with the voriconazole treatment GM was negativized and the bmGT levels decreased. bmGT was positive earlier than GM allowing early initiation of treatment. In the second patient, GM was negative and bmGT positive in both serum and BAL, so treatment with voriconazole was started. Treatment decreased bmGT values coinciding with the patient clinical improvement.

**Conclusion:** BmGT has allowed us the early diagnosis and successful treatment of pediatric IA, being a promising tool for early detection of IA in neutropenic pediatric patients.

P153

Identification for major Aspergillus species in paraffin-embedded tissue: newly developed multiplex real-time PCR method using hydrolysis probes

J.-K. Choi¹,², S.-Y. Cho¹,²,³, C. Park², D.-G. Lee¹,²,³

¹Division Of Infectious Diseases, Department Of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea, Republic of, ²Vaccine Bio Research Institute, Seoul, Korea, Republic of, ³Catholic Hematology Hospital, Seoul, Korea, Republic of

**Objectives:** Species identification is crucial for determining appropriate antifungal agents in treatment of fungal infections. However, current culture-based methods are insensitive due to low positive rate and slow process. Microscopic examination of tissue specimens has limitations in distinguishing fungal organisms. Detection of fungal DNA by polymerase chain reaction (PCR) method in tissue samples is expected to be useful for the diagnosis of fungal infections. Herein, we describe the performance and clinical impact of a multiplex fungal PCR assay of formalin-fixed paraffin-embedded (FFPE) sample for the diagnosis of *Aspergillus* infections.

**Methods:** FFPE samples previously collected from patients diagnosed as fungal infections by histopathologic finding between June 2009 and June 2012 were used for PCR analysis, and retrospective chart review was also performed for correlation with clinical diagnosis. Real-time PCR primers and multiplex hydrolysis probes were newly designed for four major *Aspergillus* species (*A. fumigatus*, *A. niger*, *A. terreus*, and *A. flavus*) based on molecular analysis data of β-tubulin gene (*benA*) in *Aspergillus* isolates. The method of real-time PCR was qualified and quantified using the reference genomic DNA (gDNA) of *Aspergillus* species. DNA was extracted from FFPE sample using TaKaRa DEXPAT kit (TaKaRa Biomedicals, Shiga, Japan). The amplified *benA* amplicon was analyzed by comparing with the standard curve of the reference gDNA. Multiplex PCR was performed on 72 FFPE samples. The sample quality was validated by human globulin detection, and 61 qualified samples were analyzed.

<table><thead><tr><td>primer & probe</td><td>Sequence</td></tr></thead><tbody><tr><td>&gt;benA_f3</td><td>TCGG TGTA GTGA CCCT TGG</td></tr><tr><td>&gt;benA_r2deg</td><td>GCTGGAGCGYATGAACGTCT</td></tr><tr><td>&gt;Asco_1F9Deg_FAM</td><td>AVACGAAGTTGTCGGGRC</td></tr><tr><td>&gt;Flavi_1F18 HEX</td><td>CGGTCAGGAGTTGCAAAGCGTTTCA</td></tr><tr><td>&gt;Fumi_1R2 HEX</td><td>CGGCAACATCTCACGATCTGACTCGC</td></tr><tr><td>&gt;Nig_1R26_FAM</td><td>ACTTCAGCAGGCTAGCGGTAACAAGT</td></tr><tr><td>&gt;Ter_1R29 Deg_FAM</td><td>ACCATCCTGGGACAGATTCTYCACGC</td></tr></tbody></table>

**Results:** Twenty-seven of 35 cases clinically suspected as aspergillosis presented positive results in this real-time PCR method (sensitivity: 77.1%). The positive rate was higher than that of the fungal culture (12/35, 34.3%) or galactomannan test (20/33, 60.6%). Among the 23 cases with culture-negative proven/probable invasive aspergillosis, 17 cases were confirmed as positive by real-time PCR. *Aspergillus* gene was identified in 5 of 9 IFI cases that causative fungal organisms were not confirmed in clinical and pathological examination. In cases with non-*Aspergillus* infection, *Aspergillus* gene was detected in 2 of 13 cases. By real-time PCR for FFPE specimen, additional informations, not only detection of *benA* gene, but also species-level identification, were obtained in 51.7% (32 of 61) cases included in this study.
<table><thead><tr><th></th><th>Clinical Dx.</th><th>Serum GM</th><th>Pathology Dx.</th><th>Microbiology Dx.</th><th>Molecular Dx.</th><th>A. fumigatus probe</th><th>A. niger probe</th><th>A. terreus probe</th><th>A. flavus probe</th></tr></thead><tbody><tr><td>Case 1</td><td>IA</td><td>-</td><td>IA</td><td>A. fumigatus</td><td>A. fumigatus</td><td>32.41</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 2</td><td>IA</td><td>+</td><td>IA</td><td>A. fumigatus</td><td>A. fumigatus</td><td>32.28</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 3</td><td>IA</td><td>-</td><td>IA</td><td>A. fumigatus</td><td>A. fumigatus</td><td>31.83</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 4</td><td>IA</td><td>+</td><td>IA</td><td>A. fumigatus</td><td>A. fumigatus</td><td>31.49</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 5</td><td>IA</td><td>+</td><td>IA</td><td>A. fumigatus</td><td>A. fumigatus</td><td>26.93</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 6</td><td>IA</td><td>+</td><td>IA</td><td>A. flavus</td><td>A. fumigatus</td><td>30.58</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 7</td><td>IA</td><td>-</td><td>IA</td><td>A. restrictus</td><td>A. fumigatus</td><td>29.22</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 8</td><td>IA</td><td>+</td><td>Aspergilloma</td><td>A. flavus</td><td>A. fumigatus</td><td>31.48</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 9</td><td>Aspergilloma</td><td>-</td><td>Aspergilloma</td><td>A. fumigatus</td><td>A. niger</td><td>-</td><td>34.39</td><td>-</td><td>-</td></tr><tr><td>Case 10</td><td>Aspergilloma</td><td>-</td><td>Aspergilloma</td><td>A. sp</td><td>A. fumigatus</td><td>30.28</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 11</td><td>IA</td><td>+</td><td>IA</td><td>A. flavus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 12</td><td>IA</td><td>+</td><td>IA</td><td>A. sp</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 13</td><td>IA</td><td>+</td><td>IA</td><td>-</td><td>A. fumigatus</td><td>32.52</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 14</td><td>IA</td><td>+</td><td>IA</td><td>-</td><td>A. flavus</td><td>-</td><td>-</td><td>34.94</td><td>-</td></tr><tr><td>Case 15</td><td>IA</td><td>+</td><td>IA</td><td>-</td><td>A. niger</td><td>-</td><td>34.98</td><td>-</td><td>-</td></tr><tr><td>Case 16</td><td>IA</td><td>-</td><td>IA</td><td>-</td><td>A. flavus</td><td>-</td><td>-</td><td>-</td><td>30.79</td></tr><tr><td>Case 17</td><td>IA</td><td>+</td><td>IA</td><td>-</td><td>A. fumigatus</td><td>34.29</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 18</td><td>IA</td><td>+</td><td>IA</td><td>-</td><td>A. fumigatus</td><td>33.99</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 19</td><td>IA</td><td>-</td><td>IA</td><td>-</td><td>A. flavus & A. terreus</td><td>-</td><td>-</td><td>25.98</td><td>26.85</td></tr><tr><td>Case 20</td><td>IA</td><td>+</td><td>IA</td><td>-</td><td>A. flavus</td><td>-</td><td>-</td><td>-</td><td>29.95</td></tr><tr><td>Case 21</td><td>IA</td><td>+</td><td>IA</td><td>-</td><td>A. niger</td><td>-</td><td>34.67</td><td>-</td><td>-</td></tr><tr><td>Case 22</td><td>IA</td><td>+</td><td>IA</td><td>-</td><td>A. niger</td><td>-</td><td>32.85</td><td>-</td><td>-</td></tr><tr><td>Case 23</td><td>IA</td><td>+</td><td>IA</td><td>-</td><td>A. flavus</td><td>-</td><td>-</td><td>-</td><td>27.02</td></tr><tr><td>Case 24</td><td>Aspergilloma</td><td>-</td><td>Aspergilloma</td><td>-</td><td>A. niger</td><td>-</td><td>34.71</td><td>-</td><td>-</td></tr><tr><td>Case 25</td><td>Aspergilloma</td><td>-</td><td>Aspergilloma</td><td>-</td><td>A. fumigatus</td><td>34.92</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 26</td><td>Aspergilloma</td><td>NC</td><td>Aspergilloma</td><td>-</td><td>A. fumigatus</td><td>30.6</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 27</td><td>Aspergilloma</td><td>NC</td><td>Aspergilloma</td><td>-</td><td>A. fumigatus</td><td>34.78</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 28</td><td>Aspergilloma</td><td>-</td><td>Aspergilloma</td><td>-</td><td>A. fumigatus</td><td>31.54</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 29</td><td>Aspergilloma</td><td>+</td><td>Aspergilloma</td><td>-</td><td>A. fumigatus</td><td>32.36</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 30</td><td>IA</td><td>+</td><td>IA</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 31</td><td>Aspergilloma</td><td>-</td><td>IA</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 32</td><td>Aspergilloma</td><td>-</td><td>Aspergilloma</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 33</td><td>IA</td><td>+</td><td>IFI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 34</td><td>IA</td><td>+</td><td>IFI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 35</td><td>IA</td><td>+</td><td>IFI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 36</td><td>IA</td><td>-</td><td>IA</td><td>Mucorales (Absida)</td><td>A. fumigatus</td><td>30.72</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 37</td><td>IA</td><td>-</td><td>IA</td><td>Mucorales (Absida)</td><td>A. fumigatus</td><td>29.85</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 38</td><td>IA</td><td>-</td><td>IA</td><td>Mucorales (Absida)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 39</td><td>IA</td><td>-</td><td>IA</td><td>Mucorales (Absida)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 40</td><td>Inv. Scadosporiosis</td><td>+</td><td>IA</td><td>Scadosporium</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 41</td><td>IFI</td><td>NC</td><td>IFI</td><td>-</td><td>A. fumigatus</td><td>29.72</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 42</td><td>IFI</td><td>-</td><td>IFI</td><td>-</td><td>A. fumigatus</td><td>21.86</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 43</td><td>IFI</td><td>-</td><td>IFI</td><td>-</td><td>A. niger</td><td>-</td><td>31.51</td><td>-</td><td>-</td></tr><tr><td>Case 44</td><td>IFI</td><td>-</td><td>IFI</td><td>-</td><td>A. fumigatus</td><td>30.56</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 45</td><td>IFI</td><td>-</td><td>IFI</td><td>-</td><td>A. fumigatus</td><td>31.22</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 46</td><td>Mucormycosis</td><td>+</td><td>IFI</td><td>Mucorales (Rhizopus)</td><td>A. terreus</td><td>-</td><td>-</td><td>26.28</td><td>-</td></tr><tr><td>Case 47</td><td>Inv. Fusariosis</td><td>NC</td><td>IFI</td><td>Fusarium</td><td>A. fumigatus</td><td>32.33</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 48</td><td>Inv. Fusariosis</td><td>NC</td><td>IFI</td><td>Fusarium</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 49</td><td>IFI</td><td>-</td><td>IFI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 50</td><td>IFI</td><td>-</td><td>IFI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 51</td><td>IFI</td><td>NC</td><td>IFI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 52</td><td>IFI</td><td>-</td><td>IFI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 53</td><td>Mucormycosis</td><td>+</td><td>Mucorales</td><td>Mucorales</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 54</td><td>Mucormycosis</td><td>+</td><td>Mucorales</td><td>Mucorales</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 55</td><td>Mucormycosis</td><td>-</td><td>Mucorales</td><td>Mucorales</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 56</td><td>Mucormycosis</td><td>-</td><td>Mucorales</td><td>Mucorales</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 57</td><td>Mucormycosis</td><td>-</td><td>Mucorales</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 58</td><td>Mucormycosis</td><td>+</td><td>Mucorales</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 59</td><td>Mucormycosis</td><td>-</td><td>Mucorales</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 60</td><td>Mucormycosis</td><td>-</td><td>IFI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Case 61</td><td>Mucormycosis</td><td>-</td><td>IFI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

GM, galactomannan; IA, invasive aspergillosis; NC, Not checked; Inv, invasive; IFI, invasive fungal infection

**Conclusion:** This study revealed that this newly developed real-time PCR method for FFPE tissue could improve diagnostic accuracy of aspergillosis. Further study is needed to understand mixed mold infections or false positive cases with additional assays including PCR for non-*aspergillus* mold.
Rostov-on-Don, Russian Federation, ³Rostov Research Institute of Oncology, Rostov-on-Don, Russian Federation, ⁴Oncohematology Department, Rostov Research Institute of Oncology, Rostov-on-Don, Russian Federation, ⁵National Research Center for Hematology, Moscow, Russian Federation

**Objectives:** To evaluate the prognostic significance of procalcitonin and mannan antigen of *Candida spp* (MA) for early and accessible verification of the causative agent of bloodstream infection (BSI).

**Methods:** We examined 349 patients with suspected BSI from 16 intensive care, oncology and oncohematology units of 9 hospitals in the Southern Federal District of Russia. The diagnostic algorithm included sterility blood testing and a study of biochemical markers with immunoenzymatic assays. Sterility blood testing was performed using `BacT/ALERT3D` analyzer. Identification of strains and detection of their antimicrobial drugs sensitivity was made with Vitek-2 automatic analyzer (BioMerieux, France). Simultaneously we measured the levels of procalcitonin and MA with the use of Procalcitonin kit ELISA-BEST (Russia) and Platelia™ Candida Ag Plus kit (France) correspondingly. Only high values of procalcitonin (10 and above ng/ml) were considered as expedient in the diagnosis of bacterial sepsis. The measurement of MA made it possible to judge about the involvement of *Candida spp.* in the infectious process. The result of MA testing was regarded as positive with the concentration ≥125 pg/ml.

