patients (positive influenza polymerase chain reaction (PCR)), 315 of them being EORTC negative. As a control group, 315 influenza negative and EORTC negative patients, admitted to the ICU with severe community-acquired pneumonia (CAP), were selected. In the 432 severe influenza patients, IPA was diagnosed in 19% (83/432), a median of 3 (IQR 0-7) days after ICU admission. The IPA incidence in the 117 immunocompromised (EORTC positive) influenza patients was as high as 32% (38/117), while 14% (45/315) of the non-immunocompromised (EORTC negative) influenza patients developed IPA. In contrast, only 5% (16/315) of the non-immunocompromised influenza-negative controls developed IPA (p<0.0001). The 90-day mortality in influenza patients with and without IPA was 51% and 28%, respectively (p<0.0001). Moreover, in this retrospective cohort study, influenza was found to be independently associated with IPA (aOR 5.2, 95% CI 2.6-10.3, p<0.0001), besides a higher APACHE II score, male sex and use of corticosteroids. Though awareness of IAA is rising, it remains unclear why patients with severe influenza infection develop IPA. Influenza-induced necrosis of the bronchial tree might provide a gateway for *Aspergillus* infection. Further, severe influenza might alter innate and adaptive host facilitating fungal infection and genetic variation might also influence the susceptibility to IPA. Finally, corticosteroid (CS) therapy is a known independent risk factor for the development of IPA at the ICU. In summary, IAA is an early and frequent complication of influenza pneumonia and increases the probability of death with a factor 2 to 3 in critically ill patients with severe influenza. An aggressive diagnostic approach should be pursued, but early diagnosis is difficult due to unspecific clinical and radiological presentation. Future studies should evaluate whether a faster diagnosis and/or antifungal prophylaxis could improve outcome of influenza-associated aspergillosis. Reference: Schauwvlieghe AFAD, Rijnders BJA, Philips N et al. Dutch-Belgian Mycosis study group. Invasive aspergillosis in patients admitted to the intensive care unit with severe influenza: a retrospective cohort study. Lancet Respir Med. 2018;6(10):782-792.

## S15.2

### Invasive aspergillosis in patients with underlying liver cirrhosis

J. Prattes

Section Of Infectious Diseases And Tropical Medicine, Medical University of Graz, Graz, Austria

**Abstract:** Invasive aspergillosis (IA) has increasingly reported in critical ill patients without classical risk factors like hematological malignancy, neutropenia or solid organ transplantation. Liver cirrhosis represents the main underlying disease in some of these patients, highlighting the relevance of cirrhosis as a risk factor for IA development. Whereas the overall incidence of IA in patients with liver cirrhosis is very low (<1%), the risk for IA increases when liver function deteriorates and patients are in need for intensive care. Reasons for susceptibility of cirrhotic patients for developing IA are diverse. Cirrhosis-associated immune dysfunction summarizes both, systemic inflammation due to cirrhosis and cirrhosis associated immunodeficiency. The latter one may be due to systemic glucocorticoid treatment but also due to alterations of the innate as well as adaptive immune functions. Impaired expression of Fcγ-receptor on neutrophils, impaired neutrophil mobilization, phagocytic activity, chemotaxis, bacterial phagocytosis and killing are present in cirrhotic patients. However, the degree of immune dysfunction correlates with the stage of the liver disease putting patients with end stage liver disease at highest risk for IA development. Awareness for IA in cirrhotic patients has to be warranted as diagnosis may be challenging. Blood biomarkers, like galactomannan and beta-d-glucan, lack sensitivity and clinical presentation may be atypical. In case of IA suspicion early chest CT scan and bronchoscopy may be needed.

## S15.3

### Aspergillus in the ICU – Management of IA in Renal Failure

R. Rautemaa-Richardson<fnref n="1" />,²,³,⁴

<footnote n="1">¹Division Of Infection, Immunity And Respiratory Medicine, University of Manchester, Manchester, United Kingdom, ²Mycology Reference Centre, Excellence Center For Medical Mycology (ecmm), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United</footnote>
Kingdom, ³National Aspergillosis Centre, Manchester, United Kingdom, ⁴Department Of Infectious Diseases, Manchester University NHS Foundation Trust, Manchester, United Kingdom

**Abstract:** Renal failure is a common complication in critically ill patients. In addition, angioinvasive aspergillosis is associated with renal lesions and renal failure. The challenge is that the main intravenous drugs used to manage invasive aspergillosis (IA), namely amphotericin B and voriconazole (with cyclodextrin) are nephrotoxic. Also, in critically ill patients with multi organ failure, the pharmacodynamics and pharmacokinetics are highly variable, which results in a significant risk for toxicity as well as suboptimal dosing and treatment failure. The impact of renal failure on the management of IA depends on whether the patient is on renal replacement therapy (RRT) or not. Once on RRT, the main consideration is the risk for suboptimal drug levels until the system is saturated with the drug. However, it is important to keep in mind that the initiation of RRT does not remove the risk for further or permanent drug-induced kidney injury. Once the patient starts to respond to the treatment, their haemodynamics stabilise and their kidney function improves, there is a risk for both toxicity and subtherapeutic levels. Therefore, in the case of azoles, critically ill patients on intensive care unit (ICU) require regular therapeutic drug monitoring (TDM) and repeated dose adjustments. This also applies to azoles not associated with kidney toxicity (isavuconazole). Whilst TDM is an important part of monitoring the safety and efficacy of antifungal treatment, microbiological diagnostics are another important tool in patient management. Sputum high-volume culture, PCR and galactomannan measurements can be used to monitor treatment response whereas blood galactomannan is less useful in non-neutropenic setting. However, it is not rare that these tests remain positive for prolonged periods despite good clinical response.

S15.4

**Fungal pneumonia in critically ill cirrhotics: Spectrum, Outcomes, Comparison of diagnostic methods and Biomarkers**

P. Kale¹, V. Khillan¹, S.K. Sarin²

¹Clinical Microbiology, Institute of Liver and Biliary Sciences, New Delhi, India, ²Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India

**Objectives:** Liver cirrhosis causes immune dysregulation and increased susceptibility to fungal infections. Further there are associated co-morbidities like diabetes mellitus, renall impairment and malignancy which predispose to fungal infections. Hence early diagnosis of fungal infections and initiation of appropriate empiric or pre-emptive antifungal is essential to reduce morbidity and mortality associated with it. We studied the epidemiology, spectrum, risk factors of fungal infections and antifungal susceptibility pattern for guiding empiric therapy. We also compared the rapid diagnostic methods and biomarkers for early diagnosis fungal pneumonia in critically ill cirrhotics.

**Methods:** Single-center, prospective cohort study of 100 critically ill cirrhotics with fungal pneumonia between January to September 2018. All patients met the diagnostic criteria for liver cirrhosis by clinical, biochemical, and ultrasonography findings. Respiratory samples were processed for fungal culture and real time PCR. Antifungal susceptibility testing was done by Broth microdilution method in accordance with CLSI guidelines. Biomarkers; bronchoalveolar lavage (BAL) and serum Galactomannan, and serum procalcitonin measured on day 1, 3, 7. Mortality within one month of diagnosis or discharge was analyzed.

**Results:** *Aspergillus flavus* was the most common species (70/100,70%). Risk factors for fungal pneumonia included neutropenia (p 0.03), steroids prior to ICU admission (p 0.02), prolonged (>21 days) hospitalization (p<0.05). Culture positivity was 80%. Culture was not inferior to real time PCR for diagnosis of fungal pneumonia. Antifungal susceptibility testing revealed, all *A.flavus* and *A.fumigatus* isolates sensitive to azoles, amphotericin B and echinocandins. BAL galactomannan was early prognostic marker with median rise >3.5 over the index value. Median PCT level was higher from day 1in the fungal pneumonia non-survivor than survivor group (3.29 vs. 0.8ng/ml), with higher 30-day mortality (72%). Baseline PCT at admission to ICU was higher in non-survivors; levels on D3 and D7 were persistently higher. Higher PCT level was associated with bacterial co-infection (48%), antibiotic (74%) and antifungal therapy and renal failure and mortality.
**Conclusion:** Cirrhotics who are neutropenic, have prolonged hospitalization and exposed to steroids have a high risk of fungal pneumonia. *Aspergillus flavus* predominate as in consensus with Asian epidemiology. Culture methods are reliable and combination of molecular test with BAL galactomannan is useful for rapid diagnosis. However the cutoff should be defined with epidemiological correlation. High serum procalcitonin level is an independent prognostic biomarker of mortality risk in fungal pneumonia which reaches nearly 70%. In our study the baseline PCT at admission to ICU was higher in non-survivor group, levels on D3 and D7 were persistantly higher. High index of suspicion and early detection is required in advanced cirrhosis

S15.5

**IP AFLU survey: Invasive Aspergillosis among Patients with Severe Influenza in Intensive Care Units**

M. Holtappels¹, C. Jacobs¹, T. Chiller², J. Fortenberry³, B. Jackson⁴, K. Lagrou⁵, M. Toda⁴, F. Van De Veerdonk⁶, P.E. Verweij⁷, J. Wauters⁸

¹Laboratory For Clinical Infectious And Inflammatory Disorders, KU Leuven, Leuven, Belgium,
²Mycotic Diseases Branch, Centre for Disease Control, Altanta, United States of America, ³Pediatric Critical Care, Children's Healthcare of Atlanta, Atlanta, United States of America, ⁴Mycotic Diseases Branch, Centre for Diseases Control, Altanta, United States of America, ⁵Department Of Microbiology, Immunology And Transplantation, KU Leuven, Leuven, Belgium, ⁶Infectieziekten, Afdeling Interne Geneeskunde, Nijmegen, Netherlands, ⁷Medical Microbiology, RadboudUMC, Nijmegen, Netherlands, ⁸Medical Icu, UZLeuven, Leuven, Belgium

**Objectives:** Invasive aspergillosis usually occurs in people with weakened immune systems. However, several reports describe fatal *Aspergillus* pulmonary infections in previously healthy patients who were hospitalized with a severe influenza virus infection. Recently, a retrospective cohort study, conducted at seven intensive care units (ICUs) in Belgium and the Netherlands during 2009–2016, found that invasive pulmonary aspergillosis occurred in 19% of patients with severe influenza requiring admission to the ICU. We conducted an international survey to determine broader knowledge and awareness of influenza-associated aspergillosis (IAA), as well as the diagnostic use of galactomannan (GM) in bronchoalveolar lavage (BAL) and serum in critically ill patients with severe influenza.

**Methods:** Members of the Extracorporeal Life Support Organization (ELSO), the Society of Critical Care Medicine (SCCM), and the European society of intensive medicine (ESICM) network (n = 20,093) were invited to participate in an online 11-question survey. Participants were asked about their roles in the ICU, ICU size, number of patients with influenza admitted to the ICU per influenza season, use of neuraminidase inhibitors (NAIs), frequency of obtaining lower respiratory specimens and GM testing on serum and BAL samples, and their knowledge on the occurrence of IAA.

**Results:** A total of 468 (2.3%) members responded, with 225 (48%) residing in the United States, 151 (32%) in Europe, and 92 (20%) in other countries, including Australia and countries in South America, Asia and Africa. Most (89%) participants were critical care physicians. Large ICU departments were more common in the United States compared with Europe. The majority of participants reported <30 influenza cases per season, and NAIs were the preferred antiviral treatment (92%). European participants reported obtaining lower respiratory specimens and determining GM on BAL and serum more commonly than U.S. participants (p < 0.001) (Figure 1). Globally, 66% (n = 303) of respondents denied hearing of or seeing IAA cases in the past five years (Figure 2). However, 57% of European participants reported seeing ≥1 patient with IAA compared with only 17% of U.S. participants (p < 0.001) and 36% of participants in other countries (p = 0.007).
<figure><img src="image_10.png" /></figure>

**Conclusion:** More participants in Europe reported having seen IAA cases in compared with the United States and other countries. Although the observed differences in IAA cases could be explained by true variation in IAA prevalence, the condition might be underdiagnosed outside Europe. The results suggest that such underdiagnosis might partly explain the differences in IAA awareness in Europe compared with the United States and other countries.

S15.6

Trend of Candidemia with Bloodstream infection in Intensive care units from 2006 to 2017: Results
from the Korean National Healthcare-associated Infections Surveillance System

Y.H. Choi¹, E.J. Kim¹, Y.G. Kwak², H.M. Yoo³, J.Y. Choi⁴, M.J. Shin⁵, S.R. Kim⁶, S.-Y. Yoo⁷, N.-H. Cho⁸

¹Department Of Infectious Diseases, Ajou University School of Medicine, Suwon-si, Gyeonggi-do, Korea, Republic of, ²Department Of Internal Medicine, Inje University Ilsan Paik Hospital, Goyang, Korea, Republic of, ³Infection Control Office, Inje University Sanggye Paik Hospital, Seoul, Korea, Republic of, ⁴Infection Control Unit, Chung-Ang University Healthcare System, Seoul, Korea, Republic of, ⁵Infection Control Office, Seoul National University Bundang Hospital, Seongnam, Korea, Republic of, ⁶Infection Control Office, Korea University Guro Hospital, Seoul, Korea, Republic of, ⁷Adjunct Assistant Professor, College of Nursing, The Catholic University of Korea, Seoul, Korea, Republic of, ⁸Department Of Infection Control, Gangnam severance hospital, Yonsei university, Seoul, Korea, Republic of

**Objectives:** Candidemia is an important healthcare-associated infections (HAIs) in intensive care units (ICUs). The incidence trend and distribution of candidemia over a 12-year period through the Korean National Healthcare-associated Infections Surveillance System (KONIS) data were analyzed.
**Methods:** The KONIS system was established in 2006, and has performed prospective surveillance for HAIs including bloodstream infection (BSI) and causative pathogen in ICU. We evaluated yearly trends of the frequencies of the causative pathogens and candidemia. All statistical analyses were 2-sided and performed the Cochran-Armitage test for trend and the Cochran-Mantel-Haenszel mean score test, by use of SAS software.

**Results:** From 2006 until 2017, 2,248 candidemia cases occurred in 9,184,264 patients-days. For BSI, the proportion of candidemia gradually increased (for BSI from 15.2% in 2006 to 16.6% in 2017, *P* < 0.05) (Figure 1). The pooled mean incidence rate of candidemia was 0.24 per 1,000 patient days. Most frequent pathogen for BSI was *Staphylococcus aureus* from 2006 to 2012, however, *candida* species emerged as the most frequent pathogen since 2013. *Candida albicans* (39.9%) was the most frequent pathogen for candidemia, followed by *C. tropicalis* (20.2%) and *C. parapsilosis* (18.2%) (Figure 2). There was no significant change in the distribution of *candida* species by year (*P* = 0.29). Central-line associated BSI were the majority (92.5%). In subgroup analysis, there was a significant association with the increase in the proportion of candidemia by year in the presence of organ transplant wards (from 18.9% in 2006 to 21.1% in 2017), hospitals with less than 500 beds (from 2.7% in 2006 to 13.6% in 2017), or surgical ICUs (from 16.2% in 2006 to 21.7% in 2017) (*P* < 0.05).
<figure><img src="image_11.png" /></figure>

**Conclusion:** The proportion of candida as nosocomial pathogen for BSI has increased in Korea. Especially, the proportion in hospitals with less than 500 beds and surgical ICUs is increasing, so appropriate infection control program is needed. This work was supported by the Research Program funded (M2018A060000011) by the Korea Centers for Disease Control and Prevention.

Symposium 16 Dermatology

S16.1

Cutaneous Aspergillosis: Is it so rare?

S. Arikan-Akdagli

Medical Microbiology, Mycology Laboratory, Hacettepe University Medical School, ANKARA, Turkey

**Abstract:** Invasion of skin by *Aspergillus* is an uncommonly encountered clinical picture. Primary cutaneous aspergillosis (PCA) indicates lesions due to direct inoculation of the fungus at the
otherwise injured site. The lesions usually develop following trauma, surgery or burn wounds. One
significant feature of PCA is that it may be observed in immunosuppressed as well as
immunocompetent individuals. Premature neonates also constitute a unique group of predisposed
hosts due to skin fragility. PCA may disseminate and be lethal particularly in immunocompromised
patients. Secondary cutaneous aspergillosis (SCA), on the other hand, develops either due to
hematogenous spread or direct invasion of the skin from adjacent infected structures, such as
paranasal sinuses. Skin and visceral involvement or multiple skin lesions in two or more
noncontiguous areas suggest disseminated *Aspergillus* infection. It is estimated that skin involvement
in invasive aspergillosis occurs at rates of 1-5%. The skin lesions in CA appear to have variable
presentations, including macule, papule, plaque, subcutaneous nodule, ulceration with necrosis, and
hemorrhagic bulla. The exact prevalence of CA is unknown and there are limited number of
dedicated series and case reports. Cases of CA are probably underdiagnosed and underreported due
also to difficulties in diagnosing fungal infections in general. In addition, the affected patient
population is heterogeneous and complex with multiple comorbidities and this further complicates
the issue. For optimal management of patients with CA, skin biopsy should immediately be taken
from suspected lesions particularly in predisposed individuals for histopathological and direct
microscopic examinations and mycological culture.

S16.2

Is resistance a problem for dermatophytosis?

P. *Nenoff*¹, M. *Monod*², S.B. *Verma*³, A. *Burmester*⁴, A. *Ebert*¹, A. *Singal*⁵, S. *Gupta*⁶, C. *Wiegand*⁴, U.-C. *Hipler*⁷, F. *Wittig*¹, C. *Krüger*¹, D. *Koch*¹, R. *Vasani*⁸, A. *Saraswat*⁹, R. *Madhu*¹⁰, S. *Panda*¹¹, A. *Das*¹¹, M. *Kura*¹², A. *Jain*¹³, Y. *Graeser*¹⁴, S. *Uhrlaβ*¹

¹Partnership Dr. C. Krueger & Prof. P. Nenoff, Laboratory of medical microbiology, Roetha OT Moelbis, Germany, ²Dermatology Service, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, ³"Nirvan" and "In Skin" Clinics, Vadodara, India, ⁴Klinik Für Hautkrankheiten, Universitätsklinikum Jena, Jena, Germany, ⁵Department Of Dermatology & Std, University College of Medical Sciences & GTB Hospital (University of Delhi), Delhi, India, ⁶Department Of Dermatology And Venereolgy, Maharishi Markandeshwar Institute of Medical Sciences and Research, Mullana, India, ⁷Klinik Für Hautkrankheiten, Universitätsklinikum, Jena, Germany, ⁸Bhojani Clinic, Mumbai, India, ⁹Dermatology, Indushree Skin Clinic, Lucknow, India, ¹⁰Department Of Dermatology (mycology), Madras Medical College, Chennai, India, ¹¹Department Of Dermatology, KPC medical college, Kolkata, India, ¹²Department Of Dermatology, Grant Medical College, Mumbai & Sir JJ Group of Hospitals, Mumbai, India, ¹³12415, Doctor's Nest, New Rajeev Gandhi Nagar, Kota, India, ¹⁴Institut Für Mikrobiologie Und Hygiene, Universitätsmedizin – Charité, Berlin, Germany

**Objectives:** Resistance of dermatophytes against antifungal agents was not perceived as a problem for decades. Therapy failure in onychomycosis or tinea capitis was never related to a reduced *in vitro* susceptibility of the causative species of dermatophytes. The situation has changed dramatically. The starting point, was, most likely, India. An incredible increase in chronic recalcitrant dermatophytoses over the past few years has been noted in India. The main causative pathogen is the zoophilic dermatophyte *Trichophyton* (T.) *mentagrophytes* ITS genotype VIII.

**Patients and methods:** Three epidemiologic exercises, for the purpose of speciation and also to look into the possibility of antifungal resistance were undertaken with cooperation from nine Indian centers with German and Swiss mycologists. A total of 291 isolates (278 *T. mentagrophytes* VIII, and 13 *T. rubrum*) were included in the terbinafine antifungal susceptibility testing and genetic point mutation analysis of the squalene epoxidase (SQLE) gene. Itraconazole and voriconazole minimal inhibitory concentrations (MICs) were determined.

**Results:** High resistance rates of 66.7% (*T. mentagrophytes* VIII), and 27.3% (*T. rubrum*) to terbinafine were found in the first multicentric study from India. 76.0% *T. mentagrophytes* VIII and 57.1% *T. rubrum* strains of two further studies (New Delhi and Mullana) showed *in vitro* resistance to terbinafine. The *T. mentagrophytes* VIII strains collected from multiple geographic regions of India showed high frequency of single point mutations in the SQLE gene leading to terbinafine resistance.
All resistant *T. mentagrophytes* strains (200) harboured missense mutations with subsequent amino acid substitutions, most frequently Phe³⁹⁷Leu (90.0%), either as a single substitution or in combination with Ala⁴⁴⁸Thr. On the other hand, terbinafine sensitive strains (78) showed almost exclusively a SQLE wild type (23.1%) or a single Ala⁴⁴⁸Thr substitution (73.1%). Substitutions less commonly encountered included Leu³⁹³Phe, Leu³⁹³Ser, Ser³⁹⁵Pro, Gln⁴⁰⁸Leu, His⁴⁴⁰Tyr and Ser⁴⁴³Pro. The number of terbinafine resistant dermatophytes in Northern India has increased significantly from 2017 to 2019 (69.0% in 2017; 72.0% in 2018; 78.0% in 2019). Additionally, we noted a rise in SQLE double mutants, which shows a selection advantage for this genotype combination.

**Conclusion:** The dramatic increase in terbinafine resistant *T. mentagrophytes* ITS VIII from all over India within such a short period of time underscores the issue of development of resistance in patients with chronic dermatophytoses. A strong association was found between *in vitro* terbinafine resistance of *T. mentagrophytes* and the occurrence of single point mutations of the SQLE gene and distinct amino acid substitutions, respectively. Alterations at position Phe³⁹⁷Leu, Leu³⁹³Ser, Leu³⁹³Phe, Gln⁴⁰⁸Leu, His⁴⁴⁰Tyr, of the SQLE were associated with terbinafine resistance. Increasing number of patients in Europe (Germany, Finland, Estonia) and from United States have been seen to be presenting with chronic recalcitrant dermatophytosis due to terbinafine-resistant *T. mentagrophytes* VIII from India. Transmission of the Indian *T. mentagrophytes* VIII to other countries due to travel, migration and in general, globalization, appears to be a serious issue even from a public health perspective. Further studies are the need of the day to understand this phenomenon better and find answers to combat it.

