¹Department I Of Internal Medicine, Ecmm Excellence Centre Of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), University Hospital Cologne, Cologne, Germany, ²Department I For Internal Medicine, European Excellence Center For Medical Mycology (ecmm), Germany And Cecad Cluster Of Excellence, University of Cologne, Cologne, Germany, ³Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany, ⁴Department I Of Internal Medicine, Ecmm Excellence Centre Of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), German Centre For Infection Research, Partner Site Bonn-cologne, University Hospital Cologne, Cologne, Germany, ⁵Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), German Centre for Infection Research, Partner Site Bonn-Cologne, Cologne, Germany, ⁶Department I Of Internal Medicine, Ecmm Excellence Centre Of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), German Centre For Infection Research, Partner Site Bonn-cologne, Clinical Trials C, University Hospital Cologne, Cologne, Germany

**Introduction:** Invasive aspergillosis (IA) is a serious hazard to hematological and critical care patients.

**Objectives:** To understand prognostic variables we analyzed original articles identifying baseline factors that predict treatment outcome in patients with IA.

**Methods:** PubMed database was searched for publications since database inception until May 2018. Inclusion criteria were published baseline prognostic factors present at the diagnosis of IA.

**Results:** In total, 58 studies from 267 centers reported 7,320 patients with IA, and 40 different predictors. Unfavorable predictors in medical history were kidney (7.4%, 10/136) and liver failure (3.7%, 5/136), ICU admission (3.7%, 5/136), and uncontrolled underlying disease (3.7%, 5/136). Regarding state of immunosuppression, negative outcome predictors were prolonged neutropenia (12.5%, 17/136), corticosteroid treatment (8.1%, 11/136), and graft-versus-host disease (3.7%, 5/136). On the pathogen side, relevant predictors were galactomannan positivity (8.1%, 11/136), *Aspergillus terreus* infection (2.2%, 3/136), and lack of amphotericin B susceptibility (1.5%, 2/136). IA-specific predictors were disseminated disease (5.1%, 7/136) and CNS involvement (2.9%, 4/136). Imaging results associated with negative outcome were multiple consolidations (2.9%, 4/136), bipulmonary lesions (2.2%, 3/136), and pleural effusion (2.2%, 3/136).

**Conclusion:** At diagnosis of IA, most frequently identified predictors of outcome were neutropenia, corticosteroid use, elevated galactomannan, renal failure, and disseminated disease.

P092

EQUAL Aspergillosis Score 2018: An ECMM score derived from current guidelines to measure QUALity of the clinical management of invasive pulmonary aspergillosis

O.A. Cornely¹, P. Köhler², D. Arenz³, S. Mellinghoff²

¹Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), Cecad Cluster Of Excellence, Clinical Trials Centre Cologne (zks Köln), University of Cologne, Cologne, Germany, ²Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), Cecad Cluster Of Excellence, University of Cologne, Cologne, Germany, ³Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), University of Cologne, Cologne, Germany

**Introduction:** Invasive pulmonary aspergillosis is a serious threat to immunocompromised and critical care patients. Recent detailed guidelines and treatment algorithms lead microbiologists and clinicians in diagnosis and treatment of invasive aspergillosis.

**Objectives:** Currently, there is no tool available that allows to measure guideline adherence and quality of managing invasive pulmonary aspergillosis.

**Methods:** To develop such a tool, we reviewed current guidelines provided by 5 scientific societies: European Society for Clinical Microbiology and Infectious Diseases, European Confederation of Medical Mycology, the European Respiratory Society, Infectious Diseases Society of America (IDSA), and Infectious Diseases Working Party of the German Society for Hematology and Medical
Oncology. We selected the strongest recommendations for management as key components for our scoring tool, broke down guidelines, and grouped recommendations into 3 groups: diagnosis, treatment, follow-up. First, we selected "AI" and "AII" recommendations. Then, we added aspects that are part of a complete work-up, but are either recommended with a lower grade of evidence or not specifically recommended in current guidelines. In a last step, we allocated score points according to levels of evidence and clinical importance.

**Results:** We developed the EQUAL Aspergillosis Score 2018 to measure the quality of clinical management of invasive pulmonary aspergillosis. We integrated diagnostic measures (chest computed tomography, bronchoalveolar lavage with galactomannan, fungal culture, fungal polymerase chain reaction analysis, species identification, and susceptibility testing; histology with silver stain, Periodic acid-Schiff staining, and molecular diagnostics), treatment (antifungal choice and therapeutic drug monitoring), and follow-up computed tomography. For ease of use, we have summarized the EQUAL scores on laminate pocket cards, which will be translated in 15 different languages. The EQUAL Aspergillosis Score 2018 can be used to evaluate guideline adherence in daily clinical practice and thus aims to support antifungal stewardship.

**Conclusion:** The EQUAL Aspergillosis Score 2018 aggregates and weighs the components recommended in the ideal management of invasive pulmonary aspergillosis and provides a tool supporting antifungal stewardship and quantifying guideline adherence.

P093

EQUAL Mucormycosis Score 2018: An ECMM Score Derived From Current Guidelines to Measure QUALity of Mucormycosis Management in Hematology

P. Köhler¹, S. Mellinghoff¹, A. Alanio², D. Arenz³, M. Hoenigl⁴, K. Lagrou⁵, C. Lass-Flörl⁶, J. Meis⁷, M.D. Richardson⁸, O.A. Cornely⁹

¹Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), Cecad Cluster Of Excellence, University of Cologne, Cologne, Germany, ²Paris-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, ³Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), University of Cologne, Cologne, Germany, ⁴Division Of Infectious Diseases, Department Of Medicine, UCSD, San Diego, United States of America, ⁵Laboratory Of Clinical Bacteriology And Mycology, Department Of Microbiology And Immunology, Excellence Center For Medical Mycology (ecmm), KU Leuven, Leuven, Belgium, ⁶Division Of Hygiene And Medical Microbiology, Excellence Center For Medical Mycology (ecmm), Med. Univ. Innsbruck, Innsbruck, Austria, ⁷Department Of Medical Microbiology And Infectious Diseases, Excellence Center For Medical Mycology (ecmm), Canisius Wilhelmina Hospital, Nijmegen, Netherlands, ⁸Mycology Reference Centre, Excellence Center For Medical Mycology (ecmm), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom, ⁹Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), Cecad Cluster Of Excellence, Clinical Trials Centre Cologne (zks Köln), University of Cologne, Cologne, Germany

**Introduction:** Mucormycosis is a serious infectious threat to immunocompromised patients in general and in hematological malignancy in particular. Detailed standards and treatment algorithms guide microbiologists and clinicians in the diagnosis and treatment of mucormycosis.

**Objectives:** Currently, there is no tool available that allows the measurement of guideline adherence and the quality of managing mucormycosis.

**Methods:** To develop such a tool, we reviewed current guidelines provided by four scientific societies: the European Society for Clinical Microbiology and Infectious Diseases (ESCMID), the European Confederation of Medical Mycology (ECMM), the European Conference on Infections in Leukemia (ECIL), and the Infectious Diseases Working Party of the German Society for Hematology and Medical Oncology. We selected the strongest recommendations for management as key components for our scoring tool, broke down guidelines, and grouped recommendations into three groups: diagnosis, treatment, follow-up. First, we selected "AI" and "AII" recommendations. Then
we added aspects that are part of a complete work-up, but are either recommended with a lower grade of evidence or not specifically recommended in current guidelines. In a last step, we allocated score points to the various levels of evidence and clinical relevance of diagnostic and therapeutic procedures.

**Results:** We developed the EQUAL Mucormycosis Score 2018 to measure the quality of mucormycosis management. We integrated diagnostic measures (chest computed tomography, bronchoalveolar lavage with fungal culture, fungal polymerase chain reaction analysis, species identification, susceptibility testing, histology with silver stain, Periodic acid-Schiff staining, and molecular diagnostics), treatment (antifungal choice, surgical debridement and therapeutic drug monitoring), and follow-up computed tomography. For ease of use, we have summarized the EQUAL scores on laminate pocket cards, which will be translated in 15 different languages. The EQUAL Mucormycosis Score 2018 can be used to evaluate guideline adherence in daily clinical practice and thus aims to support antifungal stewardship.

**Conclusion:** The EQUAL Mucormycosis Score 2018 aggregates and weighs recommendations for management of mucormycosis and provides a tool supporting antifungal stewardship and quantifying guideline adherence.

P094

**Compliance with recurrent vulvovaginal candidiasis guidelines – An audit of RVVC referrals**

L. Brown¹, R. Rautemaa-Richardson¹,²

¹Faculty Of Biology, Medicine And Health, University of Manchester, pl, United Kingdom, ²Division Of Infection, Immunity And Respiratory Medicine, University of Manchester, Manchester, United Kingdom

**Objectives:** Recurrent vulvovaginal candidiasis (RVVC) is a debilitating, chronic condition which affects over 138 million (6%) women of reproductive age annually. It is defined as four or more episodes in a 12-month period. Symptoms include vulval itch, soreness and discharge. Diagnosis is often difficult, as it is based on a combination of these non-specific clinical symptoms and detection of *Candida* by high vaginal swab (HVS) microscopy, which is part of the normal vaginal flora in many women. A number of other infectious and non-infectious conditions can present similarly. Treatment of RVVC comprises of vulval skin care with the use of emollients for personal hygiene and antifungal treatment combined with long-term antifungal suppression for 6 months or longer. The British Association for Sexual Health and HIV (BASHH) has published guidelines on the management of RVVC. The aim of our study is to evaluate the compliance of referring doctors to the current guidelines.

**Methods:** This retrospective audit was undertaken in the Infectious Diseases (ID) department of Wythenshawe Hospital, Manchester from October 2017 to May 2019. All patients referred to the tertiary clinic with suspected RVVC were included in the analyses. Electronic patient records were analysed to determine whether appropriate investigations and treatments had been offered prior to referral. The type and pattern of recorded symptoms were assessed against the typical clinical picture of RVVC and whether alternative differentials should have taken precedence.

**Results:** Patient records from a total of 47 RVVC referrals were analysed. Patient age at the time of referral ranged from 17-66 years with a mean age of 37.7 years. Symptom duration prior to referral ranged from 4 months to 24 years, with an average duration of 6.7 years. "Dry", "sensitive" skin or "eczema" were mentioned in 36% of the cases. RVVC was the final diagnosis in 80% of cases. 19% of referrals were deemed inappropriate due to repeatedly negative HVS for *Candida* and lack of response to antifungal therapy, or reported symptoms not fitting with RVVC. 57% of patients had a secondary diagnosis, the most common being vulvodynia and vulval dermatitis. 94% of the patients were appropriately investigated by high vaginal swab (HVS) microscopy or culture prior to referral. Appropriate antifungal regimens, including long term suppression, were trialled in 53% of patients prior to referral. Only 11% patients were practicing appropriate vulval skin care before attending clinic. Excessive or otherwise inappropriate personal hygiene likely to irritate vulval skin, including
the use of soap and shower gel, was mentioned in 21% of cases. MBL deficiency was diagnosed in 28% of the patients.

**Conclusion:** RVVC is often complicated by coexisting vulval pathologies and vulval symptoms labelled as RVVC are often due to alternative diagnoses. Education on vulval skin care is the mainstay of RVVC management and prevention but healthcare professionals are failing to deliver this. Also, there is lack of awareness of the efficacy and safety of long term antifungal suppression. The morbidity related to this debilitating condition could be easily reduced by following the principles summarised in the BASHH guidelines.

P095

**Validation of a clinical decision rule for selecting empiric treatment for invasive aspergillosis in a setting with high triazole resistance.**

R. Van De Peppel, R. Van Grootveld, B. Hendriks, J. Van Paassen, J. Koopmans, P. Von Dem Borne, M. Van Der Beek, M. De Boer

Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands

**Objectives:** World-wide, increasing triazole resistance complicates treatment of invasive aspergillosis (IA). In case of detected triazole resistance, the first choice treatment is replaced by lipid formulations of Amphotericin B (LAmB). In settings with substantial (>10%) prevalence of triazole resistance, empiric combination therapy with both a triazole and LAmB has been suggested. However, the expected benefits of this strategy need to be weighed against a higher rate of serious adverse events associated with combination therapy, as well as higher costs. To evade unnecessary toxicity while optimizing outcome, a clinical decision rule guiding to monotherapy with either voriconazole or LAmB was designed and validated.

**Methods:** In 2015, all medical specialties involved in the diagnosis and treatment of patients with IA participated in constructing a treatment algorithm that provided rationale for empiric monotherapy with either voriconazole or LAmB (figure 1). The designed algorithm was approved by the institution's antimicrobial steering committee. Protocolized CT-scanning and bronchoalveolar lavage (BAL) were performed upon suspicion of IA. BAL samples were examined by direct microscopy, culture, galactomannan assay (cut off at 0.5 OD) and from 2017 onwards also by PCR. Triazole resistance was routinely tested by four well agar plate. All adult patients diagnosed with IA and treated for this condition in the Leiden University Medical Center from 2015 to 2019 were included. Data about underlying diseases, age, gender, results of diagnostic tests and outcomes, were extracted from the hospital's electronic patient files and anonymized. Primary outcomes were 30- and 100-day crude- and attributable mortality rates.
<figure><img src="image_30.png" /><figcaption>**Figure 1.** Clinical decision rule for selecting empiric treatment for invasive aspergillosis</figcaption></figure>

**Legend figure 1.**

An expert-based decision rule that was primarily based on the selecting patients with a higher probability of a favorable outcome and lower a priori risk of resistant *Aspergillus*. The rationale of the algorithm was strengthened by in particular taking into account host factors that could aggravate the clinical course of infection, such that this would not allow timely evaluation and switch of therapy to an effective regimen in case of resistance.

**Results:** At first evaluation, 102 patients had been treated for IA with monotherapy according to the designed algorithm and 3 patients received off-protocol treatment regimens (figure 2). In the period of study, prevalence of voriconazole resistance in all *Aspergillus* isolates varied per year between 16.3% and 24%. Of included patients, 65 (64%) were male; the median age was 64 (range 18-82), and 80 (78%) patients were treated for an underlying hematological malignancy. Fifty-nine patients (58%) were started on voriconazole and 43 (42%) were started on LAmB. Cultures were positive in 16 (15.7%) patients and phenotypical voriconazole resistance was detected in 7/16 (44%). Upon clinical evaluation of empiric therapy and/or resistance data if available, a switch was made to LAmB in 23/59 (39%) of patients started on voriconazole. Crude mortality rates per treatment stratum are listed in figure 2. Later adaptation of antifungal therapy to LAmB was not associated with a higher crude mortality rate (p = 0.73). Definite therapy consisted of voriconazole in 41 (40%) and of LAmB in 61 (60%) of cases.
<figure><img src="image_31.png" /><figcaption>**Figure 2.** Patients following the clinical algorithm and the total number of Aspergillus isolates with successful susceptibility determination by either culture or PCR</figcaption></figure>

**Conclusion:** By applying a comprehensive clinical decision algorithm in our area with high triazole-resistance rates, 58% of patients were empirically treated with monotherapy voriconazole, without excess crude mortality even if a later switch to LAmB was needed. In 40% of patients therapy with LAmB could be safely avoided during the entire course of treatment. As treatment allocation was not randomized, and confounding by indication plays a role, it remains unclear whether patients directed to initial LAmB monotherapy would have benefited from the addition of a triazole.

P096

**Multicenter study on antifungal prophylaxis in high risk hematology patients - The SAPHIR Study**

J.P. Gangneux¹, C. Padoin², M. Michallet³, E. Saillio⁴, A. Kumichel⁵, R. Peffault De La Tour⁶, P. Ceballos⁷, T. Gastinne⁸, A. Pigneux⁹

¹Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France, ²Pharmacy Department, CHU de Martinique, Fort-de-France, France, ³Clinical Haematology Department, CH Lyon-Sud, Pierre-Bénite, Iyon, France, ⁴MSD France, courbevoie, France, ⁵ClinSearch, Malakoff, France, ⁶Saint-Louis Hospital, APHP, Paris, France, ⁷CHRU Lapeyronie de Montpellier, Montpellier, France, ⁸CHU de Nantes, Nantes, France, ⁹Hospital group Haut Leveque de Pessac, Pessac, France

**Objectives:** The main objective of the SAPHIR study was to describe the diagnostic and therapeutic management of hematology patients at high risk of Invasive Fungal Disease (IFD) under antifungal prophylaxis (AFP) in France. The secondary objectives were the description of (1) parameters and diagnostic procedures concomitant to AF therapy management and (2) preventive procedures adopted in French hematology services.

**Methods:** SAPHIR was an observational, prospective, multicenter, non-comparative French study. The included patients were ≥18 years, in a context of myelosuppressive chemotherapy for acute myeloid leukemia (AML) and initiated an AF prophylactic treatment.

**Results:** The 23 participating hematology services included 404 patients, whose mean age was 56.4±14.0 years and of whom 51.2% were male. Duration between AML diagnosis and inclusion was 65.4±178 days with 65.6% being newly diagnosed. A previous chemotherapy had been undergone by 26.7%. The index chemotherapy, was started 1.06±4.49 days before inclusion and its nature was either induction 79.0%, consolidation 12.6% or relapse 8.4%. Among the 404 patients, 91.6% had experienced a profound neutropenia period with 1.08±0.51 periods per patient and a duration of
23.3±16.6 days. For 373 patients (92.4%), the initially prescribed AFP was posaconazole tablets (90.6%) or suspension (1.73%). Considering all patients, 8.17% changed AFP between 1 and 3 times, with the first change 17.3±23.9 days after inclusion mostly due to absorption issues (65.0%). The mean AFP period was 24.2±32.1 days after which 267 patients (66.8%) stopped because they were at the end of the high-risk period and 126 (31.2%) switched to a non-prophylactic AF treatment (2/3 empirical, 1/3 preemptive/curative), mainly because of fever (67.1%). A total of 18/404 patients (4.6%) have received a curative treatment based on positive mycological exams, imaging, antifungal sensitivity tests and/or blood cultures. Among them, 9/404 (2.23%) were true probable IFDs: 5 probable invasive aspergillosis, 2 pulmonary pneumocystosis, 1 mucormycose and 1 fungemia due to *Saccharomyces* sp. At 7-15 days after AFP discontinuation, 365/387 patients (94.3%) showed no signs and symptoms of infection. In the course of the study, 20 patients (5.0%) had deceased within 57.6±50.3 days after inclusion. Of those, 7 patients (35.0%) showed signs and symptoms of infection and the death of 3 patients was directly linked to IFD infections (1 candidiasis and 2 aspergillosis). In order to prevent IFD, additionally to AFP, French haematology services provide the patient with sterile/protected food (94.3%), digestive decontamination (35.8%), and accommodation in a sterile/isolated area (93.5%). To gain a better understanding of the patient characteristics possibly indicating the necessity for a consecutive empirical/preemptive/curative AF treatment, data of patients with and without such a consecutive treatment were compared. Several parameters proved to be significantly different, for instance: cytogenetics and molecular biology prognosis, AML onset, previous chemotherapy treatment but also duration between chemotherapy initiation and inclusion and the type of current chemotherapy cycle used.

**Conclusion:** AF prophylactic treatment is frequently prescribed in French hematology services. Only few prophylactic treatment changes are necessary which reflects treatment tolerance and effectiveness regarding decreased numbers of intercurrent infections and mortality.

P097

EQUAL Cryptococcus Score 2018: A European Confederation of Medical Mycology Score Derived From Current Guidelines to Measure QUALity of Clinical Cryptococcosis Management

A. Spec¹, C. Mejia-Chew¹, W. Powderly¹, P. Köhler²,³, O.A. Cornely²,⁴

¹Internal Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO, United States of America, ²Department I For Internal Medicine, European Excellence Center For Medical Mycology (ecmm), Germany And Cecad Cluster Of Excellence, University of Cologne, Cologne, Germany, ³Department I Of Internal Medicine, Ecmm Excellence Centre Of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), University Hospital Cologne, Cologne, Germany, ⁴German Centre For Infection Research, Partner Site Bonn-cologne, Cologne, Germany And Clinical Trials Centre Cologne (zks Köln), University of Cologne, Cologne, Germany

**Objectives:** Cryptococcosis is an opportunistic fungal infection with high morbidity and mortality. Guidelines to aid clinicians regarding diagnosis, management, and treatment can be extensive and challenging for clinicians. Currently, there is no tool available that allows the measurement of guideline adherence and the quality of cryptococcosis management.

**Methods:** We reviewed current guidelines from the Infectious Diseases Society of America, the World Health Organization, the American Society of Transplantation and recent significant publications to select the strongest recommendations as vital components of our scoring tool.

**Results:** Items included diagnostic tests (blood, tissue, and cerebrospinal fluid cultures, Cryptococcus antigen, India ink, histopathology with special fungal stains, central nervous system imaging), pharmacological (amphotericin B, flucytosine, azoles) and nonpharmacological treatments (intracranial pressure management, immunomodulation, infectious disease consultation), and follow-up of central nervous system complications. For ease of use, we have summarized the EQUAL scores on laminate pocket cards, which will be translated into 15 different languages.
**Conclusion:** The EQUAL Cryptococcus Score 2018 weighs and aggregates the recommendations for the optimal management of cryptococcosis, providing a tool that could measure guideline adherence or facilitate clinical decision-making.

P099

**An ongoing malady, mucormycosis from a tertiary care centre at Chandigarh, India.**

J. Chander¹, A. Bhagat¹, N. Gulati¹, N. Singla¹, R.P.S. Punia², D. Agarwal³, A.K. Attri⁴, A. Dass⁵

¹Microbiology, Government Medical College Hospital, Chandigarh, India, ²Pathology, Government Medical College Hospital, Chandigarh, India, ³Pulmonary Medicine, Government Medical College Hospital, Chandigarh, India, ⁴General Surgery, Government Medical College Hospital, Chandigarh, India, ⁵Ent, Government Medical College Hospital, Chandigarh, India

**Objectives:** Mucormycosis is an emerging necrotizing angioinvasive infection caused by commonly encountered filamentous fungi of the class Mucormycetes. In our institution, as the awareness is gradually increasing, newer and newer species causing mucormycosis are being recognized. In this very reference a study was undertaken to further enhance our knowledge on risk factors, species distribution and their antifungal susceptibility testing.

**Methods:** The study was conducted on forty-four cases of mucormycosis over a period of eighteen months from January 1, 2017 to June 30, 2018. The samples were subjected to microbiological examination like KOH/CFW wet mount and fungal culture and histopathology. The isolates obtained were identified phenotypically and confirmed by DNA sequencing of internal transcribed spacer region. The antifungal susceptibility testing was done for amphotericin B, posaconazole, itraconazole and terbinafine by microbroth dilution as per CLSI Reference Method. Study performed by Almyroudis, et al, was referred to define susceptibility for some of the breakpoints for Mucorales.