**Results:** Positive blood cultures were obtained in 84 (24.1%) patients. Bacteria accounted for 77.4% (65 strains), *Candida spp.* - 22.6% (19 isolates). In 3 cases (3.6%) bacterial-candidal associations were detected which worsened the condition of the patients. In a parallel study of biochemical markers, an increased level of one of them was in 205 (58.7%) patients. High values of procalcitonin accounted for 68 (33.2%) that was in favor of a severe bacterial infection. Positive results for MA were in 118 (57.6%) patients. Positive results of MA testing in the presence of clinical symptoms and risk factors for the development of invasive candidiasis suggested a candidal BSI. In 19 (9.2%) patients two biochemical markers were elevated indicating a possible mixed infection (picture 1,2). For 144 (41.3%) patients with initial negative blood culture testing and normal biomarker levels additional studies were needed to exclude or confirm BSI. As a result, in 26 patients a negative results were regarded as a cancer relapse. 118 patients needed dynamic blood testing. 70 patients received a positive results. 14 of them had high procalcitonin and 56 had a positive MA. 18 patients also had a positive blood culture (4 patients had bacteria isolated, 14 - candida). 52 patients were observed with only positive biomarkers (10 with procalcitonin, 42 with MA). The rest 48 patients were with fever of unknown
<figure><img src="image_50.png" /><figcaption>Pic.1. Comparison of two methods for diagnosis of BSI</figcaption></figure>

origin.

<figure><img src="image_51.png" /><figcaption>Pic.2. Comparative values of bacteria and candida diagnosis</figcaption></figure>

**Conclusion:** A comprehensive approach to the diagnosis of BSI has increased the percentage of pathogen verification to 58.7%. It should be taken into consideration that both blood culture and biomarkers testing of bacterial infection revealed almost the same diagnostic significance. However, MA testing improved dramatically the early diagnosis of a candidal BSI despite negative blood culture testing which is probably due to the prolonged cultivation of *Candida spp.* in the blood. The inclusion of biochemical markers testing in the diagnostic algorithm for suspected BSI allowed to identify the causative infectious agent as early as possible and to start adequate antibacterial or antifungal therapy.

P155

**Validation and ongoing evaluation of a real time PCR assay for Pneumocystis jirovecii detection in bronchoalveolar fluid specimens; data from Pakistan**

K. Jabeen¹, N. Ghanchi², J. Farooqi², K. Iqbal Masood², O. Ali Siddiqui², A. Humayun², Z. Hasan²

¹Pathology And Laboratory Medicine, Aga Khan University, Karachi, Pakistan, Pakistan, ²Pathology And Laboratory Medicine, Aga Khan University, Karachi, Pakistan

**Objectives:** Pneumocystis pneumonia (PCP) is a frequent opportunistic infection in HIV-positive and immunocompromised individuals. Healthy individuals have been shown to harbor the organism without clinical signs of infection and may play a role in the transmission of PCP. PCR based assays for detection of *P. jirovecii* are reported to be more sensitive compared to conventional immunofluorescence assays (IFA). We report experience of a tertiary care hospital laboratory in Pakistan which recently shifted from IFA based to PCR based diagnosis for PCP pneumonia.

**Methods:** The study was conducted at the clinical laboratory of Aga Khan University Hospital, Karachi Pakistan. In the first phase (January-May 2018), a real time PCR assay was validated against twenty archived PCP positive or negative bronchoalveolar lavage (BAL) specimens. Once validation was completed satisfactorily, PCR based assay was introduced and offered to clinicians in June 2018. All BAL specimens submitted for detection for PCP from June 2018-May 2019 after the introduction of test were included and evaluated. Clinical data collected as routine part of reporting was assessed. Positivity rate for *Pneumocystis* detection was evaluated for PCR during this period. Additionally PCP
data using IFA was retrieved for the year 2016-2017 to compare the positivity rate of IFA with PCR over these two time periods.

**Results:** Initially twenty samples from patients clinically suspected of PCP were compared using both IAF and PCR. PCR confirmed five (25%) positive cases compared to IAF which confirmed 3(12%). After introduction of PCR in the laboratory, a total of 145 suspected patient BAL samples were tested for *P. jirovecii* of which 25 (17%) were PCR positive. Majority of the PCR confirmed patients were male (72%). Clinical data available on 18/25 (72%) positive patients revealed 4 (16%) patients positive for human immunodeficiency virus; 3 (12%) with underlying autoimmune disease; 6 (20%) with malignancy, 2 (4%) on corticosteroid therapy and 1 patient on immunosuppressive therapy post renal transplant. PCP detection increased from 4.5% during 2016-2017 using IFA based assay to 17% using PCR based assay during June 2018-May 2019.

**Conclusion:** A high positivity rate of 17% was seen in clinically suspected PCP patients using PCR based diagnosis in this study. PCP cases were reported in both HIV positive and negative patients. There was an improvement in case detection using PCR compared to IFA. However, high cost of PCR compared to IFA limits wider availability of PCR based PCP diagnosis in resource limited settings.

P156

A comparative genome approach to identify specific DNA sequence for PCR diagnosis of
*Madurella mycetomatis*

W. Lim¹, S. Smit², A.H. Fahal³, W. Van De Sande⁴

¹Microbiology And Infectious Diseases, Erasmus Medical Center, Rotterdam, Netherlands,
²Department Of Plant Science - Bioinformatics, Wageningen University & Research, Wageningen, Netherlands, ³Mycetoma Research Center, University of Khartoum, Khartoum, Sudan, ⁴Department Of Microbiology And Infectious Diseases, Erasmus Medical Center, Rotterdam, Netherlands

**Objectives:** The current gold standard of identifying eumycetoma causative agents is to culture the isolate and assess the morphological characteristics. This method of identification is time consuming and can often lead to misidentifications. Precise and early detection of the causative agent is crucial for clinical management. Currently precise species identification can only be achieved by sequencing the internally transcribed spacer (ITS) regions or by using species-specific PCRs. Only for *M. mycetomatis* a species specific PCR was developed, but recently it was demonstrated that this PCR did cross-react with other *Madurella* species. We therefore looked into the sequenced *M. mycetomatis* genome in search for unique and specific protein coding sequences which could be transformed into a species specific diagnostic PCR.

**Methods:** We used a comparative genome approach to identify unique sequences for *M. mycetomatis* based on its predicted protein coding sequences. The homology of these sequences were identified using blastclust, localization of the proteins was identified using WolfPSORT and SignalP was used to predict the secretion signals in these predicted proteins sequences. Based on these sequences, PCR primers were developed and validated in a collection of 61 *M. mycetomatis*, 4 *Madurella tropicana*, 3 *Madurella pseudomycetomatis*, 3 *Madurella fahalii*, 2 *Falciformispora senegalensis*, 3 *Trematosphaeria subthermophilia*, 3 *Thielavia terrestris*, 3 *Trematosphaeria grisea*, 2 *Chaetomium globosum*, 1 *Aspergillus fumigatus* and 1 *Aspergillus terrus* isolates using conventional PCR.

**Results:** By using a comparative genomic approach, we were able to identify at least 32 *M. mycetomatis* unique protein sequences. For the top 16 most promising candidate sequences, PCR primers were developed. Of these primer pairs, only 12 were able to detect all *M. mycetomatis* isolates tested. Of these 12, only 1 did not react with any other fungal species tested.

**Conclusion:** Based on this comparative genomics approach, we identified a novel *M. mycetomatis* specific PCR primer pair to detect *M. mycetomatis*.

P157

Performance of T2Candida Panel for the diagnosis of intra-abdominal candidiasis

F. Lamoth¹, M.H. Nguyen², F. Tissot¹, K. Squires², P. Eggimann¹, U. Flückiger³, M. Siegemund³, S. Zimmerli⁴, T. Calandra¹, O. Marchetti¹, C.J. Clancy², P.-Y. Bochud¹
¹Infectious Diseases Service, Lausanne University Hospital, Lausanne, Switzerland, ²University of Pittsburgh Medical Center, Pittsburgh, United States of America, ³University Hospital of Basel, Basel, Switzerland, ⁴Department Of Infectious Diseases, University Hospital of Bern, Bern, Switzerland

**Objectives:** Intra-abdominal candidiasis (IAC) is a frequent and life-threatening infection in critically ill patients following complicated abdominal surgery. Diagnosis of IAC is difficult because of the lack of sensitivity of blood cultures and the limited specificity of indirect markers, such as the beta-D-glucan (BDG) test. The T2Candida Panel (T2C, T2Biosystems) is a magnetic resonance-based molecular diagnostic test that identifies the five most common pathogenic *Candida* spp, (C. albicans and C. tropicalis [CA/CT], C. glabrata and C. krusei [CG/CK], and C. parapsilosis) directly in whole blood specimens within 7 hours. T2C demonstrated excellent performance for the diagnosis of candidemia, but data for non-candidemic invasive candidiasis are lacking. We tested T2C's efficiency in detecting IAC.

**Methods:** We used the T2C panel to test frozen whole blood samples from critically-ill patients previously enrolled in a prospective cohort study of the Fungal Infection Network of Switzerland (FUNGINOS), who were at high risk for IAC due to at least one of the following conditions: i) recurrent gastro-intestinal tract perforation, ii) abdominal surgery and *Candida* colonization, iii) necrotizing pancreatitis or iv) burns (>25% body surface area). IAC was defined as growth of *Candida* spp. in a per-operative culture sample from an intra-abdominal abscess or peritoneal fluid. The samples were collected within 72h of IAC diagnosis or between days 7-10 from study inclusion for patients without IAC. Results of concomitant serum BDG (Fungitell) determined previously were included in the analysis.

**Results:** Whole blood samples from 46 patients were available for testing. Most patients had *Candida* colonization of ≥1 non-sterile site (98%) and *Candida* score ≥3 (89%). Eighteen (33%) of them had IAC: 13 were due to CA, 2 to CT, 1 to mixed CA/CG and 2 to *Candida* spp. not included in the T2C Panel (C. kefyr and C. lusitaniae). Antifungal therapy was ongoing at time of IAC diagnosis and blood sampling in 2 (11%) cases. Table 1 shows the comparative diagnostic performance of blood cultures, T2C and BDG. Blood cultures were positive in 2/18 (11%) IAC cases. T2C was positive in 6/18 (33%) IAC cases (including the 2 candidemic patients) with *Candida* spp. matching those isolated in cultures. Analysis limited to IAC cases due to *Candida* spp. of the T2C panel yielded a sensitivity of 37.5%. A positive T2C result was found in 2 of the 28 patients who did not fulfill IAC criteria (specificity 93%). While discordant results for T2C and BDG were observed in 21 (46%) cases, concordant positive results were associated with IAC in 100% of cases and concordant negative results with absence of IAC in 90% of cases.

<table><caption>**Table 1.** Comparative performance of blood cultures, T2Candida (T2C) and beta-D-glucan (BDG) for the diagnosis of intra-abdominal candidiasis (IAC)</caption><thead><tr><th></th><th>Blood cultures</th><th>T2C</th><th>BDG<br>(cut-off ≥80 pg/mL)</th></tr></thead><tbody><tr><td>Sensitivity</td><td>11%</td><td>33%</td><td>83%</td></tr><tr><td>Specificity</td><td>100%</td><td>93%</td><td>71%</td></tr><tr><td>PPV</td><td>100%</td><td>75%</td><td>65%</td></tr><tr><td>NPV</td><td>64%</td><td>68%</td><td>87%</td></tr></tbody></table>

PPV = positive predictive value, NPV = negative predictive value

**Conclusion:** T2C has higher sensitivity than blood cultures and higher specificity than BDG for IAC diagnosis. T2C and BDG are anticipated to be most useful in guiding management of patients at risk for IAC, in particular when they provide concordant results to confirm or exclude IAC in cases for which intra-abdominal cultures are not feasible or delayed.
P158

Performances of the GenMark's ePlex® blood culture identification fungal pathogen (BCID-FP) panel: a prospective French evaluation

A.-A. Nawel¹, F. Foulet¹, C. Angebault¹, S. Suyyagh¹, D. Maubon², F. Botterel¹

¹Parasitologie-mycologie, CHU Henri MONDOR, Créteil, France, ²Univ. Grenoble Alpes, CNRS, CHU Grenoble Alpes, Grenoble INP*, TIMC-IMAG, 38000 Grenoble, France *Institute of Engineering Univ. Grenoble Alpes, GRENOBLE Cedex, France

**Objectives:** Fungemia is associated with high rate of morbidity increasing length of hospital stay and mortality. Delay of effective antifungal treatment is associated with unfavorable outcome and rapid species identification contributes to adapt antifungal therapy. ePlex® Blood Culture Identification fungal pathogen panel (ePlex® BCID-FP) developed by GenMark® Diagnostics, Inc. (Carlsbad, CA, USA) is a fully automated easy-to-use cartridge designed to detect 15 fungal pathogens from positive blood culture (BC) including the emerging pathogen *Candida auris*. The objective of this study was to evaluate prospectively the performance of ePlex® BCID-FP test using clinical BC samples.

**Methods:** The evaluation was performed prospectively on consecutive fungal BC over 2 years-period (from April 2017 to Mars 2019) in Henri Mondor University Hospital in France. BCs taken from patients were incubated in BacT/ALERT® 3D automated system (Biomérieux®). Once detected positive for fungi, the BC underwent classical subcultures. Then, species identification was performed on MALDI-TOF MS Andromas® system (identification within 24h). In parallel to this conventional procedure, ePlex® BCID-FP was tested (hands-on-time < 5 min and time-to-result: 1.5h).

**Results:** A total of 58 BC, corresponding to 52 patients, were tested on ePlex® using the BCID-FP. Invalid results due to technical issues were obtained in 10/58 tests (17.2%). Among the 48 valid tests, results were fully concordant with classical identification process in 45/48 BC isolated species (94%) including one fungemia caused by two species with *C. tropicalis* and *C. lusitaniae*. Identifications were available the day of BC positivity in less than two hours. This technique saved at least 24 hours compared to conventional methods. The non-concordant results consisted on one *C. parapsilosis* missed by ePlex and two species absent from the BCID-FP panel (1 *C. metapsilosis* and 1 *Saccharomyces cerevisiae*). Species distribution on valid tests was: 19 *C. albicans* (41%), 12 *C. parapsilosis* (26%), 8 *C. glabrata* (18%) and 7 others (*C. krusei* (n = 3), *C. tropicalis* (n = 2), *C. dublininiensis* (n = 1) and *C. lusitaniae* (n = 1) (15%). Thus, fluconazole could be introduced in first intention in 76% of cases, saving the use of caspofungin.