## S16.4

### Blastomycosis

I.S. Schwartz

University of Alberta, Edmonton, Canada

**Abstract:** Among the main endemic mycoses of North America, blastomycosis is least understood. Until recently, blastomycosis was thought to be a single clinical disease caused by a single pathogen, *Blastomyces dermatitidis*. However, recent re-analyses of isolates from North American and global collections have revealed substantial genetic and phenotypic diversity in *Blastomyces*. Classical blastomycosis, now known to be caused by *B. dermatitidis* and the cryptic species *B. gilchristii*, is limited primarily to eastern and midwestern parts of North America. In addition, *B. helicus*, a species morphologically and physiologically distinct from *B. dermatitidis* species complex, has been described as the cause of atypical, disseminated blastomycosis in immunocompromised hosts from western and mountainous parts of the United States and Canada. Moreover, blastomycosis from Africa and the Middle East - long recognized to have clinical differences from the disease in North America - has been determined to be primarily caused by *B. percursus* and an additional novel species. Clinicians and microbiologists should be aware of these additional species, how they differ from *B. dermatitidis*, and the clinical and epidemiological spectrum of blastomycosis.

## S16.5

### Galleria mellonella as a novelty model to study host-pathogen interaction in *Malassezia furfur*
CBS 1878

A.M. Celis Ramírez¹, C.M. Parra Giraldo², E.N. Pinzón Pineda¹

¹Biological Sciences, Universidad de los Andes, Bogotá, Colombia, ²Microbiology, Pontificia Universidad Javeriana, Bogotá, Colombia

**Objectives:** *Malassezia furfur* is a lipid-dependent yeast that is part of the animal and human skin mycobiota. This species can cause dermatological infections in the immunocompetent host until fungemia in immunosuppressed patients and neonates with parental lipid nutrition. However, aspects related to virulence traits and antifungal resistance is not entirely understood. Thus, the establishment of a model to study host-pathogen interaction has been a matter of attention in the last decade. The silkworm *Galleria mellonella* is a useful animal model characterized by its tolerance to a wide range of temperatures, including human temperature 37 °C; also, the immune response against pathogens is similar to the innate immunity in humans. Hence, this exciting model has been used for
the large-scale screening of fungal pathogens such as *Candida* spp., *Cryptococcus neoformans*, and the activity of antifungal drugs in dermatophytes. This study aim to evaluate the feasibility of using *G. mellonella* larvae as an invertebrate model for infection with *Malassezia furfur* CBS 1878

**Methods:** *M. furfur* CBS 1878 (Westerdijk institute, Utrecht, The Netherlands) was used for all experiments. An inoculum was adjusted to four different concentrations (1,5 x 10⁹ UFC/mL to 1,5 x 10⁶ UFC/mL) and 10 µl were injected in the last pro leg of *G. mellonella*. Survival analysis was performed for statistical processing using Prism software (GraphPad Software, Inc.). Infected larvae and controls were maintained at 33°C and checked daily. Hemolymph analysis was conducted to evaluate the capacity of phagocytosis of hemocytes. Experiments to corroborate the fungal load were also conducted. Each treatment was conducted in triplicate (technical replicates), and the whole experiment was repeated three times (biological replicates). Statistical analysis were performed using R package.

**Results:** Survival test reveals that at least all silkworms injected with *M. furfur* CBS 1878 at 1,5x10⁹UFC/mL died within five days, whereas all silkworms injected with saline were alive. Fungal load data were in agreement with the inoculums tested. Besides, activation of the immune system of *G. mellonella* and the interaction between the yeast with different cells of the hemolymph was observed.

**Conclusion:** This study is the first approach to the implementation of an invertebrate model *Galleria mellonella* to the future study of the host-pathogen interaction and the antifungal activity in *M. furfur* CBS1878. We demonstrated that injection of this yeast into silkworm hemolymph killed silkworms. We found the most effective inoculum was 1,5x10⁹UFC/mL with a short time of survival on average of five days. This model promise to be an efficient infection model to conduct easy and reliable approaches that perform to underpin our knowledge in *Malassezia* genus.

# Symposium 17 HIV associated cryptococcal meningitis

## S17.3

### Towards having antifungal drugs in low-resource areas

I. Kolte¹, A. Sturny Leclère², T. Boyer-Chammard², S. Lesikari Kivuyo³, S. Mfinanga³, C. Kanyama⁴, C. Kouanfack⁵, O. Lortholary⁶, F. Dromer⁷, T. Bicanic¹, S. Molloy¹, T. Harrison¹, A. Loyse¹

¹Infection & Immunity, St Georges University of London, London, United Kingdom, ²Molecular Mycology, IP Paris, Paris, France, ³National Institute for Medical Research, Dar es Salaam, Tanzania, ⁴UNC Project-Malawi, Lilongwe, Malawi, ⁵Hôpital Central de Yaoundé, Yaoundé, Cameroon, ⁶Infectious Diseases, APHP, Hopital Necker-Enfants malades, Paris, France, ⁷National Reference Center For Invasive Mycoses & Antifungals, Molecular Mycology Unit, Cnrs Umr2000, Institut Pasteur, Paris, France

**Abstract:** The results of the ACTA trial underpinned the development of new 2018 WHO guidance for the treatment of cryptococcal meningitis with short course amphotericin B deoxycholate (AmBd) + flucytosine (5FC) becoming the new gold standard for resource limited settings. The alternative recommended regimen is a two-week oral course of 5FC and fluconazole. However, access to key diagnostic tests such as the cryptococcal antigen lateral flow assay (CrAg LFA) and essential antifungal medicines including 5FC for the treatment of HIV-related cryptococcal meningitis is severely lacking in African low-and middle-income countries (LMICs) where disease burden and mortality is highest. This talk will highlight longstanding advocacy and implementation efforts to ensure access to these lifesaving tools and train frontline healthcare workers (HCWs) working on their optimal use so as to effectively reduce mortality in resource limited settings. We will focus in particular on illustrating the respective work of the cryptococcal meningitis action group (cryptoMAG), the DREAMM project, and outline the recently announced Unitaid/CHAI project on advanced HIV disease. The cryptoMAG group is an international group of stakeholders including St George's University of London (SGUL), Centers for Disease Prevention & Control (CDC), WHO, Médecins sans Frontières (MSF) and Institut Pasteur amongst notable others that has been advocating for access to diagnostic tests and essential medicines for cryptococcal meningitis since 2013.
DREAMM (Driving REduced AIDS-associated Meningo-encephalitis Mortality) is an implementation project currently ongoing in Tanzania, Cameroon and Malawi. It uses mixed methodology including co-designed training programs and local health system strengthening centred around African leadership to effectively and sustainably reduce mortality linked to HIV-related meningitis, a leading cause of advanced HIV disease. Lastly, in January 2019 Unitaid announced its program on AHD in partnership with the Clinton Health Access Initiative (CHAI). This program will enable access to the CrAg LFA and essential antifungal medicines including 5FC in 7 African LMICs, cryptococcal meningitis being a leading cause of death from advanced HIV disease.

## S17.4

### Fluconazole resistance in cryptococcal meningitis

T. Bicanic

Infection & Immunity, St Georges University of London, London, United Kingdom

In much of sub-saharan Africa, where Cryptococcus is the most common cause of community acquired meningitis, the gold standard treatment of Amphotericin B with flucytosine is not available, and fluconazole monotherapy is the norm for all phases of CM treatment: induction, consolidation and maintenance. Even at higher doses of 800-1200 mg/d, fluconazole is slow to clear Cryptococcus from CSF. Although primary resistance is rare in global surveys using MIC testing, the intrinsic mechanism of fluconazole resistance, reversible disomy of chromosome 1, is unstable: in the absence of drug pressure, it occurs in only a very small subpopulation of colonies and will be missed by conventional MIC testing. In a study in Tanzania in patients with HIV-associated cryptococcal meningitis, we performed serial lumbar punctures in 20 patients receiving fluconazole monotherapy versus fluconazole combined with flucytosine, plotting the rate of clearance of the total population and subpopulation able to grow on fluconazole, and saving colonies for subsequent whole genome sequencing. Fluconazole heteroresistance was detectable in all clinical Cryptococcal isolates from CSF of all patients prior to initiation of therapy. The proportion of resistant colonies in CSF increased during the first two weeks of treatment in all patients receiving Fluconazole at 800-1200 mg/d, but was suppressed in those receiving combination with flucytosine. Genomic analyses revealed high rates of aneuploidy in heteroresistant colonies, as well as in clinical isolates from patients experiencing relapse. The predominant mechanism in human infection was disomy of chromosome 1 (containing the drug target gene ERG11 and the efflux pump AFR1) and heteroresistance positively correlated with in vitro drug efflux pump activity. We also undertook fluconazole PK analyses in CSF for a subpopulation of patients: Monte Carlo simulations from the clinical PK/PD model predicted that only a minority of patients (13%) receiving doses of fluconazole at 1200 mg/d sterilise their CSF after 2 weeks, with 83% having a persistant subpopulation resistant to fluconazole. Our findings demonstrate that fluconazole when used alone is a sub-optimal agent for treating cryptococcal meningitis due to rapid selection of a resistant sub-population which becomes predominant within 2 weeks. Efforts are underway to make the combination oral therapy of fluconazole with flucytosine more widely available and implementable in Africa.

## S17.5

### Current and future clinical trials on HIV-associated cryptococcal meningitis.

T. Harrison

Centre For Global Health, Institute Of Infection And Immunity, St George's University of London, London, United Kingdom

The ACTA trial demonstrated that in centres in Sub-Saharan Africa, an induction regimen of 1 week amphotericin B (AmB) and flucytosine (5FC), followed by fluconazole (FLU) 1200 mg/d in the second week, was associated with reduced 10-week mortality compared with the then internationally recommended regimen of 2 weeks of AmB+5FC. 2 weeks of the oral combination of FLU plus 5FC was non-inferior to this standard; and 5FC was superior to FLU, as the partner drug given with AmB. The results have been further supported through 12-month outcome and economic analyses, and
have driven both a change in WHO guidelines for the treatment of HIV-associated cryptococcal meningitis, and increasing access to 5FC through a Unitaid programme. The ongoing AMBITION-CM trial is testing single high dose (10mg/kg) liposomal amphotericin B (L-AmB), given with a 2-week oral FLU+5FC backbone, against the new standard of 1 week AmB+5FC. A randomized phase II study had shown non-inferior early fungicidal activity (EFA) with single high dose L-AmB vs daily L-AmB at 3 mg/kg/d for 14 days. The hope is that the L-AmB arm will be at least as effective and better tolerated and easier to deliver than one week of AmB deoxycholate. The trial includes a pre-planned health economic evaluation. Over 300 participants have been enrolled. Any further improvements in treatment may depend on introduction of new drugs into induction regimens, or novel host-directed or mechanical strategies. Viamet-1598, a fungal Cyp51 inhibitor, and a compound series from Amplyx Pharmaceuticals that target Gwt1, an enzyme required for fungal cell wall localization of GPI-anchored mannoproteins, are 2 promising candidates, and it is hoped both can advance to phase II EFA studies. Immunotherapeutic approaches should be further investigated but will need to be carefully targeted based on rapid assays of patient specific immunity. Investigators at Duke University are exploring the possibility of using neurapheresis to filter fungal cells from the CSF and rapidly reduce the organism load. Meanwhile efforts continue to prevent the development of clinical cryptococcal infection in patients with advanced HIV disease, and improve the results of the cryptococcal antigen (CrAg) screen and pre-emptive treatment strategy for patients with low CD4 cell counts. Despite pre-emptive fluconazole, mortality for those screening CrAg positive is significantly higher than for those who are CrAg negative; many of those with high CrAg titre have evidence of sub-clinical meningitis; and fluconazole failures have been increasingly well documented. A study looking at single high dose L-AmB to treat CrAg positive patients in screening will take place in Uganda, and a trial to test the oral FLU+5FC combination against FLU alone for CrAg positive patients identified at screening is planned in South Africa and Tanzania.

# Symposium 18 Antifungal stewardship in the era of resistance

## S18.1

### Stewardship and azole resistant aspergillosis: a challenge for farmer or physician?

P.E. Verweij

Center of Expertise in Mycology Radboudumc/CWZ, Nijmegen, the Netherlands
Aims of antibiotic stewardship include treating infectious diseases with the appropriate drug, preventing overuse and minimizing resistance selection. These goals are frustrated by the emergence of azole resistance in Aspergillus fumigatus. Recent studies showed that voriconazole-treated patients with invasive aspergillosis have a 20% lower survival when the infection was caused by a voriconazole-resistant isolate compared with voriconazole-susceptible infection. Appropriate initial therapy is thus critical but diagnosis of resistance is slow when based on fungal culture and MIC-testing. To ensure appropriate initial antifungal therapy combination antifungal therapy is recommended if resistance frequencies exceed 10%. The Dutch guideline for management of invasive aspergillosis indeed recommends voriconazole/isavuconazole in combination with an echinocandin of liposomal amphotericin B is the azole-susceptibility is unknown as resistance surveillance indicated a resistance rate of 15%. Therefore, many patients are exposed to increased toxicity and drug interactions, while the majority still have an infection due to an azole-susceptible isolate. To improve antifungal stewardship programs accurate resistance surveillance is required as well as rapid and sensitive diagnostic resistance tests. As the main driver of resistance to medical azoles in A. fumigatus is azole fungicide use in the environment, interventions are needed to reduce the burden. Recent studies have identified specific conditions that facilitate resistance selection and the next step is to design and evaluate interventions that preclude resistance selection in the environment.

The azoles represent an important class for both medical treatments and food production. Maintaining this class for both application is therefore critical and requires investment in rapid diagnostic tests and stewardship programs.
S18.2

Rapid diagnosis of fungal infections: Impact on stewardship

S. Kanj

Department Of Internal Medicine, Division Of Infectious Diseases, American University of Beirut Medical Center, Beirut, Lebanon

Invasive fungal infections (IFI) remain a challenge to the treating physician because of difficulty in diagnosis and high rates of morbidity and mortality associated with these infections, which occur predominantly in the immunosuppressed and critically ill patients. The incidence of invasive fungal infections, especially candidiasis, aspergillosis, and mucormycosis, continues to rise. With the excessive use of azole antifungal agents, we have witnessed a global shift of fungal population to more resistant species and genera. In addition, the increased consumption of antifungal agents in agriculture have selected for resistant *Aspergillus* species. There is a wide variation in the epidemiology of fungal infections worldwide. It is no longer acceptable to manage IFI without identifying the species of the infecting organism and its susceptibility profile to the various antifungal agents. Neighboring countries might have a different epidemiology depending on antifungal practices in humans and the environment. Whereas guidelines can be of great help in the management of patients, making the correct diagnosis is of paramount importance. Studies have shown that delay in the initiation of appropriate antifungal therapy is associated with a significant increase in mortality. Therefore, there is an urgent need to make a prompt diagnosis of IFI. In the past, histopathology, fungal cultures, and Candida scores in the right clinical setting guided the diagnosis and therapy of IFI. More recently, the availability of newer diagnostic tools such as Aspergillus galactomannan, β-D glucan, and polymerase chain reaction, has been of great help in speeding the diagnosis of IFI. These tools have also been used for screening and guiding pre-emptive therapy in high-risk patients. Studies have shown that combining these new tools increases the sensitivity and specificity with an impact on fungal infection-free survival in some patients and allows for early discontinuation. More recently, the development of new molecular tools such as Matrix-Assisted Laser Desorption/Ionization – Time of Flight, FilmArray, Light Cycler, T2Candida assay, and others, allows the rapid detection of fungal pathogens and determines resistance markers with a turnaround time of only a few hours. Investigational tools such as Proximity Ligation Assay, Breath Fungal Secondary Metabolite Signature, and Siderophore-based Molecular Infection Imaging are also promising in facilitating the diagnosis of IFI in the future. In addition, the development of point-of-care testing is helpful, especially in diagnosing cryptococcal meningitis in some developing countries where access to microbiology laboratory is limited, or in diagnosing invasive Aspergillosis using Lateral Flow Devices. All these new diagnostic tools have a tremendous impact on antifungal stewardship as they allow physicians to discontinue antifungal agents earlier when not needed or to de-escalate them to narrower-spectrum agents. These efforts will ultimately translate into decreasing antifungal resistance and reducing costs to healthcare, which are essential elements of stewardship.

S18.5

Optimising antifungal stewardship: An evaluation of candidaemia guideline compliance and clinical outcome

L. Cottom¹, B. Jones²

¹Department Of Medical Microbiology, Glasgow Royal Infirmary, NHS Greater Glasgow & Clyde, Glasgow, United Kingdom, ²Department Of Medical Microbiology, Glasgow Royal Infirmary, Glasgow, United Kingdom

**Objectives:** Candidaemia remains an important cause of nosocomial infection, associated with significant morbidity and mortality. Over the last decade there has been a growing awareness that improvements to stewardship and robust surveillance is needed to ensure the judicious use of antifungal therapy. A guideline should never be construed or serve as a standard of care, rather recommendations should always be interpreted alongside individual patient findings and be guided by local epidemiology and antifungal susceptibility knowledge. The aim of this study was to evaluate
compliance with the current 2016 Infectious Diseases Society of America (IDSA) guidelines and assess the impact on clinical outcome. Adherence to management recommendations was assessed using the EQUAL Candida Score of the European Confederation of Medical Mycology (ECMM). A secondary aim was to assess clinical outcome (30 day, 90 day and all-cause mortality) with an echinocandin compared to fluconazole as the initial treatment (primary therapy) for candidaemia.

**Methods:** A retrospective analysis was performed over 18-months (January 2017 to June 2018). Data was analysed from five large university teaching hospitals within Greater Glasgow and Clyde; the largest NHS organisation in Scotland serving a population of 1.2 million. Patients were identified using our laboratory information management system and electronic clinical record systems. Only cases where a diagnosis of candidaemia had been confirmed and the patient had been actively treated were included for analysis. The source of infection and antifungal management was reviewed for each patient. The EQUAL candida score was calculated for each case. The 30-day, 90-day and all-cause mortality rate was determined for the study population.

**Results:** A total of 131 patients were identified as having a candidaemia. The most common *Candida* species isolated was *C.albicans* (40%; 53/131) followed by *C.glabrata* (27%; 35/131). An echinocandin was initiated as primary therapy in 52% (68/131) of cases. In 62% of cases the patient had an indwelling line that was attributed as the direct source/nidus of infection. 21% of cases were related to a urinary tract source and/or urological instrumentation. The mean EQUAL Candida Score was found to be 13 (range 9-17), with the maximum score for guideline adherence being 16 for non-CVC patients and 19 for CVC carrier (score adjusted as initial blood culture volume was not known for study population). No statistically significant difference in clinical outcome (30 day mortality) was found when comparing an EQUAL Candida Score of ≤13 with > 13 (chi-square calculation; *p*-value 0.76). For the study population, a statistically significant difference in clinical outcome (30 day mortality) was found when comparing fluconazole to an echinocandin as initial treatment/primary therapy with a survival benefit being found in favour of fluconazole (chi-square calculation; *p*-value 0.039).

**Conclusion:** Our study provides a valuable evaluation of compliance. Interesting, increased compliance was not found to improve clinical outcome. Furthermore, the results from our study suggest that fluconazole as initial therapy was non-inferior to an echinocandin for the treatment of candidaemia, with a survival benefit being observed. This study supports the importance of an effective program of surveillance to promote antifungal stewardship and the judicious use of antifungal agents.

# Symposium 19 Pneumocystis

## S19.1

### Recent diagnostic strategies in Pneumocystis pneumonia

#### A. Alanio

Paris-diderot, Sorbonne Paris Cité University, Institut Pasteur, Molecular Mycology Unit, Cnrs Cmr2000, Parasitology-Mycology Laboratory, Lariboisière Saint-Louis Fernand Widal Hospitals, Assistance Publique-Hôpitaux de Paris, Paris, France

Pneumocystis jirovecii pneumonia (PCP) is one of the most commonly diagnosed opportunistic infection in HIV-positive patients. In addition, the rising number of immunocompromised HIV-negative patients at risk of P. jirovecii infection (immunosuppressive therapies, allogeneic bone marrow or solid organ transplantation) is an emerging concern. PcP is difficult to diagnose in HIV-negative patients owing to the non-specific pulmonary symptoms and signs associated. Microscopic visualization of cysts or trophic forms in respiratory specimens based on immunofluorescence stainings is the most sensitive microscopic method and still considered as the gold-standard test to diagnose this infection. Bronchoalveolar lavage fluids are the most sensitive specimen type, since P. jirovecii is an organism associated to lung alveoli. PCR-based methods play an increasing role in the lab, initially developed to circumvent decreased fungal load in HIV-negative patients and detection in upper respiratory specimens. Real-time quantitative PCR (qPCR) is definitely the only format
adapted to detect P. jirovecii since the risk of contamination is minimal and quantification is possible. A negative qPCR in BAL, but not in non-invasive respiratory specimens, allows to rule out PCP diagnosis. Quantitative results have been used for years to try to discriminate PCP (high fungal load) from carriage/colonization (low fungal load). However, these methodologies have limited use since intermediate fungal load are inconclusive. Recent advances in the comparison of the performances of the different qPCR assays showed that standardization is certainly one of the clues to move forward with this question. In parallel, the combination with (1-3)-β-D-Glucan (BG) detection in serum helps but do not completely resolve the problem. BG detection has a high sensitivity and a high negative predictive value for diagnosis of PCP in immunocompromised HIV-positive and -negative patients, but a positive result may also indicate the presence of other invasive fungal infections. The clinical utility of these diagnostic tests and the diagnostic strategies in HIV or non-HIV patients should be further assessed in prospective, randomized interventional studies. In the meantime, the status of the host should be better defined with regard to the evolution of P. jirovecii carriage. New screening and prophylaxis strategies to prevent transmission in hospital settings should be proposed and discussed

S19.2

**Management of *Pneumocystis Pneumonia* in HIV-infected Patients**

J. Kovacs

Associate Clinical Professor of Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, United States of America

*Pneumocystis* pneumonia (PCP) remains one of the most common life-threatening opportunistic infections in HIV-infected patients, despite the tremendous benefits seen with combination antiretroviral therapies. PCP continues to be seen in the current era primarily because at-risk patients are unaware of their HIV infection or are not in continuous medical care. Infection appears to represent recent acquisition in many cases rather than reactivation of latent infection. Primary clinical manifestations include fever, shortness of breath, and a non-productive cough. CXR will typically show diffuse infiltrates though it may be normal in 10-20% of cases; chest CT scans will almost invariably be abnormal, however. Diagnosis is based on detection of the organism in respiratory samples such as induced sputum or BAL. Because the organism cannot be cultured, organism detection relies on colorimetric or immunofluorescent staining, or PCR assays. The latter are highly sensitive but less specific than the former, in part because PCR can identify colonization or subclinical infection not requiring therapy. Assays to measure (1-3)-β-D-glucan levels have poor specificity and should not be relied on as the exclusive diagnostic modality. The preferred first-line drug for treatment is trimethoprim-sulfamethoxazole, which targets 2 steps in *Pneumocystis* folate metabolism. Alternatives for patients with mild to moderate disease (A-a O₂ gradient <45 mm Hg) include clindamycin-primaquine, trimethoprim plus dapsone, and atovaquone. Alternatives for patients with more severe disease requiring parenteral therapy include clindamycin-primaquine (primaquine is available only for oral administration) and pentamidine. Caspofungin should not be used as a single agent since it affects only the cyst form and not the more numerous trophic form of the organism; further, there are no controlled studies evaluating its efficacy. Corticosteroids should be administered in patients with PaO₂<70 mm Hg or (A-a) O₂ gradient >35 mm Hg, ideally within 3 days of starting therapy, and be continued using a tapering regimen while anti-*Pneumocystis* therapy is administered. Although there are no randomized trials, meta-analyses suggest that clindamycin-primaquine is more efficacious than pentamidine. For patients failing therapy, there are no randomized trials to address optimal management, and whether it is better to add drugs or to change regimens. PCP can be effectively prevented in at-risk patients (e.g. CD4 <200 cells/mm³) by using trimethoprim-sulfamethoxazole, or alternatively dapsone, dapsone-pyrimethamine, atovaquone, or aerosol pentamidine. In patients recovering from an episode of PCP, secondary prophylaxis should be initiated after completion of therapy. Prophylaxis can be discontinued after the immune system has reconstituted to CD4 >200 cells/mm³ for at least 3 months. In patients with poor immune reconstitution (CD4>100 but <200 cells/mm³) but with HIV suppression to below detection limits of
commercial assays (typically <20 copies/ml) for 3-6 months, prophylaxis can also be discontinued. Prophylaxis should be continued in patients with CD4 <100 cells/mm³ even with HIV suppression. Mutations have been identified in the dihydropteroate (DHPS) gene of *Pneumocystis*, the target of sulfamethoxazole and dapsone, primarily in patients receiving those drugs for prophylaxis. Although these mutations appear to confer at least low level resistance, their clinical significance is uncertain.