**Results:** The mean age of patients of mucormycosis was 48.89 ± 17.1 years. The proportion of female patients (54.5%) was slightly more than male patients (45.5%). Rhino-orbito-cerebral mucormycosis (ROCM) was the commonest presentation (61.4%, 27) followed by pulmonary (20.4%, 9) and cutaneous (15.9%, 7). One case of mucormycosis of the middle ear was also seen. The most common risk factor associated with mucormycosis was diabetes mellitus (77.3%, 34/44), which was found to be statistically significant (P = .003). Tooth extraction was seen as a risk factor in 18.5% (5/27) of ROCM cases. The commonest isolate was *Rhizopus arrhizus* (32.3%, 10), followed by *Rhizopus microsporus* (29%, 9) and *Apophysomyces variabilis* (12.9%, 4). *Rhizopus homothallicus* was isolated from two cases, one of middle ear and other from pulmonary mucormycosis. A case of cutaneous mucormycosis caused by *Thamnostylum piriforme*, which is the second case of this genus and first of this species, is being reported. Amphotericin B was the most effective drug with an MIC range of 0.0313-4 µg/ml giving 94.7 % isolates as sensitive. The only isolate resistant was *Rhizopus homothallicus* with an MIC of 4 µg/ml. Posaconazole gave an MIC range of 0.0625-4 µg/ml with 68.4% isolates as sensitive. Dual management with surgery and antifungals proved to be significantly better (P< 0.001) than amphotericin B alone. Treatment with liposomal amphotericin B (LAmB) was associated with a higher survival rate (81%), compared to 28.6% survival when patients were treated with conventional amphotericin B deoxycholate. Twelve (27.3%) patients either left against medical advice or were referred. A total of 12 patients expired giving a mortality rate of 27.3% and 37.5% among the treated patients.

**Conclusion:** India has second largest diabetic population globally with nearly 70% being found with uncontrolled diabetes leading to high incidence of mucormycosis. Due to an ever-growing population at risk and the fatal outcome, need for more prospective studies in this field are emphasized. Increased awareness among clinicians, timely diagnosis and prompt treatment are the key to the successful management.

P100

**The Protective Role of Cinnamaldehyde against Tenuazonic acid-induced Mycotoxicity in Murine model**
A. Kumari¹, K. Singh²

¹Animal Mycology Laboratory, Zoology, Mmv, Banaras Hindu University, Varanasi, India, ²Animal Mycology Laboratory, Zoology, Mmv, Banaras Hindu University, VARANASI, India

**Objectives:** Cinnamaldehyde is a natural product obtained from cinnamon and has been reported to have insecticidal, antimicrobial, anti-inflammatory, immunomodulatory, anticancer, anti-angiogenic and potential anti-fungal effects. The present study investigated the possible protective effect of cinnamaldehyde against tenuazonic acid-induced mycotoxicity in murine model. Tenuazonic acid (TeA), a toxin produced by *Alternaria alternata* is a common contaminant in tomato and tomato based products.

**Methods:** The *in-vivo* experimental plan was as follows.

<figure><img src="image_32.png" /></figure>

All the experimental groups were administered with TeA for 8 weeks. After 2 weeks, the prophylactic group was administered with cinnamaldehyde along with TeA for the next 6 weeks. Haematological (differential leukocyte count), histopathological (haematoxylin and eosin), biochemical (superoxide dismutase, catalase, malondialdehyde, alanine aminotransferase, aspartate aminotransferase) and cell apoptotic factor (caspase-3) analyses of the experimental and control mice were performed.

**Results:** Behavioral observations- Unlike mycotoxicosis induced groups, no behavioural abnormalities were observed in the animals who received cinnamaldehyde as prophylactic agent. Weights of the mice of the prophylactic group showed reduced body weight gain as compared to that of the mycotoxicosis induced groups. Morphological observations- Hair loss indicating mycotoxicosis was not observed in the prophylactic groups. Anatomical observations- Spleenomegaly, hepatomegaly and gross lesions on liver and kidney were noted on autopsy in the mycotoxicosis induced groups. On the other hand, the prophylactic groups showed normal weights of liver and spleen. The body:organ weight indices of all the organs remained at par to control group in the prophylactic groups except for the lungs and brain where the weights of the organs were significantly high. Haematological analysis- Neutrophilia was observed in IP (mycotoxicosis induced) while neutropenia was observed in the oral (mycotoxicosis induced) group while the differential leukocyte count of the prophylactic groups were found to lie in normal range. Biochemical analyses- Acute intoxication of mice with TeA showed elevated malondialdehyde (MDA), reduced catalase (CAT) and superoxide dismutase (SOD) production; abnormal levels of aspartate transaminase (AST) and alanine transaminase (ALT). Treatment with cinnamaldehyde reversed TeA-induced alterations of antioxidant defense enzyme activities. Histological observations- Histopathological changes characterised by non alcoholic fatty liver, nuclear pyknosis and infiltration of immune cells were observed in the mycotoxicosis induced groups while cinnamaldehyde significantly prevented the TeA-induced organ damage. Cell apoptosis factor- Reduced activity of caspase-3 enzyme was noted in the mycotoxicosis induced groups while significantly high activity of the enzyme was observed in the prophylactic groups.
**Conclusion:** Thus, from the present study we concluded that, cinnamaldehyde showed therapeutic effects and toxicity reduction in TeA induced mycotoxicosis.

<figure><img src="image_33.png" /></figure>

P101

**Candi-SoL: A multi-site retrospective audit and analysis of Candidaemia across three South London Teaching Hospitals**

C. Logan¹, A. Fife², A. Goodman³, C. Ward², T. Bicanic¹

¹Infection & Immunity, St Georges university, London, London, United Kingdom, ²Department Of Microbiology, Kings College Hospital, London, United Kingdom, ³Department Of Infection, Guy's & St Thomas' Hospital, London, United Kingdom

**Objectives:** Candidaemia is associated with high mortality, particularly in critical care and immunocompromised patients. Emergence of multi-resistant candida species, notably C. glabrata and C. auris, pose further challenges in treatment. This retrospective surveillance and audit study reviews both adult and paediatric candidaemia cases between January 2012- December 2018 across three Tertiary South London Trusts; St George's Hospital in London, Kings College hospital London and Guys & St Thomas' Hospital. Collectively these trusts have over 3500 beds, including over 220 ICU beds (adult surgical, neurological, cardiac and general ICUs and paediatric and neonatal ICU) and onsite specialist surgical, cardiothoracic and medical services, including liver, bone marrow and renal transplant. The aims of this study were to assess trends in candidaemia epidemiology across the South London region, categorise at risk groups, antifungal therapy used and review clinical outcomes.

**Methods:** Blood cultures for adults and children isolating Candida species between January 2012- December 2018 were retrospectively identified from the Microbiology database. Species and antifungal susceptibility were reviewed. Persistent candidaemia (identical Candida sp. isolated ≤ 30days after the initial blood culture) was analysed as a single episode. Demographics (age, sex); location; risk factors; focus of candidaemia; antifungal treatment received and duration; ECHO performed and outcome; ophthalmology performed and outcome; line removal and culture result; length of stay; 30-day and in-hospital mortality was collected from electronic patient records. Analyses were conducted in Microsoft Excel 2013 and GraphPad Prism V7.

**Results:** Between January 2012 – December 2018, 509 candidaemia episodes were identified across the three Trusts. In total 42% were C. albicans, 30% C. glabrata, 12% C. parapsilosis and 1% of cases were due to C. auris following an outbreak in 2016. There was no significant trend towards an increasing proportion of non-albicans species over the 7-year period (Chi-squared test for trend, p = 0.22). Regarding age at the time of candidaemia, 4% were neonates (< 1 month), 9% were children (aged 1 month - <18 years), and 86% were adults (> 18 years). Patients on an intensive care unit (adult, paediatric or neonatal) accounted for 43% of cases. Clinical data regarding the candidaemia foci, the ECHO and ophthalmology findings, antifungal therapy and duration, B-D-glucan result if available, length of stay and mortality will also be presented. Susceptibility profile of isolates will be interrogated and presented to assess if there has been a change over time across the three trusts.

**Conclusion:** This retrospective audit of candidaemia across three large South London Trusts illustrates the changing regional candida epidemiology, with non-albicans species accounting for over half of all isolates and highlights the emergence of multi-drug resistant species such as C. auris in the London. The remaining analyses of this large dataset will highlight which are the dominant causes of candidaemia across the South London Trusts and examine clinical outcomes with candidaemia over a diverse group of patients.

P102

**Antifungal Efficacy of Isavuconazole and Liposomal Amphotericin B for Treatment of**
# Experimental Exserohilum rostratum Meningitis and Therapeutic Monitoring with CSF (1→3)-β-D-glucan: A Preclinical Paradigm for Treatment of CNS Phaeohyphomycosis

V. Petraitis, R. Petraitiene, E. Naing, B.B.W. Maung, A. Garcia, P. Kavaliauskas, T.J. Walsh

Infectious Diseases/medicine, Weill Cornell Medicine of Cornell University, New York, United States of America

**Objectives:** Phaeohyphomycosis of the central nervous system (CNS) is a life-threatening infection with serious morbidity and mortality. Treatment options for CNS phaeohyphomycosis are limited. This severity of CNS phaeohyphomycosis was tragically illustrated in the contamination of a corticosteroid injection preparation that resulted in epidemic *Exserohilum rostratum* meningitis (ERM), resulting in meningoradiculitis, brain abscess, neurologic deficits, and death despite antifungal therapy with voriconazole with or without liposomal amphotericin B (LAMB). We hypothesized that isavuconazole may be an alternative approach for patients with ERM and other causes of CNS phaeohyphomycosis, particularly those with severe infection or intolerance to initially high dosages of voriconazole therapy. We therefore investigated the *in vitro* antifungal activity of isavuconazole with or without LAMB followed by treatment in a new model of experimental ERM.

**Methods:** Clinical isolates of *E. rostratum* recovered from patients suffering from ERM were used in all experiments. Broth microdilution methodology was used to prepare an inoculum for each of five clinical isolates of *E. rostratum*. Combination checkerboard plates were used to test the activity of isavuconazole and LAMB, either alone or in combination. Minimum inhibitory concentrations (MICs), minimal lethal concentrations (MLCs) and fractional inhibitory concentration (FIC) indices were determined. For *in vivo* therapeutic studies, *E. rostratum* were inoculated intracisternally with 1.0x10⁶ microconidia to anesthetized rabbits. Immunosuppression was produced with cytarabine-induced profound persistent neutropenia and methylprednisolone. Study groups consisted of isavuconazole, 60 mg/kg/d, LAMB at 5.0, 7.5, and 10 mg/kg/day and untreated controls (UC).

**Results:** As there were no *in vitro* additive, synergistic, or antagonistic interactions for combinations of isavuconazole plus LAMB against the *E. rostratum* isolates, *in vivo* studies in the rabbit model of ERM were conducted with isavuconazole and LAMB as monotherapies. Isavuconazole 60 mg/kg/d and LAMB at 5.0, 7.5, and 10 mg/kg/day demonstrated significant reductions of the residual fungal burden of *E. rostratum* in cerebrum, cerebellum, spinal cord, and paravertebral muscle (*p*<0.01) in comparison to those of UC. Reduction of the residual fungal burden correlated with significant reduction of CSF (1→3)-β-D-glucan levels in comparison to those of UC (*P*<0.05).

**Conclusion:** Isavuconazole alone or LAMB alone significantly reduced residual fungal burden in parallel with reduction of CSF (1→3)-β-D-glucan levels in treatment of experimental *E. rostratum* meningitis and may serve as alternative regimens for treatment of CNS phaeohyphomycosis.

P103

Pulmonary mucormycosis caused by *Lichtheimia ornata* (experimental model)

N. Vasilyeva<fnref n="1" />, I. Bosak<fnref n="2" />, T. Bogomolova<fnref n="1" />, I. Vybornova<fnref n="2" />, A. Stepanova<fnref n="2" />, Y. Avdeenko<fnref n="2" />, G. Chilina<fnref n="2" />, O. Aak<fnref n="2" />, G. Solovyeva<fnref n="1" />

**Objectives:** *Lichtheimia ornata* (A.K. Sarbhoy) Alastr. - Izq. & Walter due to the prevalence and virulence is one of three most common species causing mucormycosis. This species was revealed among the agents of invasive mucormycosis in Russia. The purpose of the present research was to develop a murine experimental model of pulmonary mucormycosis caused by *L. ornata*.

**Methods:** For the infection of mice we used the strain RCPF-1507 of *L. ornata*, isolated from the tissue of accessory sinuses of nose from a patient with lymphatic leukemia. For modeling the pulmonary

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

<footnote n="2">²Kaschkin Research Institute Of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation</footnote>
mucormycosis we used outbred mice, males, with body weight 18-20 g. For inducing the condition of neutropenia we intraperitoneally injected cyclophosphamide at the dose 150 mg/kg four times (-3, 0, 4 and 8 days). From fungal culture grown on Sabouraud glucose agar for 3 days at 37° C we made the spore suspension in sterile 0.85% NaCl solution at the concentration 1·10⁷ CFU/ml. Infection of animals was carried out by intranasal injection of 50 µl of fungal spores suspension to a narcotized mouse.

**Results:** Data on mice lethality and results of cultural and histological investigations of murine lungs with invasive mucormycosis were obtained. Death of animals was observed from 8 to 18 days after infection. No mice survived. Culturing of lung tissue from all dead mice revealed growth of *L. ornata*. During the histological investigation of murine lungs taken after eight days from the beginning of infection the different area of localization of hyphal cells were found. Hyphae were wide (10-12 µm), with rare branching at right angle. Hyphal cells in the lung parenchyma generally were located densely and in parallel relatively each other that is characteristic of tissue forms of mucormycetes. In lungs vessels massive aggregation of densely and chaotically oriented fungal hyphae were revealed. Also we observed the hyphal aggregation in gleams of bronchial tubes and an interstitium. The inflammatory process due to a mycotic infection was poorly expressed, provided by poor neutrophilic and lymphocytic infiltration, alteration processes prevailed.

**Conclusion:** The scheme of immunosuppression used in this work (intra-peritoneal introduction of cyclophosphamide) showed efficiency for producing infection in mice by *L. ornata*. Data on survival of the infected animals and also results of cultural and histological investigations confirm existence of mycotic damage of lungs at the infected animals. The developed model of invasive pulmonary mucormycosis can be used for future scientific research, testing of new antifungal drugs and diagnostic methods.

P107

**Case of successful treatment of chronic pulmonary aspergillosis in a patient with polycystic disease**

O. Shadrivova¹, V. Kuznetsov², E. Desyatik², Y. Borzova², S. Ignatyeva², T. Bogomolova², N. Vasilyeva², N. Klimko¹

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

**Case Report: Background.** Publications on chronic pulmonary aspergillosis (CPA) in patients with polycystic disease are limited.

**Objection:** We present a case of successful treatment of CPA in a patient with polycystic disease.

**Methods:** Diagnosis of CPA was made in accordance with the criteria ESCMID/ECMM/ERS, 2016.

**Result:** Patient V., 70 years old, was admitted to the mycological clinic with complaints of productive cough and dyspnea in September 2018. It is known that in 2015 lesions in left lung lower lobe were detected on chest CT scan. The CPA diagnosis was made on the basis of chest CT scan (multiple aspergillomas in left lung S6), elevated serum *Aspergillus* IgG level, and *A. fumigatus* and *A. niger* in BAL culture. The patient was treated with itraconazole 400 mg/day for 2 weeks. In October 2018, the patient underwent surgery removal of the left lung lower lobe, and CPA diagnosis was confirmed with histology. Therapy with itraconazole 400 mg/day was continued for 1 month. At the end of treatment, there was no data for active CPA: no specific lesions on chest CT scan, with negative BAL microscopy and culture, and serum *Aspergillus* IgG level. On the Pubmed website, we did not find a case reports of CPA in patients with polycystic disease.

**Conclusions:** Polycystic disease can be underlying disease of chronic pulmonary aspergillosis. Combinations of antimycotic therapy and surgical treatment are necessary for the successful treatment of chronic pulmonary aspergillosis in patients with polycystic disease.

P108
Chronic pulmonary aspergillosis in patients with idiopathic pulmonary fibrosis

O. Shadrivova¹, M. Tarasova¹, E. Desyatik², N. Nikolaeva³, Y. Borzova², S. Ignatyeva², T. Bogomolova², N. Vasilyeva², N. Klimko¹

¹Department Of Clinical Mycology, Allergy And Immunology, North-Western State Medical University named after I.I.Mechnikov, St. Petersburg, Russian Federation, ²Kashkin Research Institute Of Medical Mycology, North-Western State Medical University named after I.I.Mechnikov, St. Petersburg, Russian Federation, ³North-Western State Medical University named after I.I.Mechnikov, St. Petersburg, Russian Federation

**Case Report:** Introduction: Publications on chronic pulmonary aspergillosis (CPA) in patients with idiopathic pulmonary fibrosis are limited.

**Objective:** We present a case of successful treatment of CPA in a patient with idiopathic pulmonary fibrosis.

**Materials and methods:** Diagnosis CPA performed in accordance with the criteria EORTC/MSG, 2008.

**Results:** Patient V., 78 years old, was admitted to the mycological clinic with complaints of productive cough, exertional dyspnea in February 2018. It is known that the patient was diagnosed with idiopathic pulmonary fibrosis in 2017. He did not receive basic therapy. On chest CT scan for the first time was detected a cavity of 43x68x29 mm with the soft-tissue formation 12x14 mm in the apex region of the right lung. A positive titer the IgG to *Aspergillus fumigatus*1: 3200 was determined. *Aspergillus fumigatus* grow in the sputum culture was revealed. The patient received itraconazole with 400 mg per day with a positive clinical effect. Decrease of cough and exertional dyspnea was noted. Duration of antifungal treatment was 3 months. In July 2018, the chest CT scan showed a decrease in the size of the cavity and reducing of the soft-tissue formation in the right lung. The IgG titer to *Aspergillus fumigatus* was decreased to 1: 800. No fungi were found in sputum culture. The patient received antimycotic therapy for another 3 months. In October 2018, the CT scan of the chest showed stabilization of changes. IgG titer to *Aspergillus fumigatus* decreased to 1: 400. The culture study of sputum was negative. Antimycotic therapy was stopped. When analyzing the literature on the Pubmed site, we found 5 cases of aspergillosis with idiopathic pulmonary fibrosis. 1 case where the disease is associated with the intake of glucocorticosteroids, 1 case associated with primary immunodeficiency, 1 case with the influenza B virus and 2 cases in non-immunocompromised patients. Conclusion: Idiopathic pulmonary fibrosis can be an additional "background" disease of chronic pulmonary aspergillosis. Prolonged antimycotic therapy is necessary for successfully treatment of chronic pulmonary aspergillosis in patients with idiopathic pulmonary fibrosis.

P109

The performance of the BALF later-flow device test in the rapid diagnosis of nonneutropenic
invasive pulmonary aspergillosis

L. Liu, Y. Wang, Y. Gu, S. Li, B. Chen, K. Shen, X. Su

Jinling Hospital, Medical School of Nanjing University, Nanjing, China, nanjing, China

**Objectives:** The *Aspergillus* lateral-flow device (LFD) is a new method to test for *Aspergillus* antigen. We performed this study to evaluate the value of the LFD test in diagnosing nonneutropenic invasive pulmonary aspergillosis.

**Methods:** A total of 136 bronchoalveolar lavage fluid (BALF) samples from 136 suspected invasive pulmonary aspergillosis (IPA) patients were finally divided into 2 groups: the aspergillosis group and the non-aspergillosis group. We performed both the LFD and glactomannan (GM) tests on 80 BALF samples and only the LFD test on the remaining 58 BALF samples.

**Results:** After systematic clinical diagnosis, all 136 patients were divided into an aspergillosis group (n = 41, proven (n = 8) and probable (n = 33) diagnosis) and a non-aspergillosis group (n = 95, pneumonia, tuberculosis and others). The sensitivity, specificity, positive predictive value, and negative predictive value of LFD for diagnosing pulmonary aspergillosis in all 136 BALF samples were 69.7%, 72.6%, 87.3% and 46.9%, respectively. When comparing the GM and LFD test results for the 80 samples, the sensitivity (75.8% vs 74.1%, P = 0.769) was similar. The specificity of the GM test
was higher than that of the LFD test (83.0% vs 66.0%, P = 0.058), but there was no significant difference between the tests. The sensitivity and specificity of the combination of the positive results of both the LFD and GM tests were 63.6% and 89.4%, respectively, and the sensitivity and specificity of either LFD or GM positive results or both LFD and GM positive results were 90.9% and 59.6%, respectively.

**Conclusion:** LFD is a promising alternative diagnostic method to the GM test for the early detection of patients with nonneutropenic pulmonary aspergillosis.

P110

High rates of misidentification of uncommon Candida species fungemia with conventional phenotypic methods

W.-L. Liu¹,², Y.-S. Huang³, F.-D. Wang⁴, M.-H. Hsieh⁵, I.-M. Hii⁶, Y.-L. Lee⁶, M.-W. Ho⁷

¹School Of Medicine, College Of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan,

²Emergency & Critical Care Medicine, Fu Jen Catholic University Hospital, New Taipei City, Taiwan,

³Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, ⁴Medicine, Taipei Veterans

General Hospital, Taipei, Taiwan, ⁵Internal Medicine, Kaohsiung Medical University Hospital,

Kaohsiung Medical University, Kaohsiung, Taiwan, ⁶Internal Medicine, Changhua Christian

Hospital, Changhua, Taiwan, ⁷Internal Medicine, China Medical University Hospital, Taichung,

Taiwan

**Objectives:** Candidemia caused by uncommon *Candida* species is increasing. Correct identification of *Candida* isolates to species level is important for optimizing antifungal choice. In this study, we aimed to evaluate the accuracy of species-level identification of conventional methods and MALDI-TOF MS system in patients with candidemia due to the uncommon *Candida* species. Sequencing of the internal transcribed spacer (ITS) was performed for comparison and determination of accuracy.

**Methods:** From July 2011 to December 2014, uncommon *Candida* species identified by conventional phenotypic methods from candidemic patients were prospectively collected at 6 hospitals in Taiwan. Identification of these uncommon *Candida* isolates were performed using MALDI-TOF MS and DNA sequencing of the fungal internal transcribed spacer (ITS). Susceptibility of the non-duplicate isolates were determined using Sensititre™ YeastOne™ and were interpreted with cut-off values recommended by the CLSI.

**Results:** A total of 85 uncommon *Candida* isolates reported by conventional methods were included. By conventional methods, the most frequently reported uncommon *Candida* species were *C. guilliermondii* (*n* = 39), *C. sake* (*n* = 7), and *C. famata* (*n* = 4). Using ITS sequencing analysis as standard, none of the *C. sake* and *C. famata* identified by conventional methods was correct, while MALDI-TOF MS correctly identified 10 of the 11 isolates. With the exclusion of the one unspecified *Candida* isolate reported by ITS sequencing, the accuracy of conventional methods and MALDI-TOF MS in the identification of 84 *Candida* isolates were 64.3% and 86.9%, respectively (p = 0.001). Eight isolates were confirmed to be non-*Candida* yeast species. Compared with other *Candida* species, *C. guilliermondii* showed elevated MIC values of echinocandins. Based on our results, an identification algorithm for
uncommon *Candida* species was proposed (Figure 1). Figure 1. Identification algorithm for uncommon *Candida* species

<figure><img src="image_34.png" /></figure>

**Conclusion:** Conventional methods could lead to high rate of misidentification of uncommon *Candida* species, especially *C. sake* and *C. famata*. MALDI-TOF MS assisted with DNA-sequencing based methods should be considered for identification of uncommon *Candida* species.