**Conclusion:** To our knowledge, this is the first large prospective evaluation of the ePlex® BCID-FP on more than 50 fungal BC. ePlex® BCID-FP accurately identified 78% of the fungal species from positive BC. Focusing on targets which are currently present in the panel, 100% of correct identification was achieved. A prospective clinical study evaluating the time-to-result benefit on antifungal stewardship and on length of hospital stay is needed.

P159

Integrating miRNA and mRNA expression profiles for improving diagnosis of invasive aspergillosis

T. Zoran¹,², B. Seelbinder¹, P. Sieber¹, S. Schaeuble¹, J. Loeffler²

¹Pidomics, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany, ²Department Of Internal Medicine Ii, WÜ4i, University Hospital Wuerzburg, Wuerzburg, Germany

**Objectives:** Invasive aspergillosis (IA) is a life-threatening disease affecting immunocompromised patients. Due to unspecific symptoms of this disease and limitation of current methods based on fungal biomarkers, diagnosis of IA remains challenging. MicroRNAs (miRNAs) are small non-coding RNAs involved in the post-transcriptional regulation of several biological processes including various disease pathways and immune response to numerous pathogens. Due to their involvement in gene regulation, their stability and ubiquitous presence in biofluids they are considered as good biomarkers for different diseases. The aim of our *in vitro* study was to investigate miRNA profiles in immune cells following fungal and bacterial exposure and identify potential miRNAs and their target
genes that may be useful as diagnostic and prognostic human biomarkers for improving IA diagnosis.

**Methods:** We used next generation sequencing (NGS) to investigate miRNA profiles in monocyte-derived dendritic cells (moDCs) generated from healthy volunteers. Immune cells were stimulated for 12 hours with inactivated *Aspergillus fumigatus* germ tubes and a clinical isolate of *Escherichia coli*. Next, we evaluated transcriptome by RNA-sequencing of moDCs infected with *E. coli* and *A. fumigatus* and investigated *in silico* interaction between differentially expressed miRNAs (DEMs) and their targets. After correlation analysis and prediction of interactions with mRNA, selected miRNA and their immunological relevant target genes were further experimentally investigated.

**Results:** Our data showed 32 significantly DEMs in moDCs stimulated with fungal and bacterial pathogens in comparison to normal expression in unstimulated moDCs. 28 miRNAs were differentially regulated after infection with *A. fumigatus* and 8 miRNAs after infection with *E. coli*. We identified 24 miRNAs significantly differentially regulated in moDCs after fungal but not bacterial infection. Among all significantly DEMs in stimulated moDCs, the following four miRNAs were observed to be differentially expressed after infection with *A. fumigatus* and *E. coli*: hsa-miR-9-5p, hsa-miR-155-5p, hsa-miR-155-3p and hsa-miR-1303. Based on two DEMs specific to *A. fumigatus* exposure and one DEM identified in moDCs infected with *A. fumigatus* as well as *E. coli*, we further investigated potential targets that had been predicted by investigating transcriptome data. Integration of hsa-miR-9-5p and gene expression showed 45 common differentially expressed (DE) target genes in both types of infection. Furthermore, our data showed 26 unique DE target genes in moDCs after exposure to *A. fumigatus* and 13 in moDCs infected with *E. coli* (n = 13). We validated the expression of three selected DEMs and their selected immunological relevant target genes in additional donors with Real-Time PCR, where we confirmed our observations from NGS data.

**Conclusion:** Our data showed distinctive miRNA profiles between uninfected moDCs as well as moDCs after exposure to *A. fumigatus* and *E. coli*, suggesting miRNAs profiles can be used to detect and distinguish bacterial from fungal infection. Furthermore, characteristic miRNA and mRNA expression profiles may be used as a diagnostic and prognostic biomarker of IA. Further experimental validation is necessary to confirm predicted miRNA-mRNA interactions and investigate the potential use of miRNA and mRNA expression profiles for IA diagnosis.

P160

**The Fungal PCR Initiative's evaluation of in-house and commercial *Pneumocystis jirovecii* qPCR assays: towards a standard for a diagnostics assay**

M. Gits-Muselli¹,², P. White³, C. Mengoli⁴, S. Chen⁵, B. Crowley⁶, G. Dingemans⁷, E. Frealle⁸, R. Gorton⁹, M. Guiver¹⁰, F. Hagen¹¹, C. Halliday¹², G. Johnson¹³, K. Lagrou¹⁴, M. Lengerova¹⁵, W. Melchers¹⁶, L. Novak-Frazer¹⁷, R. Rautemaa-Richardson¹⁸,¹⁹, E. Scherer²⁰, J. Steinmann²¹,²², M. Cruciani²³, R. Barnes²⁴, J.P. Donnelly²⁵, J. Loeffler²⁶, S. Bretagne²⁷, A. Alanio²⁷

¹Molecular Mycology, Institut Pasteur, PARIS, France, ²Parasitology-mycology Laboratory, Lariboisière Saint-Louis Fernand Widal Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), PARIS, France, ³Microbiology, Public Health Wales Microbiology, Cardiff, United Kingdom, ⁴Molecular Medicine, University of Padova, Padova, Italy, ⁵Medicine, The University of Sydney, Camperdown, New South Wales, Australia, ⁶Department Of Virology, St James's Hospital, Dublin, Ireland, Dublin, Ireland, ⁷PathoNostics, Maastricht, Netherlands, ⁸Parasitology-mycology, CHU Lille, Lille, France, ⁹Infection Sciences, Health Services Laboratories, London, United Kingdom, ¹⁰Public Health Laboratory, National Infection Service Public Health England, Manchester University NHS Foundation Trust, Manchester, UK, Manchester, United Kingdom, ¹¹Medical Mycology, Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands, ¹²Clinical Mycology Reference Laboratory, Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, New South Wales Health Pathology, Westmead Hospital, and the University of Sydney, Australia, Sydney, Australia, ¹³Scientific Development, OLM diagnostics, Newcastle, United Kingdom, ¹⁴Laboratory Of Clinical Bacteriology And Mycology, Department Of Microbiology And Immunology, Excellence Center For Medical Mycology (ecmm), KU Leuven, Leuven, Belgium,
¹⁵Department Of Internal Medicine - Hematology And Oncology, University Hospital Brno, Brno, Czech Republic, ¹⁶Medical Microbiology, RadboudUMC, Nijmegen, Netherlands, ¹⁷Mycology Reference Centre, Excellence Center For Medical Mycology (ecmm), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom, ¹⁸Division Of Infection, Immunity And Respiratory Medicine, University of Manchester, Manchester, United Kingdom, ¹⁹Manchester Academic Health Science Centre, Manchester, United Kingdom, ²⁰Parasitology - Mycology - Umr6249 Cnrs Chrono-environnement, University Hospital Besançon, Besançon, France, ²¹Institute Of Clinical Hygiene, Medical Microbiology And Clinical Infectiology, Klinikum Nuernberg, Nuremberg, Germany, ²²Institute Of Medical Microbiology, University Hospital Essen, Essen, Germany, ²³Infectious Diseases, San Bonifacio Hospital, Verona, VERONA, Italy, ²⁴Medical Microbiology And Infectious Diseases, Cardiff University School of Medicine, Cardiff, United Kingdom, ²⁵EAPCRI, Nijmegen, Netherlands, ²⁶Department Of Internal Medicine Ii, WÜ4i, University Hospital Wuerzburg, Wuerzburg, Germany, ²⁷Paris-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:** Quantitative real-time PCR (qPCR) is increasingly used to detect *Pneumocystis jirovecii* for the diagnosis of pneumocystis pneumonia (PCP). PCP is more severe and mortality rates are significantly higher in non-HIV patients than in HIV-infected/AIDS people, despite a lower fungal load during infection. Currently, definite thresholds for Pneumocystis pneumonia (PCP) in non-HIV patients have not been established to be consensual and universally used in different diagnostic laboratories. Therefore, diagnosis of PCP remains a challenge because of the importance of quantification of the low fungal loads. An evaluation of the performance of different qPCR assays is needed to assist laboratory standardization of quantification.

**Methods:** Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, one inter- and one intra-laboratory comparison of qPCR assays was performed. For the interlaboratory study, sixteen reference laboratories in eight countries running 19 assays analysed a panel consisting of two negative and three positive specimens from a pool of three different *P. jirovecii* positive bronchoalveolar lavage (BAL) fluids at three different dilutions (pure 1:1, 1:100 and 1:1000). Participants were asked to test the panel specimens as part of their routine molecular biology workflow. The results were collected using a dedicated Google form that included requests for the qualitative result (positive/negative), the quantification cycle (Cq) values for each replicate related to each specimen and specific information regarding the methods and workflows applied. The intra-laboratory study compared 10 different assays with the same person and qPCR thermocycler, by testing 10 previously confirmed *P. jirovecii* qPCR-negative and 9 previously confirmed *P. jirovecii* qPCR-positive BALs. Those 10 assays consisted of 5 in-house assays and 5 commercial kits and included 3 RT-qPCR (1 commercial and 2 in-house) and 7 qPCR assays (4 commercial and 3 in-house)

**Results:** from 20 qPCR assays (16 qPCR amplifying DNA only and 4 RT-qPCR assays amplifying Whole nucleic acids (WNA, i.e DNA+RNA) were obtained from 16 laboratories across eight countries (including seven European countries and one Australian). The different targets evaluated were mtSSU (n = 2), mtLSU (n = 16), MSG (n = 1) and beta-tubulin (Tub, n = 1). The inter-laboratory analytical sensitivity was 100% at 1:1 genomic load, 95% at 1:100, and 82.5% at 1:1000. Analytical specificity was 100% for all assays but yielded a false-positive test (95% specificity). For both evaluations and for all genomic loads, testing whole nucleic acid (RNA plus DNA) using reverse-transcriptase RT-qPCR was superior to qPCR (p≤0.001) testing DNA only. (Figure 1 and Figure 2). The target gene mitochondrial small sub-unit (mtSSU) was significantly more sensitive than the mitochondrial large subunit, the major surface glycoprotein, or the beta-tubulin (Figure 1 and Figure
2).

<figure><img src="image_52.png" /><figcaption>Figure 1: (Inter-Laboratory study) Cq variation for each assay regarding the target gene and the type of material amplified (DNA or WNA) in all the three positive panel samples at dilutions 1:1, 1:100, and 1:1000.</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>Figure 2: Cq variation of the 9 tested BAL by the 10 qPCR assays in the intra-laboratory study including all duplicates (different targets, mtSSU, mtLSU, MSG, DHPS, and nucleic acids, DNA vs. WNA).</figcaption></figure>

**Conclusion:** Thus, RT-qPCR targeting the mtSSU gene had the best sensitivity and could serve as a basis for standardizing the *P. jirovecii* load, which is essential if qPCR is to be incorporated into clinical care pathways.

P161

Identification of Cyp51A-mediated triazole resistance in *Aspergillus fumigatus* using pyrosequencing: an audit of UK National Aspergillosis Centre cases

L. Brown¹, R. Rautemaa-Richardson¹,²,³, L. Novak-Frazer¹,³, S. Hill¹,³, D. Hassan³, M.D. Richardson¹,³

¹Division Of Infection, Immunity And Respiratory Medicine, University of Manchester, Manchester, United Kingdom, ²National Aspergillosis Centre, Manchester University NHS Foundation Trust, Manchester, United Kingdom, ³Mycology Reference Centre, Excellence Center For Medical Mycology (ecmm), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom

**Objectives:** Globally, triazole resistance rates are rising. Azole-naïve patients acquiring an azole-resistant isolate from the environment, or because of long courses of azole treatment is a growing concern. TR34/L98H within the *cyp51A* gene of *A. fumigatus* leads to pan-triazole resistance and is the most common mutation found in environmental isolates. Other mutations in this gene have also been
associated with various patterns of azole resistance. Failure of microbiological testing to detect resistance delays the initiation of appropriate antifungal therapy and negatively impacts the clinical outcomes of patients with aspergillosis. Therefore, empiric combination therapies are increasingly used which has significant stewardship implications. We compared the performance of high-volume culture (HVC) and *Aspergillus fumigatus* PCR combined with pyrosequencing (AfPCR-Pyro) for the detection of triazole resistance in aspergillosis patients.

**Methods:** We performed a retrospective audit over a 27-month period (between January 2017 and March 2019) at the Mycology Reference Centre Manchester (MRCM) and the UK National Aspergillosis Centre (NAC), UK. Respiratory samples collected from patients with chronic pulmonary aspergillosis (CPA) or allergic bronchopulmonary aspergillosis (ABPA) and analysed by both HVC and AfPCR-Pyro were included in the study. Hospital and laboratory databases were searched for patient history, laboratory findings and if the pyrosequencing result triggered a change in antifungal treatment.

**Results:** Three hundred and forty-nine samples from 162 patients were analysed for susceptibility using AfPCR-Pyro. Of these 28% (96/349) were positive for *A. fumigatus* by HVC while 68% (236/349) were reported with no growth after 14 days. The remaining 3% (9/349) of samples were positive for other *Aspergillus* spp, but excluded from further analysis as pyrosequencing is limited to *A. fumigatus*. Resistance was found by pyrosequencing in 29% (99/340) of samples compared to 15% (52/340) by HVC. Resistance was found by both culture and pyrosequencing in 9% (30/340) of cases, with agreement in susceptibility profiles between the two methods in 66% (63/96) of cases. Resistance was identified by pyrosequencing in 23% (54/236) of culture negative samples. If pyrosequencing had not been used in these patients, 46 resistant cases would have been missed. Pyrosequencing identified the environmental TR34/L98H mutation in 20% (69/340) of samples. Importantly, resistance was recognised by pyrosequencing before HVC results in 17% (57/340) of cases. A change in antifungal therapy was initiated in 19% of cases (58/299) due to pyrosequencing results.