## S19.4
### Pneumocystosis in neonates

**E. Calderon**

Instituto de Biomedicina de Sevilla and CIBER de Epidemiología y Salud Pública, Seville, Spain Pneumocystosis has been well recognized as a severe pulmonary disease and an important cause of morbidity and mortality in premature infants or debilitated neonates since the mid-twentieth century when *Pneumocystis* was acknowledged as the causing agent of interstitial plasma cell pneumonia. This form of pneumocystosis was observed before, during and after World War II in Europe, where it reached epidemic proportion with more than 500 cases confirmed by autopsy. Unexpectedly, this epidemic pneumocystosis disappeared in the same manner it had been emerged in Europe. Although data on pediatric *Pneumocystis* pneumonia (PcP) from developing countries are scarce mostly based on small series of cases, PcP in infants seem to remain as an important medical problem in those areas. Nowadays, the interest in *Pneumocystis* infection goes beyond PcP because a new spectrum of *Pneumocystis*-related disease seems to emerge in immunocompetent infants. Serological studies and use of molecular techniques to identified *Pneumocystis jirovecii* have shown that this microorganism probably is one of the more frequent infectious agents faced by humans in everyday life and that the first exposition to this pathogen happen in most children early in life, probably in the neonatal period. However, primary *Pneumocystis* infection currently goes unrecognized because it has been presumed to be an asymptomatic or mild nonspecific disease. However, recent reports indicate that it can present clinically as a self-limiting upper or lower acute respiratory tract infection. *Pneumocystis* infection was identified by specific PCR in 24% of infants with bronchiolitis from France and in 29.4% of Cuban infants and toddlers with whooping cough. In another study of 422 children hospitalized with acute respiratory tract infection, *Pneumocystis* was detected in 16% of infants. However, a marked difference occurred in the age distribution, as the prevalence was 48% in infants ages 50 to 112 days, 13% in infants ages 113 to 265 days, and 2% in infants younger than 49 days. In a study carried out in Santiago de Chile, *Pneumocystis* DNA was detected in specimens from 51.7% of infants who died unexpectedly in the community, but only 15% had pneumonia. This study also showed that *Pneumocystis* infection is more frequent than viruses before the age of 6 months, and that it has a consistent peak between 2 and 5 months of age. The significance of these findings is unclear but shows the high prevalence of *Pneumocystis* infection in infants. Recently, in Spain, a high prevalence of *Pneumocystis* infection has been described in preterm neonates and this infection was associated to higher risk to develop neonatal respiratory distress syndrome. Increasing evidence suggests that the most common respiratory infection-affecting infants is the mild and sneaky, primary infection by *Pneumocystis*. This infection goes currently unrecognized and has been neglected as a subclinical irrelevant infection by contrast with the severe PcP affecting the immunocompromised patients. However, compelling new evidence suggests that this infection may be pathogenic to certain infant age groups and that microbiome host interactions in early life may condition the development of altered immune responses in older infants or adults.

## S19.5
### Evaluation of a Novel Commercial Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of *Pneumocystis jirovecii*

D. Schmidt, U. Scharmann, J. Buer, P.-M. Rath

Institute Of Medical Microbiology, University Hospital Essen, Essen, Germany
**Objectives:** *Pneumocystis jirovecii* pneumonia (PCP) is an increasing problem in immunocompromised patients. Detection of *P. jirovecii* in respiratory samples is usually based on microscopy or PCR. Both methods require experienced technical personnel and are either insensitive or time-consuming. In this study, a prototype of the eazyplex *Pneumocystis jirovecii* assay (Amplex Diagnostics, Germany) was established. The assay is based on LAMP technique (loop-mediated isothermal amplification). It requires minimal hands on time for sample manipulation (below 5 minutes) and performance of the assay itself (25 minutes) and could be a reliable technique to detect *P. jirovecii*.

**Methods:** In total, 145 respiratory samples from 134 critically ill patients (65% male, median age = 64 years) were investigated, using the LAMP assay in comparison with the RealStar Pneumocystis jirovecii PCR 1.0 (altona Diagnostics, Germany). For the LAMP assay 25µl of sample were mixed with 125µl RALF buffer (resuspension and lysis fluid, Amplex) and incubated for 3 minutes at 99°C. 25µl of this suspension were added to the wells of the test strip afterwards. The LAMP assay was performed in a Genie II Mk2 device (Amplex). For qPCR, 300µl of sample were extracted using the Maxwell16 nucleic acid extraction platform (Promega, Germany). 10µl of DNA were used for qPCR according to the manufacturer's recommendations. QPCR was performed on a RotorGeneQ cycler (Qiagen, Germany) in a final volume of 30µl. To determine the detection limit of the LAMP assay, compared to the qPCR, tenfold serial dilution of extracted P. jirovecii DNA and of a high positive P. jirovecii respiratory sample were investigated in both assays. Additionally, for each serial dilution time-to-positivity of the LAMP assay was plotted against cycle threshold values (ct values) of the qPCR. For quantification of the fungal load the quantification standards of the RealStar qPCR were used (1x10⁴ -1x10¹ copies/µl).

**Results:** The LAMP assay detected 17 of 19 qPCR positive samples and 117 of 117 qPCR negative samples. The assay was invalid in nine samples that were negative by qPCR. Time-to-positivity of the LAMP assay ranged from 11-22 minutes in patient samples. Detection limit of the assay was approximately 15-20 copies of genomic DNA/µl in patient samples and in extracted DNA which corresponded with ct values of 28-29 in the qPCR.

**Conclusion:** The LAMP assay is an accurate and time saving tool for the detection of P. jirovecii in respiratory samples. Two samples that were positive by qPCR were not detected by the assay which is probably due to the significantly lower sample volume and the crude extraction method the assay uses. Nevertheless, the convenience of the performance of the assay and interpretation of the results allows laboratories with less experience and less technical equipment to perform molecular assays on a high standard.

## Symposium 20 Candida auris

S20.1

Schizophrenic gram negative yeast conquering the world

J. Meis

Department Of Medical Microbiology And Infectious Diseases, Excellence Center For Medical Mycology (ECMM), Canisius Wilhelmina Hospital, Nijmegen, Netherlands

*Candida auris* has been named as the new "fungal superbug" which poses a significant threat to public health during outbreaks. *C. auris* was first named and described only 10 years ago. Follow-up genomic analysis has revealed that *C. auris* has emerged almost simultaneously in this short period on 3 different continents. At present 4 major clades have been identified and last month a fifth clade was discovered. In the beginning *C. auris* was often mistaken for other Candida or yeast species when using phenotypic assimilation/fermentation identification tests. Wide introduction of MALDI-TOF technology has significantly improved accurate identification in the past few years and is now the gold standard in addition to molecular sequencing. *C. auris* has also shown resistance to several different antifungal drugs and classes of drugs. While its resistance profile varies geographically, this new yeast pathogen has acquired resistant to fluconazole, a key drug in the azole class of antifungal drugs. *C. auris* spreads in healthcare facilities in a similar way as bacteria while the occurrence and
niche in the environment is elusive. Infections have been reported in a range of countries across
continents, but current estimates are probably inaccurate. Impervious to both antiseptics and the
three major classes of anti-fungal medications, *C. auris* has been quietly transmitted within hospitals
and nursing homes in at least 37 countries such as the UK, Spain, India, Pakistan, Middle East, South
Africa, Latin America and parts of the USA. Patients can be colonised with *C. auris* without becoming
sick, but the pathogen seems to prefer to colonise patients who are already sick or
immunocompromised (eg, cancer or transplant patients), and the very young or very old among
hospitalised inpatients. Among at-risk patients who have *C. auris* candidemia or invasive candidiasis,
depending on the world region, high mortality rates (~ 30–60%) have been observed. But because
infection typically occurs when a person is already very sick, it may be difficult to disentangle the
attributable mortality. It is important to realize that *C. auris* is predominantly a healthcare-associated
infection. Prior broad-spectrum antibiotics and antifungal use can influence the risk of infection.
Finally an important question is still unanswered *C. auris: Unde venis et quo tendis?* Reference Meis JF,
Chowdhary A. *Candida auris*: a global fungal public health threat. Lancet Infect Dis. 2018;18(12):1298–
1299

S20.3

**Outbreak control: "Controlling a multidrug-resistant *Candida auris* outbreak"**

A. Ruiz-Gaitan¹,²

¹Severe Infection Research Group, Medical Research Institute La Fe, Valencia, Spain, ²Department Of Clinical Microbiology, La Fe University and Polytechnic Hospital, Valencia, Spain

**Background:** *Candida auris* is an emerging, multidrug-resistant yeast causing hospital outbreaks. The outbreaks by *C. auris* described in Spain as well as in other countries with large outbreaks, are characterized by an exponential increase in the number of cases in a short period, suggesting a high transmission rate. **Objectives** We report the first 24 months of the ongoing *C. auris* outbreak in a tertiary hospital in Spain. The epidemiological, clinical and microbiological characteristics of candidemia episodes and environmental samples by *C. auris* were also analyzed. **Results:** 228 patients were involved in the case-control study (114 colonized/candidemia and 114 controls). All candidemia episodes were observed in adult patients (21–81 years old) and 87.8% of them were admitted to SICU. The most common underlying condition observed in both colonized and candidemia patients was polytrauma (n = 13, 32%) followed by cardiovascular disease (n = 10, 25%) and cancer (n = 7, 17%). Indwelling CVC (odds ratio {OR}, 13.48), parenteral nutrition (OR, 3.49), and mechanical ventilation (OR, 2.43) were the more frequently invasive procedures observed in these two groups and remained significant predictors of *C. auris* colonization/candidemia. All *C. auris* isolates were resistant to fluconazole (MICs >64 mg/L) and had significantly reduced susceptibility to voriconazole (GM, 1.8 mg/L). All isolates were susceptible to itraconazole, posaconazole, isavuconazole, and echinocandins. Environmental sampling showed presence of the *C. auris* on sphygmomanometer cuffs (25%) patient tables (10.2%), keyboards (10.2%), and infusion pumps (8.2%). **Conclusions:** - Predictor conditions to *C. auris* colonization/candidemia are similar to other Candida species. *C. auris* colonizes multiple patient's environment surfaces. All isolates are resistant to fluconazole and had significant reduced susceptibility to voriconazole. - Due to its high transmissibility and survival in the hospital environment, *C. auris* can cause long duration outbreaks that are difficult to detect in early stages, and it makes it difficult to control and eradicate. - The implementation of early and strict surveillance and control measures is essential to preventing the spread of the outbreak representing a significant risk to critical patients. - Immediate notification of *C. auris* to clinical and infection control teams, as well as to health authorities and institutions, is essential to implementing infection control precautions at all levels in a timely way, to prevent transmission inside and outside the hospital and to prevent the development of infections in patients who are already colonized. - This report is intended to demonstrate not only the complexity of handling and containing a *C. auris* outbreak, but also how, with the use of a series of effective infection prevention measures, the spread of this pathogen can be successfully controlled.
S20.4

Candida auris: in developing world

Anuradha Chowdhary

Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India Candida auris a multidrug-resistant(MDR) yeast that exhibits resistance to fluconazole and markedly variable susceptibility to other azoles, amphotericin B, and echinocandins has globally emerged as a nosocomial pathogen that can cause invasive infections. Candida auris was first described in 2009 by Satoh et al. as a novel Candida species, in the Candida haemulonii complex(Metchnikowiaceae), from a patient in Japan after its isolation from the external ear canal. Subsequent to the first nosocomial outbreak in South Korea in 2011, several hospitals associated outbreaks have been reported in developing world especially in India, Pakistan and Africa. Alarmingly, in less than a decade this yeast, which is difficult to treat, has become widespread across several countries causing a broad range of healthcare associated invasive infections that display clonal inter- and intra-hospital transmission. C. auris in routine microbiology laboratories remains an unnoticed pathogen as 90% of the isolates characterized by commercial biochemical identification systems such as API 20C, Vitek 2 (bioMérieux), Phoenix (BD), and MicroScan (Beckman Coulter), misidentify as a range of other Candida species. Most commonly, these isolates have been misidentified as C. haemulonii, but also C. famata, C. sake, Rhodotorula glutinis, Rhodotorula mucilaginosa, and Saccharomyces species. Rarely, C. auris has been identified as C. catenulate, C. lusitaniae, C. guilliermondii, or C. parapsilosis. However, MALDI-TOF MS is considered a more rapid and robust diagnostic technique for C. auris identification. Mass spectra can be easily added to the MALDI-TOF MS database, leading to accurate identification of C. auris to the species level. Sequencing of genetic loci, including D1/D2, RPB1, RPB2, and internal transcribed spacer (ITS) domains of the rRNA, has proven useful in the identification of C. auris, but it is not routinely used.

However, in the routine microbiology laboratories this yeast remains unidentified especially in
developing world due to lack of fully equipped mycological diagnostic facilities. Further, the true
burden of C. auris remains unexplored as effective surveillance of Candida spp is not available in
several countries in the developing world. Antifungal susceptibility data for C. auris is of primary
concern as C. auris exhibit consistently high fluconazole MICs and variable susceptibility to the other
azoles, echinocandins and amphotericin B. No antifungal clinical breakpoints reported for C. auris.
Studies examining the susceptibility of this organism to antifungals have used a variety of methods,
including Clinical and Laboratory Standards Institute (CLSI) broth microdilution, Etest, and the Vitek
2 yeast susceptibility system. Regarding azole resistance mutations in Erg11 associated with the
development of fluconazole resistance in C. albicans have also been detected in C. auris isolates. The
fact that this yeast exhibit MDR clonal strains which are nosocomially transmitted is unusual in other
Candida species. Therefore, the possible threat of its rapid spread in affected countries and its
emergence in unaffected countries will not only challenge clinicians for its effective therapeutic
management but will also bring high economic burden to countries especially in resource limited
settings where modern identification facilities and access to antifungals other than FLU are limited.

S20.5

Understanding Echinocandin Activity Towards *Candida auris*

M. Kordalewska¹, A. Lee¹, R. Garcia-Rubio¹, S. Park¹, I. Berrio², A. Chowdhary³, Y. Zhao¹, D.S. Perlin¹

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

**Objectives:** *Candida auris* is a recognized cause of invasive infections and health care associated outbreaks around the world. Development of echinocandin resistance has been documented in *C. auris* isolates recovered from patients treated with these drugs. Thus, a thorough understanding of *C. auris* echinocandin susceptibility profiles and resistance mechanisms is crucial for improved
management. In this study, we performed a comprehensive analysis of *C. auris* echinocandin susceptibility and molecular resistance determinants.

**Methods:** A total of 106 *C. auris* isolates (Colombia, India, AR Bank) were investigated in this study. Antifungal susceptibility testing (AFST) with echinocandins (anidulafungin, caspofungin, micafungin) was performed in accordance with CLSI document M27-A3. *FKS1* gene, encoding the target enzyme for echinocandin class antifungal drugs, was amplified and sequenced. Mutation prevention concentration (MPC) determination assay was performed for echinocandin-susceptible (micafungin MIC range 0.03-0.5 mg/l), *FKS1* wild-type *C. auris*, *C. albicans* and *C. glabrata* control strains. Isolates were cultured in RPMI-1640 containing 2-fold increasing concentrations (0.03 to 128 mg/l) of micafungin, then culture aliquots were plated onto YPD plates and number of surviving cells were calculated. Recovered colonies were screened for the presence of *FKS1* mutations. Moreover, *in vivo* drug response of *C. auris* isolates to caspofungin was assessed in a murine model of invasive candidiasis.

**Results:** Four Indian isolates from a total of 106 isolates (3.8%) exhibited highly elevated MICs ≥4 mg/l at 24 h and were considered presumptively resistant to all tested echinocandins. These isolates were found to harbor an S639F amino acid substitution in Fks1. The remaining 102 echinocandin-sensitive isolates presented wild-type genotype. Micafungin was the most potent echinocandin (MIC₅₀ = 0.125 mg/l). We encountered a significant challenge obtaining an accurate MIC readout for caspofungin since all tested isolates exhibited an Eagle effect (paradoxical growth effect), with the intensities of the Eagle effect varying among the isolates. In MPC determination assay with micafungin, *C. auris* isolates did not present a classical bi-modal killing pattern characteristic for fungicidal drugs activity against other *Candida* species. Response of *C. auris* to micafungin differed between isolates, but micafungin did not reach the fungicidal endpoint for any of the isolates, even when high drug concentrations (128 mg/L) were tested. Since no *FKS1* mutation was revealed by sequencing, we hypothesize the presence of extreme tolerance mechanism in *C. auris*. Echinocandins were effective *in vivo* in the treatment of invasive murine candidiasis caused by *FKS1* wild-type *C. auris* isolates (significant (P < 0.05) ~1 log10 CFU/g kidney burden reduction upon caspofungin treatment relative to vehicle controls), despite the presence of the Eagle effect *in vitro*. Mice challenged with *fks1* S639F mutants failed to respond to the drug and their kidney burdens were not much different between caspofungin treated and untreated groups.

**Conclusion:** Without established breakpoints for *C. auris*, AFST results interpretation and conclusions regarding resistance, especially when testing caspofungin, should be made with caution. The high echinocandin tolerance of *C. auris* observed *in vitro* should be further explored. Presently, the only validated determinant impacting *C. auris* pharmacodynamic response to echinocandins is the *FKS1* genotype.

# Symposium 21 Immunotherapy for opportunistic fungal infections

## S21.1

### CAR T cell and NK cell therapy of invasive mycoses

D. Kontoyiannis

Ut Md Anderson Cancer Center, 1515 Holcombe, Houston, United States of America

Invasive fungal infections remain a major threat to the survival of immunocompromised patients and no curative therapies are currently available to treat drug resistant opportunistic fungi. In this brief talk, I will provide a conceptual overview of recent developments to use specific chimeric antigen receptor (CAR) expressing T cells and NK cells as adoptive immunotherapy. CAR T cells have been successfully used to treat B-cell malignancies. To render CAR T cell therapy specific for fungi, we designed a CAR which combines the carbohydrate-binding domain of Dectin-1 with T cell signaling domains, designated as Dectin-1-CAR (D-CAR). The pattern recognition receptor Dectin-1 was selected based on its property to bind selectively to β-1,3-glucan present on the cell wall of life threatening fungal species. I will be discussing our efforts to manufacture clinical grade D-CAR+ T cells and to increase T cell persistence. However, allogeneic CAR T cells carry a significant risk of
graft-versus-host disease, and logistical problems of rapid and robust production remain. As the role of natural killer (NK) cells in fungal immunity has been increasingly deciphered, I will be discussing the potential benefits of cord-blood derived NK cells as an attractive, allogeneic, off-the-self source for future, logistically feasible immunotherapeutic approaches to treat invasive mycoses.

## S21.2

**WBC transfusions** Granulocyte Transfusions As Adjunctive Treatment Of Invasive Fungal Diseases
In Neutropenic Patients

L. Pagano

Fondazione Policlinico Universitario A. Gemelli - IRCCS - Universita Cattolica del Sacro Cuore,
Rome, Italy

The degree and duration of neutropenia are crucial prognostic factors in hematological patients (pts) with invasive fungal infections (IFIs). Since the introduction of granulocyte colony stimulating factor (G-CSF), there has been a renewal of interest in granulocyte transfusions (GTX). Granulocyte transfusions (GTX) are seldom used as a life-saving therapy for neutropenic patients with severe infections. Despite several compelling evidences of GTX efficacy in retrospective and prospective case series, no study has been successful in demonstrating a definite advantage for recipients in controlled clinical trials. Some specific issues relevant to the efficacy of this therapeutic approach, such as the primary infection, the delivered doses and schedules, and the immunological effects of GTX, are still subject to discussion today. Importantly, the awareness of biological effects accompanying the transfusion of neutrophils might support their use at standardized doses and may definitely convey significant advantages to the recipient patients. At present, despite statistical evidences are lacking, GTX are still perceived as a lifesaving tool to support neutropenic patients with life threatening IFIs until their bone marrow recovery. Sharing procedures for donor identification and cell mobilization, pursuing common criteria to identify which patients will benefit of GTX during febrile neutropenia and define indications and therapeutic cell doses are absolutely urgent to pinpoint the true advantage of using GTX. On the other hand, adopting equal end points and outcomes to evaluate both clinical response to treatment and biological functions of neutrophils and chemokines during IFI, need to be clarified.