P112

**Cross-sectional study of respiratory Aspergillus spp. colonization or infection in patients with various stages of Chronic Obstructive Pulmonary Disease (COPD) using culture vs non-culture based technique.**

S. Waqas¹,², K. Dunne², A. Fe Talento³,⁴,⁵, G. Wilson¹, I. Martin-Loeches¹, J. Keane¹,², T. R Rogers¹,²

¹St. James's Hospital, Dublin, Ireland, ²Trinity College Dublin, Dublin, Ireland, ³Our Lady Of Lourdes Hospital, Drogheda, Ireland, ⁴Royal College of Surgeons Ireland, Dublin, Ireland, ⁵Beaumont Hospital, Dublin, Ireland

**Objectives:** (*1st and 2nd Authors are equal contributors) COPD patients are now recognized to be at increased risk of colonization by *Aspergillus* spp. which may progress to invasive pulmonary aspergillosis (IA). Published data on the frequency of *Aspergillus* detection in COPD are limited. The objective of this study was to determine frequency of *Aspergillus* colonization or infection in COPD patients.

**Methods:** A cross-sectional study was undertaken to determine *Aspergillus* colonization or infection in COPD patients undergoing bronchoscopy for any indication. Culture as well as galactomannan antigen (GM) and *Aspergillus* nucleic acid detection (PCR) were performed on bronchoalveolar lavage fluid (BAL).

**Results:** One hundred and fifty patients were included (44.7 % female, mean age 68.2 years). 21.3% were inpatients, 74.7% outpatients and 4% were ICU patients. Investigation of lung masses was the most common indication (43.3%) for bronchoscopy. Most patients (81.3 %) were either GOLD stage 1 or 2 COPD. Cancer was the most frequent co-morbidity (60.48%). 12 % and 48.7 % were on systemic and inhaled steroids respectively. Lung mass was the most common (28.43%) CT imaging finding. Seventeen patients (11.3%) had a positive result for *Aspergillus* (Culture ±Galactomannan ± PCR). 76.4% out of these seventeen were in the early stages (GOLD stage 1 or 2) of COPD.

**Conclusion:** *Aspergillus* sp. was detected in 3.3% of patients by culture, which increased to 11.3% if culture was combined with either a positive GM or PCR result. Overall the frequency of *Aspergillus* detection in this population of COPD patients was low which may reflect the predominance of Gold stages 1 and 2 among the study population.

P113
## Evaluation of new multiplex PCR-based blood assays for detection of candidemia

M. Jobstmann¹,², J. Prattes², E. Stelzl¹, C. Zurl¹, M. Hoenigl³, J. Rabensteiner¹, R. Krause², H. Kessler¹
¹Medical University of Graz, Graz, Austria, ²Section Of Infectious Diseases And Tropical Medicine, Medical University of Graz, Graz, Austria, ³Division Of Infectious Diseases, University of California, San Diego, United States of America

**Objectives:** Blood cultures have been considered as diagnostic gold standard for candidemia. Compared to cultures, assays based on polymerase chain reaction (PCR) may be useful to shorten the time to diagnosis of invasive candidiasis and to initiation of antifungal therapy. Recently, the new CandID® and CandID PLUS® kits (OLM Diagnostics, Newcastle Upon Tyne, England) for molecular detection of different *Candida* spp. have been developed. In this study, the analytical and clinical performance of the new kits was investigated. Reference material and clinical specimens were used.

**Methods:** Nucleic acid extraction was performed on the EMAG® platform (bioMérieux, Marcy-l'Étoile, France) using the specific B protocol. Real-time PCR (qPCR) and detection with the CandID® and CandID PLUS® kits were performed on the Light Cycler® 480 II CE/IVD instrument (Roche Diagnostics, Penzberg, Germany). Both of the kits are based on multiplex qPCR providing detection of three different *Candida* spp. each and an internal control: *Candida albicans*, *Candida glabrata*, *Candida parapsilosis* with the CandID® and *Candida tropicalis*, *Candida krusei*, *Candida dubliniensis* with the CandID PLUS®. The accuracy of the new kits was determined utilizing the Quality Control for Molecular Diagnostics (QCMD) 2018 *Candida* spp. EQA Programme. The panel consisted of 10 members including *Candida albicans*, *Candida auris*, *Candida glabrata*, *Candida krusei*, and vials without *Candida* spp. The clinical performance of the new kits was determined with specimens obtained from three groups of patients: patients with culture-proven candidemia (n = 23; EDTA whole blood samples), patients with bacteremia (n = 31; plasma samples), and healthy controls (n = 25; EDTA whole blood samples). Whole blood samples from candidemic and bacteremic patients were drawn at the same day as the corresponding blood cultures.

**Results:** With the quality control program, all panel members were correctly identified by the new PCR assays. With the clinical study, 2 EDTA whole blood samples obtained from patients with candidemia and 7 EDTA whole blood samples obtained from healthy controls were found to be inhibited and thus excluded from further analysis. In patients with candidemia (mean age, 67 years, range 20 to 89 years), 14 of 21 samples (67%) gave a positive result when employing the new PCR assays. In patients with bacteremia (mean age, 62 years, range 23 to 91 years), 1 of 31 samples gave a false-positive result with the CandID assay. All healthy controls (mean age, 57 years, range 45 to 66 years) remained negative with the new PCR assays. All *Candida* spp. from candidemic patients were correctly identified with the PCR assays.

**Conclusion:** When compared to blood culture, two thirds of samples obtained from patients with candidemia were found positive with the new CandID® or CandID PLUS® kits. While in samples obtained from patients with bacteremia one false-positive result was observed with the new PCR assays, results of samples obtained from healthy controls were all found to be correct.

## Development of a New Quantitative Real-Time PCR Assay for the Detection and Identification of Aspergillosis and Mucormycosis Infections

S. Godichaud¹, J. Verrier¹, R. Boisson¹, G. Lagardere¹, P. Flori²

¹ADEMTECH, PESSAC, France, ²CHU ST ETIENNE, ST ETIENNE, France

**Objectives:** Mucormycosis is associated with high mortality rates, especially in haematological patients, and remains difficult to diagnose. The early distinction with invasive aspergillosis is of importance because the antifungal treatment for each is different. Unfortunately, the underlying conditions of both infections are similar. Moreover, detection of circulating antigens such as galactomannan and β-D-glucan provides no help for diagnosing mucormycosis, and cultures are often delayed or negative, preventing early management. Delayed directed antifungal treatment impacts the outcome of mucormycosis. The goal of this study is to evaluate a new quantitative PCR assay allowing the simultaneous detection of *Aspergillus* spp. and Mucorales spp.
**Methods:** The MycoGENIE Aspergillus-Mucorales spp. real-time PCR kit (Ademtech, France) is a triplex PCR assay targeting specifically *Aspergillus* spp., Mucorales spp. (including *Lichtheimia*, *Mucor*, *Rhizomucor*, *Rhizopus* and *Cunninghamella* species), and an exogenous Internal Control. This study was performed on a reference panel of 30 biopsy specimen, including 10 positive for *Aspergillus* spp. and 12 positive for Mucorales spp. previously identified by direct examination and culture. Twenty negative serum samples were also included in this study to evaluate the specificity of the assay. DNA extraction was performed using the MycoGENIE DNA Extraction kit (Ademtech, France) and 10 µL of pure DNA was used as a template for the PCR assay. Mucorales-infected samples were also evaluated with other external laboratory-developed PCR tests targeting only *Mucor*, *Rhizomucor*, *Rhizopus* and *Lichtheimia* species.

**Results:** On the 30 samples, the MycoGENIE Aspergillus-Mucorales spp. PCR kit successfully detected 19 *Aspergillus*-positive samples including *A. fumigatus*, *A. niger* and *A. nidulans* identified by culture. Mucorales spp. were also detected in 18 samples. Notably, the PCR assay efficiently detected Aspergillosis-Mucormycosis co-infections in 9 samples. All serum samples returned negative.

**Conclusion:** This new CE-IVD compliant real-time PCR assay, combined with an automated DNA extraction process is a sensitive and specific molecular diagnostic tool for the diagnosis of aspergillosis and mucormycosis infections in biopsy samples. The possibility to efficiently detect aspergillosis/mucormycosis co-infections is highlighted in this study and is of importance for a better therapeutic management.

P115

**Comparison of multiplex nested PCR and Maldi tof to identify clinically isolated candida from Sénégal, a preliminary study**

A. Badiane, A. Sene, A. Gaye, K. Diongue, M.C. Seck, M. Ndiaye, J.P. Gangneux, D. Ndiaye
Parasitology And Mycology, Universite Cheikh Anta Diop de Dakar, Dakar, Senegal

**Objectives:** The objective of this study is to compare the identification of clinically isolated candida species using Maldi tof and a multiplex nested PCR.

**Methods:** A total of 38 clinically isolated strains of Candida from superficial mycosis and vaginal infection were conserved in brain heart broth additioned with 15 % glycerol and stored at – 20 °C. The strains were then sub cultured in CHROMagar™ candida (Becton Dickinson), incubate at 37 °C for 24 to 48 hours and identified using Maldi tof (Bruker) at University of Rennes / France. A multiplex nested PCR were performed directly from the conservative media at Cheikh anta Diop University of Dakar/ Senegal.

**Results:** Thirty five (35) samples gave interpretable results for both methods. 51.43 % of the samples showed the same result with Maldi tof and multiplex PCR, among which *Candida albicans* (45.71 %) and *C. parapsilosis* (2.85%). The species identified by both methods were *C. albicans*, *C. tropicalis* and *C. parapsilosis*. *C. albicans* alone or mix with other species was identified 71.73 % by Maldi tof and 61.70% by PCR nested multiplex. *C. tropicalis* alone or mix with other species was identified 13.04 % with Maldi tof and 8.51% with multiplex nested PCR. *C. parapsilosis* alone or mix with other species was identified 2.17 % with Maldi tof and 23.40% with multiplex nested PCR. *C. glabrata*, *C. pintolepesii*, *C. orthopsilosis* and *C. pararugosa* were identified only by Maldi tof. *C. lusitania* and *C. kefyr* were only identified by Multiplex nested PCR. Seven (7) mix species were identified with multiplex nested PCR and five (5) with Maldi tof. Among the samples, identified as *C. albicans* using multiplex PCR three were identified as *C. tropicalis* by Maldi Tof. *C. parapsilosis* genotype 2 identified by multiplex PCR has been identified as *C. orthopsilosis* with Maldi tof. Three strains identified as *C. albicans* by multiplex PCR were identified as *C. tropicalis* by Maldi tof.

**Conclusion:** The multiplex nested PCR and the Maldi tof identified the most prevalent Candida species, but there is discordance for the identification of the non albicans species. The multiplex PCR identified more multiple strains infections than the Maldi tof. The differences observed between these two methods could be due to the absence of these strains in the Maldi tof database and the subculture of predominant strains. Also genetically close species might not be differentiated using PCR.
P117

Utilization of MALDI-TOF MS for identification of filamentous fungi: a comparative study

A. Vavrova¹, L. Svobodova¹, J. Mrazek², P. Hamal¹

¹Department Of Microbiology, Palacký University, Faculty of Medicine and Dentistry and University Hospital Olomouc, Olomouc, Czech Republic, ²Department Of Bacteriology And Mycology, Institute of Public Health in Ostrava, Ostrava, Czech Republic

**Objectives:** Identification of filamentous fungi based on morphological characteristics is the most simple and available approach used in diagnostic clinical mycology laboratories. In many cases, however, it requires considerable experience of the rater and the ability of strains to produce spores. For its rapidity and accuracy, MALDI-TOF mass spectrometry is invaluable for identification of microorganisms including fungi. It is more challenging to use this method for identification of filamentous fungi than bacteria or yeasts given differences in cell wall structures and a limited spectrum of species in the database. The study aimed to find the optimal way of identifying filamentous fungi in mycology laboratories using MALDI-TOF MS.

**Methods:** The tested group of moulds comprised 193 isolates obtained from clinical specimens. The identification started with morphological assessment (appearance of colonies, micromorphology from slide culture). Then isolates were identified using MALDI-TOF, both directly from culture and following culture in liquid media with extraction; in the latter case, both supernatant and sediment were tested. Finally, all the methods were compared as to the rate of agreement. Subsequently, a small sample of selected isolates was identified by sequencing.

**Results:** Based on morphological criteria, 17 genera were identified; due to a lack of sporulation, another 5 isolates could not be identified. Further, 13 species were identified, mostly within the genus *Aspergillus*. With MALDI-TOF MS performed directly from culture, nine isolates were identified to the genus level and 184 were identified to the species level, with a total of 75 species being noted. With MALDI-TOF MS after culture in liquid media with extraction, 190 isolates were identified to the species level, with 43 species being noted; in only three cases, identification of filamentous fungi was limited to genera. Comparison of identification from supernatant and sediment showed that identical results were always obtained at both the genus and species level and there were only minimal differences in identification scores. The rates of agreement between isolate identification using morphology and MALDI-TOF MS from culture were 58.55% at the genus level and 22.24% at the species level. The rates of agreement between isolate identification using morphology and MALDI-TOF MS after culture in liquid media with extraction were 84.97% at the genus level and 46.11% at the species level. Based on disagreeing results of the above identification methods, 20 isolates from the sample were selected to be identified by sequencing. The highest agreement rate (70%) was found for identification with MALDI-TOF MS after culture in liquid media with extraction and only 35% using MALDI-TOF MS directly from culture.

**Conclusion:** The results suggest that the optimal approach to identification of filamentous fungi in diagnostic medical mycology laboratories is a combination of morphological characteristics and MALDI-TOF MS. For species identification of morphologically typical isolates, assessing the appearance of colonies and slide cultures is sufficient; in the other cases, MALDI-TOF MS after culture in liquid media with extraction is recommended. If identification with the above combination is ambiguous or impossible to perform, molecular genetic methods need to be used, in particular sequencing.

P118

Risk Factors for Candidemia: A Prospective Matched Case-Control Study

J. Poissy¹, L. Damonti², A. Bignon³, N. Khanna⁴, M. Von Kietzell⁵, K. Boggian⁵, D. Neofytos⁶, F. Vuotto⁷, V. Coiteux⁸, F. Artru⁹, S. Zimmerli², J.-L. Pagani¹⁰, T. Calandra¹¹, B. Sendid¹², D. Poulain¹², C. Van Delden⁶, F. Lamothe¹³, O. Marchetti¹¹, P.-Y. Bochud¹¹

¹Intensive Care Department, University Hospital of Lille, Lille Cedex, France, ²Department Of Infectious Diseases, Inselspital, Bern University Hospital, University Of Bern, University Hospital of Bern, Bern, Switzerland, ³Surgical Intensive Care Department, University hospital of Lille, Lille
Cedex, France, ⁴Division Of Infectious Diseases And Hospital Epidemiology, University Hospital of Basel, Basel, Switzerland, ⁵Department Of Infectious Diseases, Cantonal Hospital of Saint Gallen, Saint Gallen, Switzerland, ⁶Infectious Diseases, University Hospital of Geneva, Geneva, Switzerland, ⁷Infectious Diseases Department, University Hospital of Lille, Lille Cedex, France, ⁸Hematological Disorders Department, University Hospital of Lille, Lille Cedex, France, ⁹Digestive Intensive Care Department, University Hospital of Lille, Lille Cedex, France, ¹⁰Intensive Care Department, University hospital of Lausanne, Lausanne, Switzerland, ¹¹Infectious Diseases Department, University Hospital of Lausanne, Lausanne, Switzerland, ¹²Laboratory Of Mycology And Parasitology, University Hospital of Lille, Lille Cedex, France, ¹³Microbiology Institute, University Hospital of Lausanne, Lausanne, Switzerland

**Objectives:** Candidemia is an opportunistic infection associated with high morbidity and mortality in hospitalized patients, both inside and outside intensive care units (ICUs). Identification of patients at risk for preemptive approach and early detection is crucial. Prospective control-matched studies and comparison between ICU and non-ICU patients are lacking in this field. We aim to identify and compare specific risk factors in ICU and non ICU patients.

**Methods:** This was a prospective multicenter matched case-control study assessing risk factors for candidemia and death in candidemic patients, both outside and inside ICUs from 6 teaching hospitals in Switzerland and France. Controls were matched to cases based on age, hospitalization ward, hospitalization duration and, when applicable, type of surgery. Risk factors were analyzed by univariate and multivariate conditional regression models, as a basis for a new scoring system for prediction of candidemia.

**Results:** The study included 183 cases and 378 matched controls. 44% were hospitalized inside ICU and 56% outside. Independent risk factors for candidemia within the ICU population included total parenteral nutrition (TPN) (OR = 9.24, 95%CI 3.39-25.2, p<0.001), acute kidney injury (OR = 6.48, 95%CI 2.39-17.6, p<0.001), solid cancer (OR = 4.59, 95%CI 1.23-17.2, p = 0.02), heart disease (OR = 4.26, 95%CI 1.37-13.3, p = 0.01), invasive mechanical ventilation (OR = 3.37, 95%CI 1.17-9.67, p = 0.02), exposure to fluoroquinolones (OR = 3.05, 95%CI 1.14-8.16, p = 0.03) and exposure to aminoglycosides (OR = 2.47, 95%CI 1.07-5.69, p = 0.03). Independent risk factors for candidemia within the non-ICU population included central venous catheter (CVC) (OR = 10.20, 95%CI 3.87-27.2, p<0.001), TPN (OR = 3.37, 95%CI 1.53-7.41, p = 0.003), exposure to glycopeptides (OR = 4.10, 95%CI 1.57-10.7, p = 0.044), and to nitroimidazoles (OR = 5.14, 95%CI 1.59-16.6, p = 0.006). The weighted ICU-score was as follows : TPN, +2; AKI, +2; heart disease, +1.5; solid cancer, +1.5; invasive mechanical ventilation, +1.0; fluroquinolone: +1.0. aminoglycoside: +1. AUC of the ROC curve was 0.773. The optimal cut-off was ≥5 (sensitivity = 76%, specificity = 64%). The best cut-off to optimize specificity was ≥7 (sensitivity = 23%, specificity = 95%). The weighted non-ICU score was as follows : CVC : +3.5; nitroimidazole : +2.5; TPN, +2; Glycopeptide : +2.0. AUC of the ROC curve was 0.717. The optimal cut-off was ≥4 (sensitivity = 83%, specificity = 49%). The best cut-off to optimize specificity was ≥6 (sensitivity = 49%, specificity = 82%). Independent factors for death in candidemic patients in the whole population were septic shock (OR = 7.17, 3.06-16.8, p<0.001), acute kidney injury (OR = 5.04, 2.16-11.8, p<0.001), number of antibiotics (OR = 1.42, 1.15-1.75, per unit, p<0.001). After stratification between ICU and non-ICU patients, only septic shock (OR = 4.49, 1.53-13.1, p = 0.006), acute kidney injury (OR = 3.13, 1.02-9.17, p = 0.04), the number of antibiotics to which patients were exposed before candidemia (OR = 1.35, 1.05-1.75 per unit, p = 0.02) remain significantly and independently associated with death in ICU patients. For non-ICU patients, acute kidney injury (OR = 10.7, 2.16-52.5, p = 0.004) and septic shock (OR = 10.50, 2.80-39.7, p<0.001) were the only variables significantly associated with death

**Conclusion:** Risk factors for candidemia are variable in the ICU versus non-ICU setting, including different patterns of antibiotic exposure. Weighted scores predictive of candidemia can be built based on these risks, with better performances for ICU-patients. An improved prediction of the risk of candidemia may contribute to guide targeted preventive and therapeutic antifungal strategies.
# Inter-laboratory robustness of screening tests for the detection of *Aspergillus* antibodies for chronic aspergillosis: finding from a French External Quality Assessment

F. Persat¹,², D. Dupont³, J.-C. Eynard², B. Poggi², M. Wallon³

¹Parasitology And Medical Mycology, Hospices Civils de Lyon / Université Lyon 1, Lyon, France,
²Association Pro.Bio.Qual, Lyon, France, ³Parasitology And Medical Mycology / Umr 5292,
Université Lyon 1 / Hospices Civils de Lyon, Lyon, France

**Objectives:** Six million people worldwide are estimated to have chronic aspergillosis or/and allergic broncho pulmonary aspergillosis. Serum *Aspergillus* assay is an important parameter to detect and follow the immune host response against this fungus. Reliable tests are crucial for patients' care but also to perform much needed multicentre-studies. However, the current tests are not standardized and may lack in robustness. We have analysed the results of a French External Quality Assessment (EQA) programme to assess the inter-laboratory robustness of four *Aspergillus* antibody techniques among those recommended in France as first-line screening tests.

**Methods:** The study was based on the answers provided by 40 laboratories that took part at least once to the EQA organised by ProBioQual between 2013 to 2018 for *Aspergillus* serology. A total of 24 sera were tested over this time period, 18 positive from patients with chronic or allergic bronchopulmonary aspergillosis and six negative sera from blood donors. The results of the following techniques were analysed: 1) Platelia™*Aspergillus* IgG EIA (Bio-Rad, France); 2) ELISA Classic™A. *fumigatus* IgG (Virion/Serion, Germany); 3) ELISA *Aspergillus fumigatus* IgG (Bordier Affinity Products, Switzerland) since 2014; 4) Aspergillose Fumouze indirect hemagglutination (Biosynex Fumouze, France).

**Results:** reported by the participants for the Bio-Rad test were all negative for five of the six negative sera and all positive for 14 of 18 positive sera. Those reported for the Virion/Serion ELISA were all negative for four of the six negative sera and all positive for 10 of the 18 positive sera. The results reported for the Bordier test were all concordant but only based on five positive sera and one negative serum. Results provided with the hemagglutination test were all negative on all negative sera, but none of 18 positive sera were found by all laboratories to be positive, and 10 of the positive sera were even found by all laboratories to be negative.

**Conclusion:** Hemagglutination technique appears lacking too much in sensitivity to be used as single screening test, compared to the ELISA techniques, probably explaining its decreasing use. ELISA Bio-Rad appeared as more robust than Virion/Serion ELISA. The potentially better performances of ELISA Bordier have to be confirmed.

P120

# Usefulness of conventional PCR through induced sputum to diagnoses *Pneumocystis* pneumonia in HIV infected patients

O.J. Chagas¹, R. Buccheri², G. Naves¹, P.L. Silva Pinto³, V.L. Pereira Chioccola⁴, G. Benard¹, G.M.B. Del Negro¹

¹Laboratório De Investigações Médicas 53, Insituto de Medicina Tropical, São Paulo, Brazil, ²Hospital Ward, Instituto de Infectologia Emílio Ribas, São Paulo, Brazil, ³Intitution: Intituto Adolfo Lutz - Núcleo de Enteroparasitas, São Paulo, Brazil, ⁴Intituto Adolfo Lutz - Laboratório de Biologia Molecular de Parasitas e Fungos do Centro de Parasitologia e Micologia, São Paulo, Brazil

**Objectives:** The present study aims to determine the usefulness of conventional PCR in induced sputum to diagnoses *Pneumocystis* pneumonia in HIV infected patients.