**Conclusion:** Compared to HVC, AfPCR-Pyro has a higher sensitivity for detecting triazole resistance. The technology allows for prompt recognition of resistant organisms and the selection of appropriate antifungal treatment. Increased availability of pyrosequencing would likely lead to improved patient outcomes and better antifungal stewardship.

P162

Evaluation of two mass spectrometry platforms, the Bruker MALDI Biotyper and the MSI online application, for the identification of clinically important moulds

L. Kristensen¹, J.B. Gertsen¹, T.M. Petersen¹, K. Handberg¹, A.C. Normand², M. Hendrickx³, N. Nørskov-Lauritsen¹

¹Department Of Clinical Microbiology, Aarhus University Hospital, Aarhus N, Denmark, ²Ap-hp, Groupe Hospitalier Pitié-salpêtrière, Service de Parasitologie Mycologie, Paris, France, ³Service Of Mycology And Aerobiology, Bccm/ihem Fungal Collection, Sciensano, Brussels, Belgium

**Objectives:** The matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) method has emerged as a rapid identification method of filamentous fungi. The aim of this study was to evaluate the performance of the Bruker MALDI Biotyper and the recently introduced MSI (mass spectrometry identification) platform to identify a collection of moulds, followed by an evaluation in the clinical setting.

**Methods:** Thirty-four well-characterised mould isolates (18 species from 10 genera) were selected from our collection of fungal strains. For the subsequent evaluation, 75 clinical isolates were prospectively collected. *Aspergillus fumigatus* and *Aspergillus flavus* with typical morphology were excluded. The isolates were cultured 2-3 days on Sabouraud glucose agar (Oxoid). Prior to MS analysis, a simple ethanol-formic acid-acetonitrile inactivation/extraction procedure was used. Acquisition of mass spectra was performed with the Bruker MALDI Biotyper, software v. 3.1. Data analysis was performed using the general Bruker library (MBT 7311 MSP) combined with the Bruker Fungi Library 1.0, and with the MSI online library after uploading of spectra. In the initial assessment,
identification thresholds of 2.0 and 1.7 for species and genus, respectively, were used. The data were
compared to a species identification threshold of 1.7. Based on these results the 1.7 threshold for
species identification was applied in the subsequent evaluation.

For MSI online identification, a species identification threshold of 20 was used. Identification by
sequencing of internal transcribed spacer (ITS2) was used as gold standard.

**Results: Initial evaluation**

ITS sequence identified 25 isolates to the species level and nine isolates to the species complex level (insufficient separation of closely related species).

Bruker (species threshold 2.0): 22 of 34 isolates (65%) were correctly identified to the species or species complex level, six isolates (18%) were correctly identified to the genus level, six isolates (18%) were not identified.

Bruker (species threshold 1.7): 28 of 34 isolates (82%) were correctly identified to the species or species complex level while six isolates (18%) were not identified.

MSI: 31 of 34 isolates (91%) were correctly identified to the species or species complex level. One *Fusarium oxysporum* was misidentified as *Fusarium acutatum*, two isolates (6%) were not identified.

**Clinical evaluation**

The clinical isolates comprised 19 species from 11 genera.

ITS sequence identified 55 isolates to the species level and 20 isolates to the species complex level.

Bruker: 60 of 75 isolates (80%) were correctly identified to the species or species complex level. Fifteen isolates (20%) were not identified.

MSI: 70/75 isolates (93%) were correctly identified to the species or species complex level. Five isolates (7%) were not identified.

**Conclusion:** Both the Bruker library (species identification threshold 1.7) and the MSI platform were accurate and reliable in the initial evaluation. The results were confirmed in the clinical setting, however, compared to Bruker the MSI library obtained the highest degree of identification to the species or species complex level (93%) with a considerably lower number of unidentified isolates.

P163

**Rapid identification of a rare fungi: A skillful mycologist against the state of the art methods**

C. Gronfors Seeth, K. Gieng Axelov, M. Ullberg, V. Özenci

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

**Case Report:** Modern diagnostics methods in clinical mycology have become widely available. However, it is important to acknowledge that these methods cannot replace the conventional methods requiring theoretical knowledge and hands on experience. The patient was 52 y old, female with a history of cystic fibrosis. Previous sputum samples showed growth of Staphylococcus aureus and Pseudomonas aeruginosa. On 16 April 2019 the patient contacted cystic fibrosis clinic and presented decreased lung capacity. The Forced expiratory volume in one second (FEV1) was 53% expected value. Sputum sample was taken for both bacterial and fungal culture. Bacterial cultures showed growth of Pseudomonas aeruginosa on 17 April 2019. Growth of mold was observed after 6 days on Sabouraud agar at 30 C. Microscopy from agar plates showed hyphi without septa. In combination with the presence of abundant and relatively rapid growth the preliminary laboratory diagnosis was reported as Zygomycetes spp. on 23 April 2019. One day later new microscopy from the plates showed the presence of spherical-, multi-spored- sporangia without columellae. The laboratory technician who had never seen a similar morphology reported the isolate preliminary as *Mortierella* spp. The report was solely based on her theoretical knowledge in combination with clinical experience. The isolate could not be diagnosed by repetitive MALDI-TOF MS tests and was sent to internal transcribed spacer (ITS) sequencing. The results from the ITS sequencing showed that the isolate was Umbelopsis isabellina (99%) on 2 May 2019. The literature search showed that Umbelopsis isabellina is used as synonym for *Mortierella* spp. Mortierella spp. is a mold belonging to the order Mortierellales according to the most recent taxonomy and is considered a pathogen solely of animals. To our knowledge, there has been only one other case report describing an invasive disease in an immunocompromised patient with Mortierella wolffii, isolated from the liver biopsy and blood
culture (Layios et al., 2014). In that case the isolate was identified by ITS sequencing. Therefore the present case is the first describing the preliminary identification of *Mortierella* spp. with a conventional method. It is important to note that the conventional method could identify *Mortierella* spp. seven days earlier than ITS sequencing. It is still not clear if the present case with *Mortierella* spp. is colonization or an infection. The patient is currently being followed up clinically and further lower respiratory samples will be taken up. In the laboratory it feels comfortable to have access to both conventional and modern diagnostic methods. The present study describes the importance of hands on experience in diagnosis of rare fungal pathogens.

P164

**An inter-laboratory multi-centre evaluation of the performance of Mucorales PCR assays when testing serum specimens: A study by the ISHAM Fungal PCR Initiative and the ModiMucor study group**

**FPCRI Mucor Laboratory Working group (A. Alanio, M. Cogliati, S. Fuchs, F. Hagen, C. Halliday, R. Hare, C. Klaassen, M. Lackner M. Lengerova, W. Posch, B. Sendid, J. Springer, B. Willinger)**

**ModiMucor study group (F. Botterel, M. E. Bougnoux, S. Bretagne, C. Damiani, F. Dalle, J. Denis, M. Gitts-Muselli, X. Iriart, F. Morio, P. Poirier, E. Scherer)**

S. Rocchi¹, C. Mengoli², P. White³, R. Barnes⁴, J.P. Donnelly⁵, J. Loeffler⁶, L. Millon¹

¹Parasitology - Mycology - Umr6249 Cnrs Chrono-environnement, University Hospital Besançon, Besançon, France, ²Molecular Medicine, University of Padova, Padova, Italy, ³Microbiology, Public Health Wales Microbiology, Cardiff, United Kingdom, ⁴Medical Microbiology And Infectious Diseases, Cardiff University School of Medicine, Cardiff, United Kingdom, ⁵Division Of Infectious Diseases, San Antonio Center for Medical Mycology, San Antonio, United States of America, ⁶Department Of Internal Medicine Ii, WÜ4i, University Hospital Wuerzburg, Wuerzburg, Germany

**Objectives:** Real-time qPCR detection of Mucorales DNA in serum, plasma and BAL fluid has been shown to be sensitive and early tool for diagnosing mucormycosis. Several qPCR assays have been developed, but little comparison and no standardization is available. In 2016, the Mucorales Laboratory Working Group of the ISHAM Fungal PCR Initiative (FPCRI) organised inter-laboratory evaluations of Mucorales PCR assays currently in use, with the objectives of: 1) determining the uniformity of qualitative detection (positive/negative) and 2) assessing qPCR performance. Participants were members of the FPCRI, laboratories involved in the ModiMucor* study and three other French laboratories which have already implemented this tool to diagnose mucormycosis.

**Methods:** For the 1st panel (A), four sera (2ml/sample) were inoculated with genomic DNA (between 27 to 116 pg of DNA/mL of serum) from four different mucorales species (*Rhizomucor pusillus*, *Lichtheimia corymbifera*, *Cunninghamella bertholetiae*, *Rhizopus oryzae*). For the 2nd panel (B), six sera (2 ml/sample) were inoculated with three concentrations of *R. pusillus* and *L. corymbifera* equivalent to one, 10 and 100 genomes/mL of serum. For each panel, a negative control serum was also sent. Twenty laboratories analysed the panel A (10 FPCRI, two Modimucor/FPCRI, seven Modimucor, three others French laboratories) and 22 laboratories analysed the panel B (two more FPCRI laboratories). All participants were requested to use their own DNA extraction methodology. The 12 French participants used the same qPCR technique (combination of three qPCR assays targeting the four most frequent genera: *Lichtheimia*, *Mucor*, *Rhizopus*, *Rhizomucor*) (1). For panel B, only quantitative PCR results were analysed (two techniques that used conventional PCR were excluded). Twenty-two laboratories returned multiple datasets, leading to 26 different datasets for analysis: - 16 datasets using a single technique (1) - 10 datasets using other techniques (Four Pathonostics MucorGenius®, Six "in-house" assays, (2-5), three of which were not previously described).

**Results:** For panel A, all datasets were negative when testing the negative control serum. Conversely, 85-90% of the sera inoculated with "common" Mucorales (*R. pusillus*, *R. oryzae*, *L. corymbifera*) were positive. Detection of sera containing *C. bertholetiae* DNA was lower (12%). For panel B, the probability of qPCR positivity increased predictably with DNA quantity, generating a mean PCR efficiency of 0.85. Detection of *Lichtheimia* DNA was optimal, irrespective of the technique. Regarding the qPCR systems, Millon et al. and MucorGenius® techniques generated greater positivity rates, with earlier Cq values.
**Conclusion:** Despite the diversity of techniques, Mucorales DNA detection in sera was very reproducible with little inter-laboratory variability providing support for including Mucorales PCR in the EORTC/MSG definitions of invasive fungal disease and for its use in the clinical diagnosis of mucormycosis *ModiMucor*\*: *French prospective multicenter study for evaluation of Mucorales PCR (PHRC (Projet Hospitalier de Recherche Clinique) national-Modimucor 2014-A00580-47.)* **References** 1- Millon et al. CID 2013 2- Springer et al. J. Med. Microbiol. 2016 3- Lengerova et al. J Clin Microbiol 2014 4- Machouart et al. J Clin Microbiol 2006 5- Hrncirova K., J Clin Microbiol 2010

P165

**Comparison of a point-of-care (PreventID®) with the DermaGenius® Nail real-time PCR kit for the detection of dermatophytes species in nail samples**

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

PathoNostics, Maastricht, Netherlands

**Objectives:** Compare the performance of a point-of-care test for the detection of dermatophyte fungi (PreventID®) with the DermaGenius® 2.0 Nail real-time multiplex PCR assay on a set of clinical nails. Both methods are compared to microscopy as the gold standard method.

**Methods:** Left over nail samples were split into different parts. The culture and microscopy of all nail samples was performed by an external clinical microbiology laboratory. One part of the nail sample was used for DNA-extraction with the PathoNostics Extraction kit and identification of the pathogen was enabled by using the DermaGenius® 2.0 Nail real-time multiplex PCR. Finally, an immunochromatographic point-of-care test (PreventID® Dermatophyte) was applied to determine the presence of dermatophyte-derived antigens in the nails.

**Results:** Microscopy of the nails was considered as the gold standard. The DermaGenius® 2.0 Nail real-time multiplex PCR was able to detect all positive samples, resulting in a sensitivity of 100%. Specificity was however lower (83%) due to detection of dermatophyte DNA in microscopy-negative samples. Interestingly, these samples were culture-positive. Culture resulted both in a reduced specificity (67%) and sensitivity (57%). Both the sensitivity (79%) and specificity (83%) for the PreventID® Dermatophyte test were promising. Although the PCR assay is sensitive, the clinical relevance is still a matter of debate. High Ct-values indicate limited amounts of DNA and do not always represent a clear infection. Interestingly, the Ct-values for samples which were negative with the PreventID® test were between 26-33 with the DermaGenius® real-time PCR. In addition, readability of the test strips was limited in some samples while Ct-values of 22-28 with the DermaGenius® PCR was obtained, indicating a high fungal load. These samples were also positive with microscopy.

**Conclusion:** Microscopy and the DermaGenius® 2.0 Nail real-time PCR showed a good correlation and are useful for rapid identification of dermatophyte infection. For specific pathogen identification, the DermaGenius® PCR can be used. Culture can result in false negatives and in combination with a long turnaround time, it has limited value in routine diagnostics. The use of an immunochromatographic point-of-care test is uncommon in dermatophyte diagnostics but was included in this study to determine its suitability. Although the PreventID® Dermatophyte test results in a sensitivity of 79%, some dermatophyte infections with low Ct-values were missed. The difference in sensitivity could be explained by the fact that the PreventID® test is using a protein target while the DermaGenius® PCR detects DNA, which is normally not influenced by treatment. But more important is that the interpretation of the PreventID® test is based on visual inspection which makes the test less reliable. The PreventID® should be compared in a larger sample set to determine suitability for routine dermatophyte diagnostics. Currently, microscopy and the DermaGenius® 2.0 Nail real-time PCR seem to be the most reliable and suitable methods for diagnosis of dermatophyte infections in nails.