## S21.4

**Novel chimeric antigen receptor T cells for invasive aspergillosis immunotherapy**

M. Seif<fnref n="1" />, T. Nerreter<fnref n="1" />, M. Machwirth<fnref n="1" />, F. Ebel<fnref n="2" />, H. Einsele<fnref n="1" />, M. Hudecek<fnref n="1" />, J. Löffler<fnref n="1" />

**Objectives:** Immunocompromised patients are susceptible to invasive fungal infections mainly caused by *Aspergillus fumigatus* (Af). Adoptive transfer of *Aspergillus*-specific T cells has been shown to reduce the burden of invasive aspergillosis. Such T cells are hard to isolate and expand. An alternative option is the use of T cells modified to express a chimeric antigen receptor (CAR). CARs are recombinant receptor constructs composed of an extracellular targeting element linked to an intracellular signaling module. A recent study using Dectin 1 as targeting element proved the applicability for antifungal CAR T cells *in vitro* and *in vivo*. One main limitation of Dectin 1 is the differential expression of its target β-glucan on Af morphotypes cell wall. β-glucan is mainly exposed on the surface of swollen conidia and early germ tubes but less on hyphae. Thus, better specificity would be highly valuable. Here we propose a highly specific and effective way of redirecting T cells towards Af.

**Methods:** For the construction of AF-specific CARs, we fused an scFv, derived from an antibody directed against the Af hyphal cell wall, to extracellular IgG4-Fc spacer domains of different lengths followed by CD28 and CD3-ζ signaling domains. We non-virally expressed the CARs on the surface of primary human CD4⁺ and CD8⁺ T cells using the Sleeping Beauty retrotransposon system and co-

<footnote n="1">¹Medizinische Klinik Und Poliklinik Ii, Universitätsklinikum Würzburg, Würzburg, Germany,</footnote>

<footnote n="2">²Institut Für Infektionsmedizin Und Zoonosen, Ludwig-Maximilians-Universität München,
München, Germany</footnote>
cultured CAR T cells with Af germ tubes to evaluate specific T cell activation and direct hyphal damage.

**Results:** We could show that our CAR T cells are specific for *Aspergillus fumigatus*, as no cross-reactivity with other Aspergilli was observed. Activated CD8⁺ CAR T cells secreted mainly Th1-related cytokines (IFN-γ, TNFα, IL-8 and GM-CSF) and chemokines (mainly CCL3 and CCL4), and only one Th2 cytokine (IL-13). Similarly, CD4⁺ CAR T cells secreted mostly Th1 cytokines and chemokines, but also some Th2 cytokines (mainly IL-13 and IL-4). CARs equipped with a long extracellular spacer containing the full hinge-CH2-CH3-motif conferred superior T cell activation as compared to CARs having a short "hinge-only" spacer. Noteworthy, mainly CAR T cells containing a long spacer underwent proliferation upon activation. Upon binding to the target, the cytolytic machinery of CD8⁺ CAR T cells was activated, leading to the release of perforin and granzyme B and up to 45% hyphal damage. Furthermore, when cocultured with macrophages, both CD4⁺ or CD8⁺CAR T cells enhanced antifungal activity of macrophages.

**Conclusion:** Taken together, our results show that we successfully redirected CD4⁺ and CD8⁺ CAR T cells against Af hyphae and that fine-tuning of CAR constructs might allow the control of fungal infections in high-risk patients.

## S21.5

### Comparison of circulating lymphocyte populations CD4+ and CD8+ T cells, B and NK lymphocytes according to the favorable or worsening evolution of patients with pneumocystosis

E. Charpentier¹,², C. Marques¹, S. Menard¹, N. Blanchard¹, P. Chauvin², E. Guemas¹, S. Cassaing², J. Fillaux², A. Valentin², A. Berry¹,², X. Iriart¹,²

¹Center for Pathophysiology of Toulouse Purpan - INSERM UMR 1043, Toulouse, France, ²Parasitology And Mycology, CHU de Toulouse, Toulouse, France

**Objectives:** Pneumocystosis is a severe opportunistic disease in which the host inflammatory response to the infection is decisive for the disease evolution. Although a reduced count of CD4+ T lymphocytes has been associated to a major risk of pneumocystosis, especially in HIV-positive subjects, little is known about other lymphocyte populations (CD8+ T cells, B and NK lymphocytes) or about CD4+ and CD8+ T cells subpopulations. This study aimed to compare circulating lymphocyte populations in patients suffering from pneumocystosis according to the patient evolution.

**Methods:** Levels of circulating lymphocyte populations were evaluated in the blood of 24 patients diagnosed with a pneumocystosis at Toulouse Teaching Hospital between 2016 and 2018. Among them, five were solid organ transplant (SOT) recipients, seven had a haematological malignancy (HM), five had a solid cancer (SC), three had an inflammatory disease (ID) and four were HIV-positive. CD4+ and CD8+ T cells, B lymphocytes and NK cells were evaluated with flow cytometry as well as Th1, Th2, Th17, Treg and naive, effector, memory subpopulations. The lymphocyte populations were compared between patients with favorable evolution (n = 16) and deceased patients (n = 8; 2 SOT, 2 HM, 3 SC, 1 HIV).

**Results:** Interestingly, the most significant results concerned CD8+ T cell subpopulations. Although there was no difference in global CD8+ T lymphocyte count, the proportions of memory CD8+ T lymphocytes (both central memory and effector memory) were lower in deceased subjects. At the opposite, the proportions of naive and effector CD8 lymphocytes were increased in this group. Effector memory CD8+ lymphocytes of deceased patients harboured less CD127 (IL7 receptor that induces lymphocyte survival and proliferation) whereas their CD8+ effector cells contained more CD127+ CD25+ (IL2 receptor and activation marker) cells in comparison to the CD8+ effector cells of surviving patients. These differences in CD8+ T cells were associated with reduced Th1 CD4 subpopulation and Th1/Th2 ratio in deceased patients. NK cells were also reduced in this group. There was no significant difference, either in CD4+ T cells count or in other CD4 subpopulations (Th2, Th17, Treg, naive, effector, memory) or in B cells count. Patients age, fungal load and CMV presence or absence were not significantly different in the two groups.
**Conclusion:** The implication of CD8+ T cells in pneumocystosis is currently a controversial topic with a discussed role of these cells in the fungus clearance and a suspected association with alveolar lesions. According to this study, high CD8+ memory subpopulations would be associated with a better evolution of the disease, along with Th1 CD4+ T cells. However, high naive and effector CD8+ populations would be associated to a worsening evolution of the patient. The disparity of CD8+ lymphocytes subpopulations might be related to a difference of immunity stimulation by *Pneumocystis jirovecii* antigens or it could be connected with a difference of CD8+ subpopulations homeostasis possibly affected by some therapeutics or living conditions. Despite the dogma that establishes CD4+ T lymphocytes as the center of pneumocystosis pathogenesis, CD8+ T cells may also have an important role in the disease pathogenesis.

## S21.6

### Glucosylceramides from Lomentospora prolificans Induce Cytokines Production and Increase the Microbicidal Activity of Macrophages

M.I.D.S. Xisto¹, J.E. Muñoz², L.S. Dias³, G.M.P. Santos², R.O.R. Calixto¹, M.C. Bernardino¹, C.P. Taborda⁴, E. Barreto-Bergter¹

¹Instituto De Microbiologia Paulo De Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, ²Instituto Biomédico, Departamento De Microbiologia E Parasitologia, Universidade Federal Fluminense, Niterói, Brazil, ³Department Of Pediatric, School Of Medicine And Public Health, University of Wisconsin-Madison, Madison, United States of America, ⁴Institute Of Biomedical Sciences, department Of Microbiology, University of São Paulo, São Paulo, Brazil

**Objectives:** *Lomentospora prolificans* is an emerging opportunistic fungus with a high resistance to antifungal agents and it can cause localized infections in immunocompetent patients and disseminated infections with a high mortality rate in immunosuppressed patients. Glucosylceramides (GlcCer) are synthetized in the majority of known fungal pathogens. They are bioactive molecules presenting different functions such as involvement in fungal growth and morphological transitions in several fungi. In this study, we report the characterization of GlcCer species in *L. prolificans* and the role of these molecules in the activation of the innate immune response.

**Methods:** GlcCer species were isolated from mycelium and conidia forms of *L. prolificans* and their chemical structures were elucidated by mass spectrometry (ESI-MS). The reactivity of *L. prolificans* GlcCer species to anti-GlcCer Mab was analyzed by ELISA. The GlcCer distribution on the surface of *L. prolificans* was analyzed by immunofluorescence using anti-GlcCer Mab. The cytokine analysis was performed by the GlcCer injection in BALB/c mice and the cytokines were quantified by ELISA according to the manufacturer's instructions. Recruitment of Cells to the Peritoneum Cavity was analyzed FACS.

**Results:** GlcCer purified from both forms presented a major species at m/z 750 that corresponds to N-2-hydroxyhexadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against GlcCer could recognize *L. prolificans* GlcCer species from mycelium and conidia, suggesting a conserved epitope in fungal GlcCer. In addition, *in vivo* assays showed that purified GlcCer species from both forms was able to induce a high secretion of pro-inflammatory cytokines by splenocytes. GlcCer species also promote the recruitment of polymorphonuclear, eosinophils, small peritoneal macrophage (SPM) and mononuclear cells to the peritoneal cavity. GlcCer species were also able to induce the oxidative burst by peritoneal macrophages with NO and superoxide radicals production, and to increase the killing of *L. prolificans* conidia by peritoneal macrophages.

**Conclusion:** Our results indicate that GlcCer species from *L. prolificans* are potent immune response activators. These molecules induce a strong production of NO in peritoneal macrophages with a high killing activity. *In vivo*, GlcCer species induce an immune response composed by a Th1 and Th17 cytokine profile, with recruitment of inflammatory cells to the peritoneum cavity. In this way, we believed that these molecules are very important to the immune response against *L. prolificans*, and could be used to produce antibodies, vaccines or as an adjuvant.

### Symposium 22 NGS and mycobiota
S22.1

Bacterial-Fungal Interactions at the Airway Mucosa and Implications for Chronic Rhinosinusitis

E. Cope, O. Kask, S. Kyman, I. Zhang, K. Lee

The Pathogen And Microbiome Institute, Northern Arizona University, Flagstaff, United States of America

Emerging studies of the human microbiome (the trillions of bacteria, fungi, viruses, and archaea that inhabit the human body) have demonstrated the myriad of ways that host-associated microbial communities contribute to host health. In the airways, the composition and diversity of the mucosal microbial communities are related to health status. However, the relationship between airway microbiota and inflammation is not well understood. Chronic rhinosinusitis (CRS) is a significant healthcare issue; current estimates suggest that CRS is responsible for 5% of the total healthcare costs in the US and affects up to 16% of the population. In a prior study of the bacterial microbiota of CRS patients, we've shown that CRS patients have decreased sinonasal bacterial diversity compared to healthy individuals. CRS patients were characterized by enrichment of one of four bacterial pathobionts, including *Staphylococcaceae* or *Pseudomonadaceae*. The objectives of this study were to determine whether bacterial and fungal microbiota interact in the airways to influence host immune response. We used ITS2 sequencing in a cohort of 100 CRS patients and healthy controls and found that, although bacterial community composition is variable across CRS patients, *Malassezia* is the predominant fungal genus in the upper airways of healthy individuals and CRS patients. We then hypothesized that species-specific bacterial-fungal interactions differentially influence host mucosal immune response. Thus, we investigated *in vitro* and *in vivo* interactions between *Malassezia sympodialis*, *P. aeruginosa*, and *S. aureus*. *In vitro* interactions were evaluated using the modified Kirby-Bauer Assay and Crystal Violet assay for biofilm quantification. A murine model of acute sinusitis was used to investigate relationships with the host immune response. *S. aureus* and *P. aeruginosa* were intranasally instilled in the presence or absence of *M. sympodialis*. 16S rRNA gene sequencing and FungiQuant were used to evaluate changes in microbiota composition and burden, and host immune response was measured using quantitative real-time PCR (qRT-PCR). *In vitro*, late stage planktonic and biofilm *P. aeruginosa* inhibited *M. sympodialis* growth. Co-infection of mice with *M. sympodialis* and *P. aeruginosa* or *S. aureus* differently influenced the immune response. In co-infected mice, expression of fungal sensing receptors (*Dectin-1*), allergic responses (*IL-5* and *IL-13*) and inflammation (*IL-10*, and *IL-17*) in the murine sinonasal cavity depended on the bacterial species that was co-instilled with *M. sympodialis* (p<0.05, Mann Whitney). Mice infected with *M. sympodialis* alone did not have increased Th1, Th2, or Th17 gene expression, suggesting that this taxon can act as a commensal. However, mice that were co-infected with *M. sympodialis* and *S. aureus* had increased *IL-5* gene expression (p = 0.01, Mann Whitney) compared to *M. sympodialis* or *S. aureus* alone. These results show that species-specific interactions in airway associated microbiota relate to distinct host immune response. These interactions may be implicated in some subsets of CRS disease. Our understanding of the role of bacterial-fungal interactions in CRS will contribute to development of novel therapies toward manipulation of the airway microbiota.

S22.3

Characterization of indoor dust microbiota in homes of asthma and non asthma patients

J.P. Gangneux¹, M. Sassi¹, P. Le Cann², P. Lemire²

¹Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France, ²Inserm Irset- Umr 1085, EHESP, Rennes, France

**Objective:** The exposure of occupants of housing to indoor air pollutants has increased in recent decades. Among microbiological contaminants, bacterial and fungal aerosols remain poorly studied and the debate on the impact of these aerosols on respiratory health is still opened. This study aimed to assess the diversity of indoor microbial communities in relationship with the health of occupants.

**Methods:** Measurements were taken from dwellings of 2 cohorts in Brittany (France), one with children without any pathology and the other with children and adults with asthma. Thirty dust
samples were analyzed by next generation sequencing with a 16S and 18S targeted metagenomics approach. Analysis of sequencing data was performed using qiime 2, and univariate and multivariate statistical analysis using R software and phyloseq package. Dust samples were collected by vacuuming the floor in the child's bedroom using a Duststream Collector (Indoor biotechnologies, United Kingdom) sampler-fitted vacuum cleaner (40 µm mesh nylon filter, domestic vacuum cleaner).

**Results:** A total of 2,637 prokaryotic (589 at genus level) and 2,153 eucaryotic taxa were identified (856 fungal taxa (39%) and 573 metazoa (26%)). Among Fungi, only 136 taxa were identified at genus level. The four main bacterial phyla were identified: *Proteobacteria* (53%), *Firmicutes* (27%), *Actinobacteria* (11%), *Bacteroidetes* (8%). The three main fungal phyla identified were: *Ascomycota* (84%), *Basidiomycota* (12%) and *Mucoromycota* (3%). No bacterial nor fungal taxa were significantly associated with asthma versus control group.

A trend of over representation in Asthma group versus control was observed for *Corynebacterium* (p-value = 0.013, adj. p-value = 0.09) and *Streptococcus* (p-value = 0.025, adj. p-value = 0.112) genus. No correlations between the populations of fungi and the asthma conditions as well as the habits of the occupants/the characteristics of the dwellings or other environmental characteristic were observed.

**Conclusion:** Our findings provide evidence that dust samples harbor a high diversity of human-associated bacteria and fungi. Molecular methods such as NGS are reliable tools for identifying and tracking the bacterial and fungal diversity in dust samples, a less easy strategy for the detection of eucaryots at least using 18S metagenomics approach. This study showed that the detection of some bacteria may be associated to indoor air of asthmatic patients. Regarding fungi, a higher number of samples and sequencing with more depth could allow to reach significant signatures.

### S22.5

#### An ECMM-ESCMID survey on goals and practices for mycobiota characterization using Next Generation Sequencing in European laboratories

J.P. **Gangneux**¹, H. **Guegan**¹, L.-E. **Vandenborgh**², S. **Buffet-Bataillon**¹, R. **Enaud**³, L. **Delhaes**⁴, F. **The Esghami-Efisg** (Escmid) **And ecmm NGS** Study Group¹

¹Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France, ²Genoscreen, Lille, France, ³University of Bordeaux, Centre de Recherche Cardio-Thoracique de Bordeaux, U1045, CHU de Bordeaux, CRCM Pédiatrique, CIC 1401, Bordeaux, France, ⁴Parasitology Mycology, Univeristy of Bordeaux, Centre de Recherche Cardio-Thoracique de Bordeaux, U1045, F-33000, Bordeaux, France, Bordeaux, France

**Objectives:** Although substantial efforts have been made to investigate about the composition of the microbiota, fungi that constitute the mycobiota play a pivotal role in maintaining microbial communities and physiological processes in the body. Next generation sequencing (NGS) techniques have clearly renewed methodology to characterize host-associated microbiota in a more comprehensively way, and have consequently revealed a much more diverse microbiota than was previously thought to exist.

**Methods:** We constituted a panel of experts from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID-ESGHAMI and -EFISG study groups) and the European Confederation of Medical Mycology (ECMM) to address an international-survey focusing on laboratory's current procedures regarding their goals and practices of mycobiota characterization using NGS.

**Results:** The laboratories reported their main reason of using NGS to study the mycobiota primary to understand the pathophysiology of a dysbiosis (n = 20), at a second level, to contribute to a diagnosis (n = 16) to implement a therapeutic strategy (n = 12), or to evaluate the exposome (environmental studies)(n = 10). Respiratory tract represented the most studied site, followed by the digestive tract, Sample originated in half of the laboratories from sputum (n = 14), or from stool (n = 12), broncho-alveolar lavage (n = 11), environmental (n = 6), oral wash (n = 4) and skin (n = 1). Target used for mycobiota analysis was equal among 18S rRNA (n = 11), ITS1 (n = 11) and ITS2 (n = 11), while some centers used the shotgun approach (n = 5). When asked about data analysis, database used for
alignment were mostly UnitE (n = 8 labs), and both Genebank and CBS databases (n = 7 labs). Other details will be presented.

**Conclusion:** We can therefore report a photography of mycobiota analysis in 28 laboratories from 19 countries, affiliated to working groups from ESCMID and ECMM and draw some conclusions of the diversity of approaches. NGS to study mycobiota is a great challenge for understanding pathophysiological questions in experimental studies as well as for driving diagnostic strategies. Techniques will rapidly develop, moving from targeted NGS to shotgun approaches and whole genome sequencing will also be very useful in the next years in the field of mycology, but also for an holistic reasoning.

## Symposium 23 The funny life of C. albicans in the oral tract

### S23.3

#### Modulation of the Fungal-Host Interaction by the Intra-Species Diversity of C. albicans.

A. Kannan¹, G. Laval², M.-E. Bougnoux³, C. D'Enfert⁴, D. Sanglard¹

¹Microbiology Institute, University Hospital Lausanne, University of Lausanne, Lausanne, Switzerland, ²Center For Bioinformatics, Institut Pasteur, Paris, France, ³Laboratoire De Parasitologie Mycologie, APHP Hôpital Necker-Enfants-Malades, Paris, France, ⁴Fungal Biology And Pathogenicity, Institut Pasteur, INRA, Paris, France

*Candida albicans* clinical isolates intrinsically display high diversity in their population genetic structure. Regulation of several host factors and development of *C. albicans* diseases can be influenced by diverse genetic backgrounds of the fungus. Using a collection of 96 genome-sequenced isolates, we aim here to understand by so-called Expression Quantitative Trait Loci (eQTLs), the influence of *C. albicans* genome diversity on fungal transcriptomic profiles in contact with human keratinocytes (TR146 cells). The impact of the fungal genomes on the host transcriptome can thus be probed. Pilot experiments using 5 distinct commensal clinical isolates (including the reference isolate SC5314) in contact with TR146 cells at different time points (0.3-, 1-,2- and 6h) revealed distinct transcriptional differences between the isolates. After 6h co-culture, only 194 *C. albicans* genes were found to be commonly differentially regulated (2-fold threshold) as compared to SC5314 in all four isolates. These data suggest a heterogeneous response between these different isolates, even with a small number of isolates. This heterogeneous response underpins the large genome diversity existing between the different *C. albicans* clades. Based on the overall summary of top 200 variable *C. albicans* genes among all the five isolates, we observed that the isolate CEC3665 most varied from SC5314 upon infection. This isolate differed from the others principally by genes involved in hyphal development and adherence. Consistently, CEC3665 exhibited low adherence to TR146 cells as compared to the others. The use of these 5 initial isolates allowed the optimization of several other experimental parameters (optimal exposure time to TR146 cells, variations within biological triplicates) that was used for probing the transcriptional profiles of the entire 96 genome-sequenced collection. The current state of the 96 genome-sequenced collection analysis will be presented.

### S23.4

#### Intestinal Th17 drives airway inflammation in ABPA

P. Bacher

Institute of Clinical Molecular Biology & Institute of Immunology, Kiel, Germany

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses are poorly understood. In humans, Th17 responses are particularly important against Candida albicans, a mucocutaneous fungal pathobiont. In contrast, the role of Th17 cells for other pathogenic fungal species is unclear. We used antigen-reactive T cell enrichment (ARTE) for the ex vivo analysis of human T helper cell responses against 30 common members of the human mycobiome. We identified the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungal species are induced by T cell cross-reactivity to C. albicans. Intestinal
inflammation expands total C. albicans- and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, such as asthma, COPD and cystic fibrosis and especially during acute allergic bronchopulmonary aspergillosis (ABPA), suggesting their specific contribution to lung pathology. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identified heterologous immunity to a single, ubiquitous member of the mycobiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a risk factor for pulmonary inflammatory diseases.

S23.5

## Persistence of clonal azole-resistant isolates of *Candida albicans* from a patient with Chronic Mucocutaneous Candidiasis in Colombia

A. Ceballos-Garzon¹,², L.M. Wintaco³, C. Hernández Padilla⁴, A. De La Hoz⁴, S.L. Valderrama Beltrán⁴, C. Alvarez Moreno⁴, P. Le Pape¹, J. Ramirez³, C.M. Parra-Giraldo², N. Velez²

¹Department Of Parasitology And Medical Mycology Of The University Of Nantes., Universite de Nantes, Nantes, France, ²Unidad de Proteómica y Micosis Humanas, Grupo de Enfermedades Infecciosas Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá D.C, Colombia, Bogota, Colombia, ³Grupo De Investigaciones Microbiológicas-ur (gimur), Universidad del Rosario, bogota, Colombia, ⁴Grupo De Investigación En Enfermedades Infecciosas, Unidad De Infectología, Hospital Universitario San Ignacio (husi) Pontificia Universidad Javeriana, Hospital Universitario San Ignacio, bogota, Colombia

**Objectives:** To evaluate and to characterize phenotypically and molecularly clinical *Candida albicans* isolates, obtained from a patient with Chronic Mucocutaneous Candidiasis.