**Methods:** Study design was of a prospective-cohort with systematic collection of data. Ninety-five HIV-infected patients presenting cough and dyspnoea for more than 7 days were included. They should not have received antiretroviral therapy (ART) or a history of inadequate treatment. Induced sputum, obtained through concentrated saline solution (NaCl 3%-5%) inhalation, was obtained at admission and submitted to O-toluidine staining and conventional PCR for *Pneumocystis*. Clinical, laboratorial, imaging records and clinical outcome from PCR positive and PCR negative patients were compared. We also compared these data in PCR positive patients and confirmed pulmonary tuberculosis (TB) (presence of bacillus-acid-alcohol-resistant or positive rapid-molecular-test).
**Results:** PCR was positive in 34/95 (35.7%) of the patients. This group presented higher rates of dry cough (55.8% X 22.9%, P = 0.003), interstitial infiltrate on chest X-ray (94.1% X 61%, P = 0.001), ground grass on chest tomography (CT) (89.2% X 42.8%, P = 0.0002) and higher lactate dehydrogenase (LDH) levels (488U/L x 329U/L, P<0.0001), when comparing with the PCR negative patients. PCR positive patients had also more non-invasive ventilation (NIV) (47% X 15%, P = 0.002) and more admissions at the intensive care unit (ICU) (35.2% X 13.5%, P = 0.02). Both groups did not differ regarding n. of days with symptoms, PaO2, HIV viral load (VL) and *Candida* infection during hospitalization. There were 32 PCR positive patients without TB and 11 patients with confirmed pulmonary TB. TB was associated with CT budding tree (57,1% X 0, P = 0.0005), CT micronodules (85,7% X 23%, P = 0.008); trend toward higher CD4 cell count (129 X 47 cell/mm³, P = 0.01) and lower use of NIV (6,2% X 50%, P = 0.0417), and less CT ground grass (14,2% X 92,3%, P = 0.0001). There was no difference between LDH, PaO2, HIV VR, and ICU hospitalization between the two groups. Of note, 5/34 (14.7%) of the PCR positive patients did not receive any antimicrobial treatment for PCP during hospitalization but improved their initial symptoms, hence the difficult to differentiate between colonization and infection solely based on the conventional PCR. In addition, only two PCR positive patients did not display ground grass at CT: both had pulmonary consolidation.

**Conclusion:** Our PCR assay was useful to define a subgroup of patients with clinical, laboratorial and imaging aspects related to PCP. Besides it, pulmonary CT is a fundamental tool, since suggestive alterations in this exam strongly contribute to the diagnosis, helping to differentiate the pulmonary involvement of PCP from that of TB, the main differential in our country, where TB is highly prevalent. Of note, five PCR positive patients did not receive specific treatment during hospitalization, pointing to either the existence of false-positive cases or to *P. jiroveci* colonization. The possibility that differentiation between these patients could be achieved by quantitative real-time-PCR remains an open question, since the literature is controversial, due mainly to the different primers and techniques employed. Another discriminatory assay would be the β-D-Glucan test, which, combined with PCR assay, would be able to discriminate colonized from infected patients.

P121

**Performance characteristics of the MucorGenius® real-time PCR assay for the detection of clinically relevant Mucorales species**

G. Gaajetaan, T. Kampermann, D. Van Tegelen, G. Dingemans

PathoNostics, Maastricht, Netherlands

**Objectives:** Determine the performance characteristics of the commercially available MucorGenius® real-time PCR assay for the detection of clinically relevant Mucorales species. For this purpose, several verification studies were performed including sensitivity, specificity and EQA evaluation.

**Methods:** The analytical sensitivity or limit of detection (LOD) was determined by testing serial dilutions of all reference Mucorales culture extracts including *Rhizopus oryzae*, *Rhizopus microsporus*, *Cunninghamella bertholletiae*, *Lichtheimia corymbifera*, *Rhizomucor pusillus*, *Mucor hiemalis* and *Rhizomucor miehei* on various real-time PCR instruments. The LOD dilution of a Mucorales species was accepted if at least 19 out of 20 reactions were detected. This dilution was finally quantified for each species by using droplet digital PCR (ddPCR). Analytical specificity was determined by testing multiple high concentrations of bacterial and fungal DNA strains that can be present in the respiratory tract. The seven serum samples of the FPCRI Muc Panel (2018-1) were tested with the MucorGenius® PCR after DNA-extraction with the NucliSENS® easyMag system (bioMérieux).

**Results:** The LOD was successfully determined for all seven Mucorales targets on 5 different real-time cyclers. All results of the LOD study are listed in table 1. Table 1. Overview of MucorGenius® LOD results.
<table><thead><tr><th rowspan="2">Target</th><th>Tested conc</th><th>LC480 II</th><th>RGQ</th><th>CFX96</th><th>Mic</th><th>QS5</th></tr><tr><th>(c/µl)</th><th>Ct</th><th>Ct</th><th>Ct</th><th>Ct</th><th>Ct</th></tr></thead><tbody><tr><td><i>R. oryzae</i></td><td>1.5</td><td>32-35</td><td>33-35</td><td>31-32</td><td>32-33</td><td>34-40</td></tr><tr><td><i>R. microsporus</i></td><td>1.7</td><td>33-34</td><td>34-43</td><td>32-33</td><td>34-36</td><td>31-38</td></tr><tr><td><i>R. miehei</i></td><td>1.1</td><td>33-34</td><td>34-39</td><td>33-35</td><td>33-37</td><td>34-40</td></tr><tr><td><i>R. pusillus</i></td><td>2.2</td><td>33-34</td><td>34-37</td><td>32-33</td><td>33-34</td><td>31-36</td></tr><tr><td><i>C. berthollatiae</i></td><td>1.2</td><td>32-33</td><td>32-38</td><td>34-40</td><td>32-35</td><td>31-38</td></tr><tr><td><i>L. corymbifera</i></td><td>0.5</td><td>32-33</td><td>33-37</td><td>33-41</td><td>32-35</td><td>31-38</td></tr><tr><td><i>M. hiemalis</i></td><td>2.1</td><td>32-33</td><td>32-37</td><td>31-32</td><td>32-33</td><td>33-40</td></tr></tbody></table>

No cross-reactivity was observed with any of the clinically relevant fungal or bacterial strains. Mucorales DNA was successfully detected in 6 out of 7 serum DNA-extracts from the FPCRI panel. All samples showed an internal control signal indicating reliable DNA-extraction performance. These results were in agreement with the data of the FPCRI.

**Conclusion:** The MucorGenius® real-time PCR assay can detect all Mucorales species at low DNA-concentrations of 1-2 copies/µl which corresponds with high Ct-values (Ct 31-43). This indicates reliable analytical sensitivity. No cross-reactivity was observed for any of the tested fungal or bacterial strains, reducing the chance of a false positive result. Although 100% agreement was obtained with the FPCRI Muc Panel, this is a limited data set and only provides an indication of the performance. Clinical validation studies are now ongoing on different sample types to determine the clinical performance and suitability for routine diagnostics.

P122

Time to and differential time to blood culture positivity for assessing catheter-related candidemia: A longitudinal, 7-year study in a single university hospital

M. Gits-Muselli¹,², S. Villiers³, S. Hamane², B. Berçot⁴, J.-L. Donay⁴, B. Denis⁵, N. Guigue², A. Alanio⁶, S. Bretagne⁶

¹Molecular Mycology, Institut Pasteur, PARIS, France, ²Parasitology-mycology Laboratory, Lariboisière Saint-Louis Fernand Widal Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), PARIS, France, ³Anesthesiology Department, Lariboisière Saint-Louis Fernand Widal Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), PARIS, France, ⁴Microbiology Department, Lariboisière Saint-Louis Fernand Widal Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), PARIS, France, ⁵Tropical And Infectious Diseases Department, Lariboisière Saint-Louis Fernand Widal Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), PARIS, France, ⁶Paris-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

**Objectives:** Central venous catheters (CVCs) significantly increase the risk of candidemia, and inversely, candidemia is often associated with intravascular catheters. Most guidelines for candidiasis management recommend removal of existing intravascular catheters, if feasible. Although the recommendation is strong, various authors have recommended a re-consideration of CVC removal on an individual basis. Practically, one must consider the possible deleterious consequence of CVC removal, especially in patients who require long-term CVC for treatment or nutrition. This raises the question of systematic early removal of CVC in the management of candidemia since some authors did not report differences, knowing that patients who cannot tolerate CVC removal have often a worse prognosis. Time to positivity (TTP) and differential time to positivity (DTTP) between central and peripheral blood cultures are commonly used for bacteremia to evaluate the likelihood of CVC related bloodstream infection. Few studies have addressed the application of these approaches to candidemia. This study aimed to evaluate TTP and DTTP to assess CVC-related candidemia (CVC-RC).

**Methods:** We retrospectively analyzed the results from 105 adult patients with incident candidemia, with CVC removed and cultured, collected from 2010 to 2017. The bottles were incubated in a
BioMérieux bact/ALERT 3D and kept for at least 5 days. Patients with CVC removed and cultured at the time of fungemia were divided into two groups as previously proposed: (i) CVC-related candidemia (CVC-RC) when the same yeast species was recovered from at least one BC and from the CVC culture; (ii) non-CVC-related candidemia (NCVC-RC) when a yeast species was recovered from at least one BC, and the CVC culture was negative or yielded a different species.

**Results:** Of the 105 patients included, most were oncology patients (85.7%) and had of long-term CVC (79.6%); 32 (30.5%) had a positive CVC tip with the same species as in BC, defined as CVC-RC. The main species involved were *C. albicans* (46%), *C. parapsilosis* (19%), *C. glabrata* (15%), and *C. tropicalis* (9%), with no statistical difference between NCVC-RC and CVC-RC. Considering all species, the median TTP of the first bottle collected from CVC was statistically shorter in the CVC-RC group (16.8 h [9.7-28.6]) compared to the NCVC-RC group: median (29.4 h [20.7-41.3], *p* = 0.001, Figure 1). When analyzing TTP regarding the *Candida* species, a significant shorter TPP was only observed in *C. albicans* with a median of 12.9 h [10.0-22.7] vs. 32.3 h [24.4-41]) for CVC-RC and NCVC-RC, respectively (*p* = 0.001, Figure 1). A TTP < 10 h had the best positive likelihood ratio (21.5), although the sensitivity was only 28%. DTTP was available for 52 patients. A DTTP > 5 h had a sensitivity of 100% and a specificity of 71%.

<figure><img src="image_35.png" /><figcaption>**Figure 1:** Comparisons of time to positivity (TTP) between positive (CVC+) and negative (CVC-) central venous catheter for all yeast cultured and according to the four more frequent *Candida* species (*: *p* < 0.0001)</figcaption></figure>

**Conclusion:** Even if some cut-offs supported the diagnosis of CVC-RC, they did not allow the prompt decision on CVC removal, since the median TTP was 17 h. More rapid methods for detecting infected catheters should be tested to avoid withdrawal of non-infected CVC (69.5% of CVCs in our study).
P123

Diagnostic value of immunological markers of allergic bronchopulmonary aspergillosis in patients
with asthma

Y. Kozlova¹, E. Frolova², A. Uchevatkina³, L. Filippova¹, G. Solovjeva¹, A. Sobolev¹, N. Vasilyeva¹, N. Klimko¹

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

**Objectives:** Allergic bronchopulmonary aspergillosis (ABPA) is a severe lung disease caused by hypersensitivity to *Aspergillus* spp. The study of the role of different immunological mediators in the formation of chronic allergic inflammation in patients with ABPA is necessary to identify potential targets for therapeutic intervention and timely diagnosis of the disease. To study of the role of immunological mediators in the development of allergic bronchopulmonary aspergillosis in patients with asthma.

**Methods:** A prospective study conducted 58 patients with severe asthma (median of age - 45 years, men - 13, women - 45). The control group consisted of 16 apparently healthy people without allergic disease in history (median age - 24 years old, male - 4 women - 12). All patients underwent allergological (skin tests with fungal allergens, determination of serum levels of total IgE ("Polygnostus", Russia) and specific IgE (sIgE) for fungal allergens ("AlcorBio", Russia). Computed tomography of the chest was performed according to the indications. Diagnostic criteria of ABPA 2013 [R. Agarwal et al] were used. The concentration of TARC ("R&D Systems", USA), TSLP ("R&D Systems", USA), IL-8 ("Vector-Best", Russia) in the serum were determined by ELISA. All patients were performed respiratory function and used the AST questionnaire (Asthma Control Test). The obtained data was processed using the STATISTICA 10 software system.

**Results:** ABPA patients demonstrated significantly lower scores on the questionnaire AST and worse lung function FVC and FEV1 compared to patients with asthma. The analysis of the content of TSLP in serum did not establish statistically significant differences between patients with ABPA 13.0 (9.70 ÷ 24.70) and asthma 19.0 (15.0 ÷ 29.0), and data from the control group 10.5 (9.0 ÷ 25.0) pg/ml. It was revealed that the level of IL-8 in patients with ABPA 35.0 (23.0 ÷ 49.0) was significantly higher than in patients with asthma 22.0 (14.4 ÷ 28.0; p = 0.002) and in healthy individuals (12.0 (4.5 ÷ 15.5); p = 0.000) pg/mg. The content of TARC was higher in patients with ABPA 733.5 (599.0 ÷ 909.0) compared with asthma patients 336.1 (208.0 ÷ 571.3; p = 0.000) and control 224.5 (184.0 ÷ 265.0; p = 0.000) pg/mg. The importance of TARC in the development of allergic inflammation in patients with fungal sensitization was confirmed by a positive correlation of the content of the proinflammatory chemokine with the levels of total IgE (r = 0.35, p <0.05), sIgE to *A. fumigatus* (r = 0.39, p <0.05), the number of eosinophils (r = 0.34, p <0.05) and a negative correlation with the indicators of the respiratory function of FEV1 (r = -0.44; p <0.05). The high content of TARC and IL-8 suggests a mixed eosinophilic-neutrophilic type of inflammatory response in patients with ABPA.

**Conclusion:** An increase in serum TARC can serve as a diagnostic biomarker for the development of ABPA in patients with asthma.

P124

Comparison of serum beta-D-glucan levels obtained by Fungitell Assay and Wako beta-Glucan Test

I. Mareković, S. Pleško, V. Rezo Vranješ, M. Jandrlić

Department Of Clinical And Molecular Microbiology, University Hospital Centre Zagreb, Zagreb, Croatia

**Objectives:** Fungitell Assay (FA) is the most widely used test for determining serum beta-D-glucan (BDG) levels in Europe. However, the disadvantage of this test is the need for samples' cummulation
to collect appropriate number of samples assuring optimal test usability. It poses a problem in laboratories with limited number of samples because of prolonged turnaorund-time and questionable impact on the diagnostic process and antifungal treatment of invasive fungal infections (IFI). Wako β-Glucan Test (WBG), recently launched in Europe, offers the possibility of individual sample testing. The objective of this study was to compare serum beta-D-glucan levels obtained by these two assays in patients with suspected IFI.

**Methods:** The study was performed at the University Hospital Centre Zagreb, Department of Clinical and Molecular Microbiology. The Fungitell Assay (Associates of Cape Cod Inc.) and Wako β-Glucan Test (FUJIFILM Wako Chemicals Europe Gmbh) were performed on patients' sera and the results were interpreted according to manufacturers' instructions. Two to three serum samples were tested in each patient on two or three consecutive days. Discrepant results between two tests were explained and commented in the context of patients' clinical data.

**Results:** Forty-four serum samples in 24 patients were tested. FA was positive in 13 and WBG in 9 patients. The results obtained with two different tests regarding positivity and negativity were correspondent in 83,3% (20/24) of patients. In 16,7% (4/24) of patients, the results were discordant, with positive FA and negative WBG result. Results for four patients with discordant FA and WBG results are shown in Table 1. When cut-off value for WBG was lowered from manufacturer's cut-off value of 11 pg/mL to 3,8 pg/mL, as suggested in a recent publication, all the results, except one result in Patient 1, were concordant. Moreover, in Patient 1 diagnosis of IFI was excluded after diagnostic work-up as well as in Patient 2. In Patient 3, with a recent heart transplant, chest HR-CT was done eight days after last serum sample tested with both tests, demonstrated pulmonary infiltrates suspected by radiologist to be of fungal etiology and subsequently determined levels of BDG with FA only showed further increase. *Aspergillus fumigatus* grew in a culture of sputum and patient was treated with voriconazole. Patient 4 had a cystic fibrosis with *A. fumigatus* isolated in sputum and treated with voriconazole. In Patients 1, 2, and 4 the highest level of BDG determined with WBG was 5,1 pg/mL, while in a Patient 3 the highest level was 9.62.
<table><caption>Table 1 - Patients with discordant Fungitell Assay and Wako β-glucan results</caption><thead><tr><th>Patient</th><th>Sample</th><th>FA (pg/mL)</th><th>Interpretation</th><th>WBG (pg/mL)</th><th>Interpretation</th></tr></thead><tbody><tr><td rowspan="2">1</td><td>Sample 1</td><td>116,63</td><td>positive</td><td>4,46</td><td>negative</td></tr><tr><td>Sample 2</td><td>87,74</td><td>positive</td><td>3,3</td><td>negative</td></tr><tr><td rowspan="3">2</td><td>Sample 1</td><td>&lt;7,81</td><td>negative</td><td>&lt;2,359</td><td>negative</td></tr><tr><td>Sample 2</td><td>90,49</td><td>positive</td><td>4,107</td><td>negative</td></tr><tr><td>Sample 3</td><td>10,54</td><td>negative</td><td>&lt;2,359</td><td>negative</td></tr><tr><td rowspan="3">3</td><td>Sample 1</td><td>168,57</td><td>positive</td><td>6,86</td><td>negative</td></tr><tr><td>Sample 2</td><td>205,63</td><td>positive</td><td>9,62</td><td>negative</td></tr><tr><td>Sample 3</td><td>191,93</td><td>positive</td><td>7,67</td><td>negative</td></tr><tr><td rowspan="2">4</td><td>Sample 1</td><td>110,52</td><td>positive</td><td>4,494</td><td>negative</td></tr><tr><td>Sample 2</td><td>175,76</td><td>positive</td><td>3,837</td><td>negative</td></tr></tbody></table>

**Conclusion:** In our study FA was more frequently positive in comparison to WBG test. Among patients with positive FA and negative WBG was a also a patient with IFI. This is in concordance with so far conducted studies showing superior sensitivity of FA. Therefore, cut-off value for WBG should be reconsidered. As the results of any of these tests should be evaluated in the context of clinical picture and microbiological and radiological findings, WBG test remains a useful alternative for laboratories with low number of samples.

<footnote>*FA - Fungitell Assay; WBG - Wako β-Glucan</footnote>
A.L. Tomás¹,², F. Cardoso¹, M.P. De Almeida³, E. Pereira³, R. Franco², O. Matos¹

¹Medical Parasitology Unit, Group Of Opportunistic Protozoa/hiv And Other Protozoa, Global Health And Tropical Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal, ²Departamento De Química, UCIBIO/REQUIMTE, Universidade NOVA de Lisboa, Caparica, Portugal, ³Departamento De Química E Bioquímica, REQUIMTE/LAQV, Faculdade de Ciências da Universidade do Porto, Porto, Portugal

**Objectives:** *Pneumocystis jirovecii* is an ubiquitous opportunistic fungus capable of causing fatal pneumonia (PcP) in immunocompromised patients worldwide. The diagnosis relies on microscopic visualization of the *P. jirovecii* organisms or on DNA detection, in respiratory specimens obtained by invasive and costly techniques. Thus, the search for a specific serological biomarker and for a faster, cost-effective and less-invasive diagnostic approach is an old ambition of the scientific community. This work aims to develop a point-of-care strip-based platform for anti-*P. jirovecii* antibodies detection in human serum specimens, applying the innovative association of recombinant synthetic antigens (RSA) technology with functionalized gold nanoparticles (AuNPs).

**Methods:** *P. jirovecii*'s surface proteins such as the major surface glycoprotein (Msg) and kexin-like serine protease (Kex1) present highly reactive antigenic properties. Thus, newly recombinant synthetic (multi-epitope) antigens from these proteins were designed based on immunogenicity of their sequences. After synthesis and purification by immobilized metal affinity chromatography, these RSA were applied in ELISA tests as biomarkers of the disease to identify circulating anti-*P. jirovecii* antibodies in serum specimens of patients previously classified in distinct clinical groups. After confirming their applicability as biomarkers of infection, these RSA were conjugated with spherical AuNPs previously functionalized with mercaptoundecanoic acid. The interaction of such RSA-AuNP bionanoconjugates with specific anti-*P. jirovecii* antibodies in patient's sera, was characterized by agarose gel electrophoresis (AGE). The same RSA-AuNP bionanoconjugates were then utilized in two strip-based lateral flow immunoassays (LFIA) prototypes, projected to detect the presence of human IgM reactive to each of the *P. jirovecii*'s RSA. These prototypes were optimized and tested with positive and negative clinical samples.

**Results:** A synthetic antigen with three reactive Msg epitopes and another with three Kex1 reactive epitopes were produced. ELISA results with both RSA showed that IgM anti-*P. jirovecii* levels were significantly increased in patients with PcP compared with patients colonized by *P. jirovecii* (*p* = 0.002 with Kex1 RSA; *p*<0.001 with Msg RSA) or patients without *P. jirovecii* infection (*p*≤0.001 with both RSA). AGE assays with the RSA-AuNPs bionanoconjugates confirmed that these bionanoconjugates reacted with anti-*P. jirovecii* antibodies present in patient's serum. The LFIA prototypes developed were tested with sera pools from patients with active PcP (positive sample) and from patients without *P. jirovecii* infection (negative sample). Both samples showed the expected performance, namely, test and control colored lines with the positive sample and only a control colored line with the negative sample.

**Conclusion:** Our results support the possibility to diagnose PcP and distinguish active disease from colonization using RSA as biomarkers of the disease and IgM anti-*P. jirovecii* antibodies as targets for immunonanodiagnosis. Further improvement and validation of the LFIA prototypes developed will help in the global effort to reduce high costs of medical diagnosis and consequently treatment of PcP. This applies not only to more economically advanced regions, but can also be a huge contribution, in terms of public health and economy, in communities with low-income and scarce access to technology.

R. Piarroux¹, J.-C. Dubus²,³, M. Reynaud-Gaubert³,⁴, M. Gouitaa⁵, S. Ranque⁶,⁷, J. Vitte⁸

¹Research & Development, LDBIO Diagnostics, Lyon, France, ²Pneumo-pédiatrie, Crcm Enfants, Aix-Marseille Université AP-HM Hôpital Timone Enfants, Marseille, France, ³Ihu Méditerranée Infections Mephi, Aix-Marseille Université IRD AP-HM, Marseille, France, ⁴Pneumologie Crcm Adultes, Aix-
Marseille Université AP-HM Hôpital Nord, Marseille, France, ⁵Clinique Des Bronches Et Allergies Du Sommeil, Aix-Marseille Université AP-HM, Marseille, France, ⁶IHU-Méditerranée Infection, Marseille, France, ⁷Vitrome, Aix Marseille Univ, IRD, AP-HM, SSA, Marseille, France, ⁸Ihu Méditerranée Infections Mephi, Aix-Marseille Université IRD AP-HM SSA, Marseille, France

**Objectives:** Diagnosis of Allergic Broncho Pulmonary Aspergillosis (ABPA) is complex and based on a multi-criteria definition. One of the major ABPA criteria, the detection and quantification of immunoglobin E (IgE) responses to *Aspergillus fumigatus* (*Af*), is also associated with *Af*-sensitization. In the past few years, a novel approach assaying IgE responses to molecular antigens enhanced the discrimination between *Af* sensitization and ABPA, but without allowing for a clear-cut distinction between these two diseases. We recently reported that a Western Blot detecting *Af*-specific IgE antibodies could be helpful in separating those two entities. This work aimed to strengthen the evaluation of the *Af* IgE WB assay as a differentiating tool between *Af*-sensitization and ABPA.