P166

**Polymerase chain reaction on respiratory tract specimens of immunocompromised patients to diagnose Pneumocystosis - A systematic review**
L. Brown¹, R. Rautemaa-Richardson¹,², E. Rowbotham¹, S. Chen³, B. Jones⁴,⁵

¹Division Of Infection, Immunity And Respiratory Medicine, University of Manchester, Manchester, United Kingdom, ²Mycology Reference Centre, Excellence Center For Medical Mycology (ecmm), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom, ³Medicine, The University of Sydney, Camperdown, New South Wales, Australia, ⁴NHS Greater Glasgow and Clyde Health Board, Glasgow, United Kingdom, ⁵Institute Of Infection Immunity & Inflammation, University of Glasgow, Glasgow, United Kingdom

**Objectives:** *Pneumocystis jirovecii* pneumonia (PCP) is a severe, life-threatening infection affecting immunocompromised patients. The highest rates are seen in those with HIV infection, but other groups are increasingly affected such as solid organ recipients and those receiving long-term corticosteroid therapy. Polymerase Chain Reaction (PCR) is highly sensitive for the detection of *P. jirovecii* when compared to microscopy, but may not differentiate between infection and colonisation. The Fungal PCR Initiative group (FPCRI) working group of the international Society for Human and Animal Mycology (ISHAM) aims to develop international standards for *P. jirovecii* PCR. A systematic review to determine the diagnostic accuracy of PCR testing in respiratory tract specimens for PCP was undertaken.

**Methods:** Following protocol registration with PROSPERO, a systematic review of the literature was performed using PubMed, Scopus, Embase and Cochrane without applying any language or date limits. Studies where PCR techniques were used on respiratory specimens for the diagnosis of PCP in humans were included if they (i) compared the PCR test results with the diagnosis made using standard laboratory methods and clinical presentation; (ii) sufficient information was provided to assess the strength of diagnosis; (iii) results were reported as false-positive, true-positive, false-negative and true-negative; and (iv) evaluation of the test(s) was performed in prospective cohorts of patient populations, defined as groups of individuals at high risk for PCP. Where relevant, the diagnostic performance of PCR techniques to the beta-d-glucan (BDG) assay, was compared. Breakpoint analyses for differentiating colonisation from infection will also be investigated.

**Results:** A total of 2843 unique records were identified. Abstracts of these records were sent out to six pairs of reviewers to be assessed against the inclusion and exclusion criteria. The reviewers were in agreement on 2371 abstracts; 472 abstracts with reviewer discrepancies were sent out for a third review. After screening, 2509 records were excluded and 334 full-text articles are now being assessed for eligibility by pairs of reviewers. Papers meeting the inclusion criteria after reading in full will be included in the meta-analysis to be carried out by the group. Risk of bias will be assessed using the QUADAS-2 tool.

**Conclusion:** This systematic review will provide essential information about the diagnostic accuracy of PCR tests in respiratory tract specimens for PCP required for the development of a standardised method for *Pneumocystis* PCR which will be later validated in clinical trials.

P167

**Evaluation of the LDBio Aspergillus ICT lateral flow assay as a point-of-care test for serological antibody detection in allergic bronchopulmonary aspergillosis**

E. Hunter¹, M.D. Richardson¹,², D. Denning¹,³

¹Division Of Infection, Immunity, And Respiratory Medicine, University of Manchester, Manchester, United Kingdom, ²Mycology Reference Centre, Excellence Center For Medical Mycology (ecmm), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom, ³National Aspergillosis Centre, Manchester University NHS Foundation Trust, Manchester, United Kingdom

**Objectives:** Allergic bronchopulmonary aspergillosis (ABPA) is an immunological pulmonary disorder complicating asthma, as a result of hypersensitization to *Aspergillus* species (predominantly *A. fumigatus*). It is estimated to affect 2.5% of adults with asthma, which accounts for approximately 4.8 million patients worldwide. Early recognition and diagnosis of ABPA is critical to improve patient symptoms and prevent or delay progression of bronchiectasis and development of chronic pulmonary aspergillosis (CPA). It is especially important to make the distinction between asthma
and *Aspergillus* sensitization. LDBio Diagnostics has recently commercialized a lateral flow assay (*Aspergillus* ICT) that detects *Aspergillus* antibodies less than 30 minutes, requiring minimal laboratory equipment. Herein, we evaluate this assay for specific diagnosis of ABPA as compared to asthma controls.

**Methods:** A retrospective, ongoing study was performed using 94 patient sera collected at the National Aspergillosis Centre (NAC, Manchester). Patient sera were selected based on a clinical diagnosis of ABPA and classified as 'proven' (n = 70) or 'probable' (n = 24). Control sera (n = 93) were obtained from the Manchester Allergy, Respiratory and Thoracic Surgery (ManARTS) biobank. LDBio *Aspergillus* ICT was performed as per manufacturer's instruction. Results were read at intervals, and determined after 20 to 30 minutes as recommended (any line at the "T" marker considered positive), both manually and using the Qiagen ESEQuant LR3 lateral flow reader. Serological *Aspergillus*-specific IgG and IgE, and total IgE titres were measured by ImmunoCAP.

**Results:** We found the LDBIO *Aspergillus* ICT to have a sensitivity of 88.6% across for 70 proven ABPA cases, and 89.4% for all cases ('proven' and 'probable') combined. Specificity was 91.4% for 93 control sera (Table 1). A comparison of ICT result with *Aspergillus*-specific IgG and IgE titres showed no evident immunoglobulin isotype bias (Figure 1). Using digital measurements obtained using the Qiagen LR3, we found no correlation between ImmunoCAP *Aspergillus*-specific IgE level and *Aspergillus* ICT test line intensity. Finally, assessment of 3 assay lots using 30 samples showed perfect agreement between lots when tests were read visually.

<table><thead><tr><th></th><th>(n)</th><th>ICT + (n)</th><th>Sensitivity (%[95% CI])</th><th>Specificity (%[95% CI])</th><th>Accuracy (%[95% CI])</th><th>DOR</th></tr></thead><tbody><tr><td>ABPA (proven)</td><td>70</td><td>62</td><td>88.6 (78.7, 94.9)</td><td>---</td><td>90.2 (84.6, 94.3)</td><td>82.34</td></tr><tr><td>ABPA (all)*</td><td>94</td><td>84</td><td>89.4 (81.3, 94.8)</td><td>---</td><td>90.4 (85.2, 94.2)</td><td>89.25</td></tr><tr><td>Asthma (control)</td><td>93</td><td>85</td><td>---</td><td>91.4 (83.8, 96.2)</td><td>---</td><td>---</td></tr></tbody></table>

*proven + probable

<figure><img src="image_54.png" /></figure>

**Conclusion:** The LDBio *Aspergillus* ICT lateral flow assay exhibits good sensitivity for serological screening for ABPA. The assay is easy to perform and interpret, using minimal equipment and resources. As such, it provides a valuable point-of-care screening resource to rapidly distinguish between asthma and *Aspergillus* sensitization.
United Kingdom, ²National Aspergillosis Centre, Manchester University NHS Foundation Trust, Manchester, United Kingdom, ³Mycology Reference Centre, Excellence Center For Medical Mycology (ecmm), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom

**Objectives:** Prevalence of chronic pulmonary aspergillosis (CPA) is estimated at approximately 3 million cases worldwide, and serological detection of *Aspergillus*-specific antibody is a critical component in the CPA diagnostic pathway. Some patients with CPA, however, have subtle immune deficits, including low CD4 or CD19 (B cell) counts, mannose binding lectin deficiency and/or low gamma IFN or IL12 production. It is possible these deficits contribute to poor *Aspergillus* antibody production to one or more ordinarily immunodominant antigens, and may lead to false negative results by diagnostic assays used to detect *Aspergillus*-specific antibodies. Herein, we examine the potential effect of patient immunodeficiencies on the accuracy and performance of CPA serological diagnostics.

**Methods:** We analyzed patient data, as available, from 167 cases of clinically confirmed CPA that were previously evaluated by ImmunoCAP *Aspergillus*-specific IgG EIA and LDBio *Aspergillus* ICT lateral flow assay, to determine the presence of deficiencies in the following: mannose binding lectin (MBL), IgG, IgA, IgM, IL12 production, IL17 production, and/or cell markers (CD4, CD19, CD56). We compared patient data with test results (ImmunoCAP, ICT) as well as for ImmunoCAP 'seronegative' patients -- defined as cases for which ImmunoCAP *Aspergillus* IgG was consistently and repeatedly positive for up to 3 years of patient history. Seronegative cases accounted for 12.6% of all cases evaluated (n = 21). 'Seropositive' was defined as all other CPA cases (n = 146).

**Results:** We found the rate of false negatives by ImmunoCAP *Aspergillus* IgG EIA to be more prevalent in patients with low levels of IgG, IgA, or IgM. The performance LDBio *Aspergillus* ICT appears to relatively unaffected by these (Figure 1). Similar findings were observed for IFN gamma production. Additionally, though not significant, we observed a trend in lower levels of CD4, CD19, or CD56 cell markers amongst seronegative CPA patients as compared to seropositive CPA patients, according to ImmunoCAP results (Figure 2).

<figure><img src="image_55.png" /></figure>
<figure><img src="image_56.png" /></figure>

**Conclusion:** In select cases of CPA, ImmunoCAP EIA yields a false negative result, making serological diagnosis difficult and requiring increased diagnostic sensitivity. Our previous findings demonstrated that LDBio *Aspergillus* ICT show greatly improved sensitivity and ability to detect *Aspergillus*-specific antibody in patient sera, especially for ImmunoCAP IgG-negative ('seronegative') cases. Our analysis of patient immunodeficiencies reveals that ImmunoCAP false negatives are more prevalent in patients with certain immunodeficiencies, and that diagnostic performance of ImmunoCAP *Aspergillus*-specific IgG EIA may be more adversely affected by these patient conditions, as compared to LDBio *Aspergillus* ICT.

P169

**The diagnostic value of a molecular assay for Fusarium infections recently introduced in the**
**Danish national reference laboratory**

R. Hare¹, N. Abou-Chakra¹, R. Datcu¹, M.C. Arendrup¹,²,³

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

**Objectives:** *Fusarium* keratitis and other infections remain difficult to diagnose even in the modern mycology laboratory. Molecular methods have enabled genus and species specific detection independent of culture and have improved diagnostic speed, sensitivity and accuracy of fungal infections. A specific diagnostic assay for *Fusarium* infections has been introduced in the Danish mycology reference laboratory and this study sought to evaluate the clinical performance.

**Methods:** Clinical samples from patients suspected for fusariosis were subjected to *Fusarium* PCR (detecting *Fusarium solani*, *Fusarium dimerum* and *Fusarium oxysporum*) during the period October-2017 to April-2019. The real-time multiplex *Fusarium* PCR assay using Taqman probes was slightly modified from that described by Salehi et al. 2016 (Pubmed ID 27605714). Patient samples were DNA extracted using the Qiacube (Qiagen, Denmark) standard protocol for blood and tissue samples, with up to 200 µL input volume and elution in 100 µL. Bronchoalveolar lavage samples were processed on the NucliSENS easyMAG (Biomérieux, Denmark) DNA extraction platform with up to 1 mL input volume and elution in 60 µL. Specimens were subjected to PCR in combination with culture and Blankophor microscopy, except for one patient (PCR only) due to insufficient amount of material. PCR was performed on the Applied Biosystems 7500 or the Quantstudio 5 thermocyclers (Thermo Fisher Scientific, Denmark). Positive and negative template controls were applied but not PCR inhibition or DNA extraction controls. PCR results were compared for each patient to our other
laboratory results and to results from the Danish Microbiological Database (MiBa), which captures clinical microbiological results from every laboratory in the entire country.

**Results:** In total, 71 samples from 34 patients, among which 50 samples were from 29 patients with suspected keratitis, were included. Five patients (14.7%) accounted for 19/33 (57.6%) samples positive for *Fusarium* spp. DNA (Table). Four PCR positive patients provided 12/32 (37.5%) samples microscopy positive (septate hyphae) and 10/32 (31.3%) culture positive with *Fusarium* species (Table). The fifth PCR positive patient, was culture negative, but clinically diagnosed with a fungal keratitis and responded to targeted antifungal treatment. The remaining 29 patients provided 38 PCR negative samples. The majority of those patients (18/29) had culture positive samples for other potential culprits (clarification pending) and none were culture and microscopy positive for *Fusarium*.