**Methods:** sixteen C. albicans were isolated from a 25-year-old male with several recurrent fungal infections and admitted to the San Ignacio Hospital in Bogota, Colombia. The isolates were recovered during four-year from a different anatomical sites. The isolates were typified by MultiLocus Sequence Typing, and evaluate for susceptibility to triazoles, Caspofungin and Amphotericin B by microdilution method (CLSI). Their pathogenic capacity was evaluated in the *Galleria mellonella* model.

**Results:** Genotyping of all clinical isolates showed the persistence of the same Diploid Sequence Type (DST). Isolates changed their susceptibility profile over time acquiring resistance to azole drugs. In contrast there were no significant statistical differences in pathogenicity during the period.

<figure><img src="image_12.png" /><figcaption>**Figure 1.** Characteristics of *Candida spp* isolates **A.** Antifungal treatment over time **B.** Antifungal susceptibility results.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Figure 2.** Survival curve of average according to anatomical origin. Kaplan-Meier plots of G. mellonella survival after infection with C. albicans 3x10⁵ UFC/ml. Inside the brackets the number of samples.</figcaption></figure>

**Conclusion:** The 16 samples collected, allow us to identify the same DST918 which belongs to Clade 9, being this DST widely reported worldwide. It is important to highlight that among the isolates reported to date with the same DST, none had been previously associated with Chronic Mucocutaneous Candidiasis (CMC). The persistent clonal strain C. albicans strain (DST 918) in a patient with CMC, showed changes in its susceptibility profile after several antifungal treatments acquiring gradual resistance to the azole drugs, which did not affect their pathogenicity.

# Symposium 24 Fungal neglected tropical diseases

## S24.1

### Updates on mycetoma

W. Van De Sande

Department Of Microbiology And Infectious Diseases, Erasmus Medical Center, Rotterdam, Netherlands

**Abstract:** Mycetoma is a subcutaneous inflammatory and infectious disease of mainly the foot. It is endemic in tropical and subtropical regions where it causes a high morbidity. It was the first fungal infection recognized as a Neglected Tropical Disease by the World Health Organization by resolution WHA69.21 in 2016. Within this resolution, endemic regions were encouraged to assess the burden of mycetoma and the scientific community was encouraged to develop tools for early diagnosis and to improve treatment of mycetoma. It is now more than 3 years since this resolution and within these three years new information became available on all these subjects. In terms of assessing the burden of infection, case reports and single center studies were published from different regions in the world. Studies from Brazil, China, India, Mexico, the Philippines, Senegal, Sudan and Togo. Also reports from non-endemic regions such as Italy, Spain, Switzerland and the United States of America demonstrated that immigrants and refugees can also take mycetoma to their new homelands where they often cause problems in diagnosis due to lack of awareness. Within these reports it became evident that there are still new mycetoma causative agents discovered every year. To identify the causative agents, morphological identification and/or histology often leaded to misidentification demonstrating the need for more reliable diagnostic tools. To speed up diagnosis and make it more reliable, techniques were developed to isolate DNA directly from mycetoma grains and novel PCR primers were developed to be able to identify more causative agents. Isothermal alternatives were also explored and for histology sections an in situ hybridization probe was developed. For identification on cultures, a Maldi-Tof database was generated. To optimize treatment, an open source drug discovery platform was founded (MycetOS) which identified the fenarimols as a new class of potent anti-mycetoma drugs and other classes such as the orotomides were tested in vitro for their activity against mycetoma causative agents. Based on the in vitro potency of ravuconazole, the first ever clinical trial on a new drug was started at the mycetoma research centre in Khartoum. It is
obvious that since the recognition as a Neglected Tropical Disease the mycetoma field has moved
forward and will continue as such in the upcoming future.

S24.2

Chromoblastomycosis: A true neglected tropical disease

F. Queiroz-Telles

Public Health, Federal University of Parana, Curitiba, Brazil

Several infectious diseases, including helminthic, protozoal, bacterial, and viral infections but not
fungal diseases other than mycetoma and chromoblastomycosis, are currently defined as "neglected
diseases" by the WHO. After sporotrichosis, chromoblastomycosis is the second most prevalent
implantation mycosis in the world. Chromoblastomycosis (CBM) is defined as a chronic and indolent
granulomatous fungal disease caused by the transcutaneous inoculation of propagules from several
species of melanized fungi from the Herpotrichiellaceae family. The most prevalent species causing
chromoblastomycosis are *Fonsecaea pedrosoi*, *F. monophora*, and *Cladophialophora carrionii*. Advances in
molecular taxonomy have shown that the biodiversity of the CBM agents has increased without
substantial correlation with specific type of lesions or severity of the disease and response to therapy.
Because several CBM sibling etiologic agents are found in soil and plant microbiota, this disease is
strongly associated with cutaneous macro or micro traumas during diverse outdoor activities, such
as agro pastoralism, hunting, eco-tourism, etc. Chromoblastomycosis mainly occurs in tropical and
subtropical zones in Latin America, Africa, Asia and Oceania. According to recent data, the highest incidence density of CBM (number of reported cases /10 million), occurs in the Dominican Republic (50.1), Madagascar (26.4), Gabon (23.5), Congo and Costa Rica (11.8), respectively (Santos
DW, PhD Thesis) Melanized cell wall, muriform cell architecture, hydrophobicity and cell adhesion
are considered virulence factors leading to increased pathogenicity, a key point for disease
development. After an uncertain period of incubation, the initial lesion starts at the site of inoculation.
With time, these can evolve to polymorphic cutaneous lesions. The clinical forms of the disease might
be associated with cytokine cellular-mediated response and in severe cases, the production of TNF α
and interleukin 10 has been detected. To confirm diagnosis, the visualization of pathognomonic
muriform (sclerotic) cells is the most simple, inexpensive, rapid and 100% sensitive method. In
culture, the organisms are fastidious. Molecular methods are mandatory for species identification,
but they remain expensive and are not important for therapeutic guidelines. Although serologic tests
(immunodiffusion and ELISA) have been validated, they are not commercially available. The
therapeutic options, for CBM, include physical methods and chemotherapy. Without a doubt,
excisional surgery is the best method for the treatment of CBM but only for initial small and well-
delimitated cutaneous lesions. According to a few open non-comparative clinical studies,
itraconazole (ITZ) is currently the best therapeutic option for CBM, at the daily dose of 200 to 400 mg
from 8 to 36 months. The success rate ranges from 15 to 80%. The duration and cure rates are related
to the severity of the disease, ITZ gut absorption and the etiologic agent. Terbinafine (TBF) at the
daily dose of 250 to 500 mg is the second most used drug. For refractory cases, posaconazole may be
used as well as the combination of ITZ with TBF or 5-flucytosine. The only way to prevent CBM is to
avoid trauma Individual under occupational risk should use protective equipment such as adequate
clothes, shoes and gloves. Similarly, to other implantation mycoses, there are no available
immunoprophilaxis for CBM.

S24.3

Sporotrichosis: past and future

L. Lopes Bezerra

CIETEC/USP, São Paulo, Brazil

The description of new cryptic species with different pathogenic potentials has changed the perspectives on sporotrichosis. The Pathogenic Clade of the *Sporothrix* genus has four pathogenic species: *S. schenckii*, *S. brasiliensis*, *S. globosa* and *S. lurei*. The new emerging pathogen, *S. brasiliensis*, shows higher virulence and is mainly related to zoonotic transmission while *S. schenckii* and *S. globosa*
are sapronotic agents. After 20 years of the zoonotic outbreak of sporotrichosis in Brazil the challenges to manage the disease caused by *S. brasiliensis* are enormous, including clinical-epidemiological aspects, treatment and diagnosis. The cat-transmitted disease has already spread from the South to the Northeast of Brazil with impressive number of cases in either humans or felines being reported every year. The increment of cases reported by Centers of Zoonotic Control account for over 500% between 2016 and 2018. For this reason, a national effort among scientists, clinicians and veterinaries was established to propose a guideline for the management of *S. brasiliensis* infection. This group of experts from several endemic areas is working since 2018. The data and conclusions achieved by the Feline and Human Brazilian Working Groups will be presented.

S24.4

Novel diagnostic and treatment strategies for the Southeast Asian fungus Talaromyces marneffei

T. Le

Duke University School of Medicine, DURHAM, United States of America

Novel diagnostic and treatment strategies for the Southeast Asian fungus talaromyces marneffei – Thuy Le, MD DPhil *Talaromyces marneffei* (Tm) is one of seven endemic human fungal pathogens that causes substantial global morbidity and mortality. Over just two decades, Tm has emerged as a leading cause of opportunistic infection and death in adults and children with advanced HIV disease in southern China and Southeast Asia. The mortality on antifungal therapy in both HIV and non-HIV infected patients is as high as 30%. The current diagnosis still relies on microscopy of skin lesions (which is a late manifestation and is absent in 40% of patients) and on cultures of the pathogen which takes up to 14 days. Late diagnosis is the most challenging clinical problem and drive mortality from 24% to 50%. The current treatment options are suboptimal. Intravenous amphotericin B deoxycholate is recommended for induction therapy but has serious side effects and is still not widely available. Itraconazole tablets are cheap and are widely available, but has low oral bioavailability, variable pharmacokinetics and is inferior to amphotericin B in efficacy. This talk presents new data on the comparative performance of real-time PCR assays versus antigen detection assays for rapid diagnosis of talaromycosis. Both assays have excellent specificity of >95%; however, the antigen tests are significantly more sensitive than the real-time PCR tests (>90% compared to 70-80%). Tm antigen can be detected up to 16 weeks prior culture positivity in symptomatic hospitalized AIDS patients, and Tm antigen independently predicts 12-month mortality in patients with a CD4 cell count <100 starting antiretroviral therapy. This talk will also present results of the IVAP trial, a multi-center clinical trial comparing amphotericin B and itraconazole for induction therapy in Vietnam, which demonstrated a 50% mortality reduction in the amphotericin B group. The trial showed that 98% of patients in the amphotericin B group cleared fungemia within 8 days of therapy, paving the road for a planned phase II clinical trial testing short courses of liposomal amphotericin B therapy to reduce toxicity. Efforts to test novel antifungal compounds are ongoing and should offer new and less toxic treatment options in the coming years.

S24.5

Urban sporotrichosis: neglected micro-epidemia in the State of Minas Gerais, Brazil

M.A. Resende Stoianoff, D.B. Silva Navarro, D.L. Silva, D. Assis Santos

Dept. Of Microbiology, Federal University of Minas Gerais, Belo Horizonte MG, Brazil

**Objectives:** Sporotrichosis is a subcutaneous mycosis caused by dimorphic fungus of the *Sporothrix* complex that can affect both humans and other animals, being more common and severe in cats, and occurring more frequently in regions of tropical climate, as is the case in Brazil. Because it is not a compulsory notification disease in most Brazilian states, the true incidence of the disease is unknown. The aim of this study was to report the occurrence of cases of human sporotrichosis in the state of Minas Gerais, Brazil. **Methods:** The secretions of the ulcerated lesions were collected with the aid of sterile swab and the material was subjected to Gram staining, as well as to the culture at 28°C and
37°C. It was confirmed by the micromorphological examination that the samples belong to the genus *Sporothrix*. The molecular identification of the species is underway and the patients were referred for treatment. **Results:** It was reported the occurrence of 11 cases of human sporotrichosis between August 2018 and May 2019, nine of them from the city of Ribeirão das Neves and two from Belo Horizonte, state of Minas Gerais, Brazil. Among the patients, nine belonged to the female gender and two to the male. The patients' ages ranged from two to 61 years and all of them reported contact with sick cats. The mean time between contact with the animals and the appearance of the lesions was 15 days. Nine patients presented lymphocutaneous form and two presented fixed cutaneous form. The patients presented lesions in the arms, forearms, fingers or in the heel. The Gram staining revealed presence of yeasts of elongated and/or rounded form, sometimes with single bud. The thermal dimorphism and the micromorphology confirmed the identification of *Sporothrix* spp. **Conclusion:** The results suggest that there may be a sporotrichosis microepidemia in the municipality of Ribeirão das Neves, with marked importance of the zoonotic transmission of the disease. According to official information, the conditions of infrastructure and sanitation are precarious in this region. The notification of this disease and studies that relate the socio-environmental and behavioral conditions that can determine the variation in sporotrichosis transmission are of great importance, in order to adopt the appropriate prevention and control measures. The contact of individuals with cats at home environment may be increasing in the last years, a fact that contributes to the increase of the possibility of infection of the population. The control of the source of infection is fundamental in this situation, with priority being given to the investigation of factors involved in the transmission dynamics of the disease, to assist in the surveillance and control measures necessary to contain its growth. Key words: Sporotrichosis, *Sporothrix* spp., Urban sporotrichosis Financial Support: CNPq, CAPES

## Posters

P001

**induction of antifungal resistance exerted by the exposure of paclobutrazol to Aspergillus fumigatus**

E. Marques De Araujo¹,², P. Le Pape², R. Gonçalves De Lima-Neto¹, T. Gonçalves¹, N. Buarque De Gusmao¹

¹Biologia De Fungos, universidade federal de pernambuco, Recife, Brazil, ²Ea1155 licimed, Nantes University Hospital, NANTES, France

**Objectives:** The resistance of Aspergillus fumigatus to azolic drugs occurs mainly through two routes: in vivo as a consequence of antifungal therapy, and in the enviroment as a consequence of the use of fungicides in agriculture. Placobutrazol is a plant growth retardant used in agriculture, which inhibits cellular growth and hormones composition. Since it is excessively applied and presents chemical stability, paclobutrazol accumulates in the soil and can affect the existing microbiota. The present study aimed to induce to the resistance and consequently the induction of CYP51 gene mutation on azoles-susceptible isolates of Aspergillus fumigatus after exposition against paclobutrazol.

**Methods:** he A. fumigatus (2 from environmental origin and 1 ATCC) were submitted to antifungal susceptibility testing by the microdilution method (MIC-EUCAST) to prove that all strains were sensitive at concentrations of 32 µg/mL of paclobutrazol and 0.5 µg/mL of voriconazole and itraconazole. For the resistance induction test, these fungi were exposed to paclobutrazol for five weeks in RPMI culture medium at 37°C in initial concentration of 4 µg/mL, and increasing to a concentration of 64 µg/mL, it was also checked whether concentration progression influenced induction using the same method, without elevation in concentration of the molecule in the medium. Over the five weeks of the test, the MIC was verified through the micro dilution method established by EUCAST and the yeastone sensitrite commercial method in order to evidence the elevation of MIC after exposure.
**Results:** he MICs obtained over the five weeks of the test showed a decreasing of sensitivity in all strains (100%) against antifungals used in the hospital routine after exposure to the paclobutazol.

**Conclusion:** Thus, it is possible to affirm the exposure of the Aspergillus fumigatus to drugs used on agriculture may induce resistance against azoles usually prescribed. These findings demonstrating the need of a better and more rigid regulation in the use of this molecule in agriculture. The next steps in this research are to confirm that placobutazol exposure may induce a permanent mutation on resistance gene.

P002

**Oral Candidiasis in HIV Infected Patients and its Antifungal Susceptibility Pattern by Disc Diffusion Method.**

P. Chaudhary

Microbiology, Sagarmatha chaudhary eye hospital, lahan, siraha, Nepal

**Objectives:** Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection that is commonly found in (Human Immunodeficiency Virus) HIV infected patients. Acquired Immunodeficiency syndrome (AIDS), a disease of human immune system caused by HIV, has emerged as a global crisis since its discovery in summer of 1981 in United States. The low absolute CD4+ T-lymphocyte count has traditionally been cited as the greatest risk factor for the development of OPC and current guidelines suggest increased risk once CD4+T-lymphocyte counts fall below 200 cells/mm³. Hence, a cross-sectional study was conducted in Sukraraj Tropical and Infectious Disease Hospital (STIDH), Teku, Kathmandu over 3 months period with a primary objective, to isolate and identify *Candida* species among HIV infected patients and susceptibility pattern of isolates by disc diffusion method and secondary objective, to estimate the frequency of oral candidiasis according to age, sex, and other factors (CD4+ count, tobacco consumption, URTI, recent antibiotic consumption) in HIV infected patients.

**Methods:** A total of 408 throat swab samples were collected from patients with and without active lesion visiting ART center of STIDH. Flow chart for sample processing and identification of isolates is shown below.

**Results:** 114 isolates were obtained from total sample processed. Among them, 65 isolates were *Candida* species and remaining 49 was bacterial flora and other yeasts. Based on various phenotypic methods, *Candida albicans*, 53 (81.5%), was the most commonly isolated organism followed by *Candida tropicalis* 3 (4.6%), *Candida krusei* 2 (3.1%), *Candida glabrata* 1 (1.5%) and 6 (9.2%) were other *Candida* species. Of the 65 yeast isolates from HIV seropositive individuals, 26 patients had CD4+ ≤ 200 cells/mm³ and 39 patients had CD4+ >200 cells/mm³. 14 patients exhibited severe immune suppression with CD4+ count less than 100 cells/mm³. There was a significant association between oral *Candida* carriage and CD4+ cell count ≤ 200 cells/mm³ (p<0.001). Antifungal susceptibility test of *Candida* species showed highest sensitivity with Amphotericin B and resistance with Fluconazole respectively. Table below depicts about the susceptibility pattern of the *Candida* spp.

**Conclusion:** *C. albicans* was the predominant species for oral yeast colonization in our study. Different isolates presented different susceptibility to various antifungal agents. Among the antifungal discs tested, Fluconazole was found to show resistance in most of the isolates and Amphotericin B was the most sensitive drug which is an excellent antifungal to control *Candida* spp. since it showed the best result among all antifungal tested. Oral yeast colonization was associated with low CD4+ count (<200 cells/mm³). Thus, oral lesion serves as early marker for HIV infection. The increase incidence of oral candidiasis in HIV patients may be due to low CD4+ count, URTI and recent antibiotic consumption.

P003

**The validation of VIPcheck™ plates to screen Aspergillus fumigatus isolates for phenotypic resistance to triazole antifungal agents in St. James's Hospital, Dublin.**

A. Griffin
Microbiology, St. James's Hospital, Dublin, Dublin, Ireland

**Objectives:** Triazole resistance is an emerging problem in Aspergillus fumigatus (AF) resulting in failure of azole therapy. Triazole resistant AF is acquired through one of two routes - previous exposure to triazole therapy or an environmental source. In vitro antifungal susceptibility testing (AFST) on all AF strains isolated in a microbiology laboratory would be both labour intensive and impractical. A method to screen for triazole resistance would be more favourable. The objective was to validate VIPcheck™ plates (Mediaproducts BV, Groningen, Netherlands) in SJH to investigate whether they would be a suitable screening method for triazole resistance in AF clinically isolated from patients in a large tertiary hospital with a significant cohort of immunocompromised patients.

**Methods:** A total of 18 retrospective isolates (clinical and environmental) of AF were tested using the VIPcheck™ plates (n=2 wild type, n=16 resistant to ≥ 1 triazole drug as previously determined by AFST and/or molecular methods). VIPcheck™ plates are a simple agar based screening method, each 4 well plate containing a growth control (GC) well and 3 wells containing itraconazole (4mg/L), voriconazole (2mg/L) and posaconazole (0.5 mg/L). Briefly, 25ul of a 0.5-2McF suspension AF is inoculated into each well and plates are read after 48hrs incubation at 37°C. Any growth in a triazole containing well is suggestive of resistance prompting full AFST be carried out.

**Results:** A total of 18 isolates (clinical and environmental) of AF were tested using the VIPcheck™ plates as described above. The wild type isolates showed growth only in the GC well while the resistant strains all showed growth in one or more of the triazole containing wells.

**Conclusion:** VIPcheck™ plates are not intended to give an exact MIC; the preparation of the inoculum has a broad McFarland range of 0.5-2 and only one concentration of each azole drug is included in the plates. Rather, they are useful as a screening method to determine the need for further AFST and/or molecular testing and more importantly for earlier detection of triazole resistance in patients who will require treatment. Currently in SJH, AFST is carried out using gradient strips (Liofilchem™) and results are interpreted using EUCAST breakpoints We validated the VIPcheck™ plates with the intention to include this screening method as part of our AFST for AF isolated from clinical samples and our results suggest that they provide a reliable screening method for triazole resistance.

P004

In vitro susceptibility to isavuconazole of Fusarium clinical isolates; comparison between eucast and etest methods

A. Broutin1, J. Bigot1, A.C. Normand2, Y. Senghor3, A. Moreno3, M. Hendrickx4, J. Guitard1, C. Hennequin3

1Parasitology-mycology, APHP, hôpital St Antoine, Paris, France, 2Parasitology-mycology, APHP, Hôpital Pitie-Salpêtrière, Paris, France, 3Parasitology-mycology, APHP, Hôpital St Antoine, Paris, France, 4Mycology, Scientific Institute of Public Health, Brussels, Belgium

**Objectives:** To evaluate the in vitro susceptibility to isavuconazole of a panel of precisely identified clinical *Fusarium* isolates and to assess the value of Etest as an alternative method in this indication.

**Methods:** We randomly selected 75 clinical isolates previously identified as *Fusarium sp.* The strains were identified using MALDI-TOF technology (MSI System). They were then tested comparatively against isavuconazole (ISA) either using the broth microdilution method proposed by the Eucast consortium according to the recent document E.DEF 9.1 and the Etest technique as recommended by the manufacturer (Liofilchem).

**Results:** All strains could be identified at the cryptic species-level revealing the existence of 14 different species in the panel (Table 1). *F. solani* (FSSC), *F. oxypsum* (FOSC) and *F. fujikuroi* (FFSC) were the species complexes the most frequent, encompassing 22, 17 and 31 strains, respectively. Overall, ISA exhibited a limited activity against the *Fusarium* strains with MICs90 >16 µg/ml whatever was the species complex. Strains of the FOSC appeared a little bit more susceptible with a geometric mean at 9.41 µg/ml versus 14.02 µg/ml and 13.68 µg/ml for FSSC and FFSC,
respectively. Considering the diversity of species, it was not possible to test a possible difference in susceptibility between the cryptic species within a given complex. Finally, we observed MIC between 2 to >16 µg/ml for strains of the single species of the FOSC, suggesting that precise identification may not be enough to predict the susceptibility to ISA. The concordance between Etest and Eucast results was calculated at 100% within an interval +/- 2 dilutions. The Intraclass Correlation Coefficient was calculated at 0.91, indicative of a strong concordance between the 2 methods.