**Methods:** 221 sera with known sIgE reactivity (21 ABPA, 200 *Af*-sensitized) were assayed with the LDBIO Asp II WB. Sera were collected 2014-2018 and previously assayed with ImmunoCap® to *Af* extract and molecular antigens. We evaluated the ability of WB LDBIO to detect *Af* sensitization and to discriminate between ABPA and *Af*-sensitized patients, relying on ImmunoCap® and clinical chart conclusions as a reference.

**Results:** Samples displayed 0 to 10 bands in the 10-37 kDa range. Among those bands, four (16, 18-20, 22 and 30 kDa) were most frequent and therefore considered as major bands, while the others were considered as minor bands. *Af* IgE WB positivity was defined by the presence of at least 2 major bands. The *Af* IgE WB was positive in 21/21 ABPA and 115/200 *Af*-sensitization sera (61% sensitivity). *Af* IgE WB positivity was strongly correlated to IgE level and was positive in 95% (75/79) of the sera with IgE to *Af* >2kUa/l. Band patterns formed various profiles for discrimination between ABPA and *Af*-sensitization. Of those profiles, one (5 detectable bands, regardless of minor/major classification) had 100% sensitivity (21/21) and 91% specificity (201/221) for ABPA diagnostic. However, by using a more specific profile with 2 major bands and 2 minor bands outside of the 16-30 kDa range, the WB yielded 90% (19/21) sensitivity and 96% (211/221) specificity.

**Conclusion:** This study shows the potential of WB in the work-up of IgE responses to *Af* in asthmatics and cystic fibrosis patients: *Af* IgE WB was able to discriminate between ABPA and *Af*-sensitized patients, with a sensitivity of 90% and a specificity of 96%. In comparison, the ISHAM criteria (tIgE>1000 kUI/l and *Af* IgE>0.35 kUa/l) had 48% sensitivity (10/21) and 98.2% specificity (217/221) in the same population Noteworthy, among the *Af* IgE WB negative patients, 4 were sensitized to another mould (*Alternaria alternata*), thus *Af* IgE WB could avoid unnecessary diagnostic difficulties due to such cross-reactivity. A multicenter evaluation is now needed to confirm those results, but *Af* IgE WB shows a great potential for second-line diagnostic tool when ABPA is suspected.

P127

Rapid Identification of *Candida* spp. from positive blood cultures

V. Özenci, K. Ininbergs, C. Gronfors Seeth, K. Jansson, M. Ullberg

Clinical Microbiology, Karolinska University Hospital, karolinska Institutet, Stockholm, Sweden

**Objectives:** Candidemia is related to high mortality, morbidity. Rapid and reliable identification of *Candida* spp. and antifungal susceptibility testing is essential for early appropriate antifungal treatment. The aim of the present study was to evaluate two rapid methods for identification of Candida spp. from blood cultures (BC) using matrix assisted laser desorption ionization (MALDI-TOF MS). The methods tested were (i) the Sepsityper kit and (ii) short-term culture followed by MALDI-TOF MS. The effect of using Bact/ALERT FA Plus and BACTEC Mycosis-IC/F blood culture bottles were compared for the performance of both of the rapid methods. The performance of short-term culture on faster antifungal susceptibility testing was also analyzed. Conventional identification and antifungal susceptibility testing methods were used as reference.

**Methods:** In total, 52 clinical Candida spp. were inoculated in two types of BC bottles and incubated in respective BC systems. Each bottle was inoculated with 8 mL human blood from a healthy donor which had been spiked with ~ 125 colony forming units (CFU) per mL. When the BC bottles signaled
positive, the samples were analyzed directly from BC bottles by the Sepsityper kit as well as after 6h short-term incubation on sabouraud agar and chrome plates followed by MALDI-TOF MS if growth in 6h was observed.

**Results:** Overall, the Sepsityper kit and short-term culture method performed similarly and could identify Candida spp. in 66/101 (65.3%) and 63/85 (74.1%), respectively (NS). Mycosis-IC/F had higher identification rates than FA Plus BC bottles using the Sepsityper kit 39/51 (77%) and 27/50 (54%), respectively (p<0.05). Similarly Mycosis-IC/F performed better than FA Plus BC bottles using the short-term culture method 38/43 (88%) and 25/42 (60%), respectively (p<0.01). Six hours short term culture followed by Sensititre YeastOne method was tested on ten positive BC bottles. On average the MIC levels for antifungal agents were within one titre difference compared to conventional Sensititre method which was performed after overnight culture plates for both BC bottles (NS).

**Conclusion:** The present data shows that Sepsityper kit and short-term culture method can be used in rapid identification of Candida spp. from blood cultures. Mycosis-IC/F has higher performance in identification of yeast with both methods compared to FA Plus BC bottles. Preliminary data suggest that six hours short term culture followed by Sensititre Yeast One method can be used as a reliable rapid antifungal susceptibility testing method for positive blood cultures.

P128

**Comparison of Bactec Mycosis IC/F and BacT/Alert FA plus bottles in laboratory diagnosis of candidemia**

V. Özenci<fnref n="1" />, N.I.M. Lopez<fnref n="1" />, C. Gronfors Seeth<fnref n="1" />, M. Ullberg<fnref n="1" />, P. Dinnetz<fnref n="2" />

**Objectives:** The aim of the present study was to compare the clinical performance of Bactec Mycosis IC/F and BacT/Alert FA blood culture bottles in detection and time to detection of Candida spp. over eight years period in a tertiary care hospital setting.

**Methods:** During a period of eight years, all blood samples that were concurrently taken from patients with suspected candidemia and cultured paralleled in Bactec Mycosis IC/F, BacT/Alert FA, and BacT/Alert FN blood culture bottles were included in the study. Data collection was retrieved from the laboratory information system and from the respective blood culture system. Bottles that did not signal positive in 10 days in the blood culture system were regarded as negative. Time to growth was counted from the time that the incubation occurred until the time the system gave a positive signal. Results analyzed with use of R, conducting a mixed linear model and Chi-square test.

**Results:** In total, 2,748 positive blood cultures with Candida spp. i.e Bactec Mycosis IC/F, BacT/Alert FA Plus, and BacT/Alert FN Plus bottles from 810 patients were analyzed. Triplicates of blood culture bottles that were taken in 24 h were included. There were 274 blood cultures from 261 patients with all three blood culture bottle types. The BacT/Alert FN bottles had too few positives and excluded from further analysis. The Bactec Mycosis IC/F Bottles could detect Candida spp. in 216 (78.83%) of 274 samples, whereas BacT/Alert FA aerobic bottles were able to detect in 233 (85%) of 274 samples. The Mycosis IC/F and BacT/Alert FA aerobic Bottles did not differ in detection of Candida spp. (NS). There was no species-specificity difference between the two bottles in growth of different Candida spp (NS). There was no difference in time to detection between the two bottles (NS).

**Conclusion:** The present study shows that the performance of Bactec Mycosis IC/F and BacT/Alert FA blood culture bottles in detection of Candida spp. are similar. Further analysis of the discrepant results and the patient characteristics will be performed.

P129

**Direct identification of 81% of candidemia from blood culture bottles by MALDI-TOF MS**

M. Vannini, E. Chandemerle, A. Landreau, L. Simon, C. Pomares, L. **Hasseine**

Laboratoire De Parasitologie Mycologie, CHU DE NICE, NICE, France

<footnote n="1">¹Clinical Microbiology, Karolinska University Hospital, karolinska Institutet, Stockholm, Sweden,</footnote>

<footnote n="2">²School Of Natural Sciences, Technology And Environmental Studies, Södertörn University,
Stockholm, Sweden</footnote>
**Objectives:** Candidemia are associated with high mortality rate (40%). Rapid yeast species identification is crucial to introduce as soon as possible appropriate treatment which is associated with an increase of patients survival rate. Because of the emergence of echinocandin resistance, strategies that allow targeted use of antifungals are essential, notably to reduce the duration of echinocandins exposition. With the conventional method, 24 to 48 hours are required to obtain enough fungal colonies for species identification. In this study, we developed a method to directly identify yeast from blood cultures bottles within one hour. In the context of Laboratory Accreditation we performed method validation according to the ISO 15189 norm.

**Methods:** This work was done in the laboratory of parasitology and mycology at the University Hospital Center of Nice, France. We used 37 positive blood cultures obtained from 25 patients from 4th January 2019 to 28 Mars 2019. 1.3 ml of positive blood cultures was transferred into 1.5 ml microtube and each sample was extracted according to the SDS lysis-based protocol (Jeddi et al 2017). Supernatants were spotted four times on the target plates that were read by the MADLI-TOF mass spectrometer (Bruker®). We validated species identification when a log (score) from Bruker either ≥ 1.6 or ≥ 1.1 three-times repeatable was found, since we did not observe any species misidentification above these thresholds. In case of failure, spectra were analyzed online with the MSI-Users® database (Mass Spectrometry Identification, Normand et al 2017) and identifications were validated when the score was ≥ 17%. We compared our results with the conventional culture and identification methods.

**Results:** 30 blood cultures (81%) were correctly identified with both databases. Only 68 % with Bruker database and 73 % with MSI User database. This method is reproducible and reliable. We tested two mixed infections but they were not totally identified.

**Conclusion:** Our study shows that direct identification of candidemia from blood culture with the SDS protocol allows rapid identification of the causative yeast in 81% of cases. It allows early identification of yeast species and thus appropriate antifungal treatment. This will improve patient's healthcare in the context of candidemia. Mycological culture remains necessary when our protocol doesn't work and in case of mixed infections.

P130

**The Use Of Coffee Agar As An Alternative Medium For The Presumptive Identification Of Cryptococcus neoformans In Resource - Limited Settings.**

P. Chidebelu, E. Nweze

Microbiology, University of Nigeria, Nsukka., Enugu, Nigeria

**Objectives:** Cryptococcus neoformans is presumptively identified by its characteristic brown - black colonies on media containing caffeic acid (Katiyar et al., 2011). In low income regions, neither the conventional identification media nor their basic component(s) is readily affordable and available. We proposed an alternative coffee agar media made of coffee which is rich in caffeic acid, for differential recovery of C.neoformans colonies from mixed cultures of various sample sources.

**Methods:** The coffee agar media which is a modification of caffeic acid agar was composed with coffee as follows: ammonium sulphate (5.0g), glucose (5.0g), yeast extract (2.0g), dipotassium phosphate (0.8g), magnesium sulphate (0.7g), coffee (18.5g), chloramphenicol (0.05g), ferric citrate solution (4.0ml), agar (15.0g). The final pH of the solution in 1 litre of de-ionized water was adjusted to 6.5 ± 0.3. After sterilization at 121°C and 15psi pressure for 15 minutes, the solution was cooled at 45°C, dispensed in Petri dishes and stored at refrigeration temperature until use. Test isolates (n = 12) of Cryptococcus neoformans VNI (ST32) identified by MALDITOF MS and MLST, were streaked on the Coffee agar plates and monitored for pigmentation. Reference strains of Cryptococcus neoformans var. grubii VNI H99 and Candida albicans SC5314 were cultured parallel as the control. A set of the plates was incubated at 30°C, and the other at 37°C for up to 1 week.

**Results:** The colonies of Cryptococcus neoformans (both the test and reference strains) turned brown - black on the coffee agar plates, while Candida albicans SC5314 remained as cream - white colonies. Melanization was more conspicuous at 30°C after 5 days of incubation.

**Conclusion:** The brown - black pigmentation observed for C. neoformans indicated that coffee is rich in caffeic acid and thus, provided the substrate for the phenoloxidase enzyme laccase, which is absent
in C. albicans. Expression of melanin especially at 30°C remains integral for Cryptococcus' environmental survival. After the necessary validation, we suggest that coffee agar can provide an alternative detection media especially in resource-limited settings where absence and cost of conventional identification media pose a barrier to research and diagnostic studies of Cryptococcus neoformans and Candida albicans.

P131

Prospective comparison of colometric and turbidimetric assays for diagnosing invasive fungal infection in a routine laboratory

N. Guigue¹, M. Gits-Muselli¹,², S. Hamane¹, A. Alanio³, S. Bretagne³

¹Parasitology-mycology Laboratory, Lariboisière Saint-Louis Fernand Widal Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), PARIS, France, ²Molecular Mycology, Institut Pasteur, PARIS, France, ³Paris-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

**Objectives:** The detection of serum (1→3)-β-D-glucan (BDG), an antigen common to several fungi, is part of the definition of invasive fungal infections (IFI). The turbidimetric or the colorimetric detection is the basis of two different commercial kits, the Fungitell assay (FA) and the Wako assay (WA), respectively. The FA has been used in Europe since the 2000's, whereas the WA has been only recently CE approved. We wondered how the WA compares to the FA on a routine use.

**Methods:** We prospectively tested 321 serum samples (median: 1[1-11]) from 171 patients (sex ratio M/F = 60%, mean age 48+/-16 yrs) mainly with hematological conditions (64%; 110/171) using both assays from December 21st 2018 to April 23rd 2019. The tests were performed according to the manufacturers' recommendations.

**Results:** Twenty-three patients had IFI (pneumocystosis *n* = 12; invasive aspergillosis *n* = 4; candidemia *n* = 3; invasive fusariosis *n* = 2; Hepato-splenic candidiasis *n* = 1; and cryptococcosis *n* = 1). Both assays provided similar AUC around 0.9 (Figure 1).

<figure><img src="image_36.png" /></figure>

Sensitivity, specificity, positive and negative predictive values were as follows: FA (positivity threshold ≥80 pg/ml): 91%, 89%, 55%, 98%; WA (positivity threshold at 11 pg/ml): 48%, 97%, 67%, 92%; WA (positivity threshold at 4 pg/ml): 83%, 94%, 68%, 97%. When removing the FA negative (<80
pg/ml) and FA overflow values (> 523 pg/ml), the analytical comparison showed a good correlation (R² = 0.81), the FA values being 28 higher in mean than the WA values (Figure 2). Among 73 samples from patients with bacterial sepsis, 21% (15/73) were FA positive, 11% (8/73) were WA positive (positivity threshold at 4 pg/ml), whose 7 positive with both methods. These possible false positive results should be taken with caution knowing that extensive work-up for diagnosing fungal infection was not homogenous between the patients due to the study design and that bacterial and fungal diseases are often concomitant.

<figure><img src="image_37.png" /><figcaption>Figure 2: correlation between Fungitell and Wako beta-D-glucan detection for patients with Invasive fungal infection.</figcaption></figure>

**Conclusion:** The WA performed similarly compared to the Fungitell assay when a lower cutoff (4 instead of 11 pg/ml) is used for the diagnosis of IFI, with a better specificity. The WA is a single sample testing which is clinically relevant when prompt therapeutic decision is on stake.

P132

The development of a real-time PCR assay for the detection of Mucormycosis infections:
Fungiplex Mucorales

J. Green, K. Dempsey

Bruker Microbiology, Glasgow, United Kingdom

**Objectives:** Mucorales have been increasingly reported as causes of invasive fungal infections in immunocompromised subjects, particularly in patients with haematological malignancies, uncontrolled diabetes mellitus or those undergoing dialysis. Mucorales are now also being reported in *Aspergillus*-positive patients who are not responding to first line treatments. Histology and culture are still the most important diagnostic approaches for mucormycosis because of the lack of molecular diagnostic methods available, and β-d-Glucan detection is not useful due to the extremely low content of the biomarker in the Mucorales order. Timely diagnosis of invasive mucormycosis is essential due to the rapid progression of the disease, and because signs and symptoms of the infection are consistent with other invasive fungal infections. PCR would improve detection of Mucorales and complement the current Bruker offering within the area of invasive fungal disease.

**Methods:** The Bruker real-time PCR assays are designed in an easy to use format with minimum hands on time and results generated in around 1 hour after extraction. Universal primer and probe sequences have been designed to target the internal transcribed spacer (ITS) region of the rRNA gene for the genera detailed in Table 1. The probes associated with each pathogen have been labelled with different fluorescent dyes enabling some differentiation within the multiplex reaction. A specific control material has been developed to give the users an option of running it as an extraction control
or a PCR inhibition control.

<table><caption>Table 1: Genera detected by the Fungiplex Mucorales Real-Time PCR Kit</caption><tr><td>Lichtheimia spp.</td><td>Rhizopus spp.</td><td>Mucor spp.</td></tr><tr><td>Cunninghamella spp.</td><td>Rhizomucor spp.</td><td>Apophysomyces spp.</td></tr><tr><td>Saksenaea spp.</td><td>Syncephalastrum spp.</td><td>Actinomucor spp.</td></tr></table>

**Results:** The coverage specified in Table 1 has been confirmed using a range of simulated samples prepared from plasmid and genomic DNA. The limit of detection for species representing the highest prevalence of IFD for each genus has been assessed over six thermocycler platforms to ensure accuracy across a variety of systems.

**Conclusion:** Bruker have developed a Mucorales assay which identifies a wide range of clinically relevant genera. The assay is being further developed to produce a kit to provide rapid detection of the main causative agents in invasive mucormycosis.

P133

The development of a real-time PCR assay for the diagnosis of *Pneumocystis pneumonia: Fungiplex Pneumocystis*

J. Green, T. Donnelly

Bruker Microbiology, Glasgow, United Kingdom

**Objectives:** *Pneumocystis jirovecii* is the causative organism in *Pneumocystis pneumonia* (PCP); a life-threatening pneumonia in immunocompromised patients. Due to a lack of methods to propagate *P. jirovecii* in vitro, the most common current practice in diagnosis of PCP is the microscopic identification of *P. jirovecii* through staining of bronchoalveolar lavage (BAL) samples. This method lacks sensitivity to detect low fungal burdens, therefore PCR methods of detection would improve diagnosis of PCP and complement the current Bruker offering within the area of invasive fungal disease. Two different *Pneumocystis* assays have been designed for initial evaluation against various sample types, with the intention of selecting the best performing assay for further development.

**Methods:** Two *Pneumocystis* real-time PCR assays were initially developed, with specific primer and probe sequences designed to target different genes of *Pneumocystis jirovecii*. Assay 1 targeted the internal transcribed spacer 2 (ITS) sequence between the 5.8S and 28S rRNA genes, with Cy5 as the fluorophore. Assay 2 targeted the mitochondrial large subunit (mtLSU) rRNA gene and used Yakima Yellow as fluorophore. During initial feasibility studies a range of plasmid concentrations were amplified in the presence of an amplification and extraction control using the standardised Fungiplex assay real-time PCR conditions. The real-time PCR assays were also tested against QCMD's *P. jirovecii* pneumonia EQA panels.

**Results:** When assessed against a range of known plasmid concentrations, both assays were found to perform equivalently, with a limit of detection of 20 ipc and a dynamic range of 20 – 2x10⁶ ipc as displayed in Figure 1. When tested against the QCMD 2017 PCP panel, the correct result was obtained in duplicate testing of all extracts using Assay 2, whereas several replicates were undetected using Assay 1. The internal control was detected in all samples, showing that there was no inhibition. Assay 2 was then assessed against the QCMD 2018 PCP panel; again, the correct result was obtained in duplicate testing of all extracts using this assay.

<figure><img src="image_38.png" /><figcaption>Figure 1: Linear amplification plots for *P. jirovecii* plasmid concentrations from 20 to 2x10⁶ ipc</figcaption></figure>
**Conclusion:** The assay designed to target the mtLSU gene, is therefore most suitable and will be developed further. The better performance of this assay is likely due to the presence of multiple copies of the mtLSU gene per organism, compared with just a single copy of ITS2.

P134

**Upgrading the level of diagnosis towards a more targeted treatment. A six year period of laboratory monitoring in a Paediatric Haematoloy-Oncology Unit.**

E. Zarkada¹, A. Tragiannidis², T. Papageorgiou², E. Hatzipantelis², K. Samaras¹, A.-M. Markantonatou¹, E. Zachrou¹, T.-A. Vyzantiadis¹

¹First Department Of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece, ²Haematology-oncology Unit, Second Department Of Paediatrics, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece

**Objectives:** Invasive fungal infections have been proved an important cause of negative prognosis in critically ill patients and notably the immunocompromised, while the paediatric haematology-oncology patients form a high risk group among them. Although several laboratory methodologies for the diagnosis of fungal infections are not completely validated in these patients, the criteria provided by the EORTC/MSG, alongside to the recommendations by ESCMID and ECIL, can be followed in order to upgrade the level of diagnosis. The main aim of this study was to show that the implementation of laboratory testing in this group of patients, served as a useful tool towards a more targeted antifungal approach by managing to upgrade the diagnosis from possible to probable or maintain the clinical suspicion in just a state of readiness.

**Methods:** Data was gathered retrospectively, over a period of six years, from the records of the forty-three patients with haematological malignancies that were hospitalised in the Paediatric-Oncology Unit and had at least one mycology result during their follow-up. All mycology studies were performed in the First Department of Microbiology and included specific cultures, testing for galactomannan, mannan and antibodies against *Candida*, as well as PCR for *Aspergillus* or *Candida*. Also, sensitivity testing or TMD for antifungal drugs, when necessary.

**Results:** Among the above 43 haematology patients the 22 were initially considered to have a possible diagnosis according to the EORTC/MSG criteria due to the presence of at least one host factor and a clinical criterion (mainly positive CT imaging). From the 22 patients, nine proved to have a probable infection by *Aspergillus* with positive galactomannan and (in one case) / or (in one case) a positive PCR for *Aspergillus*. One patient had a proven diagnosis, based on the isolation of *Rhizopus arrhizus* from several cultures of face biopsies and one was considered with a “probable” diagnosis due to the presence of positive high titers of mannan, positive *Candida* PCR in blood and kidney ultrasound imaging of a possible mycotic mass. There were also three “probable” patients with sequential positive mannan testing (with decreasing to negative titers after treatment) that although not included in the criteria, they could not be considered as just possible. Consequently, the diagnosis was considered upgraded, after the mycology findings, in almost 64% (14/22) of patients with possible diagnosis. Among the 22 patients with possible diagnosis, the rest eight, as well as all other 21 patients (from the total of 43), remained with a possible or completely negative diagnosis (respectively) for fungal infection proving the utility of negative and often excluding results.

**Conclusion:** The results of this study could argue in favor of a meticulous and well organized protocol of laboratory monitoring in this group of patients. Taking in consideration that the here-mentioned monitoring, due to several reasons, is not performed with the proposed frequency and combination, the laboratory results are already a useful adjunct towards the documentation or even the exclusion of a fungal infection, helping the clinical decision and guiding the targeted treatment.

P135

**Revision of special microscopic techniques for visualization of aspergilli structural elements**

N. Vasilyeva¹, I. Riabinin¹, Y. Mihaylova², Y. Borzova¹, L. Alieva¹

¹Kaschkin Research Institute Of Medical Mycology. Department Of Medical Microbiology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation,
²Laboratory Of Biosystematics And Cytology, Komarov Research Institute of Botany, Saint-Petersburg, Russian Federation

**Objectives:** Objective of the study is to select the optimal type of special light optics for obtaining microphotographs of *Aspergillus* spp.

