol were determined against all *Aspergillus* isolates using reference broth microdilution method (CLSI M38-A2). The interactions of farnesol with voriconazole and amphotericin B were investigated by the checkerboard method and evaluated based on the fractional inhibitor concentration index (FICI). The FICI was obtained by summing the FIC values of each drug; the FIC was calculated for each agent by dividing the inhibitory concentration of each antifungal or compound when used in combination by its MIC. Synergy was defined as a FICI of ≤ 0.5; no interaction was defined as a FICI > 0.5 but < 4; and antagonism was defined as a FICI ≥ 4.

**Results:** The MIC ranges of farnesol, voriconazole and amphotericin B were 1500-6000 µM, 0.125-1 µg/mL and 0.125-0.5 µg/mL against *A.fumigatus* isolates, 3000-≥6000 µM, 0.125-0.5 µg/mL and 0.25-2 µg/mL against *A.flavus* isolates, respectively. The most common interaction in combination tests was "no interaction" and synergistic interaction was not detected in any combination. The combination of farnesol with voriconazole had antagonistic activity against 13 of 31 *A.fumigatus* isolates with FICI values of 4.01 to 8.5, and 4 of 14 *A.flavus* isolates with FICI values of 4.03 to 5. Similarly, the combination of farnesol with amphotericin B had antagonistic activity against 7 of 31 *A.fumigatus* isolates with FICI values of 4.01 to 8.03, and 5 of 14 *A.flavus* isolates with FICI values of 4.5 to 10.

**Conclusion:** Antifungal activity of farnesol and synergistic interaction when it was combined with antifungal drugs have been reported against *Candida* spp and its biofilm. However, farnesol did not produce any significant inhibitor activity alone or any synergistic interaction in combination with voriconazole or amphotericin B against *Aspergillus* isolates in this study. Conversely, considerably antagonistic interaction was observed; 38% for farnesol with voriconazole combination, 27% for farnesol with amphotericin B combination.

P405

**Olorofim is potent against *Madurella mycetomatis* – the most common causative agent of Eumycetoma**

W. Lim¹, M. Konings¹, B. Rijnders¹, A.H. Fahal², M. Birch³, W. Van De Sande¹

¹Department Of Microbiology And Infectious Diseases, Erasmus Medical Center, Rotterdam, Netherlands, ²Mycetoma Research Center, University of Khartoum, Khartoum, Sudan, ³F2G Ltd, Manchester, United Kingdom

**Objectives:** *Madurella mycetomatis* is the main causative agent of eumycetoma, a chronic granulomatous infection of the subcutaneous tissue. Currently, the only antifungal agents with activity against *M. mycetomatis* are agents acting on ergosterol in the fungal cell membrane. Itraconazole is currently the drug of choice, but the duration of treatment is long and therapeutic failure is common. Therefore, there is an urgent need to identify more potent antifungal agents with activity against *M. mycetomatis*. One of the novel classes of antifungal agents are the orotomides and olorofim is the leading representative of this class. It inhibits fungal pyrimidine biosynthesis and it is currently being evaluated for invasive fungal infections in patients lacking treatment options. To determine if olorofim has *in vitro* activity against *M. mycetomatis*, we carried out MIC determinations against 21 *M. mycetomatis* clinical isolates with different genetic and geographical background and compared the results to those obtained for itraconazole.

**Methods:** Minimal inhibitory concentrations (MIC) were determined for Olorofim and Itraconazole against 21 *M. mycetomatis* clinical isolates from Sudan (14), Algeria (1), Mali (1), India (1), Chad (1), the Netherlands (1) and also from an unknown origin (2). The filamentous nature of *M. mycetomatis* necessitates the need for homogenization of inoculum by sonication to obtain a standardized inoculum for testing. This creates turbidity in the wells in an MIC test which complicates visual reading of the antifungal activity, tests were therefore read using XTT as a colorometric agent to
indicate growth. MICs was performed using our CLSI-based in vitro susceptibility testing method with XTT reading at 450nm at the end after a 7-day incubation period at 37°C. As a number of *M. mycetomatis* isolates produces pigments that influences colour intensity and the endpoint reading, an 80% reduction in viable fungal mass was determined instead of a 100%.