<table><caption>Table 1: Isavuconazole MICs distribution against 75 clinical isolates of *Fusarium*</caption><thead><tr><th rowspan="2">Species complex<br>(SC)</th><th rowspan="2">Cryptic species<br>(# of isolates)</th><th colspan="8">MICs (µg/ml) distribution</th></tr><tr><th>0.25</th><th>0.5</th><th>1</th><th>2</th><th>4</th><th>8</th><th>16</th><th>&gt;16</th></tr></thead><tbody><tr><td rowspan="3">F. solani SC</td><td>F. falciforme (2)</td><td></td><td></td><td></td><td></td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>F. petroliphilum (15)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>15</td></tr><tr><td>F. keratoplasticum (5)</td><td></td><td></td><td></td><td></td><td></td><td>1</td><td></td><td>4</td></tr><tr><td>F. oxysporum SC</td><td>F. oxysporum (17)</td><td></td><td></td><td>1</td><td>2</td><td>6</td><td>5</td><td>3</td><td></td></tr><tr><td rowspan="7">F. fujikuroi SC</td><td>F. verticillioides (1)</td><td></td><td></td><td></td><td></td><td></td><td>1</td><td></td><td></td></tr><tr><td>F. proliferatum (21)</td><td></td><td></td><td></td><td></td><td></td><td>2</td><td>6</td><td>13</td></tr><tr><td>F. acutatum (2)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td></tr><tr><td>F. sacchari (3)</td><td></td><td></td><td></td><td>1</td><td>1</td><td></td><td></td><td>1</td></tr><tr><td>F. anthophilum (1)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>F. thapsinum (2)</td><td></td><td></td><td></td><td></td><td>2</td><td></td><td></td><td></td></tr><tr><td>F. nygamai (1)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>F. dimerum SC</td><td>F. dimerum (2)</td><td></td><td></td><td></td><td></td><td>1</td><td></td><td>1</td><td></td></tr><tr><td>F. sambucinum SC</td><td>F. sporotrochioides (2)</td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>1</td><td></td></tr><tr><td>F. incarnatum-<br>F. equiseti SC</td><td>F. incarnatum (1)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr></tbody></table>

**Conclusion:** ISA exhibits a limited *in vitro* activity against clinical isolates of *Fusarium*, with few variations between the species complexes. In the case the drug should be used, Etest appears a valuable alternative to Eucast for evaluating the *in vitro* susceptibility of the infecting strain.

P005

## Species Distribution and Antifungal Susceptibility of Candida Ear Isolates from a Hospital in Korea

J.H. Shin, Y.J. Kwon, S.A. Byun, M.J. Choi, E.J. Won, S.H. Kim

Department Of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Korea, Republic of

**Objectives:** *Candida auris* was first isolated from the ears of Japanese and Korean patients. However, the prevalence of *Candida* species from ear cultures and their antifungal susceptibility profiles in these nations are unclear.

**Methods:** We assessed *Candida* isolates recovered from cultures of ear specimens from a university hospital in Korea over the 4-year period from January 2014 to December 2017. Species identification was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry
and/or sequence analysis. Antifungal minimal inhibitory concentrations (MICs) were determined by the broth microdilution method (M27A) of the Clinical and Laboratory Standards Institute.

**Results:** Candida species were isolated from the ears of 80 patients (75 adult and 5 pediatric patients). Most of these patients suffered from chronic otitis media (83.8%), and had a history of antibiotic use (73.8%) and ear surgery (60.0%). Among 80 non-duplicate isolates from ear cultures, *Candida parapsilosis* was the most frequently detected *Candida* species (35.0%), followed by *C. auris* (28.8%), *Candida metapsilosis* (10.0%), *Candida orthopsilosis* (8.8%), *Candida albicans* (7.5%), and other species (11.1%). The MICs of the isolates were 0.125 to > 64 µg/mL, ≤ 0.03 to 4 µg/mL, 0.25 to 1 µg/mL, 0.125 to 1 µg/mL, and ≤ 0.03 to 2 µg/mL for fluconazole, voriconazole, amphotericin B, caspofungin, and micafungin, respectively. Of the 80 isolates, 43.8% (35/80) showed decreased susceptibility to fluconazole (MIC ≥ 4 µg/mL). Of 23 *C. auris* isolates, 19 (82.6%) had a fluconazole MIC of ≥ 32 µg/mL. None of the isolates showed resistance to amphotericin B or echinocandins.

**Conclusion:** *C. parapsilosis* complex and *C. auris* were the *Candida* species identified most frequently in ear cultures and exhibited a high rate of fluconazole non-susceptibility, particularly *C. auris*.

P006

Epidemiological pattern of Malassezia, its phenotypic identification and anti-fungal susceptibility profile to azoles by broth micro-dilution method.

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:** Analyse the epidemiological pattern of disease with regards to age ,sex and site of predilection To Speciate *Malassezia* phenotypically. To study the anti-fungal susceptibility (AFST) pattern of Azoles to pathogenic *Malassezia* species by recording their MIC values (MIC₅₀ & MIC₉₀) using Broth Micro dilution method in accordance with CLSI M27-A3. To identify resistant drug/drugs to *Malassezia* species. .

**Methods: Prospective study** was done from **August 2016-October2018** with sample size of **150**
*Sample collection-Hyper/hypo pigmented skin lesions by Skin scrapings and swabs Microscopic*
*examination with 10%KOH Those showing meat ball and spaghetti appearances were included in the*
*study. Culture Samples inoculated into 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 Done by colony growth characteristics, gram staining, urease test,*
*catalase test, bile esculin with tween 60 hydrolysis and tween assimilation AFST AFST was performed*
*by the broth micro-dilution method, according to the Clinical and Laboratory Standard Institute*
*(CLSI) guidelines M27-A3 (2008) Systemic anti-fungals used-Fluconazole, Itraconazole,*
*Voriconazole Topical Anti-fungals used-Clotrimazole, Miconazole, Luliconazole Since Malassezia*
*are lipid dependant yeast RPMI 1640 was supplemented with glucose, peptone, ox bile, malt extract,*
glycerol, tween 80, tween 40 and Chloramphenicol Culture-Malassezia isolates grown on MDA for 72
h at 32°C± 2°C were used for inoculum preparation. Anti-fungal stock solutions of drugs were
prepared and stored at - 70°C Quality Control-Each microtitre plate included growth control well with
inoculum and supplemented RPMI -1640 medium without the antifungal agent. Candida krusei ATCC
6258 was used as a quality control strain for antifungal susceptibility testing. Incubation and Reading
Results-The microtitre plates were incubated at 32°C results read after 24hrs for candida isolate and
after 72-96 hours for Malassezia isolates. The minimum inhibitory concentration (MIC90) (reduction of
90% growth compared to growth control well) and MIC50 (50% reduction of growth compared to
growth control well) were recorded.

**Results:**
<figure><img src="image_14.png" /></figure>

<table><thead><tr><td>DRUG</td><td>MIC<sub>50</sub> (µg/ml)</td><td>MIC<sub>90</sub> (µg/ml)</td></tr></thead><tbody><tr><td>FLUCONAZOLE</td><td>8-32</td><td>16-&gt;64</td></tr><tr><td>MICONAZOLE</td><td>2-4</td><td>4-16</td></tr><tr><td>CLOTRIMAZOLE</td><td>0.125-2</td><td>0.5-4</td></tr><tr><td>SERTACONAZOLE</td><td>4-16</td><td>8-&gt;16</td></tr><tr><td>LULLICONAZOLE</td><td>1-4</td><td>2-8</td></tr></tbody></table>

**Age distribution-** The most common age affected was 20 to 30yrs (36%), followed by 30-40yrs (21%) and 10-20yrs (19%) extremes of age showed lower incidence. **Sex distribution-** The incidence among male and female was almost equal, showing little female predominance **Site of infection** - Among the total 150, the most common site was back (47 samples), followed by neck and shoulder (31 samples), and scalp (27 samples). **Skin lesions-** Both hypo and hyperpigmented skin scrapings were collected. But more of hypopigmented lesions were noted in our study **ANTI-FUNGAL SUSCEPTIBILITY PATTERN**-fig

**Conclusion:** Among 150 samples only 95(62%) yielded growth on both SDA with olive oil and MDA. Growth on MDA was soon than SDA with olive oil Overlay. All organisms were urease and catalase positive. Among the 95 samples grown the predominant species was *Malassezia sympodialis* 29(33%) followed by *M.globosa* 26(29.5%), *M.furfur* 21(23%), *M.obtusa* 12(13%) by conventional method. The systemic anti-fungal with the least MIC values were Itraconazole and Voriconazole. Fluconazole shows resistant Pattern with high MIC values The most effective topical anti-fungal was Clotrimazole with low MIC values *Malassezia sympodialis* responded to anti-fungal better than other species isolated

P007

Prevalence and genotype distribution of fluconazole-resistant Candida parapsilosis bloodstream isolates from a neonatal intensive care unit during a 14-year period

J.H. Shin<fnref n="1" />, I.J. Hwang², E.J. Won<fnref n="1" />, M.J. Choi<fnref n="1" />, S.-I. Jung³, E.S. Song², Y.Y. Choi²

<footnote n="1">¹Department Of Laboratory Medicine, Chonnam National University Medical School, Gwangju,</footnote>
Korea, Republic of, ²Paediatrics, Chonnam National University Medical School, Gwangju, Korea, Republic of, ³Chonnam National University Medical School, Gwangju, Korea, Republic of

**Objectives:** *Candida parapsilosis* bloodstream infection (BSI) is a significant problem in neonatal intensive care units (NICUs). *C. parapsilosis* is usually susceptible to azole antifungals; however, some recent reports have indicated the emergence of BSIs caused by fluconazole-resistant (FR) *C. parapsilosis* isolates. We investigated the prevalence and genotype distribution of FR *C. parapsilosis* BSI isolates recovered from a NICU during a 14-year period.

**Methods:** We assessed a total of 64 *C. parapsilosis* BSI isolates comprising 22 non-duplicate isolates from 22 neonates and 42 isolates from 21 neonates (two isolates from each neonate obtained on the first and the last days of *C. parapsilosis*-positive culture). All isolates were obtained at the same NICU between 2002 and 2015. *In vitro* antifungal susceptibility testing for fluconazole, amphotericin B, caspofungin, and micafungin was performed using the Clinical and Laboratory Standards Institute (CLSI) M27-A3 microdilution method. All isolates were genotyped via microsatellite typing using four specific microsatellite markers (CP1, CP4, CP6, and B). Clonal strains were defined as the isolation of two or more strains with identical genotype as determined by microsatellite typing.

**Results:** Of the 64 *C. parapsilosis* BSI isolates, 11 (17%) obtained from eight patients were FR (fluconazole minimal inhibitory concentration [MIC] ≥ 8 mg/L). All 64 isolates were susceptible to amphotericin B, caspofungin, and micafungin. Based on microsatellite typing, the 64 isolates yielded 19 different genotypes, including 12 genotypes unique to a single isolate and seven genotypes shared by 49 isolates (77%). All 11 FR isolates shared two genotypes (I and II) that displayed closer genetic relatedness than other isolates examined in the microsatellite analysis, according to as an unweighted pair group method with arithmetic mean (UPGMA) tree. Of these two genotypes, type I strains (three patient isolates) were isolated within a 6-month period, whereas type II strains (five patient isolates) were isolated over a 4-year period. Among 21 patients showing more than one *C. parapsilosis* isolate on 2 or more separate days, two sequential strains from each patient had identical types in 76% (16/21) of patients and different types in 24% (5/21). No difference in antifungal susceptibility was detected between two sequential isolates from the same patient. Among eight patients with FR *C. parapsilosis* isolates, 88% had no known prior antifungal exposure. We detected no significant difference in the overall 30-day mortality of *C. parapsilosis* BSI due to FR strains, or in that of BSI due to fluconazole-susceptible (FS) strains (25 vs. 20%).

**Conclusion:** The results of the present study suggest that clonal transmission may occur frequently among both FS and FR blood isolates of *C. parapsilosis* in a NICU setting. Increased incidence of BSI due to the FR *C. parapsilosis* strain in this NICU may be associated with clonal transmission rather than prior antifungal exposure.

P008

**Bloodstream Isolates of Azole-Resistant Candida glabrata in Korean hospitals: Resistance Mechanisms and Clinical Features**

J.H. Shin¹, E.J. Won¹, S.-I. Jung², I.J. Hwang³, M.J. Choi¹, S.A. Byun², S.H. Kim¹, Y.-J. Park⁴, M.-N. Kim⁵

¹Department Of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Korea, Republic of, ²Chonnam National University Medical School, Gwangju, Korea, Republic of, ³Paediatrics, Chonnam National University Medical School, Gwangju, Korea, Republic of, ⁴The Catholic University of Korea College of Medicine, Seoul, Korea, Republic of, ⁵University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea, Republic of

**Objectives:** *Candida glabrata* has recently become the most common non-*albicans* *Candida* species causing bloodstream infection (BSI) in South Korea, and the occurrence of azole-resistant *C. glabrata* isolates has been observed. We investigated the azole resistance mechanisms and clinical features of patients with azole-resistant *C. glabrata* BSI isolates recovered from multicenter surveillance in Korea.

**Methods:** In total, 248 *C. glabrata* BSI isolates comprising 43 azole-resistant (fluconazole minimum inhibitory concentration [MIC], ≥ 64 µg/mL and voriconazole MIC, ≥ 1 µg/mL) and 205 non-azole-resistant (non-AR) isolates (fluconazole MIC, ≤32 µg/mL and voriconazole MIC, ≤ 0.5 µg/mL) were analyzed. The isolates were collected from 10 university hospitals over 8 years (2009–2017). The *PDR1*
genes of the isolates were sequenced to identify mutations. Expression levels of *CDR1*, *CDR2*, and *ERG11* genes after fluconazole exposure were quantified by real-time PCR. The clinical features related to risk factors for BSI and the mortality rates were compared. Risk factors for BSI caused by AR isolates and survival rates in patients were assessed using multivariate logistic regression.

**Results:** Pdr1 mutations were identified in 42 of 43 azole-resistant isolates, while no Pdr1 mutation was found in 201 of 205 non-AR isolates. Azole-resistant isolates of *C. glabrata* had higher mean expression levels of *CDR1* (8.3-fold) gene than non-AR isolates (*P* < 0.05). However, there were no significant differences in the mean expression levels of *CDR2* and *ERG11* between two groups. Previous azole exposure (odds ratio [OR], 6.369; 95% confidence interval [CI], 2.434-16.664; *P* < 0.001), central venous catheter use (OR, 11.406; 95% CI, 2.462-52.837; *P* = 0.00211), and total parenteral nutrition (OR, 4.042 95% CI, 1.381-11.828; *P* = 0.011) were the independent risk factors for BSI caused by AR *C. glabrata*. *C. glabrata* BSIs caused by AR isolates resulted in a higher 30-day mortality rate (55.8%, 24/43) than BSIs caused by non-AR strains (36.6%, 75/205) (*P* = 0.019). In multivariate analyses, severe sepsis (OR, 5.592; 95% CI, 2.689-11.630; *P* < 0.001), a high age-adjusted Charlson comorbidity index (ACCI) score (OR, 1.131; 95% CI, 1.012-1.263; *P* = 0.03), lack of antifungal therapy (OR, 5.213; 95% CI, 2.487-10.930; *P* < 0.001), and azole-resistance in *C. glabrata* BSI isolates (OR, 2.856; 95% CI, 1.161-7.025; *P* = 0.022) were independent predictors of the 30-day mortality among the 248 patients.

**Conclusion:** This study provides that Pdr1 mutation with overexpression of *CDR1* may be the main resistance mechanism of *C. glabrata* in Korea, and that azole-resistance in *C. glabrata* BSI isolates is independent predictor of 30-day mortality, and is associated with antifungal exposure, central venous catheter, and total parenteral nutrition use.

P009

A case of urinary tract candidosis: a risky business

R. Barton, [K. Sethi](#)

Microbiology, Leeds Teaching Hospitals Trust, Leeds, United Kingdom

**Case Report:** A 62 year old man with a history of psoriasis treated with methotrexate, poorly controlled type 2 diabetes, and chronic obstructive pulmonary disease had a laparoscopic cholecystectomy for cholecystitis and repair of a cholecystoduodenal fistula. He then presented with severe dysuria, polyuria and complained of passing cloudy urine ever since the surgery. Following a series of urine samples which were positive for yeasts which were not identified and a course of fluconazole (details not available), he was seen at a haematuria clinic with continued dysuria, frequency and urgency, and a flexible cystoscopy showed a "snowstorm" appearance within bladder, and an ultrasound revealed mobile echogenic debris. He was then put on a 14/7 course of 200mg oral fluconazole, pending a cystoscopy and bladder biopsy which revealed erosive cystitis. A subsequent urine sample was identified as *Candida glabrata* which was resistant to fluconazole (MIC >256mg/L) but sensitive to flucytosine (MIC 0.06mg/L). Despite a further 14 day course of 800mg fluconazole, signs and symptoms of *Candida* cystitis were not resolved. The patient was then referred to a urologist at our hospital and various treatment choices were discussed with oral flucytosine and IV Amphotericin deoxycholate being the main two options, associated with the risk of resistance and toxicity respectively. It was decided to treat with a 7 day course of 25mg/kg of oral flucytosine (levels were not monitored due to the short course) but follow up urine samples 9 days after completing the flucytosine course were still positive for *C. glabrata* resistant to both fluconazole (MIC >256mg/L) and flucytosine (MIC >64mg/L). Approximately 2 weeks after completing the course of flucytosine, the patient was admitted via Accident and Emergency with suspected sepsis relating to a biliary source, and a new ultrasound showed that the left kidney was moderately hydronephrotic, the bladder was filled with echogenic debris. A nephrostomy was performed and blood cultures taken which grew *C. glabrata* with identical sensitivities to that from the most recent urine sample. The patient was prescribed a 2 week course of Anidulafungin (200mg loading dose then 100mg 24-hourly IV) to combat the systemic candidosis, and a 7 day course of intravenous Amphotericin deoxycholate at 0.6mg/kg/day. During the Amphotericin B deoxycholarte treatment, renal function was not markedly affected, though increases in alkaline phosphatase levels led to the treatment being stopped at 6 days.
Subsequent blood and urine cultures remained negative, CRP declined from 150 to 5.8 mg/L and symptoms resolved. This case is one of the examples of how the significance of the presence of yeasts in urine of patients is not clearly understood by the clinicians. Our patient presented here highlights the increasing prevalence of *C.glabrata* in funguria and non-*albicans* candidaemia in at risk settings, the challenges in its management, the risks and drug related factors which need to be assessed when treating fluconazole resistant urinary tract candidosis.

P010

Effects of efflux pump modulators on the azole antifungal susceptibility of Microsporum canis

C. Aneke, W. Rhimi, D. Otranto, C. Cafarchia

Veterinary Medicine, University of Bari, Aldo Moro, Bari, Italy

**Objectives:** This study investigated the effect of efflux pump modulators, EPMs (i.e. haloperidol-HAL and promethazine-PTZ) and their inhibiting activity on the minimum inhibitory concentrations of itraconazole (ITZ) and fluconazole (FLZ), against selected *M. canis* strains.

**Methods:** *M. canis* strains with low (≤ 1mg/ml itraconazole and <64mg/ml fluconazole) and high (>1mg/ml itraconazole and ≥64 µg/ml fluconazole) azole MIC values were tested, and data analyzed using the model-fractional inhibitory concentration index (FICI) method.

**Results:** The MIC values of ITZ and FLZ of *M. canis* decreased in the presence of sub-inhibitory concentrations of HAL and PTZ, the latter being more effective with a greater increased susceptibility. Synergism was observed in all strains with high azole MICs (FICI<0.5) and no synergism in the strains with low azole MICs.

**Conclusion:** These results suggest that the drug efflux pumps are involved in the defense mechanisms to azole drugs in *M. canis* strains. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Additionally, the most effective double combinations were those of PTZ with either FLZ or ITZ as this had a greater increase in azole susceptibility. Complementary studies on *M. canis* resistance are advocated in order to investigate the molecular mechanisms of this phenomenon.

P011

Antifungal resistance pattern of Candida species isolated from women of child-bearing age in Nsukka Nigeria suffering from different clinical conditions.

A. Mgbeahuruike

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

**Objectives:** To isolate and identify Candida species in child bearing women suffering from malaria, typhoid and diabetes To investigate the resistance partarn of the identified Candida species using conventional azoles and plant extracts To trace the evolutionary relatedness of the isolated fungi using phylogenetic analysis

**Methods:** High vaginal swabs were analyzed for *Candida* presence in women of child bearing age suffering from malaria, typhoid and diabetes using Chromogenic agar. Evolutionary relatedness of the *Candida* species was inferred using phylogenetic reconstruction. Growth rate assay was done using Sabourand Dextrose medium supplemented with NaCl and glucose. The resistance pattern of the *Candida* species was tested using fluconazole and extracts (methanol and ethyl) from *Byrsocarpus coccineus*.

**Results:** Out of the 79 fungal samples analyzed, 21 (26.6%) were *C. tropicalis*, 25 (31.6%) were *C. krusei*, 10 (12.7%) were *C. parapsilosis* and 8 (10.1%) were *C. albicans*. *C. tropicalis* was highest in the malaria patients, *C. albicans* dominated in the typhoid patients while *C. krusei* was more in the diabetic patients. The healthy women presented a very low percentage (8.9%) of *Candida* species. Phylogenetically, the sequenced *Candida* species was more closely related to *C. tropicalis*. High concentration of glucose (1g/ml) and NaCl (0.5M) decreased the growth of the fungus. From the study, fluconazole showed a higher activity than the two extracts (methanol and ethyl acetate).
**Conclusion:** In conclusion, although fluconazole showed a higher activity than the two extracts (methanol and ethyl acetate), the plant extracts demonstrated very high activity and can be used as potential antifungal agents. *C. tropicalis* and *C. krusei* were the most abundant *Candida* species found in this study.

P012

Colistin and isavuconazole interact synergistically in vitro against Aspergillus nidulans and Aspergillus niger

P. Schwarz¹,², E. Dannaoui³,⁴

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

**Objectives:** Invasive aspergillosis is a devastating disease in immunocompromised patients. It mostly affects patients with hematological malignancies, especially those with severe and prolonged neutropenia. Colistin is a polypeptide antibiotic from the class of polymyxins. Polymyxins bind to lipopolysaccharides and phospholipids of the outer cell membrane of gram-negative bacteria. Binding leads to competitive displacement of divalent cations from the phosphate groups of the membrane lipids, leading to bactericidal action by disruption of the outer cell membrane and leakage of intracellular contents. Colistin has also shown *in vitro* activity against some fungi. The aim of the present study was to assess the *in vitro* interaction of the broad-spectrum azole isavuconazole with colistin against the most important *Aspergillus* species responsible for human disease.