**Methods:** Aspergillus clinical strains belonging to 14 species from the Russian Collection of Pathogenic Fungi (Saint-Petersburg, Russia) were used: *A. fumigatus*, *A. flavus*, *A. tamarii*, *A. terreus*, *A. niger*, *A. nidulans*, *A. ustus*, *A. calidoustus*, *A. sydowii*, *A. versicolor*, *A. ochraceus*, *A. candidus*, *A. amstelodami*, *Neosartorya hiratsukae*. Original strains were previously identified by the target DNA-sequencing of ITS and β-tubulin loci according to the CLSI MM18 protocol (2nd edition). Subcultures were obtained on Czapek yeast extract agar (28°C, 10 days). Micropreparations were studied by three microscopic methods: (1) phase contrast (PCM), (2) «dark field» (DFM), (3) water-immersion light microscopy with oblique illumination (WILMOI) using an «aplanatic» condenser (Leningrad Optical-Mechanical Association, Russia). Presto T55 (Rekam, Canada) and PowerShot A2000IS (Canon, USA) cameras were used for taking photo. Digital image processing was performed in XnView 2.35 and GIMP 2.8.6 software.

**Results:** Series of microphoto for three described techniques were received. As it turned out, the phase-contrast microscopy sharply emphasizes the contours of the aspergilli thallic elements and their conidia, but the most successful microscopic pictures are obtained only in areas of location of the structural elements in single layer. With dark-field microscopy in some species (*A. fumigatus*, *A. nidulans*, *Aspergillus spp.* of Versicolores and Usti sections) structural details of the conidial heads are not clearly visible at high magnification. In dark-field microscopy, in comparison with elements of mycelium, vegetative spores and ascospores are better visualized. Dark-field and phase-contrast microscopy to varying degrees distort the natural color of the microscopic elements of the *Aspergillus* spp. cultures. In contrast to the techniques mentioned above, the use of a condenser with a shiftable iris diaphragm and a water-immersion diaphragmatic lens made it possible to take microphoto while preserving the natural pigmentation of aspergilli, sufficient contrast and creating a «relief» of a microscopic picture. In addition, by changing the setting of the aplanatic condenser, it is easy to transform the microscope in dark-field microscopy mode. Examples of taken images are shown in the figure.
<figure><img src="image_39.png" /><figcaption>**Figure.** *Aspergillus flavus* micrographs made by using three special microscopic techniques (abbreviations are placed in text).</figcaption></figure>

**Conclusion:** Water-immersion microscopy with oblique illumination allows to obtain the most natural microphoto of aspergillosis causative agents with the effect of «soft» contrasting. Moreover, the WILMOI mode is much faster adjusted by a microscopist than the DFM and PCM modes. We believe high-quality reproduction of the WILMOI technique is fundamentally available on microscopes of various manufacturers. With the help of phase-contrast and dark-field microscopy, it is possible to make quite spectacular images, but for complex representation of the strains they must be supplemented with the results of traditional light microscopy or WILMOI technique.

P136

**Evaluation of 99mTc-Amphotericin B for nuclear imaging of mould infections in a transwell *in vitro* system**

L. Page<fnref n="1" />, S. Wurster², F. Schadt³, H. Einsele⁴, A.J. Ullmann<fnref n="1" />, S. Samnick³

**Objectives:** Several studies have evaluated molecular imaging using positron emission tomography (PET) and single photon emission computed tomography (SPECT) to diagnose invasive mycoses. The

<footnote n="1">¹Internal Medicine Ii, Division Of Infectious Diseases, University Hospital of Wuerzburg, Wuerzburg, Germany, ²Ut Md Anderson Cancer Center, 1515 Holcombe, Houston, United States of America, ³Nuclear Medicine, University Hospital of Wuerzburg, Wuerzburg, Germany, ⁴Internal Medicine Ii, University Hospital of Wuerzburg, Wuerzburg, Germany</footnote>
most promising tracers described in the literature include <sup>68</sup>Ga-labelled *Aspergillus*-derived siderophores [1] or *A. fumigatus*-specific antibodies labelled with <sup>64</sup>Cu [2]. However, these PET-tracers are cost-intensive and onsite generation may not be feasible at regional medical centers. Therefore, we investigated the potential of Amphotericin B (AMB) labelled by the gamma emitter technetium-99m (<sup>99m</sup>Tc) to visualize pathogenic moulds.

**Methods:** Sodium pertechnetate (derived from a <sup>99</sup>Mo/<sup>99m</sup>Tc generator, 1000 MBq in 0.35 mL saline) was added to a suspension of 50 µg AMB in 100 µL HBSS, followed by 20 µL of a stannous chloride solution (10 mg SnCl<sub>2</sub> in 1 mL 0.1 N HCl). After 20 min incubation at room temperature, the <sup>99m</sup>Tc-AMB solution was neutralized by addition of 3 ml PBS and sterile filtered. To prepare the transwell assay, 1x10<sup>5</sup> human pulmonary artery endothelial cells (HPAEC) were seeded on the lower side of a transwell insert (24-well format, 3 µm pore size) according to previously published protocols [3-4]. The HPAEC layer was infected with 2.5x10<sup>-1</sup>-2.5x10<sup>6</sup> *A. fumigatus* conidia or a bacterial control (*Staphylococcus aureus*). Additional inserts were infected with 2.5x10<sup>5</sup> spores of different Mucorales species. After an incubation period of 15 h to obtain early mycelial biofilms, <sup>99m</sup>Tc-AMB was added to the medium at 50 ng/mL, a concentration that is significantly below commonly achieved therapeutic serum levels [5]. Transwell inserts were incubated for 120 min in 600 µL of the tracer solution (initial activity: 30 kBq per well) and were subsequently washed five times with HBSS. Thereafter, the activity was measured using a gamma counter and normalized to the activity of 600 µL tracer-supplemented medium to calculate percent uptakes. Independently prepared inserts were imaged by autoradiography.

**Results:** Radiochemical yield and purity of <sup>99m</sup>Tc-AMB, as determined by thin layer chromatography, reached >98 % and >99 %, respectively. Minimal unspecific tracer uptake was found in uninfected (0.03-0.14 %) or *S. aureus* infected inserts (0.05-0.16 %). An inoculum-dependent increase in tracer uptake was seen in *A. fumigatus* infected inserts, with a limit of detection at 2.5x10<sup>4</sup> fungal cells (Panel A). Low inter-assay variability was found (mean coefficient of variation = 0.32, n = 3). Quantitative results were validated by autoradiography imaging, confirming inoculum-dependent tracer enrichment in *A. fumigatus*-infected HPAEC monolayers (Panel B). Finally, we tested the applicability of <sup>99m</sup>Tc-AMB to detect Mucorales. Mean 4.4 and 6.2-fold <sup>99m</sup>Tc-AMB enrichment compared with an uninfected control was found in inserts infected with 2.5x10<sup>5</sup> spores of *Rhizopus arrhizus* or *Cunninghamella bertholletiae*, respectively.
<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

**Conclusion:** Our *in vitro* results suggest promising potential of ⁹⁰mTc-AMB as an easily producible, cost-efficient alternative for nuclear imaging of pathogenic moulds by SPECT. These data could encourage further *in vivo* evaluation of modified antifungal drugs as nuclear medical tracers. [1] Petrik et al., 2014, Mol Imaging Biol; [2] Rolle et al., 2016, PNAS; [3] Hope et al., 2007, J Infect Dis; [4] Belic et al., 2019, Front Microbiol; [5] Hamill, 2013, Drugs.

P137

Detection of circulating DNA in serum by PCR for the diagnosis of invasive mucormycosis

J. Savio, J. Mathew

Microbiology, St. John's Medical College, Bangalore, India

**Objectives:** Mucormycosis is an invasive fungal infection with increasing incidence in developing countries like India. Because of its rapid and fatal progression, early diagnosis is essential. Conventional methods like microbiological and histopathological investigations have limitations. In the absence of specific biomarkers for diagnosis, detection of circulating *Mucorales* DNA in serum by PCR proves a better alternative to conventional methods. The objectives of this study were to compare detection of circulating *Mucorales* DNA in serum by PCR with the conventional microbiological and histopathological investigations.

**Methods:** The Institutional Ethics Committee approved this study. 36 patients with a clinical diagnosis of mucormycosis whose samples were received in the microbiology and or histopathology laboratories were included. Three patients with laboratory finding not suggestive of mucromycosis were excluded. Blood samples from all 33 patients were collected. Serum was separated and stored until further analysis. Microbiological investigations included microscopy and culture. Isolates were identified by the conventional techniques. Histopathological examination by microscopy was carried out using H and E, PAS and GMS stains. DNA was extracted from serum by the salting out and TRIZOL methods. Conventional Pan-*Mucorales* PCR was performed on the extracted DNA.

**Results:** Of the 33 patients, 20 were males and 13 were females. Mean age was 52 years. 29 of the 33 patients had diabetes mellitus and 3 were diagnosed following admission. Clinical presentation included rhino orbito cerebral in 25, cutaneous in 4, pulmonary in 3 and renal in one. All 32 samples
received for microbiological investigations were positive by microscopy and 22 (68.8%) were culture positive. Isolates included *Rhizopus* species 17, *Apophysomyces* 2, *Lichtheimia* 1 and one is yet to be identified. 24 of the 25 samples on histopathological examination was suggestive of mucormycosis. 19 of the 33 serum samples were positive for circulating DNA. PCR was positive in 80% of the culture negative patients. PCR was positive in 42.1% of samples with no angioinvasion. PCR was positive in 50% of ROC and cutaneous manifestation and all 3 patients with pulmonary mucormycosis. 61.9% of PCR positive patients had an unfavorable outcome.
<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /><figcaption>Figure: Agarose Gel Imaging – Pan-Mucorales PCR</figcaption></figure>

**Conclusion:** Most patients were above 40 years of age and from urban and rural areas DM was the most common underlying risk factor. ROC was the most common presentation. *Rhizopus* species was the most common isolate, *Apophysomyces* and *Saksenaea* were isolated from cutaneous lesions. 57.6% of the samples were positive for circulating *Mucorales* DNA PCR was positive in 80% of culture
negative samples PCR was positive in 42.1% of samples with no angioinvasion Detection of circulating *Mucorales* DNA could serve as an indicator of angioinvasion and help in early diagnosis of 36.4% of the patients recovered Other factors that could influence the PCR results include: use of different volumes of serum for DNA extraction and commercial extraction kits A larger sample size and serial sampling during the course of illness is required to critically evaluate the utility of this test

P138

Rapid Diagnosis of Invasive Aspergillosis in Bronchoalveolar Lavage: Performance of the Aspergillus Galactomannan Lateral Flow Assay and the Aspergillus-specific Lateral Flow Test in a Mixed Patient cohort

J. Jenks, S. Reed, S. Mehta, N. Law, S. Aslam, R. Taplitz, M. Hoenig

Medicine, University of California San Diego, San Diego, United States of America

**Objectives:** Early diagnosis and treatment of invasive pulmonary aspergillosis (IPA) remains the most important factor to reduce mortality and improve outcome. Diagnosis remains a challenge, however, due to unspecific clinical presentation and radiological findings. Only very recently rapid tests for IPA have been developed. The objective of this study was to evaluate the performance of two new CE-marked tests, the Aspergillus Galactomannan Lateral Flow Assay (LFA; IMMY, Norman, Oklahoma, USA) and the Aspergillus-specific Lateral Flow Device Test (LFD; OLM, Newcastle, UK) for IPA in a mixed cohort of patients with and without hematological malignancies.

**Methods:** The LFA and LFD were retrospectively performed according to the manufacturer's instructions in 106 previously frozen bronchoalveolar lavage fluid samples from 106 patients at risk for IPA (23% with underlying hematological malignancies). Samples were collected between September 2016 and September 2018 at the University of California San Diego, United States. Performance of the LFA and the LFD were compared to Galactomannan, and BAL culture. IPA was classified according to revised EORTC/MSG criteria.

**Results:** Overall, 22 patients met criteria of probable or proven IPA, 9 criteria of possible IPA, while 75 patients did not fulfill EORTC/MSG criteria of IPA. Sensitivity of the Aspergillus Galactomannan LFA for probable/proven IPA was 77% (17/22), and thereby slightly higher than sensitivity for the Aspergillus-specific LFD (59%; 13/22). Sensitivity was similar to BAL GM (77% with a cutoff of 1.0 ODI), but higher compared to BAL culture (23%). The LFA resulted negative in 7/9 cases with possible IPA and 47/73 cases without IPA (overall specificity 66%, 54/82). The less than perfect specificity was driven particularly by non-neutropenic patients (specificity 62%, 43/69), while specificity was 85% among patients with underlying hematological malignancies. Specificity was slightly higher for the LFD (70% specificity; 67% among non-neutropenic patients). Lower specificity among non-neutropenic patients was also observed for the BAL GM (overall specificity 77%, 72% among non-neutropenic patients), and BAL culture (overall 90%, 88% among non-neutropenic patients).

**Conclusion:** Our study indicates that both the LFA and LFD may be useful for rapid diagnosis of IPA in BALF when IPA is clinically suspected. The lower specificity in non-neutropenic patients without underlying hematological malignancies may be explained by limited applicability of the EORTC/MSG criteria in non-neutropenic patients. Better consensus criteria for classifying IPA in those patients are needed.

P139

Use of the basophil surface marker CD203c in the diagnosis of fungal sensitization in patients with cystic fibrosis

Y. Kozlova<fnref n="1" />, E. Frolova², A. Uchevatkina², L. Filippova², Y. Borzova², V. Makhmutova³, T. Stepanenko³, O. Aak², T. Bogomolova², V. Spiridonova², N. Vasilyeva⁴, N. Klimko⁵

<footnote n="1">¹Department Of Clinical Mycology, Allergology And Immunology, 1.North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ²Kashkin Research Institute of Medical Mycology, Saint-Petersburg, Russian Federation, ³Multidisciplinary City Hospital №2, St. Petersburg, Russia, Saint-Petersburg, Russian Federation, ⁴Kaschkin Research Institute Of Medical Mycology. Department Of Medical Microbiology, North-Western State Medical</footnote>
University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ⁵Department Of Clinical Mycology, Allergology And Immunology, North-Western State Medical University n.a. I.I. Mechnikov, Saint-Petersburg, Russian Federation

**Objectives:** Allergic bronchopulmonary aspergillosis (ABPA) is the most common form of aspergillosis in patients with cystic fibrosis (CF). ABPA affects from 2% to 15% of patients with CF and significantly worsens the course of the underlying disease. Diagnosis of ABPA with CF is complicated because many diagnostic criteria intersect with typical manifestations of the underlying disease. Recent studies show that the sensitization to *A. fumigatus* associated with a significant reduction in lung function and an increased incidence of pulmonary exacerbations in CF patients. Thus, early detection of fungal sensitization very important for the diagnosis of ABPA, and appointment of adequate therapy which prevents the progression of lung disease. Now there is a search for new diagnostic tests that will identify fungal sensitization in these patients. To study possibility of basophil activation test use for identification of fungal sensitization in patients with cystic fibrosis.

**Methods:** The study included 190 patients with CF aged 1 to 40 years. All patients underwent allergological (skin tests with fungal allergens, determination of serum levels of total IgE ("Polygnostus", Russia) and specific IgE (sIgE) for fungal allergens ("AlcorBio", Russia)) and mycological (microscopy and culture of respiratory substrates) examination. Computed tomography of the chest was performed according to the indications. Diagnostic criteria of ABPA 2003 [Stevens et al] were used. Study of the activation of basophils by flow cytometry was performed using Allergenicity kit ("Beckman Coulter", USA). Basophils were identified by CD3+CRTH2+ marker, basophil activation - by elevated expression of CD203c. For basophil stimulation were used allergens *A. fumigatus* ("AlcorBio", Russia). The basophil activation test with the *A. fumigatus* allergen was conducted in 12 CF patients with ABPA and 25 CF patients without ABPA in addition to the standard allergological examination. The obtained data was processed using the STATISTICA 10 software system.

**Results:** In patients with cystic fibrosis the frequency of sensitization to *A. fumigatus* was 27%, the incidence of allergic bronchopulmonary aspergillosis - 5.7%. The number of eosinophils, total IgE and specific IgE levels in CF patients with ABPA were significantly higher than in CF patients without ABPA. In blood were identified 77.1 (46.3-87.8) % basophils activated by *A. fumigatus* allergen, the stimulation index was 19.2 (11.3-30.6). In the comparison group stimulation index did not exceed the value of 2.5 (p = 0.000). Direct positive correlation between the level of sIgE to *A. fumigatus* and the number of basophils activated by *A. fumigatus* allergens (r = 0,67; p<0,05) was established. In CF patients with ABPA the FVC and body mass index were significantly lower comparing with patients without fungal sensitization.

**Conclusion:** The basophil activation test applying, along with traditional methods, will allow a more differentiated approach to assessing the development of ABPA in CF patients. Timely identification of the associated with *A. fumigatus* clinical status of CF patients will facilitate early and effective assignment of specific therapy.

P140

**Comparison of In-house Real-time Assay and MucorGenius Assay performance for testing of Clinical Samples from Immunocompromised Patients**

M. Lengerova¹, P. Volfova¹, J. Langer¹, D. Pantuckova¹, J. Pospisilova¹, M. Bezdicek¹, I. Kocmanova², J. Mayer¹, Z. Racil¹

¹Department Of Internal Medicine - Hematology And Oncology, University Hospital Brno, Brno, Czech Republic, ²Department Of Clinical Microbiology, University Hospital Brno, Brno, Czech Republic

**Objectives:** Diagnostics of infections caused by mucormycetes is still quite complicated because no serological tests (unlike in aspergillosis) are available. Therefore, PCR methods are currently the methods of choice. The aim of this study was to compare MucorGenius assay results (Pathonostics) on samples that previously tested positively using our in-house PCRs.
**Methods:** In our laboratory, we use nested real-time PCR assay followed by high-resolution melting analysis (RT-HRM) for PCR screening of clinical samples from patients at risk of invasive mucormycosis. This assay enables not only detection but also identification of detected species. If the result is positive, we confirm the result and determine the fungal load with species-specific real-time quantitative PCR (RQ-PCR). In this study, we tested 29 selected clinical samples (10 tissues, 15 bronchoalveolar lavage (BAL)samples, 3 cerebrospinal fluid samples and 1 endosecret), 25 that had been tested as positive using our screening assay and 4 mucormycetes negative samples. The results were compared to tests with the MucorGenius assay.

**Results:** Out of 25 RT-HRM positive samples, 21 were positive using species-specific RQ-PCR. A significant fungal load (Ct £ 35) was detected in 10 samples. Using MucorGenius assay, 17 out of 25 samples (68%) were positive (Ct 18.1 – 37.3). RT-HRM detected *Rhizopus* sp. (17 samples), *Mucor* sp. (3 samples), *Lichtheimia corymbifera* (2 samples), *Lichtheimia ramosa* (2 samples) and *Rhizomucor* sp. (1 sample). 12 out of 17 samples with *Rhizopus* sp. detected revealed low fungal load or were negative. To analyze this discrepancy, sequencing was performed and in 10 samples and *R. stolonifer* was identified (which is not targeted by in-house RQ-PCR). The MucorGenius assay was positive in 7/10 *R. stolonifer* positive samples (Ct 31.0-37.3).

**Conclusion:** Using commercial assays for PCR diagnostics is currently preferred because of standardization; therefore we compared our in-house assay results with a commercial kit for mucormycetes detection. In this study, we also analyzed samples with discrepant results and identified *R. stolonifer* as a frequent finding when testing BAL samples. As this species is rarely described as a cause of infection, its detection most probably represents colonization or contamination. Based on these results, we added *R. stolonifer* specific RQ-PCR to our diagnostic algorithm (if RT-HRM detects *Rhizopus* sp. than *R. microsporus*, *R. oryzae* and *R. stolonifer* RQ-PCRs are used to verify the results). Due to our experience, the MucorGenius kit is easy to use, as it is a one tube real-time PCR assay and has very good sensitivity. The main disadvantage is the inability to identify mucormycetes species. Although this information is not critical to initiating treatment, it is essential for epidemiological reasons and might help to distinguish colonization/contamination from a truly ongoing infection. This study was supported by the Ministry of Health, Czech Republic - conceptual development of research organization (FNBr, 65269705) and TE02000058.

P141

**Pythium insidiosum - sporulation with leaves of grass: Two case reports**

J. Mathew, J. Savio, J. Mathew, M.S. Reddy

Microbiology, St. John's Medical College, Bangalore, India

**Case Report:** *Pythium insidiosum* is a filamentous, aquatic parasite which was till recently considered to be a fungus. It is known to cause sight-threatening keratitis. The filaments of *Pythium* resemble pauci-septate hyphae of *Mucorales*. Case history: Both patients presented with redness, irritation and reduced vision of right eye since one month. There was no history of trauma or any co-morbid conditions. Patient 1 (27 years old gentleman): Ocular examination revealed corneal ulcer with feathery margins, stromal infiltrates and endothelial plaque. Hypopyon and corneal congestion were noted. Fungal keratitis was the clinical diagnosis. KOH microscopy of corneal scraping revealed broad pauci-septate fungal hyphae. The patient was started on oral fluconazole and topical voriconazole and amphotericin-B along with antibiotics. With deterioration in the clinical course, therapeutic penetrating keratoplasty was performed and the patient was discharged on topical Natamycin. Progressive loss of vision in the eye was managed with iris repositioning and tightening of corneal suturing. The patient developed a white corneal opacity and was later lost to follow-up. Patient 2 (26 years old lady): Ocular examination revealed a corneal epithelial defect, satellite lesions, corneal scarring with stromal infiltration and no hypopyon. The impression was healing fungal ulcer/viral keratitis. Initially, she was started on topical acyclovir. On her follow-up OPD visit she still complained of pain in the right eye. KOH microscopy of corneal scraping revealed broad, pauci-septate fungal hyphae. The patient was started on oral ketoconazole and topical voriconazole. Topical linezolid, doxycycline and azithromycin were also added. On follow-up visit, the patient was
asymptomatic and ocular examination revealed healed lesions. No surgical management was undertaken. Both samples of corneal scraping on fungal culture yielded a non-sporulating mould. Colonies were flat, feathery, glabrous and light-brown coloured. Attempts were made to induce sporulation using the conventional techniques such as water agar, water culture and using non-nutrient media such as corn meal agar. With colony morphology resembling *Pythium*, literature search was made for specific sporulation techniques. The induction medium required chemicals such as K₂H₂PO₄, MgCl₂. Due to the non-availability of these in our laboratory, we set out to use the grass on our campus (the common Bermuda grass, *Cynodon dactylon*) with yeast extract and sterile distilled water with the hope to induce sporulation. To our surprise, the isolates produced sporangia and motile zoospores, which were visualized under light microscope. The isolates were sent to PGIMER, Chandigarh for confirmation by gene sequencing and the identification was confirmed as *Pythium insidiosum*. Video link-zoospore: https://drive.google.com/file/d/1cxHGWR-EwHgx0ucuTQZGy81nwfVagFEj/view Conclusion: *Pythium insidiosum* keratitis may present as chronic corneal ulcers. The simple Grass leaf culture as a technique for induction of sporulation of *Pythium insidiosum* can be performed in resource-limited settings. Experimentation can be attempted using leaves from different species of grass. Increased awareness and high index of suspicion on direct microscopy (pauci-septate, ribbon-like hyphae) and colony morphology (flat, feathery colonies) is integral to prevent misdiagnosis as fungal infection. Studies prove that prognosis of such infections is better when treated by a combination of antibiotics and antifungals than antifungals alone, which may occur when misdiagnosed as a fungal infection.