**Results:** We showed that olorofim was highly active against all tested *M. mycetomatis* isolates. MICs obtained for olorofim ranged from <0.004 µg/ml to 0.125 µg/ml and 0.06 µg/ml olorofim was needed to inhibit 90% of the isolates (see results in Table). Olorofim MICs were consistently one-dilution more potent than the MIC values for itraconazole. For itraconazole, MICs ranged from 0.008 µg/ml to 0.25 µg/ml and 0.125 µg/ml was needed to inhibit 90% of the isolates.

<table><caption>**Table.** *In vitro* susceptibility of *Madurella mycetomatis* to olorofim and itraconazole, results of 21 isolates.</caption><thead><tr><td>Antifungal agent (µg/ml)</td><td>Median</td><td>MIC range</td><td>MIC<sub>50</sub></td><td>MIC<sub>90</sub></td></tr></thead><tbody><tr><td>Olorofim</td><td>0.0156</td><td>0.0039 - 0.125</td><td>0.015</td><td>0.06</td></tr><tr><td>Itraconazole</td><td>0.0312</td><td>0.0078 - 0.25</td><td>0.031</td><td>0.125</td></tr></tbody></table>

**Conclusion:** Our study showed that *M. mycetomatis* can also be inhibited with antifungal agents with a different mode of action to the azoles. Olorofim from the novel antifungal class of orotomides showed potent *in vitro* activity against all tested *M. mycetomatis* isolates and had MICs similar to or slightly lower than those for itraconazole - the current drug of choice for therapy. Further studies, including *in vivo* models are warranted to determine if olorofim would be a suitable alternative to itraconazole therapy.

P407

**Inhibiting fungal BET protein: a new hope to treat systemic fungal infections**

S. El Kennani¹, S. Picaud², M. Champleboux¹, J. Marais¹, M. Courçon³, M. Arlotto¹, G. Rabut⁴, P. Filippakopoulos², J. Govin¹

¹Signaling Through Chromatin, INSTITUTE FOR ADVANCED BIOSCIENCES, GRENOBLE, France, ²Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom, ³Biologie à Grande échelle, CEA, Grenoble, France, ⁴Institute of Genetics and Development, Rennes, France

**Objectives:** Invasive fungal infections kill an estimated 1.6 million people each year – as many deaths as are caused by tuberculosis or malaria. Currently, only four drug classes are available to treat these infections (polyenes, azoles, flucytosine and echinocandins). This limited repertoire of antifungal drugs, combined with an alarming rise in drug-resistant fungal strains, has created an urgent need for novel therapeutic agents. In the developed world, the most common fungal disease among hospitalized patients is invasive candidiasis. Among *Candida* species, *C. albicans* and *C. glabrata* rank first and second in isolation frequency, respectively, accounting for ~70% of all systemic candidiasis. This project investigates new antifungal strategies that target the BET family of transcriptional regulators in these *Candida* species. The fungal BET protein Bdf1 contains two bromodomains (BDs) and an extra-terminal domain (ET). We recently established the proof of concept that small-molecule inhibition of Bdf1 BDs compromises the viability and virulence of *C. albicans* (Mietton et al., Nature Communications 2017).

**Methods:** This project focuses on the ET domain and is based on a combination of genetics, medicinal chemistry, structural biology and biochemistry studies.

**Results:** It shows that the ET domain is essential for the survival of *C. albicans* and *C. glabrata* *in vitro*. Furthermore, we identified a specific groove on the ET domain and demonstrate that it is also essential for growth, paving the way for the potential discovery of selective inhibitors.
**Conclusion:** To conclude, epigenetic targets remain largely unexplored in the fungal infection field. Our investigation of Bdf1 as a therapeutic target represents a novel and highly promising area of exploration in the antifungal field.