**Methods:** A panel of 31 Aspergillus isolates belonging to 5 species responsible for human invasive aspergillosis was used for the experiments (10 *Aspergillus fumigatus*, 5 *Aspergillus flavus*, 5 *Aspergillus terreus*, 6 *Aspergillus nidulans*, and 5 *Aspergillus niger*). Combinations of isavuconazole with colistin were tested by a broth microdilution checkerboard technique based on the EUCAST reference methodology. Plates were read spectrophotometrically, and ninety percent of inhibition was used as an endpoint for both, the drugs alone and in combination. Results were interpreted with the fractional inhibitory concentration index (FICI). Drug interactions were defined as synergistic (FICI≤0.5), indifferent (FICI>0.5≤4) or antagonistic (FICI>4).

**Results:** Isavuconazole MICs ranges from 0.5 to 16 µg/ml. When tested alone, colistin exhibited no antifungal activity with MICs of 128 µg/ml for all isolates. Combination of isavuconazole with colistin was synergistic for 100 and 60% of the *A. nidulans*, and *A. niger* isolates, respectively (Table). All other interactions were indifferent. Antagonism was never seen.

<table><thead><tr><th rowspan="2">Species (number of isolates)</th><th colspan="3">% interaction of colistin with isavuconazole</th></tr><tr><th>synergy</th><th>indifference</th><th>antagonism</th></tr></thead><tbody><tr><td><i>Aspergillus fumigatus</i> (10)</td><td>0</td><td>100</td><td>0</td></tr><tr><td><i>Aspergillus flavus</i> (5)</td><td>0</td><td>100</td><td>0</td></tr><tr><td><i>Aspergillus terreus</i> (5)</td><td>0</td><td>100</td><td>0</td></tr><tr><td><i>Aspergillus nidulans</i> (6)</td><td>100</td><td>0</td><td>0</td></tr><tr><td><i>Aspergillus niger</i> (5)</td><td>60</td><td>40</td><td>0</td></tr><tr><td>All (31)</td><td>29</td><td>61</td><td>0</td></tr></tbody></table>

**Conclusion:** Combination of colistin with isavuconazole showed strong synergy against all tested *A. nidulans* isolates and synergy for the majority of the tested *A. niger* isolates. *In vitro* interaction of colistin and isavuconazole will be further evaluated by checkerboard and gradient concentration strips against a larger panel of *A. nidulans* and *A. niger* isolates.

P013

Update 2016-2018 of the nationwide Danish fungaemia surveillance study: epidemiologic changes in a 15-year perspective

M. Risum¹, R. Datcu¹, H.K. Johansen², H.C. Schønheyder³, F.S. Rosenvinge⁴, I.J.D. Knudsen⁵, B.L. Røder⁶, V.S. Antsupova⁷, J.B. Gertsen⁸, L. Kristensen⁸, J.K. Møller⁹, R. Hare¹, K.M.T. Astvad¹, K.M.
Jørgensen¹, E. Dzajic¹⁰, M.C. Arendrup¹,²,¹¹

¹Mycology Unit, Statens Serum Institute, Copenhagen, Denmark, ²Department Of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark, ³Department Of Clinical Microbiology, Aalborg University Hospital, Aalborg, Denmark, ⁴Department Of Clinical Microbiology, Odense University Hospital, Odense, Denmark, ⁵Department Of Clinical Microbiology, Hvidovre Hospital, Hvidovre, Denmark, ⁶Department Of Clinical Microbiology, Slagelse Sygehus, Slagelse, Denmark, ⁷Department Of Clinical Microbiology, Herlev Hospital, Herlev, Denmark, ⁸Department Of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark, ⁹Department Of Clinical Microbiology, Vejle Sygehus, Vejle, Denmark, ¹⁰Department Of Clinical Microbiology, Esbjerg Sygehus, Esbjerg, Denmark, ¹¹University Of Copenhagen, Dept. of Clinical Medicine, Copenhagen, Denmark

**Objectives:** The objectives of this study was to provide nationwide contemporary data on the epidemiology of candidaemia, often a serious complication of hospital stay with a crude 30-day mortality rate of about 40% despite recent advantages in diagnosis and management. The motivation was that outcome depend on early appropriate therapy necessitating knowledge on susceptibility pattern of involved species. Moreover, epidemiological changes reflect current practises in prophylactic, empirical and pre-emptive use of antifungals and therefore are also informative regarding the need for adjustment of antifungal stewardship programmes or treatment guidelines.

**Methods:** As part of an active national surveillance programme, fungal isolates from blood culture were referred to Statens Serum Institut (Copenhagen) for confirmatory species identification and EUCAST E Def 7.3.1 susceptibility testing. A total of 1452 unique isolates (same patient and species isolates were regarded unique if more than 21 days apart or with different susceptibility) were included. 13 isolates (0.9%) were not referred. When MICs for echinocandins were above the clinical breakpoints, FKS sequencing of the isolates was performed. An isolate was deemed echinocandin resistant if the MIC was above anidulafungin breakpoint and/or if the isolate had a known FKS hotspot-mutation. Results were compared to previous data in a 15-year perspective.

**Results:** The mean incidence rate during 2016-2018 was 8.13/100.000 inhabitants. Candidaemia was more frequent in males than females (60% vs 40%) as in accordance with previous years. In comparison to previous data, the fungaemia rate was stable (8.64, 9.03 and 8.38 per 100.000 inhabitants in 2004-7, 2008-11 and 2012-15 respectively). In the years 2016-2018 *C. albicans* accounted for 42.1% of all unique isolates with a decline in 2017 to below 40%. *C. glabrata* accounted for 32.1%. The proportion of candidaemia in 2018 involving *C. albicans* decreased by a third from 64% in 2004 (P<0.0001), and *C. glabrata* almost doubled from 17% in 2004 (P<0.0001) (Figure). We found a fluconazole susceptibility rate across all *Candida* species of 59% of isolates which has decreased since 2004-7 from 69% (P = 0.02). Echinocandin resistance was detected in 18/1294 isolates (1.4%) including 11/459 (2.4%) in *C. glabrata* specifically. No echinocandin resistance was detected in 2004-7. FKS-mutations were identified in the following species *C. glabrata*: two L630Q/S663F, four F659S, one L662W, three S663F and one S663P; *C. krusei*: four L701M, two S659F and one S659S/F; *C. albicans* one P1354P/S; *C. dubliniensis* one S645P; and for *Candida lusitaniae* one H657Y/L1243F/I283V. H657Y/L1243F/I283V, P1354P/S and L701M are outside the hot spot, and L701M is not associated as a molecular cause of echinocandin resistance.
<figure><img src="image_15.png" /></figure>

*Before 2010 the term non- C. albicans was used

**Conclusion:** We report a stable incidence rate of candidaemia, but the proportion of C. glabrata continues to rise at the expense of C. albicans which accounted for less than 40% in 2017. The consequent decrease in fluconazole susceptibility along with the presence of FKS-mutations particularly in C. glabrata raises concern for future perspectives in the treatment of candidaemia.

P014

**In vitro potpourri: Aspergillus terreus and the activity of amphotericin B**

R. Vahedi Shahandashti

Division Of Hygiene And Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria

**Objectives:** Invasive aspergillosis caused by intrinsically resistant non-*fumigatus* *Aspergillus* species displays a poor outcome in immunocompromised patients. *Aspergillus terreus* holds an exceptional position within the *aspergilli* due to its intrinsic resistance against the polyene amphotericin B (AmB). Till now, the exact mode of action in polyene resistance is not well understood. Micro-environmental factors at the infection site influence the growth of fungal pathogens and most likely also the efficacy of antifungal drugs. The purpose of this study was to evaluate the impact of hypoxia versus normoxia on the *in vitro* activity (fungistatic and fungicidal) of AmB against *A. terreus*.

**Methods:** In total, 100 clinical strains of *Aspergillus* section *terrei*, collected from 21 various countries were analyzed in detail. Antifungal susceptibility testing was carried out according to EUCAST guideline, with normoxic (20% oxygen/5% carbon dioxide/75% nitrogen) and hypoxic (1% oxygen/5% carbon dioxide/94% nitrogen) conditions. Final drug concentrations used for AMB were 0.03–16 µg/ml. Minimum inhibitory concentration (MIC) was defined as the lowest drug concentration showing no fungal growth and minimum fungicidal concentrations (MFCs) was taken as the first well with ≥90% killing of fungi. MFC was determined by agar and broth-based (flash microbicide method) methods. MICs (µg/ml) and colony forming units were determined after 48h at 37°C. Testing was performed in duplicates.

**Results:** AmB MICs of *A. terreus* section *terrei* ranged from 0.125-2 µg/ml for hypoxia, and from 0.5-8 µg/ml for normoxia. These data indicate that some strains were more susceptible to AmB in hypoxic conditions compared to normoxia (Figure 1). The majority of strains showed that AmB had no fungicidal effect against tested *A. terreus* under any methods and atmospheric conditions (Figure 2). *A. terreus* representing lower MICs (n = 41, ≤0.5 µg/ml) did not result in modest MFCs (≥90% killing in 1-2 µg/ml). Fig.1. *In vitro* susceptibility to AMB of 100 *A. terreus* isolates in normoxia versus
hypoxia, measured by EUCAST

<figure><img src="image_16.png" /><figcaption>Fig.2.a. The effect of oxygen on MFC distributions of AMB, agar-based method measured by colony forming units.</figcaption></figure>

<figure><img src="image_17.png" /></figure>

**Conclusion:** Simulating the host environment in *in vitro* susceptibility testing will contribute to a better understanding of how these conditions influence antifungal activity. Hypoxic conditions had a marginal influence on the *in vitro* antifungal susceptibility pattern of *A. terreus* species to AmB. A slight reduction of MICs was demonstrated, however, *A. terreus* did not turn to be classified as AmB susceptible (AmB MICs < 0.125 µg/ml) under hypoxic conditions. Remarkably, AmB lacked any fungicidal activity in the majority of examined strains; comparing with MICs, MFC seems to be related more with *in vivo* outcome and might partially explain the high failure rate of AmB therapy *in vivo*.
R. Stauf¹, D. Todt², E. Steinmann², P.-M. Rath³, H. Gabriel⁴, J. Steinmann⁵, F.H.H. Brill⁴

¹Institute For Clinical Hygiene, Medical Microbiology And Infectiology, Paracelsus Medical University Nuremberg, Nuremberg, Germany, ²Department For Molecular And Medical Virology, Ruhr-University Bochum, Bochum, Germany, ³Institute Of Medical Microbiology, University Hospital Essen, Essen, Germany, ⁴Institute For Hygiene And Microbiology, Dr. Brill + Partner GmbH, Hamburg, Germany, ⁵Institute Of Clinical Hygiene, Medical Microbiology And Clinical Infectiology, Klinikum Nuernberg, Nuremberg, Germany

**Objectives:** Prevention of immunosuppressed patients against fungal pathogens is essential in times of emergence of antifungal-resistant yeasts and moulds. Therefore, the use of fungicidal disinfectants is an important measure preventing infections. In this study, the fungicidal activity of common microbial agents of chemical disinfectants against antifungal-resistant and antifungal-susceptible yeasts and moulds was tested in comparison to the reference strains *Candida albicans*, *Candida tropicalis*, *Aspergillus brasiliensis* and *Aspergillus niger*.

**Methods:** In total, nine clinical isolates from the fungal collection of the University Hospital Essen and four reference strains were included. Species identification was performed by morphological methods and sequencing of the internal transcribed spacer region (ITS). The antifungal susceptibility testing was performed via microdilution assay according to EUCAST standard 9.3 for moulds and 7.3.1 for yeasts. The sensitivity of clinical yeasts and moulds isolates in comparison to the reference isolates was tested in a quantitative suspension test against peracetic acid and ethanol as the common active ingredients of chemical disinfectants.

**Results:** Ethanol was sufficiently active with a log₁₀ reduction factor of ≥ 4.0 against all yeasts in a concentration of 50 % v/v within 1 min and against all moulds as 80 % solution within 1 min. Peracetic acid was active as 0.25 % solution in 5 min against all yeasts and as 0.5 % solution in 5 min against all moulds. Compared to the reference strains the clinical isolates showed similar or higher sensitivity to the active ingredients. In addition, antifungal-resistant strains exhibited equivalent sensitivity in *vitro* against ethanol and peracetic acid compared to the susceptible ones.

**Conclusion:** Clinical fungal isolates including multidrug resistant ones did show a similar or higher sensitivity to active ingredients of disinfectants than the reference strains.

P016

In vitro susceptibility of olorofim against 1,682 clinical Aspergillus isolates

J. Buil¹, J. Oliver², M. Birch², D. Law², J. Rex², M. Tehupeiory-Kooreman¹, H. Van Der Lee¹, W. Melchers¹, P.E. Verweij¹

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

**Objectives:** Olorofim (OLO) is a novel antifungal agent with potent activity against filamentous fungi such as *Aspergillus spp.*, *Scedosporium spp.* and *Lomentospora prolificans*. We included OLO in our routine MIC-panel for filamentous fungi and analysed the *in vitro* susceptibility of OLO against clinical *Aspergillus* isolates received in our laboratory between May 2017 and March 2019.

**Methods:** The *in vitro* activity of OLO was tested against 1,682 clinical *Aspergillus* isolates (987 azole susceptible *A. fumigatus* isolates, 414 azole-resistant *A. fumigatus*, 72 *A. flavus* species complex (SC), 32 *A. nidulans* SC, 110 *A. niger* SC, 21 *A. terreus* SC, 7 *A. versicolor* SC, 39 other species). OLO susceptibility was determined using broth microdilution based on EUCAST guidelines. Isolates were identified to the SC level using microscopy and growth characteristics.

**Results:** The olorofim MICs of all clinical *A. fumigatus*, *A. flavus* species complex, *A. terreus* species complex, *A. niger* species complex, *A. versicolor* species complex were found to be uniformly <1 mg/L. The MIC50 and MIC90 of species with >10 different isolates are displayed in the table. No significant differences were found in the OLO MICs of azole-susceptible *A. fumigatus* isolates compared to azole-resistant *A. fumigatus* isolates. Isolates of some cryptic species within the *Aspergillus aspergillus* species complex (*A. hollandicus*, *n* = 1 *A. glaucus*, *n* = 4 and *A. chevalieri* *n* = 1) were found to have elevated MIC's: 1 isolate (*A. glaucus*) with a MIC of 1 mg/L and the remainder with a MIC of >2 g/L).
<table><thead><tr><th>Species</th><th>Number of isolates</th><th>Geometric mean (mg/L)</th><th>MIC 50 (mg/L)</th><th>MIC 90 (mg/L)</th></tr></thead><tbody><tr><td><strong>Aspergillus fumigatus</strong>,<br>azole susceptible</td><td>987</td><td>0.027</td><td>0.031</td><td>0.063</td></tr><tr><td><strong>Aspergillus fumigatus</strong>,<br>azole resistant</td><td>414</td><td>0.033</td><td>0.063</td><td>0.063</td></tr><tr><td><strong>Aspergillus flavus</strong> SC</td><td>72</td><td>0.023</td><td>≤0.016</td><td>0.063</td></tr><tr><td><strong>Aspergillus terreus</strong> SC</td><td>21</td><td>0.023</td><td>≤0.016</td><td>0.063</td></tr><tr><td><strong>Aspergillus niger</strong> SC</td><td>110</td><td>0.034</td><td>0.031</td><td>0.063</td></tr><tr><td><strong>Aspergillus nidulans</strong> SC</td><td>32</td><td>0.043</td><td>0.031</td><td>0.125</td></tr></tbody></table>

**Conclusion:** OLO showed potent *in vitro* activity against a large collection of clinical *Aspergillus* species, including 1401 *A. fumigatus* isolates, with many of these resistant to one or more azoles. Interestingly, OLO was less potent against a small number of isolates belonging to the *Aspergillus aspergillus* SC (formerly *Eurotium*).

P017

Detection and Typing of Fluconazole Resistance on *Candida albicans* with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass

M. Delavy, M. Corthesy, G. Greub, D. Sanglard, A.T. Coste

Microbiology Institute, University Hospital Lausanne, University of Lausanne, Lausanne, Switzerland

**Objectives:** *Candida albicans* causes life-threatening systemic infections in immunosuppressed patients. These infections are commonly treated with fluconazole, an antifungal agent targeting the ergosterol biosynthesis pathway. Current Antifungal Susceptibility Testing (AFST) methods take time and are often subjective. An alternative to the classical AFST methods could use Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry (MS). This tool, already used in clinical microbiology for microbial species identification, has already offered promising results to detect antifungal resistance and could be used to detect specific peaks associated with fluconazole resistance.
<figure><img src="image_18.png" /></figure>

**Methods:** We analyzed MALDI-TOF MS spectra acquired, with the Bruker microflex device, from 21 C. albicans clinical strains isolated from 9 patients. Those strains were exposed for 3h to 3 fluconazole concentrations (256, 16, 0µg/mL). We optimized a protein extraction protocol that allowed the acquisition of high-quality spectra. Two steps of quality controls were implemented. First, the quality of the spectrum was assessed through its identification score. Second, the variability of the biological and technical replicates was evaluated, and outliers were removed. We then developed an R pipeline to identify MALDI-TOF MS signature peaks associated with fluconazole resistance or with TAC1 or ERG11 mutations known to affect C. albicans susceptibility level, and we designed score-based diagnostic tests with these signature peaks. Finally, we evaluated the effects of adding a protein standard (Bacterial Testing Standard, BTS, Bruker) to the proteins extracts to reduce the variability of the peaks position. And then we tested the effect of the tolerance inhibitor cyclosporin A on the susceptible strains' spectra quality.

**Results:** Up to now, in absence of cyclosporin A or BTS, we identified a 4480 m/z peak, whose intensity after exposition to 16 µg/mL fluconazole, was inversely proportional correlated with
fluconazole resistance. The intensity of the peak seems to be more inversely proportional correlated with *TAC1* gain-of-function mutation than with *ERG11* mutations. Since only a single peak was identified, it is likely that the trailing effect of fluconazole might hide other interesting peaks. We thus decide to inhibit fluconazole tolerance using cyclosporin A in further experiments. In addition, the main challenge of this first analysis was to reproducibly align the spectra. To circumvent this issue, we decided to introduce a protein standard (BTS) to the fungal proteins extracts to be analyzed. Effects of BTS or cyclosporin A are currently under investigation.

**Conclusion:** This study acts as a proof-of-concept for the use of MALDI-TOF MS for the typing of fluconazole resistance in *C. albicans* and currently offers promising results.

P018

**Azole resistance in *Aspergillus*. The first six months data from the Danish nationwide surveillance study**

M. Risum¹, R. Hare¹, F.S. Rosenvinge², J.B. Gertsen³, L. Kristensen³, J. Bangsborg⁴, B.L. Røder⁵, E.S. Marmolin⁶, S. Sulim⁷, K.M.T. Astvad⁸, M. Pedersen⁸, R. Datcu¹, M.C. Arendrup¹,⁹,¹⁰

¹Mycology Unit, Statens Serum Institute, Copenhagen, Denmark, ²Department Of Clinical Microbiology, Odense University Hospital, Odense, Denmark, ³Department Of Clinical Microbiology, Århus University Hospital, Århus, Denmark, ⁴Department Of Clinical Microbiology, Herlev Hospital, Herlev, Denmark, ⁵Department Of Clinical Microbiology, Slagelse Sygehus, Slagelse, Denmark, ⁶Department Of Clinical Microbiology, Vejle Sygehus, Vejle, Denmark, ⁷Department Of Clinical Microbiology, Ålborg University Hospital, Ålborg, Denmark, ⁸Department Of Clinical Microbiology, Hvidovre Hospital, Hvidovre, Denmark, ⁹University Of Copenhagen, Dept. of Clinical Medicine, Copenhagen, Denmark, ¹⁰Department Of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark

**Objectives:** Azole resistance (azole-R) in *Aspergillus* has been increasingly reported worldwide. The use of azole fungicides has been proposed to contribute to the emergence of azole-R *A. fumigatus*, and isolates harbouring the resistance mechanisms TR34/L98H or TR46/Y121F/T289A have been dominant among azole-R *A. fumigatus* in azole naïve patients and environmental samples. These two resistance genotypes have been sporadically found in clinical and environmental samples in Denmark, but the size of the problem remains unknown. The purpose of this study was to establish a nationwide surveillance programme of azole-R *Aspergillus* in Denmark and to report the data from the first six month's study period.

**Methods:** Unique *Aspergillus* isolates were included from all Danish clinical microbiological departments in the period October-2018 to March-2019. Isolates from same patients were defined as unique if 1) found >30 days apart, 2) confirmation of another species or 3) different susceptibility pattern. Inclusion criteria were: a) *Aspergillus* isolates regarded clinically significant, or b) any *Aspergillus* isolate detected on a Monday regardless of clinical significance. Referral practices varied depending on local mycology skills. Most laboratories referred all moulds or all *Aspergillus* isolates which then underwent species ID and susceptibility testing at the reference lab (SSI). One laboratory (Aarhus University Hospital) reported the species identification for *Aspergillus* (n = 214) and only referred part of the isolates. Thus susceptibility was performed for 46/138 *A. fumigatus* isolates from AUH at the time of writing. Referred *Aspergillus* isolates were identified to the complex level except *A. fumigatus* which was identified sensu stricto using thermotolerance testing. *A. fumigatus* isolates underwent screening for azole-R following the EUCAST E.Def 10.1 method and using VIPcheck azole agar plates (Mediaproducts BV, Grönningen, NL). Screening positive *A. fumigatus* isolates and all non-*fumigatus* *Aspergillus* isolates underwent EUCAST E.Def 9.3.1 susceptibility testing. Isolates were deemed intermediate when classified as such for >1 azole. Isolates with azole MIC(s) above the EUCAST BP(s) underwent CYP51A sequencing. Duplicates within 30 days were excluded.

**Results:** A total of 695 *Aspergillus* isolates were included of which 503 were *A. fumigatus*. Susceptibility testing was performed for 411 *A. fumigatus* isolates from 319 patients at the time of writing (Table 1). For *A. fumigatus*, 92.7% isolates were azole susceptible, 1.7% intermediate and 5.6% azole-R. Nineteen out of 319 patients harboured an *A. fumigatus* which was azole-R (6%). Among these
18/19 (95%) harboured an isolate with a CYP51A mutation (Table 2), and 14/19 (73.7%) a CYP51A mutation derived from the environment. Furthermore, 2/9 patients harboured three azole resistant *A. terreus* isolates, each with a CYP51A mutation (Table 2).