<table><caption>Table. Patient and samples, diagnostic results</caption><thead><tr><th>Patient ID<br>(no. of samples)</th><th>Clinical suspicion</th><th>Specimen type<br>chronological</th><th>Fusarium PCR<br>results</th><th>Microscopy<br>+ few, ++ some, +++ many</th><th>CULTURE</th><th>COMPARISON</th></tr></thead><tbody><tr><td>A (N=1)</td><td>Keratitis</td><td>Cornea</td><td>F. solani*</td><td>Negative</td><td>Negative</td><td>PCR+ / microscopy- / culture-</td></tr><tr><td rowspan="10">B (N=16)</td><td rowspan="10">Invasive fusariosis</td><td>Maxillary sinus</td><td>F. dimerum</td><td>Negative</td><td>F. dimerum</td><td rowspan="10">PCR: 12/16 positive<br>Microscopy: 8/15 positive<br>Culture: 6/15 positive</td></tr><tr><td>Ethmoid</td><td>F. dimerum</td><td>Negative</td><td>Negative</td></tr><tr><td>Meatus medius</td><td>F. dimerum</td><td>septate hyphae +++</td><td>F. dimerum</td></tr><tr><td>Concha media</td><td>F. dimerum</td><td>septate hyphae +++</td><td>F. dimerum</td></tr><tr><td>Meatus inferior</td><td>F. dimerum</td><td>septate hyphae ++</td><td>F. dimerum</td></tr><tr><td>Biopsy gingiva</td><td>F. dimerum</td><td>septate hyphae ++</td><td>F. dimerum</td></tr><tr><td>Biopsy septum</td><td>F. dimerum</td><td>septate hyphae ++</td><td>F. dimerum</td></tr><tr><td>Spinal fluid</td><td>F. dimerum</td><td>Not done</td><td>Not done</td></tr><tr><td>Spinal fluid</td><td>Negative</td><td>Negative</td><td>Negative</td></tr><tr><td>Biopsy crus</td><td>F. dimerum</td><td>septate hyphae +</td><td>Negative</td></tr><tr><td>Pus from crus</td><td>F. dimerum</td><td>septate hyphae +++</td><td>Negative</td></tr><tr><td>Biopsy crus</td><td>F. dimerum</td><td>septate hyphae +++</td><td>Negative</td></tr><tr><td>Biopsy crus</td><td>F. dimerum</td><td>Negative</td><td>Negative</td></tr><tr><td>Spinal fluid</td><td>Negative</td><td>Negative</td><td>Negative</td><td rowspan="2">Microscopy: 8/15 positive<br>Culture: 6/15 positive</td></tr><tr><td>Pus from nose</td><td>Negative</td><td>Negative</td><td>Negative</td></tr><tr><td>Spinal fluid</td><td>Negative</td><td>Negative</td><td>Negative</td><td rowspan="2">Microscopy: 8/15 positive<br>Culture: 6/15 positive</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>C (N=9)</td><td>Keratitis</td><td>Aqueous humour</td><td>F. solani</td><td>Negative</td><td>Negative</td><td>PCR: 3/9 positive</td></tr><tr><td></td><td></td><td>Cornea</td><td>F. solani</td><td>septate hyphae ++</td><td>F. solani</td><td rowspan="2">PCR: 3/9 positive<br>Microscopy: 2/9 positive<br>Culture: 2/9 positive</td></tr><tr><td></td><td></td><td>Aspirate</td><td>Negative</td><td>septate hyphae +</td><td>Negative</td></tr><tr><td></td><td></td><td>Aqueous humour</td><td>Negative</td><td>Negative</td><td>Negative</td><td rowspan="2">Microscopy: 2/9 positive<br>Culture: 2/9 positive</td></tr><tr><td></td><td></td><td>Corpus vitreum</td><td>Negative</td><td>Negative</td><td>Negative</td></tr><tr><td></td><td></td><td>Aspirate</td><td>Negative</td><td>Negative</td><td>Negative</td><td rowspan="2">Microscopy: 2/9 positive<br>Culture: 2/9 positive</td></tr><tr><td></td><td></td><td>Cornea</td><td>Negative</td><td>Negative</td><td>Negative</td></tr><tr><td></td><td></td><td>Corpus vitreum</td><td>Negative</td><td>Negative</td><td>Negative</td><td rowspan="2">Microscopy: 2/9 positive<br>Culture: 2/9 positive</td></tr><tr><td></td><td></td><td>Corpus vitreum</td><td>F. solani</td><td>Negative</td><td>Negative</td></tr><tr><td>D (N=1)</td><td>Cutaneous infection</td><td>Skin scrape</td><td>F. solani</td><td>septate hyphae ++</td><td>F. solani</td><td>PCR+ / microscopy+ / culture+</td></tr><tr><td>E (N=6)</td><td>Keratitis</td><td>Cornea</td><td>F. solani</td><td>Negative</td><td>Negative</td><td>PCR: 2/6 positive</td></tr><tr><td></td><td></td><td>Cornea</td><td>F. solani</td><td>septate hyphae +++</td><td>F. solani</td><td rowspan="2">Microscopy: 1/6 positive<br>Culture: 1/6 positive</td></tr><tr><td></td><td></td><td>Corpus vitreum</td><td>Negative</td><td>Negative</td><td>Negative</td></tr><tr><td></td><td></td><td>Corpus vitreum</td><td>Negative</td><td>ND</td><td>ND</td><td rowspan="2">Microscopy: 1/6 positive<br>Culture: 1/6 positive</td></tr><tr><td></td><td></td><td>Swab</td><td>Negative</td><td>Negative</td><td>Negative</td></tr><tr><td></td><td></td><td>Cornea</td><td>Negative</td><td>Negative</td><td>Negative</td><td rowspan="2">Microscopy: 1/6 positive<br>Culture: 1/6 positive</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

**Conclusion:** The diagnostic platform for *Fusarium* infections has proven accurate to the species level and was applied successfully in three incidents of *Fusarium* keratitis, one invasive fusariosis and one cutaneous infection. These data indicate a superior sensitivity of PCR compared to microscopy and culture. Of note, no clinically false positive findings were identified. Still, the specificity needs to be confirmed including a larger sample set. PCR performance may be further improved upon ongoing optimisation of the extraction procedures and quality control as well as introduction of a PCR inhibition control. In conclusion, the *Fusarium* PCR assay appears to be a significant improvement of the overall diagnostic algorithm at this reference laboratory both in speed and accuracy and thus important for those suffering from this uncommon but sight or potentially life-threatening disease.

P170

Mucorales PCR: a diagnostic improvement at the Danish national reference laboratory

R. Hare¹, N. Abou-Chakra¹, R. Datcu¹, M.C. Arendrup¹,²,³

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

**Objectives:** Invasive *Mucorales* infections are associated with a poor prognosis for affected patients due to challenging therapeutic management strategies and often inadequate diagnostic tools. Molecular methods are increasingly complementing conventional culturing and microscopy to improve the overall diagnostic performance for invasive *Mucorales* infections. In the Danish mycology reference laboratory, a molecular assay detecting *Mucorales* DNA has been introduced which in a recent EQA panel managed by the *Mucorales* subgroup of the Fungal PCR Initiative scored 100% (7/7 correct). This study sought to evaluate the clinical performance.

<footnote>*Supported by conventional PCR (targeting translation elongation factor) detecting F. solani. Good response to amphotericin B and subsequently topical natamycin. Red coloured text indicates discrepant results between PCR, culture and microscopy.</footnote>
**Methods:** Clinical samples from patients suspected for invasive *Mucorales* infections were subjected to two *Mucorales* PCR tests (1: *Rhizopus microsporus*, *Rhizopus arrhizus* and *Mucor* spp. and 2: *Lichtheimia* spp. and *Rhizomucor* spp.) during the study period October-2017 to April-2019. Both real-time multiplex *Mucorales* PCRs (Taqman probes) were slightly modified from those described by Salehi et al. 2016. Patient samples were DNA extracted using the Qiacube (Qiagen, Denmark) standard protocol for blood and tissue samples, with up to 200 µL input volume and elution in 100 µL. Formalin fixed paraffin-embedded tissue samples were processed using Neo-clear (Sigma-Aldrich, Denmark) followed by the Tissue protocol as described above. BALs were processed on the NucliSENS easyMAG (Biomérieux, Denmark) DNA extraction platform with up to 1 mL input volume and elution in 60 µL. PCR was performed on the Applied Biosystems 7500 and since mid-2018 the Quantstudio 5 thermocyclers (Thermo Fisher Scientific, Denmark). Positive and negative template controls were applied but not PCR inhibition or DNA extraction controls. PCR results were compared to culture and Blankophor microscopy whenever possible and to results from the Danish Microbiological Database (MiBa), which captures clinical microbiological results from every laboratory in the entire country.

**Results:** In total, 116 samples from 18 patients were included. Four patients diagnosed with invasive mucormycosis contributed with 91 samples of which 38 were *Mucorales* PCR-positive (41.8%, range 25-100% per patient) (Table). Among those four patients, microscopy revealed pauci-septate hyphae in 31/85 (36.5%) and *Mucorales* species was cultured from 10/81 (12.3%) samples (Table). All patients received targeted treatment and surgical interventions guided by PCR results and all survived. The remaining 14 patients had 25 PCR negative samples, of which 20/21 and 19/19 were also negative by microscopy and culture, respectively. One patient, was microscopy positive with pauci-septate hyphae and by pan-fungal ITS DNA sequencing, *Cunninghamella* spp. DNA was detected, which is not included in the current *Mucorales* tests.

<table><caption>Table. Diagnostic results from four patients with invasive *Mucorales* infections</caption><thead><tr><th>Patient ID</th><th>PCR positive/total</th><th>Microscopy positive/total<br>(pauci-septate hyphae)</th><th>Culture positive/total</th><th>Other microbes<br>detected</th><th>Samples sent for culture<br>but not PCR</th></tr></thead><tbody><tr><td>A</td><td>2/8 (25%)<br>Rhizopus microsporus</td><td>3/5 (60%)</td><td>1/5 (20%)<br>Rhizopus microsporus</td><td>C. dubliniensis<br>A. flavus<br>A. fumigatus</td><td>2/14 Rhizopus microsporus</td></tr><tr><td>B</td><td>20/60 (33.3%)<br>Rhizopus arrhizus</td><td>13/58 (22.4%)</td><td>1/54 (1.9%)<br>Rhizopus arrhizus</td><td>A. fumigatus<br>A. niger</td><td>1/1 Rhizopus arrhizus</td></tr><tr><td>C</td><td>6/13 (46.2%)<br>Rhizomucor spp.*</td><td>6/12 (50%)</td><td>0/12 (0%)</td><td></td><td></td></tr><tr><td>D</td><td>10/10 (100%)<br>Rhizopus arrhizus</td><td>9/10 (90%)</td><td>8/10 (80%)<br>Rhizopus arrhizus</td><td></td><td></td></tr><tr><td>Overall</td><td>38/91 (41.8%)</td><td>31/85 (36.5%)</td><td>10/81 (12.3%)</td><td></td><td></td></tr></tbody></table>

**Conclusion:** Both diagnostic tests for *Mucorales* proved accurate to the genus or species level and with a sensitivity comparable to that for microscopy but significantly superior to that for culture (p<0.001). No clinically false positive findings were reported suggesting a high specificity. Yet, further evaluation including a larger sample set is warranted. PCR performance may be further improved upon ongoing optimisation of extraction procedures and PCR parameters as well as introduction of a PCR inhibition control. In conclusion, the *Mucorales* PCR assay appears to improve the diagnostic armamentarium and may play a pivotal role in managing patients suffering from *Mucorales* infections.

P171

An evaluation of the analytical specificity of PCR assays for the detection of *Candida* species – A study by the ISHAM working group, the Fungal PCR initiative

R. Gorton<fnref n="1" />, P. White², J. Loeffler³, R. Barnes⁴, J.P. Donnelly⁵, M. Cruciani⁶, R. Hare⁷, C. Linton⁷,⁸, E. Johnson⁸, L. Novak-Frazer⁹, M. Cogliati¹⁰, C. Mengoli¹¹, B. Willinger¹², W. Posch¹³, C.J. Clancy¹⁴, W. Melchers¹⁵, M. Lackner¹³, P. Hamal¹⁶, M. Paholcsek¹⁷, B. Sendid¹⁸, M.H. Nguyen¹⁴, A. Arastehfar¹⁹, U. Ugoji²⁰, B. Vanderbosch¹⁵, B. Selitsch¹², S. Fuchs¹³, T. Vyzantidis²¹

<footnote>*Confirmed Rhizomucor pusillus by ITS DNA sequencing.</footnote>

<footnote n="1">¹Infection Sciences, Health Services Laboratories, London, United Kingdom, ²Microbiology, Public Health Wales Microbiology, Cardiff, United Kingdom, ³Department Of Internal Medicine Ii, WÜ4i, University Hospital Wuerzburg, Wuerzburg, Germany, ⁴Medical Microbiology And Infectious</footnote>
Diseases, Cardiff University School of Medicine, Cardiff, United Kingdom, <sup>5</sup>Division Of Infectious Diseases, San Antonio Center for Medical Mycology, San Antonio, United States of America, <sup>6</sup>Infectious Diseases, San Bonifacio Hospital, Verona, VERONA, Italy, <sup>7</sup>Unit Of Mycology, Statens Serum Institut, Copenhagen, Denmark, <sup>8</sup>Public Health England UK National Mycology Reference Laboratory, Bristol, United Kingdom, <sup>9</sup>Mycology Reference Centre Manchester, Manchester University NHS Foundation Trust, Manchester, United Kingdom, <sup>10</sup>Laboratorio di Micologia Medica, Dipartimento di Scienze Biomediche per la Salute, Universita degli Studi di Milano, Milano, Italy, <sup>11</sup>Molecular Medicine, University of Padova, Padova, Italy, <sup>12</sup>Medical University of Vienna, Vienna, Austria, <sup>13</sup>Division Of Hygiene And Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria, <sup>14</sup>University of Pittsburgh Medical Center, Pittsburgh, United States of America, <sup>15</sup>Medical Microbiology, RadboudUMC, Nijmegen, Netherlands, <sup>16</sup>Department Of Microbiology, Palacký University, Faculty of Medicine and Dentistry and University Hospital Olomouc, Olomouc, Czech Republic, <sup>17</sup>Faculty Of Medicine, Department Of Human Genetics, University of Debrecen, Debrecen, Hungary, <sup>18</sup>Laboratory Of Mycology And Parasitology, University Hospital of Lille, Lille Cedex, France, <sup>19</sup>Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands, <sup>20</sup>The Bourne Laboratory, Royal Holloway University of London, London, United Kingdom, <sup>21</sup>Medical Biopathology-microbiology, Aristotle University of Thessaloniki, Thessaloniki, Greece

**Objectives:** The use of PCR to detect *Candida* in clinical samples has been described for two decades, yet standardization is limited, hampering its widespread application. Following the successful efforts of the Fungal PCR initiative (FPCRI) in standardizing *Aspergillus* PCR the FPCRI has expanded its efforts to standardising the molecular detection of other fungi, including *Candida*. This research describes the efforts of the FPCRI *Candida* Laboratory Working Party to determining the analytical specificity (detection range and cross-reactivity) of *Candida* PCR assays currently used in clinical mycology centres.

**Methods:** A panel of 12 samples containing genomic DNA extracted from nine yeast species (*Candida albicans* (2 concentrations), *Candida glabrata* (Nakaseomyces glabrata) (2 concentrations), *Candida krusei* (Pichia krusei), *Candida parapsilosis*, *Candida tropicalis*, *Candida auris*, *Clavispora lusitaniae*, *Meyerozyma guilliermondii* and *Dipodascus geotrichum*) and commercially bought human genomic DNA was distributed to 11 centres. The recipients were requested to perform *Candida* PCR following local protocols. Qualitative results (positive/negative), together with associated $C_T$ values (for real-time PCR) were reported for each sample, along with the assay's technical details. True positivity rates (detection of the correct species) and false positivity rates (detection of species not included in the panel, or misidentification as another species in the panel) were calculated for the overall panel and for individual samples. Regression analysis has been performed to identify any technical variables associated with improved analytical specificity.