P142

An Evaluation to assess the performance of the Fungiplex Aspergillus and the Fungiplex Aspergillus Azole-R IVD Real-Time PCR Kits following the implementation of an extraction control

J. Green

Bruker Microbiology, Glasgow, United Kingdom

**Objectives:** This Performance Evaluation was designed to test contrived samples spiked into serum, plasma and BAL material to assess the impact of a new extraction control on the limit of detection of the Fungiplex Aspergillus and Fungiplex Aspergillus Azole-R IVD Real-Time PCR Kits and to ensure sensitivity was unaffected by this change. The results from this study have been evaluated to determine whether the inclusion of an extraction control has an impact on the limit of detection defined in previous studies.

**Methods:** Simulated samples were prepared and analysed by the clinical evaluators. Samples prepared in serum, plasma and BAL were spiked with specific concentrations of *Aspergillus fumigatus* or *Aspergillus terreus* genomic DNA prior to extraction. *Aspergillus fumigatus* wild type strains were used as well as resistant strains containing DNA mutations TR34 and TR46 in the *Cyp51A* gene. Samples were prepared in replicates of five and negative controls were included for each sample type. Prior to extraction 10 µL of the new extraction control formulation was added to each sample. Extraction was performed using two commercial extraction platforms; the eMAG® by bioMérieux and the BioRobot® EZ1™ by Qiagen. The PCR was prepared in duplicate for each sample and run on the Qiagen Rotor-Gene Q 5plex HRM platform.

**Results:** When tested with the Fungiplex Aspergillus IVD Real-Time PCR Kit, the limit of reproducibility for *Aspergillus fumigatus* and *Aspergillus terreus* samples was 5 ge (genome equivalents). No difference in sensitivity was observed when the results from two different extraction platforms were compared, however, on average the resultant Ct (cycle threshold) values were 1.2 cycles higher when the EZ1™ platform was used. When tested with the Fungiplex Aspergillus Azole-R IVD Real-Time PCR Kit, the limit of reproducibility for *Aspergillus fumigatus* samples containing mutations TR34 and TR46 was 50 ge. This higher limit of reproducibility was expected as the *Cyp51A* gene is single copy. The limit of reproducibility was consistent between all sample types, where in a previous study, without an extraction control, the limit of reproducibility had been reported between
50 and 75 ge depending on which sample type was used. The extraction control was detected in every sample analysed with no significant differences observed in Ct value reported.

**Conclusion:** 100% sensitivity was reported for the Fungiplex Aspergillus Kit when analysing *Aspergillus* samples at 5 ge and for the Fungiplex Aspergillus Azole-R Kit when analysing resistant strains at 50 ge. The sensitivity is not dependent on sample type and the new extraction control implemented in these products did not affect the performance of either Kit. Data has shown that results from this study are equivalent, and in some cases, superior to previous studies performed.

P143

Evaluation of the clinical usefulness of dermatophyte EQA panels using molecular tests

G. Gaajetaan, T. Kampermann, D. Van Tegelen, G. Dingemans
PathoNostics, Maastricht, Netherlands

**Objectives:** Determine the relevance of external quality assessment (EQA) panels for detection of dermatophytes by real-time PCR. For this purpose, the commercially available DermaGenius® 2.0 Complete real-time multiplex PCR was used and simultaneously its performance was assessed.

**Methods:** Samples from the Dermatophytosis EQA pilot study (years 2016-2018) from QCMD (Quality Control for Molecular Diagnostics, Glasgow, UK) and the dermatophytes EQAS program (years 2017-2018) from INSTAND e.V. (Düsseldorf, Germany) were all tested with the DermaGenius® 2.0 Complete real-time multiplex PCR after suitable DNA-extraction. The samples from QCMD were processed with a NucliSENS® easyMag® DNA extraction system (bioMérieux) and the samples from the EQA from INSTAND e.V. were processed with the PathoNostics Extraction kit.

**Results:** The Dermatophytosis EQA pilot study panel from QCMD is provided once a year and contains 8 samples. All 8 samples in each year were correctly identified with the DermaGenius® 2.0 Complete real-time PCR. The composition of the EQA panel was comparable every year and contains 1 negative, three samples with different *T. rubrum* concentrations (Ct 26-35) and three samples with different *T. interdigitale* concentrations (Ct 27-38). Remarkably, each year the panel contained one sample which was positive for *C. albicans* (Ct 34-36), which is not a dermatophyte (and not in the scope of the panel) but can cause a nail infection. The dermatophytes EQAS panel from INSTAND e.V. is provided twice a year and contains 4 samples. Most samples were correctly identified with the DermaGenius® 2.0 PCR, only *Nannizzia gypsea* was not detected because this dermatopyte target is not included in the PCR assay. Other dermatophytes included in the four panels were *T. rubrum* (4x), *T. tonsurans* (2x), *T. interdigitale* (1x), *M. canis* (1x), *T. violaceum* (1x), *T. benhamiae* (1x) and *M. audouinii* (1x). Almost all dermatophytes were detected at very low Ct-values (Ct 19-26) representing a high DNA load. A negative control was included in each panel and was also identified as negative by the PCR.

**Conclusion:** Both dermatophyte EQA panels are intended for performance evaluation of molecular detection methods. The DermaGenius® 2.0 Complete real-time multiplex PCR showed good performance on both EQA panels, but the panels from these organisations have a different scope. The EQA panel from QCMD contains variable DNA concentrations of common dermatophytes and thereby focuses on sensitivity and clinical prevalence of dermatophyte species. The EQA panel from INSTAND e.V. panel contains a very high load of common but also more rare dermatophytes and thereby focussing more on specificity rather than sensitivity and prevalence/clinical significance. More importantly, the sample type is very difficult to mimic and therefore quality control samples do not really reflect the actual situation. Nevertheless, the EQA panel from INSTAND e.V. supplies a solid sample (lyophilized) and can therefore be processed in a more realistic way in comparison with the liquid samples from the EQA samples from QCMD. Overall, it is important to select a quality assessment program which serves your purpose.

P144

Diagnosis of fungal peritonitis: optimization by evaluating two culture procedures
S. Martínez¹, E. Vallina¹, I. Costales¹, L. Milian², E. Llamas¹, M. Sandoval¹, J. Fernández¹, M.S. Zapico¹, T. Peláez¹

¹Servicio De Microbiología Y Parasitología, HUCA, Oviedo, Spain, ²Servicio De Microbiología Y Parasitología, CAUSA, Salamanca, Spain

**Objectives:** Peritonitis is one of the most serious intra-abdominal infections. Among them, the most frequent is secondary peritonitis, usually caused by intestinal perforation and with polymicrobial etiology. Fungal involvement in peritonitis is especially relevant, as fungal peritonitis is associated to a high mortality rate. Regular culture-based diagnosis of fungal peritonitis is difficult, due to bacterial growth may mask the presence of yeasts with slower growth. The use of selective culture media for these microorganisms means an improvement of the diagnosis. The aim of this study is to evaluate the improvement of the microbiological diagnosis of fungal peritonitis, comparing two culture procedures.

**Methods:** Between 2016 and 2019, a total of 1671 peritoneal fluids were retrospectively studied. Two time periods were established to evaluate two culture procedures: Period 1 (January 2016 to May 2018) and Period 2 (June 2018 to April 2019). During Period 1, if sample volume was enough, peritoneal fluids were inoculated in blood-culture bottles (BD BACTEC Plus Aerobic/F culture vials) and incubated in an automatic incubator (BACTEC FX Instruments, Becton-Dickinson). Otherwise, they were inoculated in growth media agar plates (blood agar, chocolate agar, McConkey agar and Brucella agar). During Period 2, all peritoneal fluids were also inoculated at the same time in a fungus selective culture medium (Gentamycin + Chloramphenicol Sabouraud Agar, Biomèrieux).

**Results:** A total of 1671 peritoneal fluids were evaluated, 1254 during the first period and 417 during the second period. Of them 147 (8.8%) were positive for fungal growth. The positivity rates of fungal growth during the first and the second period were, respectively: 6.7% vs 15.1%. The new culture protocol (Period 2) increased significantly the number of yeast isolates in peritoneal fluids (p = 0.0001), rising from 7% in the first period to 19.5% in the second (Table1). Comparing data by yeast species, a significant rise in incidence of *C. albicans* (4.5% vs 10.5%) and *C. parapsilosis* (0.48% vs 3.4%) was observed (Table 2).

<table><caption>Table 1:</caption><thead><tr><th></th><th>PERIOD 1</th><th>PERIOD 2</th><th>TOTAL</th></tr></thead><tbody><tr><th>Peritoneal fluid</th><td>1254</td><td>417</td><td>1671</td></tr><tr><th>Positive culture</th><td>84</td><td>63</td><td>147</td></tr><tr><th>% positive</th><td>6.7</td><td>15.1</td><td>8.8</td></tr></tbody></table>

<table><caption>Table 2:</caption><thead><tr><th></th><th>Period 1<br>%POSITIVE</th><th>Period 2<br>%POSITIVE</th></tr></thead><tbody><tr><th>C. albicans</th><td>4.55</td><td>10.49</td></tr><tr><th>C. parapsilosis</th><td>0.48</td><td>3.40</td></tr><tr><th>C. glabrata</th><td>0.72</td><td>1.85</td></tr><tr><th>C. tropicalis</th><td>0.32</td><td>0.93</td></tr><tr><th>C. krusei</th><td>0.40</td><td>1.54</td></tr><tr><th>C. dubliniensis</th><td>0.32</td><td>0.62</td></tr><tr><th>Saccharomyces cerevisiae</th><td>0.16</td><td>0.62</td></tr></tbody></table>

**Conclusion:** Our data show that the incorporation of selective culture medium for fungi improves significantly the microbiological diagnosis of fungal peritonitis, increasing the positivity rates and the recovery of fungal isolates. *C. albicans* was the most frequent species isolated, followed by *C. glabrata* and *C. parapsilosis*. The incidence of *C. albicans* and *C. parapsilosis* increased significantly during the second period of the study.
P145

Direct detection of *Candida auris* and its ERG11-associated azole resistance and FKS1-associated echinocandin resistance in clinical skin swabs

M. Kordalewska, A. Lee, Y. Zhao, D.S. Perlin

Center For Discovery And Innovation, Hackensack Meridian Health, Nutley, United States of America

**Objectives:** *Candida auris* is a multidrug resistant yeast, recognized as a cause of invasive infections and health care associated outbreaks around the world. Major obstacles impacting control of *C. auris* spread include common misidentification by diagnostic platforms available in clinical and public health laboratories; a poor understanding of resistance to antifungal drugs and disinfectants; and a high propensity to contaminate health care environments which results in transmission of clonal *C. auris* isolates. Ultimately, correct detection and identification of the pathogen and its antifungal susceptibility, followed by strict adherence to appropriate treatment and infection prevention and control strategies is crucial for limiting the spread of *C. auris*. Here, we have developed and validated platforms for direct detection of *C. auris* and its *ERG11*-associated azole resistance and *FKS1*-associated echinocandin resistance in clinical skin swabs.

**Methods:** DNA isolated from 112 de-identified clinical axilla/groin skin swab samples was tested in the recently developed *C. auris*-specific SYBR Green qPCR assay (Kordalewska et al. JCM 2017) with the addition of inhibition control. Enrichment culture (Welsh et al. JCM 2017) followed by sequencing of rDNA was used as a reference method. 24 samples, determined as *C. auris*-positive by both qPCR and reference method, were further screened in the *ERG11*-associated azole resistance and *FKS*-associated echinocandin resistance assays (Hou et al. AAC 2018). Antifungal susceptibility testing with echinocandins (anidulafungin, micafungin) and azoles (fluconazole, voriconazole) according to CLSI guidelines and sequencing of *ERG11*, and *FKS1* were used as reference methods.

**Results:** In the *C. auris*-specific SYBR Green qPCR assay 45% of swabs were *C. auris*-negative (50/112), and 55% were *C. auris*-positive (62/112). However, only 21% (24/112) of swabs were culture-positive. The finding that *C. auris* cells lost their viability in 34% of swabs, but the DNA was still present (giving a positive real-time PCR result), was possibly due to the long swabs storage (4-5 months) before processing. Detection of *C. auris* was not affected by the presence of other microorganisms present in the sample. Thus, the assay was found to produce results comparable to the enrichment culture approach currently implemented at CDC and Antibiotic Resistance Laboratory Network (ARLN) laboratories. The resistance detection assays identified all *C. auris*-positive swabs as *FKS1* wild-type, and *ERG11* wild-type. The molecular diagnostic results from the assays were 100% concordant with DNA sequencing results. Moreover, all isolates exhibited low minimal inhibitory concentration (MIC) values (MIC ranges - anidulafungin: 0.06-0.125 mg/l; micafungin: 0.06-0.125 mg/l; fluconazole: 1-2 mg/l; voriconazole: ≤0.03 mg/l) and were categorized as echinocandin- and azole-susceptible.

**Conclusion:** Overall, we found the *C. auris*-specific SYBR Green qPCR assay can be successfully applied for rapid and accurate detection of *C. auris* in patient skin swabs, thereby increasing high-throughput diagnostic options for this emerging pathogen. Moreover, the *ERG11*-associated azole resistance and *FKS1*-associated echinocandin resistance detection platform has the potential to overcome the deficiencies of existing *in vitro* susceptibility-based assays to identify azole and/or echinocandin resistant *C. auris*, and thus, it holds promise as a surrogate diagnostic method to direct antifungal therapy more effectively.

P146

Role of PCR, mannan antigen and 1,3-beta-d-glucan measurements in cerebrospinal fluid for the diagnosis and follow-up of *Candida* meningitis

E. Kampouri<fnref n="1" />, V. Scherz², P. Marquis³, J.-L. Pagani³, G. Greub<fnref n="1" />,², F. Lamoth<fnref n="1" />,²

<footnote n="1">¹Infectious Diseases Service, University Hospital of Lausanne, Lausanne, Switzerland, ²Microbiology Institute, University Hospital of Lausanne, Lausanne, Switzerland, ³Intensive Care Department, University Hospital of Lausanne, Lausanne, Switzerland</footnote>
**Objectives:** Infections of the central nervous system due to *Candida* spp. are rare and usually associated with intracranial devices. Diagnosis is challenging because of low sensitivity of cultures and lack of specificity of clinical and biological signs of meningitis. Optimal treatment is not well established and response to therapy is difficult to assess. We present two cases of *Candida albicans* meningitis occurring in the setting of craniofacial surgery without intracranial device for which different fungal markers (1,3-beta-d-glucan, mannan, PCR) in the cerebrospinal fluid (CSF) were used for diagnosis and follow-up.

**Methods:** Platelia™ Candida Ag plus (Bio-Rad), Fungitell™ (Associates of Cape Cod) and a home-made quantitative C. albicans-specific PCR (targeting 26S rDNA) were used for detection of mannan antigen (Mn-Ag), 1,3-beta-d-glucan (BDG) and fungal DNA, respectively, in serum and cerebrospinal fluid (CSF).

**Results:** *Candida* meningitis presented as a complication of neurosurgery, following transsphenoidal surgery and skull base reconstruction with CSF leakage respectively. Biological analyses of CSF were consistent with bacterial meningitis, but with negative culture and broad-spectrum 16S rDNA PCR for bacteria. C. albicans was detected by CSF culture in one case and PCR in CSF in both cases. Antifungal therapy consisted in fluconazole and liposomal amphotericin B. PCR fungal loads subsequently declined along with clinical and biological improvement. BDG levels in CSF were very high at diagnosis while negative in serum and remained elevated during follow-up in both cases. Mn-Ag was measured in one case with elevated value in CSF (>500 pg/ml vs 20 pg/ml in serum) at diagnosis and significant decline in follow-up.

<table><thead><tr><th>CSF</th><th>Diagnosis</th><th>Improvement</th></tr></thead><tbody><tr><td>1st case</td><td></td><td>Day-14</td></tr><tr><td>Culture</td><td>Sterile</td><td>Sterile</td></tr><tr><td>PCR C. albicans (cp/ml)</td><td>365</td><td>210</td></tr><tr><td>1,3-β-D-glucan (pg/ml)</td><td>>500</td><td>485</td></tr><tr><td>Mn-Ag (pg/ml)</td><td>>500</td><td>62.5</td></tr><tr><td>2nd case</td><td></td><td>Day-6</td></tr><tr><td>Culture</td><td>C. albicans</td><td>Sterile</td></tr><tr><td>PCR C. albicans (cp/ml)</td><td>2940</td><td>Negative</td></tr><tr><td>1,3-β-D-glucan (pg/ml)</td><td>>500</td><td>>500</td></tr></tbody></table>

**Conclusion:** These two cases illustrate the potential value of PCR, Mn-Ag and BDG measurements in CSF for the diagnosis of *Candida* meningitis. Further analyses are required to define a threshold of positivity for BDG and Mn-Ag in CSF or the diagnostic value of CSF/serum indexes. Moreover, PCR and Mn-Ag may be useful for monitoring of response to therapy.

P147

Usefulness of Galactomannan antigen test in Serum or Bronchoalveolar lavage sample in Kidney transplant patients

A. Xess<fnref n="1" />, I. Xess², G. Singh²

**Objectives:** Evaluation of Galactomannan in BAL and Serum samples in Kidney Transplant Patients
**Methods:** A total of 127 kidney transplant patients (KTP) suspected of invasive pulmonary aspergillosis (IPA) were enrolled for the study. Patients were further investigated radiologically and microbiologically. Galactomannan was done in serum and BAL samples Receiver Operating Curve (ROC) was generated to determine the optimal cut off value for Galactomannan in BAL and serum samples.

<footnote n="1">¹Microbiology, all india institute of medical sciences, new delhi, India, ²Microbiology, All India Institute Of Medical Sciences, new delhi, India</footnote>
**Results:** Out of 127 patients 32(25.2%) were females and 95(74.8%) were males. 76/127(59.8%) patients presented with fever not responding to antibiotics and 62/127(48.8%) presented with cough/respiratory distress. 54/127 (42.5%) presented with both fever and cough. In CT scan, 24/127(18.9%) patients showed consolidation, 10/127(7.9%) showed ground glass appearance, 5/127(3.9%) showed nodules and 3/127(2.4%) patients had only opacity. Galactomannan antigen was tested in 138 serum and 47 BAL samples of 127 patients. Forty-four serum samples and 34 BAL samples were positive for galactomannan antigen. Three patients were positive for fungal elements on KOH-calcofluor stain and two were only positive for culture. During the course of hospitalization 12/127(9.4%) received Amphotericin-B, 4/127(3.1%) voriconazole, 3/127(2.4%) caspofungin, 2/127(1.6%) posaconazole and 1/127(0.8%) itraconazole. Mortality was around 22% (28/128). Receiver operating characteristic (ROC) curve was plotted for BAL and Serum galactomannan levels. The value for BAL was found to be 1.0 and for serum it was 0.7.

**Conclusion:** Corrected BAL and serum galactomannan values for diagnosis of invasive fungal aspergillosis in kidney transplant patients were found to be 1.0 and 0.7 respectively, can be useful for diagnosis where the sensitivity of direct demonstration and culture is very low.

P148

**Potential impact of anti-mould prophylaxis on Aspergillus PCR blood testing for the diagnosis of invasive aspergillosis: a Fungal PCR initiative (FPCRI) systematic review and meta-analysis.**

M. Cruciani¹, P. White², J. Löffler³, C.O. Morton⁴, R. Barnes⁵, J.P. Donnelly⁶, L. Klingspor⁷, D. Buchheidt⁸, J. Maertens⁹, W.J. Heinz¹⁰, T. R Rogers¹¹, A. Warris¹², C. Mengoli¹³, D. Lockhart¹², C. Cordonnier¹⁴

¹Infectious Diseases, San Bonifacio Hospital, Verona, VERONA, Italy, ²Microbiology, Public Health Wales Microbiology, Cardiff, United Kingdom, ³Medizinische Klinik Und Poliklinik Ii, Universitätsklinikum Würzburg, Würzburg, Germany, ⁴western Sidney University, Sidney, Australia, ⁵Medical Microbiology And Infectious Diseases, Cardiff University School of Medicine, Cardiff, United Kingdom, ⁶EAPCRI, Nijmegen, Netherlands, ⁷Laboratory Medicine, Karolinska Insitutet, Stockholm, Sweden, ⁸Mannheim University Hospital, Heidelberg University, Mannheim, Germany, ⁹KU Leuven, Leuven, Belgium, ¹⁰University hospital, Wuerzburg, Germany, ¹¹Department Of Clinical Microbiology, St. James's Hospital, Dublin, Ireland, ¹²Mrc Centre For Medical Mycology, University of Aberdeen, Aberdeen, United Kingdom, ¹³University of Padua, Padua, Italy, ¹⁴University of Paris, Paris, France

**Objectives:** To analyse the impact of anti-mould prophylaxis on the diagnostic accuracy of *Aspergillus* PCR for the diagnosis of Invasive Aspergillosis (IA) in immunocompromised patients.