<table><caption>**Table 1.** Species distribution and susceptibility for *Aspergillus* complexes and *A. fumigatus* sensu stricto</caption><thead><tr><th rowspan="2"></th><th rowspan="2">No.</th><th colspan="2">Itraconazole</th><th colspan="2">Posaconazole</th><th colspan="2">Voriconazole</th><th colspan="2">Isavuconazole</th></tr><tr><th>Range</th><th>MIC50</th><th>Range</th><th>MIC50</th><th>Range</th><th>MIC50</th><th>Range</th><th>MIC50</th></tr></thead><tbody><tr><td><i>A. flavus</i></td><td>21</td><td>≤0.03-1</td><td>0.125</td><td>0.125-0.25</td><td>0.125</td><td>0.5-1</td><td>1</td><td>0.5-4</td><td>1</td></tr><tr><td><i>A. fumigatus</i></td><td>411</td><td>0.06-&gt;16</td><td>0.25</td><td>0.03-&gt;4</td><td>0.06</td><td>0.25-8</td><td>0.5</td><td>0.5-16</td><td>1</td></tr><tr><td><i>A. nidulans</i></td><td>3</td><td>0.125</td><td>0.125</td><td>0.06-0.125</td><td>0.125</td><td>0.125-0.25</td><td>0.25</td><td>0.125-0.25</td><td>0.125</td></tr><tr><td><i>A. niger</i></td><td>61</td><td>0.25-&gt;16</td><td>1</td><td>0.125-0.5</td><td>0.5</td><td>0.5-2</td><td>1</td><td>01-aug</td><td>2</td></tr><tr><td><i>A. terreus</i></td><td>18</td><td>0.06-&gt;16</td><td>1</td><td>0.03-0.5</td><td>0.5</td><td>0.25-4</td><td>1</td><td>0.25-16</td><td>2</td></tr><tr><td>Other <i>Aspergillus</i></td><td>8</td><td>0.06-&gt;16</td><td>0.5</td><td>0.03-&gt;4</td><td>0.25</td><td>0.25-16</td><td>0.5</td><td>0.25-8</td><td>0.5</td></tr></tbody></table>

<table><caption>**Table 2.** cyp51A alterations found in resistant *A. fumigatus* and *A. terreus* isolates</caption><thead><tr><th colspan="3">A. fumigatus</th></tr><tr><th>No. of patients</th><th>Cyp51A profile</th><th>Azole resistance</th></tr></thead><tbody><tr><td>13</td><td>TR34/L98H</td><td>Pan-azole resistant</td></tr><tr><td>1</td><td>Trip34/L98H</td><td>Pan-azole resistant</td></tr><tr><td>2</td><td>G54R</td><td>Itraconazole and posaconazole resistant</td></tr><tr><td>1</td><td>G432S</td><td>Pan-azole resistant</td></tr><tr><td>1</td><td>M220K</td><td>Itraconazole and posaconazole resistant</td></tr></tbody><thead><tr><th colspan="3">A. terreus</th></tr><tr><th>No. of patients</th><th>Cyp51A profile</th><th>Azole resistance</th></tr></thead><tbody><tr><td>1*</td><td>M217I</td><td>Pan-azole resistant</td></tr><tr><td>1*</td><td>Y491H</td><td>Intermediate resistant</td></tr><tr><td>1</td><td>G51A</td><td>Itraconazole and posaconazole resistant</td></tr></tbody></table>

*same patient with two different mutant isolates

**Conclusion:** We report a nationwide azole-R rate of 6% in *A. fumigatus* at the patient level. The underlying resistance mechanisms was target gene mutations in all but one case and notably, the vast majority were of environmental origin linked to the use of azole fungicides. The fact that such isolates have increasingly been found in Denmark since 2009 is concerning and suggests that a one-health approach involving human and environmental azole management is necessary to limit further rise in azole-R *A. fumigatus*

P019

## Multicentre study to determine the Etest® epidemiological cut-off values of antifungal drugs in Candida spp. and *Aspergillus fumigatus* SC

M. Salse<fnref n="1" />, J.P. Gangneux², S. Cassaing³, L. Delhaes⁴, A. Fekkar⁵, D. Dupont⁶, F. Botterel⁷, D. Costa⁸, N. Bourgeois⁹, B. Bouteille¹⁰, S. Houzé¹¹, E. Dannaoui¹², H. Guegan², E. Charpentier³, F. Persat¹³, L. Favennec⁸, L. Lachaud⁹, M. Sasso<fnref n="1" />

<footnote n="1">¹Laboratoire De Microbiologie, CHU Nîmes, Nîmes, France, ²Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France, ³Jean-pierre.gangneux@chu-rennes.fr, CHU de Toulouse, Toulouse, France, ⁴Laboratoire De Parasitologie-mycologie, CHU de Bordeaux, Bordeaux, France, ⁵Laboratoire De Parasitologie-mycologie, APHP La Pitié Salpétrière, PARIS, France, ⁶Laboratoire De Parasitologie-mycologie, Hospices civils de Lyon, Lyon, France, ⁷Laboratoire De Parasitologie-mycologie, APHP Hôpital Henri Mondor, Créteil, France, ⁸Laboratoire De Parasitologie-mycologie, CHU de Rouen, Rouen, France, ⁹Laboratoire De Parasitologie-mycologie, CHU de Montpellier, Montpellier, France, ¹⁰Laboratoire De Parasitologie-mycologie, CHU de Limoges, Limoges, France, ¹¹Laboratoire De Parasitologie-mycologie, APHP Hôpital Bichat, Paris, France, ¹²Laboratoire De Parasitologie-mycologie, APHP Hôpital Pompidou, Paris, France, ¹³Parasitologie Mycologie Médicale, Hospices Civils de Lyon, Lyon, France</footnote>
**Objectives:** To determine the specific Etest®-based epidemiological cut-off values (ECVs) for the main antifungal agents used in clinical practice, against the most frequent *Candida* spp. and *Aspergillus fumigatus* species complex (SC), in order to harmonise the interpretation of the Etest® minimum inhibitory concentrations (MICs) results in daily routine.

**Methods:** For each antifungal agent, the Etest® MICs in *Candida* spp. and *A. fumigatus* SC isolated from routine specimen cultures were retrospectively collected from 2004 to 2018 from the laboratories of 12 French university hospitals. To ensure that robust and comparable data were included for the ECV estimation, basic requirements and criteria have been fulfilled during the selection of MIC data: aberrant individual distributions were excluded, MIC data were pooled only if there are generated by at least three independent laboratories, and a minimum of 100 MIC values were required after data pooling for each species-antifungal agent combinations. The ECVs were then calculated using the iterative statistical method with a 97.5% cut-off.

**Results:** Forty-eight Etest® ECVs were determined for amphotericin B, caspofungin, micafungin, anidulafungin, fluconazole, voriconazole, posaconazole and itraconazole, after pooling and analysing the MICs of 9,654 *Candida albicans*, 2,939 *Candida glabrata* SC, 1,458 *Candida parapsilosis* SC, 1,148 *Candida tropicalis*, 575 *Candida krusei*, 518 *Candida kefyr*, 241 *Candida lusitaniae*, 131 *Candida guilliermondii*, and 1,526 *A. fumigatus* SC isolates. The ECV results were identical (17/32 ECVs) or within one two-fold dilution (15/32 ECVs) to the previously reported Etest®-based ECVs. They were comparable to the Clinical and Laboratory Standards Institute (CLSI) ECVs in 76.1% (similar or within one two-fold dilution: 35/46 ECVs) and strictly identical in 39.1% of cases (18/46 ECVs). They were comparable to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) ECVs in 81.2% of cases (similar or within one two-fold dilution: 26/32 ECVs) and strictly identical in 28% (9/32 ECVs).

**Conclusion:** On the basis of these and other previous results, we recommend the determination of method-dependent ECVs. As Etest® is the most widely used commercial method in French laboratories, the goal of this study was to calculate specific ECVs for this method and harmonise the interpretation of the Etest® MICs results in daily routine. Etest® ECVs should not be used in place of breakpoints but may identify non-WT isolates with a potential resistance to antifungal agents and suggest that an isolate may not respond as expected to the standard treatment.

P020

**Human cell cultures exposed to antibiotic amphotericin B: toxicity and defence**

E. Grela¹, M. Piet², R. Luchowski¹, W. Grudzinski¹, R. Paduch², W. Gruszecki¹

¹Biophysics, Maria Curie-Sklodowska University, Lublin, Poland, ²Virology And Immunology, Maria Curie-Sklodowska University, Lublin, Poland

**Objectives:** Amphotericin B (AmB) is a polyene antifungal antibiotic, which is synthetized by the bacteria *Streptomyces nodosus*. AmB is a "gold standard" in treatment of systemic fungal infections, due to its broad spectrum of action, high effectiveness and rare pathogens resistant to this drug. Unfortunately due to the low selectivity of the action of this antibiotic, it is very toxic for patients. Although, the exact mechanism of action of AmB is still unknown, it is used as a lifesaving medicine. Understanding the mechanism of action of AmB would enable to minimize its toxic side effects, and possibly increase its therapeutic effects. The aim of this study was to get to know the toxicity mechanism of action of AmB.

**Methods:** To understand molecular mechanism underlying the toxicity of AmB, human normal colon epithelial cells CCD 841 CoTr (ATCC No. CRL-1807) and human colon adenocarcinoma cells HT-29 (ATCC No HTB-38) were exposed to the drug and were imaged using Fluorescence Lifetime Imaging Microscopy (FLIM) and Raman scattering spectroscopy. Cultures were incubated for 24 h with AmB. Fluorescence lifetime imaging was carried out on MicroTime 200 (Picoquant GmbH, Germany) linked with Olympus IX71 inverted microscope. The cell samples were excited by 405 nm pulsed laser. Fluorescence lifetimes and fluorescence anisotropy values were analyzed. Raman spectral analysis and imaging on a microscale was carried out with inVia Reflex confocal Raman microscope
(Renishaw, UK) with Cobolt o8-NLD 405 nm laser (power at a sample 0.2 mW). Obtained results were analyzed by DCLS spectral deconvolution.

**Results:** The results confirmed high toxicity of antibiotic AmB for both human cell lines but lower for HT-29 cells. The results show that AmB incorporates to the lipid membrane containing cholesterol. The cell defense mechanism against the action of antibiotic was also observed, which is based on removing AmB from the membrane in the form of sterol-deficient exosomes.

**Conclusion:** The lower toxicity of AmB observed for HT-29 cells, may be associated with high proliferation rate or different membrane composition of adenocarcinoma cells compared to CCD 841 CoTr cells. AmB binds to the bilayer in the form of small aggregates, which can cause uncontrolled ion-leakage and lead to the cell death.

<figure><img src="image_19.png" /></figure>

P021

**African ST173 *Cryptococcus deuterogattii* strains are commonly less susceptible to fluconazole : a tricky mechanism of resistance**

V. Bellet¹, F. Roger¹, T. Gouveia¹, P. Drakulovski¹, F. Kassi², H. Menan², D. Krasteva¹, S. Bertout¹

¹Laboratoire De Parasitologie Et Mycologie Médicale Umi 233, University of Montpellier, Montpellier, France, ²CEDRES CHU de Treichville, Abidjan, Côte d'Ivoire

**Objectives:** Fluconazole, alone or in association, is often administered during cryptococcosis treatment especially in Sub-Saharan Africa. Its intensive use leads to the emergence of resistant strains. The mechanisms underlying resistance are poorly documented for yeasts belonging to the *C. gattii* species complex. Literature suggests that resistance could be due to mutations and/or surexpression of ERG11 gene and overexpression of efflux pumps, like MDR or AFR.

**Methods:** In Ivory Coast, we highlighted the presence of strains with a rare subtype in the molecular type VGII associated with high minimum inhibitory concentrations to FCZ compared to VGII strains originating from the Pacific Northwest. We investigated the mechanisms of fluconazole resistance of 18 ivorian clinical strains of *C. deuterogattii* with high MIC to FCZ compared to 10 strains with low MIC to FCZ.

**Results:** We demonstrated that (i) these strains exhibited no mutation in the *ERG11* gene, (ii) some strains had an increased in *ERG11* and *MDR1* mRNA expression while *AFR1* and *AFR2* were not overexpressed in high MIC to FCZ strains versus the low MIC to FCZ strains and (iii) exposure to FCZ in strains with high MIC to FCZ induced AFR1 mRNA overexpression.

**Conclusion:** We demonstrated that the FCZ-resistant mechanism commonly described in *Cryptococcus neoformans* or in *Cryptococcus gattii* from the Pacific Northwest were not responsible for the resistance to FCZ in this rare sub-type strains.

P022
Novel Technique to Measure Susceptibility to Lipid-Associated Antifungals

M. Frenkel¹, Y. Yunik¹, E. Segal²

¹Tel Aviv University, Tel Aviv, Israel, ²Sackler School Of Medicine, Clinical Microbiology And Immunology, Tel Aviv University, Tel Aviv, Israel

**Objectives:** Lipid associated antifungals, such as polyenes, are extensively used for therapy in patients with invasive fungal infections. Thus, susceptibility of fungi to the lipid- associated antifungals is of importance. The most reliable method used in susceptibility testing is the broth micro-dilution technique. However, visual- or spectral- assessment of the antifungal activity is difficult, and possibly unreliable, in lipid-associated drugs, due to turbidity caused by the lipid. Hence, the objective of this study was to adapt a method for spectral measurement of susceptibility of fungi to lipid-associated antifungal drugs.

**Methods:** The technique was adapted for Amphotericin B- Intralipid (AMB-IL) and Nystatin-Intralipid (NYT-IL) in comparison to AMB and NYT. **Drug preparation:** AMB-IL was prepared by adding to 5 mg/ml of AMB 20% Intralipid in DDW to a final concentration of 0.2 mg/ml AMB and shaken at 280 rpm for 18 h at 24⁰ C. Intralipid in DDW was used as control. NYT-IL was prepared similarly, by adding to 20 mg/ml nystatin 20% Intralipid in DMSO to a final concentration of 0.8 mg/mL NYT and shaken at 280 rpm for 18 h at 24⁰ C. Intralipid in DMSO was used as control. **Susceptibility test:** Susceptibility of the *Candida albicans* strains to AMB, NYT, AMB-IL, and NYT-IL was assessed by the micro-broth dilution assay using micro-titer plates. Susceptibility was evaluated using spectral readings in an EMax+ Microplate Reader at 560nm and 650nm, for the standard (AMB, NYT) and experimental Intralipid-associated polyene formulations (AMB-IL, NYT-IL), respectively. For AMB-IL or for NYT-IL the drug solutions and the relevant controls were diluted in RPMI. Each well contained the same concentration of RPMI, IL, DDW or DMSO, and serial dilutions of the antifungal drugs (AMB, NYT).

**Results:** The validity of the test was evaluated by testing susceptibility of 40 C. albicans isolates: 20 from human mucosal infections (M strains) and 20 from candidemia patients (S strains) vs. a control strain C. albicans CBS-562, for which susceptibility data were available. We determined 50, 90 & 100% MIC values by spectral readings (see an example of tests with the control stain – CBS 562–Fig.1). The spectral readings were compared to visual readings of the micro-titer plates using an inverted microscope. In addition, minimal fungicidal concentration (MFC) for each strain was determined (Fig.2). **Fig. 1: Spectral readings of Control strain CBS 562 treated with AMB-IL & NYT-IL**

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>
<figure><img src="image_22.png" /><figcaption>Figure 2: Spectral readings vs. Visual assessment of M and S *Candida albicans* strains.</figcaption></figure>

**Conclusion:** This study presents a spectral method for measuring susceptibility of yeasts to lipid associated polyenes which deals with the difficulties of visual assessment of susceptibility to lipid associated antifungals.

P023

Antifungal activity of antimicrobial peptides against clinical and environmental *Aspergillus fumigatus* isolates.

E. Ballard¹, R. Yucel², W. Melchers³, A. Brown¹, P.E. Verweij³, A. Warris¹

¹Mrc Centre For Medical Mycology,, University of Aberdeen, Aberdeen, United Kingdom, ²Ifcc, University of Aberdeen, Aberdeen, United Kingdom, ³Medical Microbiology, RadboudUMC, Nijmegen, Netherlands

**Objectives:** Antimicrobial peptides (AMPs) are a key defence against invading microorganisms. The activity of AMPs against human fungal pathogen *Aspergillus fumigatus* remains poorly understood. We characterised the anti-*Aspergillus* activity of specific human AMPs and determined whether in-host adaption could lead to resistance to specific AMPs.

**Methods:** The antifungal activity of lysozyme, histones, lactoferrin, β-defensin-1 and LL-37, was tested against 15 clinical isolates (from patients with cystic fibrosis, chronic and acute invasive aspergillosis), 5 environmental isolates and 13 isogenic A. fumigatus isolates obtained from a single patient over 2 years showing in-host acquired azole resistance. Hyphae grown from 5x10⁴ conidia and incubated with individual AMPs at various concentrations for 2 h at 37oC, after which metabolic activity was determined by XTT. Aspergillus conidia (1x10⁵ per well) were incubated for 10 h at 37oC with specific AMPs and fixed in 4% paraformaldehyde fixing solution. Analysis of hyphal length was performed using the Amnis ImageStreamX MK II Imaging Flow Cytometer (Luminex, USA). Data analysis was performed using IDEAS software.

**Results:** Dose-dependent decreases in hyphal metabolic activity in all isolates were shown for lysozyme (20-80 µM; 31-65% and 5-25% decrease respectively; p<0.0001) and histones (50-100 µg/ml; 35-66% and 6-45% decrease respectively; p<0.0001). No effect was observed for lactoferrin (10-40 µM), β-defensin-1 (1-10 µM) and LL-37 (5-50 µM). Imaging flow cytometry revealed that incubation with histones (100 µg/ml), β-defensin-1 (10 µM) or lactoferrin (40 µM) resulted significantly fewer cells in the hyphae gate (p<0.0001, p = 0.04 and p = 0.003, respectively). No significant differences were observed with lysozyme (80 µM) or LL-37 (12.5 µM).

**Conclusion:** Lysozyme and histones show dose dependent antifungal activity against A. fumigatus hyphae, irrespective of the isolate's azole resistance profile. In addition, histones, lactoferrin and β-defensin-1 display inhibitory activity against germinating conidia. No differences were observed in the susceptibility to AMPs between the groups of A. fumigatus isolates used. We did not identify any trends indicating increased resistance to AMPs as a result of in-host adaptation.

P024

Characterization of I1380T a new FKS1 polymorphism in *Candida parapsilosis* that confers high-level echinocandin resistance when associated to P660A
I. Accoceberry¹, S. El-Kirat-Chatel², F. Quilès², N. Biteau¹, T. Noël¹

¹Cnrs Umr5234, University of Bordeaux, Bordeaux, France, ²Cnrs Umr 7564, Université de Lorraine, Villers-lès-Nancy, France

**Objectives:** The aim of this study was to determine the impact on echinocandin resistance of a new mutation identified in a *FKS1* allele of a clinical isolate of *C. parapsilosis*.

**Methods:** To assess the role of mutations potentially implicated in echinocandin resistance, a *C. lusitaniae* strain specially engineered for the expression of a single chromosomal copy of *FKS1* was used for the expression of I1380T and P660A *FKS1* alleles. The echinocandin susceptibility of the mutants, as well as their growth rates and phenotypes of interaction with murine macrophages, were evaluated in the absence or in the presence of caspofungin. Atomic force microscopy (AFM) was used to observe the morphological changes induced by caspofungin exposition. Finally, infrared spectroscopy in attenuated total reflection mode (IR-ATR) spectroscopy was used to detect and quantify the biochemical signatures of the cell wall before and after caspofungin treatment.

**Results:** A caspofungin-resistant *C. parapsilosis* bloodstream isolate was recovered from a patient who underwent allogeneic bone marrow hematopoietic stem-cell transplantation (HSCT) for an idiopathic medullary aplasia with positive PNH clone. Full-length nucleotide sequencing of the *FKS1* gene showed that the strain harboured the heterozygous mutation I1380T (isoleucine at position 1380 replaced by threonine) located 4 amino acids beyond the HS2 region (amino acids 1369 to 1376), compared to Fks1p of the *C. parapsilosis* reference strain CBS 604, in addition to the naturally occurring P660A polymorphism responsible for increased echinocandin MICS in this species. To characterize the impact of each mutation on caspofungin resistance, *FKS1* alleles bearing I1380T associated or not to P660A were genetically engineered and expressed in *Candida lusitaniae*. When compared to a wild type allele bearing no mutation, I1380T conferred a 12-fold MIC increase, P660A a 6-fold MIC increase and the combination of both mutations a ≥ 256-fold MIC increase. Measuring the cellular interactions between *C. lusitaniae* *FKS1* mutants and murine macrophages showed that, in the presence of caspofungin, I1380T enhanced yeast phagocytosis but had no effect on intramacrophagic yeast survival rates. AFM analysis of *FKS1* mutants showed that caspofungin treatment impacts the cell surface topography in both susceptible and resistant strains, and that the single I1380T mutation leads to morphological changes even in the absence of caspofungin treatment. Infrared spectroscopy in attenuated total reflection mode (IR-ATR) showed that under high drug concentration exposure, the *FKS1* I1380T mutants exhibited a massive β-glucan synthesis inhibition that was not proportionally compensated by chitin synthesis.

**Conclusion:** We have identified I1380T, a new mutation located close to the HS2 region of the *FKS1* gene of a clinical isolate of *C. parapsilosis*. We demonstrated that the high caspofungin MIC for this isolate was depending on the combination of I1380T with the naturally occurring P660A polymorphism. Further characterization at the cell wall level suggested a potential fitness cost caused by I1380T.

P025

**Azole resistant *Aspergillus fumigatus*: surveillance from flowered patios to departments with at-risk patients**

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

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

**Objectives:** The University Hospital of Besançon is geographically located in a highly agricultural area (wood, vegetables, cereals, fruit and vines), and is very concerned by the emergence of azole resistance in *Aspergillus fumigatus* (ARAf), having an increasing number of cases especially amongst cystic fibrosis patients. For more than 20 years, the University Hospital of Besançon has been taking weekly samples (n = 25) to assess fungal air contamination in the 3 departments of hematology and corridors. Since 2015, the number of ARAf found in corridors has been increasing. This study aimed to determine whether plant, tree and flowerbeds within or near the hospital are sources of ARAf and to assess their spread into the hospital.