P408

Efficacy of intravenous posaconazole for the treatment of azole-resistant invasive aspergillosis

A. *Seyedmousavi*¹, J. *Kwon-Chung*², R.J.M. *Brüggemann*³, P.E. *Verweij*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, Bethesda, Maryland, United States of America, ³Pharmacy, RadboudUMC, Nijmegen, Netherlands, ⁴Medical Microbiology, RadboudUMC, Nijmegen, Netherlands

**Objectives:** Azole antifungals play a prominent role in the management of patients with invasive aspergillosis (IA). However, azole resistance is a growing problem in patients with aspergillus infection, which translates into treatment failure. Alternative treatments with new formulations of azoles may improve therapeutic outcome in IA even for the clinical cases caused by strains with low susceptibility to currently available azoles.

**Methods:** The *in vivo* efficacy of 0.25, 1, 4, 16 and 64 mg/kg/day recently introduced intravenous formulation of posaconazole (POS) was assessed in mice (CD-1 strain) infected with a wild type (POS MIC<sub>EUCAST</sub>, 0.031mg/L) and azole-resistant *Aspergillus fumigatus* (POS MIC<sub>EUCAST</sub>, 0.5 mg/L) harboring TR<sub>34</sub>/L98H mutation in Cyp-51A gene. Efficacy of POS treatment was assessed by tissue histopathology 3 days post infection and monitoring of survival for 14 days following intravenous inoculation.

**Results:** The survival curves for all control groups receiving 0.9% saline intravenously showed 100% mortality. The efficacy of POS treatment at the dosing regimens ≤4 mg/kg/day depended on the MIC of each isolate. However, the maximum effect (100% survival at day 14 post infection) was achieved with a POS dose of ≥ 16 mg/kg for both wild-type and TR<sub>34</sub>/L98H mutant isolates, and histopathological slides revealed limited number of inflammatory foci with or without detectable fungal elements in the Kidneys. The Hill-type model with a variable slope fitted the relationship between the dose and 14-day survival well, with R<sup>2</sup> values of 0.99 for the wild type, and 0.95 for the TR<sub>34</sub>/L98Hisolate. The 50% effective dose (ED<sub>50</sub>) based on survival was 0.89 mg/kg (95% confidence interval [CI], 0.59 to 1.4 mg/kg) for the wild type, and 4.5 (95% CI, 2.1 to 7.6 mg/kg) for the TR<sub>34</sub>/L98Hisolate.

**Conclusion:** Overall, treatment with intravenous formulation of POS improved the survival of the mice in a dose-dependent manner. A dose-response relationship was observed regardless of the underlying azole-resistance mechanism. These results show intravenous formulation of POS to be a promising therapeutic agent for treatment of azole-resistant IA.

P409

Anti-*Candida* activities of carvacrol, cinnamaldehyde, citral and thymol liposomes

K. Miranda-Cadena¹, M. Dias²,³, A. Costa-Barbosa²,³, T. Collins²,³, C. Marcos-Arias¹, *E. Eraso*¹, G. Quindós¹, P. Sampaio²,³

¹Inmunología, Microbiología Y Parasitología, Universidad del País Vasco/Euskal Herriko Unibertsitatea UPV/EHU, Bilbao, Spain, ²Centre Of Molecular And Environmental Biology (cbma), Department Of Biology, University of Minho, Braga, Portugal, ³Institute Of Science And Innovation For Bio-sustainability (ib-s), University of Minho, Braga, Portugal

**Objectives:** To develop and characterize carvacrol, cinnamaldehyde, citral and thymol-loaded DODAB:MO nanoparticles, to test their cytotoxicity on murine macrophages and to evaluate their anti-*Candida* activities in vitro.

**Methods:** Encapsulation was made using monoolein-based liposomes of DODAB:MO in a 1:2 molar ratio. DODAB:MO liposomes were prepared using thin lipid-film hydration method. Concentrations tested were 32, 64 and 128 mg/L of carvacrol and thymol; 16, 32 and 64 mg/L of cinnamaldehyde and