**Results:** Across 11 datasets ten different PCR protocols were utilized. At the high DNA concentration detection of the five main causes of invasive candidiasis (*C. albicans*, *C. glabrata*, *C. krusei*, *C. parapsilosis*, *C. tropicalis*) was 92.7%, ranging from 100% for *C. parapsilosis* to 90.9% for the other species. Detection of less common yeast pathogens (*C. lusitaniae*, *M. guilliermondii*) was 36.3%. At the lower DNA concentration the detection of *C. albicans* and *C. glabrata* was 72.7% and 54.5%, respectively. For *C. auris*, 63.6% of datasets were negative, but 36.4% generated an incorrect identification. Similarly for *Dipodascus geotrichum*, 45.5% were correctly negative but 45.5% provided an incorrect identification; only one assay identified *Dipodascus geotrichum* correctly. One assay generated a false positive result when testing human DNA. Nine centers used real-time PCR. Analysis of $C_T$ values for the five main *Candida* species indicated variance in the sensitivity of the assays with a mean $S_d$ of 5.7 (range 4.3 – 6.9) with higher variance in $C_T$ values observed for *C. parapsilosis*, *C. tropicalis* or lower concentrations of *C. albicans* and *C. glabrata*, shown in Figure 1 and Table 1.
<figure><img src="image_57.png" /><figcaption>Figure 1. Box plot detailing median and interquartile ranges for seven genomic DNA samples analysed by real-time PCR across nine centres. s1, s2, s6, s7, s8, s11, and s12 contained genomic DNA belonging, respectively, to *C. glabrata*, *C. albicans*, *C. krusei*, *C. parapsilosis*, *C. tropicalis*, *C. glabrata* (low concentration), and *C. albicans* (low concentration).</figcaption></figure>

<figure><img src="image_58.png" /><figcaption>Table 1 Analysis of real-time PCR C~T~ values reported from nine centres for genomic DNA samples for the top five *Candida* species implicated in invasive fungal disease</figcaption></figure>

<table><thead><tr><th>Sample</th><th>N</th><th>mean</th><th>Sd</th><th>IQR</th></tr></thead><tbody><tr><td>s1 C. glabrata</td><td>17</td><td>35</td><td>4.4</td><td>32.1 38.5</td></tr><tr><td>s2 C. albicans</td><td>17</td><td>34.2</td><td>4.6</td><td>32 36.3</td></tr><tr><td>s6 C. krusei</td><td>17</td><td>33.1</td><td>4.3</td><td>29.9 35.2</td></tr><tr><td>s7 C. parapsilosis</td><td>17</td><td>34.4</td><td>6.6</td><td>31.6 35</td></tr><tr><td>s8 C. tropicalis</td><td>17</td><td>35</td><td>6.6</td><td>31 36.5</td></tr><tr><td>s11 C. glabrata (low conc.)</td><td>17</td><td>42.2</td><td>6.9</td><td>35.6 50</td></tr><tr><td>s12 C. albicans (low conc.)</td><td>17</td><td>38.7</td><td>6.8</td><td>34 39.5</td></tr></tbody></table>

<figure><img src="image_59.png" /></figure>

**Conclusion:** The detection of the main *Candida* pathogens that account for >95% of invasive disease is excellent, albeit at lower concentrations detection may be reduced. The detection of less common species is poor. In particular, only one of the 11 assays evaluated was designed to detect and identify *C. auris* but failed to do so which, given the susceptibility profile and infection control concerns associated with *C. auris*, highlights the design limitations of current Candida PCR assays. Preliminary analysis of C~T~ values for the main *Candida* pathogens indicates differing sensitivity levels across real-time Candida assays.

P172

An evaluation of human β-globin gene detection to assess the quality of broncho-alveolar lavage and sputum specimens submitted for the diagnosis of Pneumocystis Pneumonia (PcP).

R. Gorton¹, E. Wey², M. Hussein¹, K. Patabendige¹, G. Johnson³

¹Infection Sciences, Health Services Laboratories, London, United Kingdom, ²Microbiology, Royal Free Hospital Foundation Trust, London, United Kingdom, ³Scientific Development, OLM diagnostics, Newcastle, United Kingdom

**Objectives:** The use of PCR to diagnose Pneumocystis Pneumonia (PcP) from clinical samples is recommended in recent guidelines for the diagnosis of PcP. One component of the diagnostic pathway, specifically sampling of broncho-alveolar lavage and sputum, is poorly controlled and often the laboratory receives specimen that is not standardised in volume or portion. This research aims to evaluate the use of a human β-globin gene (HgG) PCR from respiratory samples as a method for quality control of specimens, with the hypothesis that adequate levels of HgG present in a specimen is an indicator of a good quality sample,

**Methods:** DNA was extracted from 47 BAL and 18 sputa using the Diasorin Arrow with DNA extraction cartridges. 500µL of BAL concentrate was centrifuged at 8000g for 10 minutes. The supernatant discarded; 50µL of glass beads were added and ribolysed for 45 seconds. The lysate was suspended in 240 µL Diasorin buffer 2 and 10µl proteinase K and incubated at 56°C for 10 minutes.
Extraction was performed as per manufacturer's instructions on the Arrow platform. PCR was performed using the OLM PneumID assay with 6µL template in a total reaction volume of 20µL, following manufacturer's instructions. PCR was performed on the RotorGeneQ platform. Outliers were determined using the 1.5 x IQR rule.

**Results:** HgG $C_T$ characteristics are presented in Table 1 and Figure 1a) and b).

<figure><img src="image_60.png" /><figcaption>Figure 1 a) Scatter plot of $C_T$ values obtained from the analysis of 47 DNA extracts from BAL and b) Scatter plot of $C_T$ values obtained from the analysis of 18 DNA extracts from sputum for the detection of HgG. Red bars indicating the median and IQR and the dotted line indicating the threshold for identifying outliers.</figcaption></figure>

<table><caption>Table 1. HgG PCR $C_T$ characteristics from DNA extracts of BAL and sputum for PcP diagnosis</caption><thead><tr><th></th><th>n</th><th>Mean</th><th colspan="2">IQR</th><th>Lower limit</th></tr></thead><tbody><tr><td>BAL</td><td>47</td><td>19.49</td><td>21.73</td><td>23.49</td><td>26.13</td></tr><tr><td>Sputum</td><td>18</td><td>18.04</td><td>19.16</td><td>20.26</td><td>21.91</td></tr></tbody></table>

The mean HgG $C_T$ value from BAL was 19.49 (IQR 21.73, 23.49) and the lower threshold was determined to be 26.13. 10.6% (5/47) of the BAL samples had HgG amplification below the threshold. One sample was negative for *P. jirovecii*, two samples were sub-clinical (internal threshold 1E+5) positive for *P. jirovecii* measuring 2.3E+1 and 8.5 E+4 copies per mL. The remaining two samples were positive for *P. jirovecii* measuring 2.1 E+7 and 8.3 E+9 copies per mL. The mean HgG $C_T$ value from sputum was 18.04 (IQR 19.16, 20.26) and the lower threshold was determined to be 21.91. 16.7% (3/18) of the sputum samples had HgG amplification below the threshold. One sample was negative and two samples were positive for *P. jirovecii* at 9 E+5 and 1.9 E+6 copies per mL. From the total dataset of 65 specimens 47.7% (31/65) were positive for *P. jirovecii*. No significant difference (p = 0.38) was observed between the measured copies per mL using the OLM PneumID assay and the FTD Pneumocystis assay indicating that inclusion of the HgG PCR did not competitively inhibit the detection of *P. jirovecii*.

**Conclusion:** This study has demonstrated HgG detection enables the laboratory to quality control respiratory specimens by determining if the specimen contains sufficient host material, which can be used as an indicator of good quality specimen. In this study almost 90% of samples had measured HgG above the established threshold, therefore negative and positive P. jirovecii PCR results could be reported with confidence. Where the HgG is below the acceptable threshold, negative or low positive samples should be interpreted with caution, as results may be derived from poor quality samples, seen for >10% of samples in this study.
H. Salah¹,², A. Kolecka², J. Houbraken², S.J. Taj-Aldeen¹, T. Boekhout²,³

¹Department Of Laboratory Medicine And Pathology, Division Of Microbiology, Hamad medical corporation, Doha, Qatar, ²Westerdijk fungal biodiversity Institute, Utrecht, Netherlands, ³Institute Of Biodiversity And Ecosystem Dynamics, University of Amsterdam, Amsterdam, Netherlands

**Objectives:** Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) has been widely used in clinical laboratories for routine identification of bacteria and yeast. However, there remain difficulties when applied to filamentous fungi. The standard liquid cultivation method recommended for identification is labour intensive and time-consuming. The aim of this study was to develop a quicker and less laborious method for cultivating filamentous fungi which leads to a reliable identification using Bruker MALDI-TOF MS.

**Methods:** Reference strains of *Aspergillus* section *Fumigati* (*n* = 55) and section *Flavi* (*n* = 104), which were previously identified by molecular methods, were cultivated on top of and in between polycarbonate filters placed on Sabouraud's dextrose agar (SDA) plate, and by the Bruker standard liquid cultivation method. After 24 hours of incubation at 35°C, mycelia were harvested for ethanol/formic acid protein extraction. Main Spectra Profiles (MSPs) were created and databases were constructed for each cultivation method. These databases were validated by identification of 50 reference strains and 100 clinical strains of *Aspergillus* and *Penicillium* species which were previously characterized by DNA sequencing.

**Results: Growth:** On top of a polycarbonate filter: The strains grew as a monolayer of mycelia with some spores in the middle. In between two polycarbonate filters: The strains grew as a mycelial monolayer without spores. Bruker method: Mycelial growth without sporulation **MSP creation:** 55/55 MSPs of *Aspergillus* section *Fumigati* strains were created by cultivation in between 2 polycarbonate filters, 52/55 by cultivation on top of a polycarbonate filter and 47/55 by Bruker standard method. Out of 104 *Aspergillus* section *Flavi* strains, 94, 92 and 76 MSPs were created by cultivation on top of and in between polycarbonate filters, and Bruker standard method, respectively.

**Validation:** Identification of 50 reference strains of *Aspergillus* and *Penicillium* species against in-house databases resulted in 95%, 90% and 62% correct identifications for the strains cultivated on top of a polycarbonate filter, in between 2 polycarbonate filters and by Bruker method, respectively. Identification against Bruker database (BDAL) resulted in 79%, 64% and 61% correct identifications by 1 polycarbonate filter, 2 polycarbonate filters, and Bruker method, respectively. Identification against in-house databases and Bruker database together yielded 87%, 82% and 61% correct identifications by 1 polycarbonate filter, 2 polycarbonate filters, and Bruker method, respectively (Figure 1).
<figure><img src="image_61.png" /></figure>

**Identification of clinical strains:** Out of 100 clinical *Aspergillus* and *Penicillium* strains cultivated on top of a polycarbonate filter, 81% were correctly identified against in-house databases of strains grown on top of a polycarbonate filter, 78% with 2 polycarbonate filters and 59% by Bruker commercial database (BDAL) (Figure 2).

<figure><img src="image_62.png" /><figcaption>Figure 2. Identification of 100 clinical strains cultivated on top of a polycarbonate filter against different databases</figcaption></figure>

**Savings:** For 50 strains, the cultivation method using polycarbonate filter was 270 minutes quicker and 81 Euros cheaper than Bruker standard method.

**Conclusion:** Cultivation of filamentous fungi on top of a polycarbonate filter showed significantly improved results compared to the Bruker standard liquid cultivation method in terms of correct identification, MSP creation, time consumption, cost, and labour intensity. This can reliably be applied for identification of clinically significant filamentous fungi.
H. Guegan¹, X. Iriart², F. Robert-Gangneux¹, A. Berry², M.-E. Bougnoux³, J.P. Gangneux¹

¹Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France, ²Laboratoire De Parasitologie-mycologie, CHU de Toulouse, Toulouse, France, ³Laboratoire De Parasitologie Mycologie, APHP Hôpital Necker-Enfants-Malades, Paris, France

**Objectives:** Mucormycosis is a life-threatening fungal infection whose diagnosis remains challenging due to a lack of reliable diagnostic tests. Clinical and radiological signs are often unspecific. The contribution of histopathological and cultural procedures for early diagnosis is limited. In this context, Mucorales in-house real-time PCRs have been previously shown to be of high interest for a reliable and early diagnosis both in serum and respiratory specimens. A multiplex real-time PCR assay (MucorGenius®, Pathonostics, the Netherlands) has been newly marketed to detect the main genera involved in human mucormycosis. The aim of this study was i) to assess the diagnostic performances of MucorGenius® assay in pulmonary samples compared to in-house Mucorales PCRs, and ii) to evaluate the occurrence of Mucorales detection among invasive aspergillosis (IA) patients.

**Methods:** We retrospectively included 320 pulmonary samples between January 2012 and May 2019: bronchoalveolar lavage fluids (n = 293), tracheal aspirations (n = 13), sputum samples (n = 5), pleural fluids (n = 3), and lung biopsies (n = 6) collected from 320 patients with respiratory symptoms at Rennes, Toulouse and Paris-Necker teaching Hospitals (France). Among the 320 patients, 30 patients were classified as a proven mucormycosis based on a positive direct examination and/or a positive culture in the respiratory sample and/or a positive serum mucorales PCR. Fifty-eight patients were proven/probable IA cases according to EORTC-MSG criteria. The 232 remaining patients were considered as having no IA and no mucormycosis (negative group). In-house real-time PCRs consisted in 3 assays targeting respectively *Mucor-Rhizopus* spp, *Lichteimia* spp, and *Rhizomucor* spp. MucorGenius® targeted the same genera, including *Cunninghamella* spp. Amplification was performed according to manufacturer's instructions using a LC480 (Roche) device.