**Methods:** Systematic review and meta-analysis of prospective cohort studies evaluating *Aspergillus* PCR testing of blood from high-risk neutropenic cancer patients, haematopoietic stem cell transplant (HSCT) and solid organ transplant recipients. QUADAS-2 was used to assess methodological quality of included studies. The sensitivity and specificity of *Aspergillus* PCR was evaluated by meta-analytical pooling of logit sensitivity and logit specificity. We also calculated the diagnostic Odd Ratio (DOR). A subgroup analysis of studies using anti-mould prophylaxis compared to studies not using anti-mould prophylaxis (or using placebo or fluconazole) was performed

**Results:** Nine studies used anti-mould prophylaxis (itraconazole, voriconazole, amphotericin B or caspofungin) in the entire population or in a subset of patients, while 13 studies did not use any anti-mould prophylaxis. The incidence of IA was significantly lower in the prophylaxis group compared to the non prophylaxis group (11.43 vs 16.13; p = 0.0005). The use of anti-mould prophylaxis increased sensitivity (from 0.80 to 0.86; p = n.s) and decreased specificity (from 0.78 to 0.64; p = 0.059) and DOR (from 17.10 to 11.14; p = n.s.). In sensitivity analysis, removing 3 studies using prophylaxis with itraconazole has no significant impact on sensitivity, Sensitivity and specificity of PCR test in studies using or not anti-mould prophylaxis. TP, TN = true positive and true negative results of PCR test, compared to total cases of IA according to the reference standard (EORTC/MSG criteria) or no-IA. specificity and DOR.
Prophylaxis and Specificity

<table><thead><tr><th>Study</th><th>TN</th><th>no</th><th>IA</th><th>Spec [95% CI]</th></tr></thead><tbody><tr><td colspan="5"><strong>prophylaxis = 0</strong></td></tr><tr><td>Badiee 2010</td><td>55</td><td>67</td><td>0.82</td><td>[0.71; 0.90]</td></tr><tr><td>Badiee 2017</td><td>86</td><td>86</td><td>1.00</td><td>[0.96; 1.00]</td></tr><tr><td>Boch 2016</td><td>22</td><td>30</td><td>0.73</td><td>[0.54; 0.88]</td></tr><tr><td>Boluk 2016</td><td>22</td><td>30</td><td>0.73</td><td>[0.54; 0.88]</td></tr><tr><td>da Silva 2010</td><td>159</td><td>173</td><td>0.92</td><td>[0.87; 0.96]</td></tr><tr><td>El Mahallawy 2006</td><td>52</td><td>63</td><td>0.83</td><td>[0.71; 0.91]</td></tr><tr><td>Halliday 2006</td><td>31</td><td>82</td><td>0.38</td><td>[0.27; 0.49]</td></tr><tr><td>Hebart 2000</td><td>66</td><td>74</td><td>0.89</td><td>[0.80; 0.95]</td></tr><tr><td>Landlinger 2010</td><td>56</td><td>121</td><td>0.46</td><td>[0.37; 0.56]</td></tr><tr><td>Rogers 2013</td><td>86</td><td>102</td><td>0.84</td><td>[0.76; 0.91]</td></tr><tr><td>Schwarzinger 2013</td><td>79</td><td>174</td><td>0.45</td><td>[0.38; 0.53]</td></tr><tr><td>Springer 2016</td><td>77</td><td>151</td><td>0.51</td><td>[0.43; 0.59]</td></tr><tr><td>von Lilienfeld-Toal 2009</td><td>113</td><td>116</td><td>0.97</td><td>[0.93; 0.99]</td></tr><tr><td><strong>Total (95% CI)</strong></td><td></td><td></td><td><strong>1269</strong></td><td><strong>[0.78; 0.65; 0.87]</strong></td></tr></tbody></table>

Heterogeneity: Tau² = 1.1437; Chi² = 208.07, df = 12 (P < 0.01); I² = 94%

Specificity

<figure><img src="image_44.png" /></figure>

<table><thead><tr><th>Study</th><th>TN</th><th>no</th><th>IA</th><th>Spec [95% CI]</th></tr></thead><tbody><tr><td colspan="5"><strong>prophylaxis = 1</strong></td></tr><tr><td>Barnes 2013</td><td>276</td><td>496</td><td>0.56</td><td>[0.51; 0.60]</td></tr><tr><td>Cuenca-Estrella 2009</td><td>57</td><td>73</td><td>0.78</td><td>[0.67; 0.87]</td></tr><tr><td>Ferns 2002</td><td>4</td><td>14</td><td>0.29</td><td>[0.08; 0.58]</td></tr><tr><td>Florent 2006</td><td>69</td><td>134</td><td>0.51</td><td>[0.43; 0.60]</td></tr><tr><td>Hummel 2009</td><td>48</td><td>65</td><td>0.74</td><td>[0.61; 0.84]</td></tr><tr><td>Loeffler 2017</td><td>19</td><td>35</td><td>0.54</td><td>[0.37; 0.71]</td></tr><tr><td>Rogers 2013</td><td>80</td><td>131</td><td>0.61</td><td>[0.52; 0.69]</td></tr><tr><td>Springer 2011</td><td>27</td><td>43</td><td>0.63</td><td>[0.47; 0.77]</td></tr><tr><td>Sugawara 2013</td><td>53</td><td>55</td><td>0.96</td><td>[0.87; 1.00]</td></tr><tr><td><strong>Total (95% CI)</strong></td><td></td><td></td><td><strong>1046</strong></td><td><strong>[0.64; 0.55; 0.72]</strong></td></tr></tbody></table>

Heterogeneity: Tau² = 0.2186; Chi² = 43.19, df = 8 (P < 0.01); I² = 81%

<figure><img src="image_45.png" /></figure>

Prophylaxis and Sensitivity

<table><thead><tr><th>Study</th><th>TP</th><th>IA</th><th>Sens [95% CI]</th></tr></thead><tbody><tr><td colspan="4"><strong>prophylaxis = 0</strong></td></tr><tr><td>Badiee 2010</td><td>13</td><td>15</td><td>0.87 [0.60; 0.98]</td></tr><tr><td>Badiee 2017</td><td>3</td><td>4</td><td>0.75 [0.19; 0.99]</td></tr><tr><td>Boch 2016</td><td>36</td><td>40</td><td>0.90 [0.76; 0.97]</td></tr><tr><td>Boluk 2016</td><td>36</td><td>40</td><td>0.90 [0.76; 0.97]</td></tr><tr><td>da Silva 2010</td><td>15</td><td>20</td><td>0.75 [0.51; 0.91]</td></tr><tr><td>El Mahallawy 2006</td><td>21</td><td>28</td><td>0.75 [0.55; 0.89]</td></tr><tr><td>Halliday 2006</td><td>13</td><td>13</td><td>1.00 [0.75; 1.00]</td></tr><tr><td>Hebart 2000</td><td>13</td><td>18</td><td>0.72 [0.47; 0.90]</td></tr><tr><td>Landlinger 2010</td><td>4</td><td>4</td><td>1.00 [0.40; 1.00]</td></tr><tr><td>Rogers 2013</td><td>16</td><td>30</td><td>0.53 [0.34; 0.72]</td></tr><tr><td>Schwarzinger 2013</td><td>8</td><td>11</td><td>0.73 [0.39; 0.94]</td></tr><tr><td>Springer 2016</td><td>17</td><td>18</td><td>0.94 [0.73; 1.00]</td></tr><tr><td>von Lilienfeld-Toal 2009</td><td>3</td><td>3</td><td>1.00 [0.29; 1.00]</td></tr><tr><td><strong>Total (95% CI)</strong></td><td></td><td></td><td><strong>244</strong></td><td><strong>[0.80; 0.71; 0.87]</strong></td></tr></tbody></table>

Heterogeneity: Tau² = 0.3378; Chi² = 22.13, df = 12 (P = 0.04); I² = 46%

Sensitivity

<figure><img src="image_46.png" /></figure>

<table><thead><tr><th>Study</th><th>TP</th><th>IA</th><th>Sens [95% CI]</th></tr></thead><tbody><tr><td colspan="4"><strong>prophylaxis = 1</strong></td></tr><tr><td>Barnes 2013</td><td>49</td><td>53</td><td>0.92 [0.82; 0.98]</td></tr><tr><td>Cuenca-Estrella 2009</td><td>9</td><td>10</td><td>0.90 [0.55; 1.00]</td></tr><tr><td>Ferns 2002</td><td>2</td><td>3</td><td>0.67 [0.09; 0.99]</td></tr><tr><td>Florent 2006</td><td>29</td><td>33</td><td>0.88 [0.72; 0.97]</td></tr><tr><td>Hummel 2009</td><td>4</td><td>5</td><td>0.80 [0.28; 0.99]</td></tr><tr><td>Loeffler 2017</td><td>4</td><td>4</td><td>1.00 [0.40; 1.00]</td></tr><tr><td>Rogers 2013</td><td>13</td><td>15</td><td>0.87 [0.60; 0.98]</td></tr><tr><td>Springer 2011</td><td>2</td><td>3</td><td>0.67 [0.09; 0.99]</td></tr><tr><td>Sugawara 2013</td><td>6</td><td>9</td><td>0.67 [0.30; 0.93]</td></tr><tr><td><strong>Total (95% CI)</strong></td><td></td><td></td><td><strong>135</strong></td><td><strong>[0.86; 0.78; 0.91]</strong></td></tr></tbody></table>

Heterogeneity: Tau² = 0; Chi² = 6.4, df = 8 (P = 0.60); I² = 0%

<figure><img src="image_47.png" /></figure>

**Conclusion:** The cumulative incidence of IA was significantly lower in patients receiving anti-mould prophylaxis. The use of prophylaxis decreases the specificity of *Aspergillus* PCR testing of blood, which is of borderline statistical significance. Although an increase in sensitivity and decrease in DOR was observed, this did not reach statistical significance between the 2 groups. Anti-mould prophylaxis reduces the proportion of proven/probable cases of IA (according to EORTC/MSG criteria) which is associated with a lower specificity of the *Aspergillus* PCR testing of blood.
**Comparison of fungal cultures and *Aspergillus* PCR kits results in SOT patients: an evaluation of five new commercial kits on respiratory samples.**

N. Argy¹, C. Bonnal¹, C. Godet², J.F. Timsit³, B. Lortat-Jacob⁴, S. Houzé¹

¹Laboratoire De Parasitologie Mycologie, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, PARIS, France, ²Service De Pneumologie B, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, PARIS, France, ³Service De Réanimation Médicale Et Infectieuse, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, PARIS, France, ⁴Service De Réanimation Chirurgicale, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, PARIS, France

**Objectives:** The diagnosis of invasive aspergillosis (IA) is difficult in patients without hematological malignancies. For diagnosis, direct microscopy, histopathology and culture are strongly recommended and, in solid organ transplant patients (SOT), broncho-alveolar lavage (BAL) galactomannan measures are recommended as well. Recent recommendations suggest that BAL-PCR should be considered in conjunction with other diagnostic tests in lung transplanted patients particularly¹. Commercially *Aspergillus fumigatus* and *Aspergillus spp* PCR kits are now available but their evaluation in clinical practice remains to be performed. The aim of our study was to compare the results obtained with some of those Aspergillus PCR kits and cultures performed on respiratory samples in non-hematological patients.

**Methods:** Five kits (Ademtech MycoGENIE® *Aspergillus fumigatus* and MycoGENIE® *Aspergillus* species, AsperGenius® Species PathoNostics (*A. fumigatus*, *A. terreus*, *Aspergillus spp*), Fungiplex® IVD PCR (*Aspergillus spp*, *A. terreus*) Bruker and *Aspergillus* ELITe MGB® ElitechGroup (*Aspergillus spp*).) were evaluated on respiratory samples which were routinely analyzed for patients (including SOT) in our hospital. The PCR results were considered as not acceptable if the matching between fungal cultures and PCR results represents less than 70% of the samples tested (in bold in results).

**Results:** Forty respiratory samples (BAL, bronchial aspirates) from 36 patients were included. For 30 samples, 8 *A. fumigatus*, 14 *Aspergillus spp* (4 *A. flavus*, 4 *A. niger*, 2 *A. hiratsukae*, 1 *A. terreus*, 1 *A. nidulans*, 1 *A. calidoustus* and 1 *Aspergillus spp.*) and 8 *Penicillium spp.* were isolated. Twelve were negative. For MycoGENIE® *A. fumigatus*, MycoGENIE® *Aspergillus* species, AsperGenius® *A. fumigatus*, AsperGenius® *Aspergillus spp*, Fungiplex® and ELITe MGB® respectively, the results were: - PCR positive results/number of samples tested and *A. fumigatus* positive cultures: 7/8, 7/8, 5/8, 6/8, 5/7, 3/8, - PCR positive results/number of samples tested and *Aspergillus spp* positive cultures: 0/12, 6/12, 1/10, 6/10, 6/12, 2/12, - PCR negative results/number of samples tested and *Penicillium spp.* positive cultures: 6/7, 6/8, 8/8, 3/8, 4/7, 7/7 - PCR negative results/number of samples tested and negative cultures: 7/8, 10/12, 7/9, 5/9, 8/12, 11/12. MycoGENIE® *Aspergillus fumigatus* shows a high concordance for *A. fumigatus* positive cultures without false positive results for *Aspergillus spp* or *Penicillium spp*. AsperGenius® *A. fumigatus* does not give satisfactory results for positive cultures. Neither of the *Aspergillus spp*. PCR kits have a good correlation with the culture results.

**Conclusion:** MycoGENIE® *Aspergillus fumigatus* seems to be suitable for the diagnosis of AI due to *A. fumigatus* and must be prospectively studied in non-hematological patients. For *Aspergillus spp*. PCR kits, all except ELITe MGB® gave unsatisfactory results for negative results, mainly because of the cross reactivity with other hyalohyphomycetes. This is a real problem for lung transplanted patients who are often colonized with filamentous fungi and may have positive PCR results without AI. On the other hand, it is difficult to conclude about their correlation with positive cultures because it may reflect colonization without infection. The cohort was too small for a clinical evaluation therefore prospective evaluation of these PCRs in patients without hematological malignancies are needed. Ullmann AJ et al. Clin Microbiol Infect. 2018

**High-Volume Culture and Quantitative Real-Time PCR for the Detection of *Aspergillus* in Sputum**
P. Vergidis¹, C.B. Moore², L. Novak-Frazer²,³, R. Rautemaa-Richardson³,⁴, A. Walker³, D. Denning³,⁴, M.D. Richardson²,³

¹Infectious Diseases, Mayo Clinic, Rochester, United States of America, ²Mycology Reference Centre, Excellence Center For Medical Mycology (ecmm), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom, ³Division Of Infection, Immunity And Respiratory Medicine, University of Manchester, Manchester, United Kingdom, ⁴National Aspergillosis Centre, Manchester University NHS Foundation Trust, Manchester, United Kingdom
**Objectives:** Sputum culture is an insensitive method for the diagnosis of pulmonary aspergillosis. Growth of the organism allows identification of the causative species and susceptibility testing, both of which can inform treatment choices. The current practice is to culture an aliquot of diluted sputum. We assessed the value of culturing large volumes of unprocessed sputum, a method that we have termed high-volume culture (HVC).

**Methods:** Specimens were processed by conventional culture (using an aliquot of homogenized, diluted sputum on Sabouraud agar at 37°C and 45°C for up to 5 days) and HVC (using undiluted sputum on Sabouraud agar at 30°C for up to 14 days). A separate specimen was tested by quantitative real-time PCR (qPCR). Aspergillus MycAssay (Myconostica) was used from January 2015 until February 2016. Aspergillus FUMI (Progenie) was used from February until October 2016. Aspergillus Elite MGB (ELITechGroup) was used from October 2016 through February 2017. Antifungal susceptibility testing was performed by the EUCAST standard.

**Results:** We obtained 290 sputum specimens from 231 patients with the following conditions: Chronic pulmonary aspergillosis (67.1%), allergic bronchopulmonary aspergillosis/severe asthma with fungal sensitization (25.1%) and *Aspergillus* bronchitis (7.8%). The sensitivity of conventional culture was 16.2% (95% CI: 12.4-20.9%) and that of HVC was 52.4% (95% CI: 46.7-58.1%) (p<0.001). qPCR had an overall sensitivity of 49.0% (95% CI: 41.2%-56.9%), comparable to that of HVC (p = 0.46). Rates of growth by HVC in correlation with transformed Ct values (TCt) for MycAssay and FUMI are shown in Fig. 1. There is a correlation between higher TCt values and the percentage of growth by HVC. By combining HVC and qPCR, the sensitivity increased to 66.0% (Fig. 2. Solid bars patients not receiving antifungal treatment. Dotted bars represent specimens of patients receiving treatment). *A. fumigatus* was the most commonly isolated species (82.9%) followed by *A. niger* (9.2%), *A. montevidensis* (3.3%) and *A. flavus* (2.6%). Among *A. fumigatus* isolates, pan-azole resistance was detected in 17.2%. At the time of sputum collection, 52.1% of patients were receiving antifungal treatment. Among patients with CPA, the sensitivity of HVC was higher compared to conventional culture for those treated with itraconazole (48.0% vs 12.0%, p<0.001) as well as those not receiving antifungal drug treatment (60.5% vs 26.7%, p<0.001). This trend is replicated in patients treated with other antifungals for all three conditions. HVC allowed for detection of azole-resistant isolates that would have been missed by conventional culture.

<figure><img src="image_48.png" /><figcaption>Fig 1. MycAssay and FUMI</figcaption></figure>
<figure><img src="image_49.png" /><figcaption>Fig 2.</figcaption></figure>

**Conclusion:** The recovery rate of *Aspergillus* spp. is significantly higher for HVC compared to conventional culture. HVC can be performed in any microbiology laboratory without the need for additional equipment or technical skills. The sensitivity of HVC is comparable to that of qPCR, although susceptibility testing is not possible by the latter technique. Combination of the two methods can lead to further increase in the recovery of *Aspergillus* from sputum specimens. Use of HVC of expectorated sputum for the detection of fungi can have a significant impact in the diagnosis and management of patients with pulmonary mycoses.

P151

**Aspergillus, Mucorales, Fusarium: Detectable in BAL by real-time PCR?**

J. Springer¹, D. Teschner², J. Kessel³, O.A. Cornely⁴, T. Liebregts⁵, S.W. Krause⁶, S. Schwartz⁷, M.G. Kiehl⁸, W.J. Heinz¹, B. Willinger⁹, H. Einsele¹, V. Rickerts¹⁰, J. Löffler¹

¹University hospital, Wuerzburg, Germany, ²University Medical Center of the Johannes Gutenberg University, Mainz, Germany, ³University Hospital Frankfurt, Frankfurt, Germany, ⁴German Centre For Infection Research, Partner Site Bonn-cologne, Cologne, Germany And Clinical Trials Centre Cologne (zks Köln), University of Cologne, Cologne, Germany, ⁵University Hospital Essen, Essen, Germany, ⁶University Hospital Erlangen, Erlangen, Germany, ⁷Charité Berlin, Berlin, Germany, ⁸Klinikum Frankfurt/Oder, Frankfurt/Oder, Germany, ⁹Medical University of Vienna, Vienna, Austria, ¹⁰Robert Koch Institut Berlin, Berlin, Germany

**Objectives:** Invasive fungal infections (IFI) remain a major complication in patients with haematological malignancies (HM) and post allogeneic haematopoietic stem cell transplantation (HSCT). In these patients, invasive aspergillosis (IA) is the most common cause of mortality due to infection. Early detection of fungal infections has the potential to facilitate a more effective management of invasive disease. Bronchoalveolar lavage samples (BAL) directly obtained from the focus of infection are often used as diagnostic tool. In this study, BAL samples were tested using different real-time PCR assays and correlated to EORTC/MSG classification for IFI (DePauw 2008).

**Methods:** In a multi-centre approach, nine hospitals collected BAL samples from HM/HSCT patients comprising a CT scan suspicious for IFI. DNA was isolated from supernatant and BAL pellet and analysed for the presence of fungal DNA. Analysis was performed in Wuerzburg only by using 3 specific real-time PCR assays and one panfungal assay (Khot 2009, 28S 10f-12r). All samples were analysed for the presence of *Aspergillus* and Mucorales, additionally some for *Fusarium* and fungal DNA.

**Results:** In total, 294 samples were collected. 69/294 showed *Aspergillus* DNA, 20/294 Mucorales, 2/141 *Fusarium* and 62/69 panfungal DNA (Tab. 1). Sequencing of panfungal amplicons revealed mostly DNA of yeasts (mainly *Malassezia* sp. and *Candida* sp.) which were classified as non-invasive/contaminations. Only in 7 cases (7.9%; 6 *Aspergilli*, 1 Mucorales) the result of the specific assay could be confirmed. Therefore no further PCR analysis using the panfungal assay was performed. In correlation to EORTC/MSG criteria (6 proven, 85 probable, 130 possible IFD and 26 undetermined cases; 47 without data) *Aspergillus* DNA was detected in 42 probable/proven cases, Mucorales in 11, *Fusarium* in 1 and panfungal DNA in 24 (only 7 if excluding contaminations) (Table 2). DNA of *Aspergillus* and Mucorales was detected in just one or none control samples (undetermined cases), 10/69 samples (14.5%) showing DNA of *Aspergillus* were also positive for
Mucorales (n = 9) or *Fusarium* (n = 1). Changing the perspective revealed that almost half of the Mucorales positive samples were double-infected with *Aspergillus* (9/20; 45%).

<table><caption>Table 1: qPCR assay results</caption><thead><tr><th>PCR assay</th><th>positive # (%)</th><th>negative</th><th>sum</th><th>not done</th></tr></thead><tbody><tr><td><em>Aspergillus</em></td><td>69 (23.5)</td><td>225</td><td>294</td><td></td></tr><tr><td>Mucorales</td><td>20 (6.8)</td><td>274</td><td>294</td><td></td></tr><tr><td><em>Fusarium</em></td><td>2 (1.4)</td><td>139</td><td>141</td><td>153</td></tr><tr><td>panfungal</td><td>62 (89.9)</td><td>7</td><td>69</td><td>225</td></tr></tbody></table>

<table><thead><tr><th>EORTC/MSG</th><th>undetermined</th><th>possible</th><th>probable *</th><th>proven</th><th>na</th></tr></thead><tbody><tr><td><em>Aspergillus</em></td><td>1</td><td>15</td><td>39</td><td>3</td><td>11</td></tr><tr><td>Mucorales</td><td>0</td><td>3</td><td>10</td><td>1</td><td>6</td></tr><tr><td><em>Fusarium</em></td><td></td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>panfungal</td><td>10</td><td>26</td><td>23</td><td>1</td><td></td></tr></tbody></table>

<caption>Table 2: Distribution of qPCR positive samples correlated to EORTC classification</caption>

**Conclusion:** qPCR was able to detect DNA of specific fungi in BAL samples. Specific assays for *Aspergillus*, Mucorales and *Fusarium* proved to be helpful to detect fungal pathogen DNA, whereas the panfungal approach mainly detected non-invasive contaminations. The number of double-infections seems to be higher than expected (14.5% to 45%).

P152

**Analysis of the efficacy of bis(methyl)gliotoxin for early invasive aspergillosis detection and to guide antifungal treatment in neutropenic pediatric oncology patients.**

M.P. Domingo¹, S. Nabal², S. Redrado¹, C. Lopez², C. Rodriguez-Vigil³, A. Muñoz³, C. Calvo³, A. Rezusta², J. Pardo⁴, E. Galvez¹

¹Instituto de Carboquimica (ICB-CSIC), Zaragoza, Spain, ²Microbiology, Hospital Miguel Servet, Zaragoza, Spain, ³Oncology Pediatric, Hospital Miguel Servet, Zaragoza, Spain, ⁴Centro De Investigaciones Biomedicas, Universidad De Zaragoza, ARAID, Zaragoza, Spain

**Objectives:** Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in immunocompromised patients. This is due in part to the absence of optimal diagnostic modalities, which hamper early specific disease detection. Systemic detection of serum biomarkers is a standard noninvasive practice used to early diagnose and treat IPA in high risk population. During the last years, doubts have been raised regarding the use of galactomannan (GM) in onco-hematological population mainly due to the large number of patients with false positive test as well as the low efficacy in patients under antifungal profylaxis. In a previous study, we demonstrated the high diagnostic accuracy of bis(methyl)gliotoxin (bmGT) compared to GM for IPA diagnosis in adult oncohematological patients. However, the performance of bmGT as a diagnosis biomarker in pediatric patients is unknown. The objective of this study is to compare the ability of bmGT and GMN detection for IPA diagnosis in oncopediatric patients and to analyse the efficacy of bmGT to serve as guide for voriconazol treatment.

**Methods:** The presence of bmGT and voriconazole levels in sequential series of serum from 7 oncopediatric patients at risk of IA (prospective samples) is simultaneously quantified by High Performance Thin Layer Chromatography (HPTLC) as well as LC/MS/MS.

**Results:** We have established a fast and sensitive technique for simultaneous separation, detection and quantification of bmGT and voriconazole in serum and BAL of pediatric patients. bmGT and GM were negative in all possible IA cases (5 patients). Two patients, presenting prolonged neutropenia after chemotherapy treatment, fever, hypoxemia and pulmonary infiltration compatible with Aspergillosis in high resolution CT, were categorised as probable IA. GM and bmGT was monitored in serum and bronchoalveolar lavage. In one patient, GM and bmGT were detected in serum and

<footnote>*: probable IFI without positive BDG results; na not available</footnote>