**Results:** The global concordance of both tests was high, of 97.8% (kappa coefficient = 0.877). Discordant results were mainly related to a higher sensitivity of in-house PCRs compared to MucorGenius® assay (100% (30/30) *versus* 90.0% (27/30). The 3 cases missed by MucorGenius® PCR were related to low fungal burden (mean Cq of in-house PCRs of 39.5±1.4) and were positive for *Mucor-rhizopus* (n = 2) and *Rhizomucor* (n = 1) with in-house PCRs. The specificity of both tests was similar (98.6% (286/290) *versus* 99.3% (288/290). No Mucorales DNA was detected in IA patients using in-house PCRs whereas one was found positive with MucorGenius® assay, without other biological evidence for mucormycosis. Of the 4 positive-PCR samples in the negative group, one was probably a true positive result, from a real mucormycosis episode missed at the time of diagnosis (hematological malignancy, imaging compatible with IA during aplasia, early death under voriconazole treatment). The other could be associated with fungal colonization.

**Conclusion:** The commercial MucorGenius® assay provides the possibility to detect in one well the main mucorales responsible for human mucormycosis, and then could reduce the diagnostic delay and improve the outcome. MucorGenius® assay is an easy to use marketed kit including internal quality control, that shows good diagnostic performances with 97.8% of concordance compared to in-house PCRs, missing 3 low fungal burden cases.

P176

**Development and Validation of an In-House Candida auris database in MALDI TOF MS to improve the yeast identification.**

A. Ceballos-Garzon¹,², C.M. Parra Giraldo², D. Amado², N. Velez², C. Rodríguez³

¹Department Of Parasitology And Medical Mycology Of The University Of Nantes., Universite de Nantes, Nantes, France, ²Microbiology, Pontificia Universidad Javeriana, Bogotá, Colombia, ³Bruker Mexicana, Ciudad de México, Mexico

**Objectives:** to construct an In-house Colombian database to improve *Candida auris* identification by MALDI-TOF MS.

**Methods:** 30 clinical isolates of *C. auris* were used to construct an *in-house* data base. For each isolation, protein extraction was performed using the formic acid/ethanol method according to the
Bruker Daltonics protocol. Minimum 20 mass spectra of each one was obtained. The library was named "Candida auris Colombia". For its validation 300 Colombian isolates of C. auris were used, comparing the results between the BDAL (Bruker) and Candida auris Colombia (in house) libraries. Finally, Cohen's kappa index was used to compare the results of the classification with both libraries.

**Results:** From 30 clinical isolates, 770 mass spectra were obtained for the construction of the database. The validation was carried out with 300 isolates to compare the identification of results in the BDAL and Candida auris Colombia libraries (Figure 1). Our library allowed us to obtain an identification result for 100% of the evaluated isolates and improve significantly the scores obtained, which shows a better performance compared to the library present in the Bruker spectrometer.

<figure><img src="image_63.png" /><figcaption>**Figure 1** BDAL and *Candida auris* Colombia libraries identification results. **A** Total results and **B** Matrix of concordance identification from BDAL and Colombia databases. The MALDI-TOF MS results according to the manufacturer's technical specifications as follows: Correct genus and species identification (>2,300), correct genus and probably species identification (≥2.0), correct genus identification and (1.7–2.0) no reliable identification (<1.7).</figcaption></figure>

**Conclusion:** The current bruker database has only 3 *C. auris* isolates from Korea and Japan, what it entails with identifications problems and low punctuation. Our database had a remarkable improvement in the identification of the 300 isolates, evidencing that the strengthening of the database is a great opportunity to improve the punctuation and *C. auris* identification.

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

<footnote>**Objectives:** Chronic pulmonary aspergillosis (CPA) is a fungal disease characterised by cough, haemoptysis and weight loss present for at least for three months in non-immunocompromised people. It is caused by *Aspergillus* spp. especially *Aspergillus fumigatus* that can reach the respiratory tract by airborne transmission. The diagnosis of CPA can be made by a variety of clinical symptoms, radiological and laboratory evidence of *Aspergillus*. Most patients with CPA have positive *Aspergillus* precipitating antibodies or an abnormally high level of *Aspergillus*-specific IgG (Aspf IgG). The</footnote>
Bordier *Aspergillus fumigatus* ELISA kit is one assay that can detect Aspf IgG and the result is deemed positive when the optical density (OD) index is higher than 1.0. We have re-evaluated this cut-off in patients with CPA in the UK.

**Methods:** Aspf IgG levels in sera from 114 CPA patients and 150 British healthy controls were measured by the Bordier *Aspergillus fumigatus* ELISA kit. The results obtained were organised in MS Excel spreadsheets and analysed were performed using SPSS. Sensitivity and specificity at a number of points on the receiver operating characteristics (ROC) curve were described. The optimum diagnostic cut-off for the Bordier assay was calculated using Youden's J statistic (sensitivity + specificity - 1).

**Results:** Based on statistical analyses, we determined that the ROC area under the curve (AUC) of the Bordier *Aspergillus fumigatus* ELISA kit was 0.928. This assay has 77.2% sensitivity and 99.3% specificity using the manufacturer's cut-off (1.0). The sensitivity and specificity of this assay at various points on the ROC curve improves to 83.3% sensitivity and 97.3% specificity if the OD index cut-off is lowered to 0.9. At this cut-off, the diagnostic odds ratio and diagnostic accuracy are 145 and

Figure 1.

<figure><img src="image_64.png" /><figcaption>ROC curve for Bordier assay</figcaption></figure>

90.9%.

Table 1.

<table><caption>Bordier assay performance at various points on the ROC AUC curve</caption><thead><tr><td>Cut-off</td><td>Sensitivity (%)</td><td>Specificity (%)</td><td>Youden's J statistic</td></tr></thead><tbody><tr><td>0.5</td><td>92.1</td><td>72.7</td><td>0.648</td></tr><tr><td>0.75</td><td>86.0</td><td>92.7</td><td>0.786</td></tr><tr><td><strong>0.9</strong></td><td><strong>83.3</strong></td><td><strong>97.3</strong></td><td><strong>0.807</strong></td></tr><tr><td>1.0</td><td>77.2</td><td>99.3</td><td>0.765</td></tr><tr><td>1.5</td><td>62.3</td><td>100</td><td>0.623</td></tr><tr><td>2.0</td><td>51.8</td><td>100</td><td>0.518</td></tr></tbody></table>

**Conclusion:** We have performed a ROC AUC analysis comparing Aspf IgG levels in a large group of patients with CPA compared to healthy British controls. Diagnostic performance improves if the OD index cut-off falls from 1.0 to 0.9.

P178

Influence of Intravenous Human Immunoglobulins on the Accuracy of Aspergillus Galactomannan Antigen Assays

W.-D. Liu<fnref n="1" />, M.-C. Shih²,³, Y.-W. Wang⁴, S.-C. Lin⁴, Y.-F. Lee⁵, S.-W. Lin⁴,⁶, W.-C. Chou⁵,⁷, U.-I. Wu<fnref n="1" />,⁸, Y.-C. Chen<fnref n="1" />,⁹

<footnote n="1">¹Department Of Internal Medicine, Division Of Infectious Disease, National Taiwan University Hospital, Taipei City, Taiwan, ²Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei City, Taiwan, ³Department Of Medical Education, National Taiwan University Hospital, Taipei City, Taiwan, ⁴Department Of Pharmacy, National Taiwan University Hospital, Taipei City, Taiwan, ⁵Department Of Laboratory Medicine, National</footnote>
Taiwan University Hospital, Taipei city, Taiwan, ⁶Graduate Institute Of Clinical Pharmacy, National Taiwan University, Taipei City, Taiwan, ⁷Department Of Internal Medicine, Division Of Hematology, National Taiwan University Hospital, Taipei City, Taiwan, ⁸Department Of Internal Medicine, National Taiwan University Cancer Center, Taipei City, Taiwan, ⁹Center Of Infection Control, National Taiwan University Hospital, Taipei City, Taiwan

**Objectives:** Galactomannan (GM) enzyme immunoassay (EIA), a diagnostic tool that detects the cell wall component secreted by *Aspergillus* species, provide a non-invasive method for early diagnosis of invasive aspergillosis (IA). However, its performance in diagnosing IA can be limited by potential false positivity caused by cross-reaction with other fungal species, food consumption and administration of certain antibiotics and blood products. False positive results were also demonstrated in a few patients having received intravenous immunoglobulins (IVIG). Some cases were attributed to cross-reactivity between the GM kits and saccharose contained in some IVIG products. Recently, we observed a growing number of patients prescribed with maltose-stabilized IVIG had false positive GM results despite absence of factors reported in the literature. We thus sought to determine if the false positivity was due to the processing procedures and presence of other unrecognized cross-reactive antigens contained in IVIG.

**Methods:** Two new, unopened bottles of human IVIG (TBSF, CSL Behring, Australia) was randomly obtained from our hospital pharmacy. Each vial contained 3g (6%) human IVIG and 14.6 mmol maltose in 50ml solution. From each bottle, solution were aspirated for dilution using 0.9% saline to the concentration of 1% and 0.1%, respectively in order to simulate the peak (15.8 g/L) and trough (7.4 g/L) of serum IVIG level in an adult having received IVIG infusion at a concentration of 0.2-0.6 g/Kg. A total of three different concentrations (6%, 1% and 0.1%) of IVIG together with negative controls (0.9% saline) were prepared in duplicates for GM detection using the Platelia *Aspergillus* EIA Kits (Bio-Rad) according to the manufacturer's instructions. In order to explore if heat treatment and addition of EDTA required in standard sample processing procedures were associated with false positivity, samples were prepared with and without the investigated pretreatment, respectively for comparisons. A GM test result was considered positive if the optical density index (ODI) reached 0.5 or above. In addition, conventional bacteria and fungus cultures, pan-fungal polymerase chain reaction (PCR) were performed using undiluted samples from the bottles of IVIG to assess the presence of contaminants. All procedures were performed under strict sterile conditions.

**Results:** Using standard procedures with heat pretreatment and addition of EDTA, all IVIG samples tested positive for GM regardless of the concentrations used for the experiments (Figure 1), while all 0.9% saline controls yielded negative results. Pre-treatment of samples with boiling water-bath resulted in considerable elevation of GM ODI in both presence and absence of EDTA compared to samples without heat treatment. No fungus or other microorganisms were isolated from our IVIG
samples using PCR or conventional culture methods.

<figure><img src="image_65.png" /><figcaption>Fig 1. GM ODI of IVIG samples prepared with different concentrations and pretreatment methods.</figcaption></figure>

**Conclusion:** IVIG treatment can cause false GM positivity owing to the reaction between the contents in the IVIG products and standard pretreatment procedures of the assays. Interpretation for a positive GM assay result should be cautious in patients having received human IVIG infusion.

P179

Evaluation of the relevance of use of the BD BACTECTM mycosis IC/F, BD BACTECTM Plus Aerobic/F, BD BACTECTM Lytic/10 anaerobic/F et BD BACTECTM Peds Plus/F culture bottles system for fongemia detection: A 4-year retrospective study at the University Hospital of Dijon, France.

A. Magallon¹, L. Basmaciyan¹, S. Valot¹, C. Goulard De Curraize², M. Sautour³, J. Bador², F. Dalle⁴

¹Mycology, University Hospital of Dijon, Dijon, France, ²Bacteriology, University Hospital of Dijon, Dijon, France, ³Laboratoire Parasitology Mycology, CHU Dijon, Dijon, France, ⁴Parasitology-mycology, University Hospital Dijon, Dijon, France

**Objectives:** Fongemia are severe invasive fungal diseases that combine rapid evolution with high mortality. The early diagnosis of these pathologies is therefore fundamental in the care of patients and is a vital emergency. Currently, only the BD BACTEC™ Automated Blood Culture System (Becton Dickinson, Franklin Lakes, New Jersey, USA) has a bottle specifically dedicated to the detection of fungal agents, the BD BACTEC™ Mycosis IC/F bottle. The objective of this study was to (i) evaluate the fongemia detection performance of the BD BACTEC™ Mycosis IC/F culture bottles in comparison to the so-called "bacteriological" culture bottles (i.e. BD BACTEC™ Plus Aerobic/F bottles, BD BACTEC™ Lytic/10Anaerobic/F bottles, and BD BACTEC™ Peds Plus/F) in different clinical situations and so (ii) the relevance of their use.

**Methods:** This retrospective study was conducted over a period of 4 years (i.e. from January 2013 to December 2016) at the University Hospital of Dijon. 331 pairs of blood cultures (i.e. each pair combining a BD BACTEC™ Mycosis IC/F culture bottle with at least one of the bacteriological culture bottle). Univariate analysis by the MacNemar chi² test was carried out globally as well as in the subgroups corresponding to the various clinical situations encountered.

**Results:** Of the 331 pairs of positive for fungi blood cultures included in the study, the BD BACTEC™ Mycosis IC/F culture bottles showed a diagnostic advantage over bacteriological culture bottles in 57.4% of cases (p <0.01). In addition, for 96 pairs of blood cultures, only the BD BACTEC™ Mycosis IC/F culture bottles was positive for fungi *versus* 66 pairs in which only the bacteriological culture
bottles was positive for fungi. On the other hand, a fungus / bacterium co-infection was observed in 46 of the 331 pairs of blood cultures included in the study. In this "co-infection" subgroup, the overall "diagnostic advantage" variable was significantly in favor of the BD BACTEC™ Mycosis IC/F culture bottles (n = 30, p<0.05). Multivariate analysis revealed an association between diagnostic performance and (i) the existence of yeast-bacteria co-infections, (ii) the mode of sampling, (iii) the type of fungus and (iv) diagnostic indication or follow-up of the patient, showing the relevance of the use of the BD BACTEC™ Mycosis IC/F culture bottles in addition to those of bacteriology.

**Conclusion:** This study showed the necessity of the use of the BD BACTEC™ Mycosis IC/F culture bottles in the diagnosis of candidemias, in particular in case of bacterial co-infections.

P180

Gliotoxin (GT) and bis-methyl-Gliotoxin (bmGT) characterization during Aspergillus fumigatus infection.

A. Gomez Lopez, C. Rueda Hernández, R. Pando Pozo

Reference Laboratory On Mycology, CNM-ISCIII, MAJADAHONDA-MADRID, Spain

**Objectives:** To develop a new method based on liquid-liquid extraction and chromatographic separation to specifically and simultaneously characterize both mycotoxins. We also describe the primary bmGT and GT's role as defence molecule during *Aspergillus* progression in two cell lines infection model of IA that mimic human disease by measurement of GT and bmGT amount and their cytotoxic effect.

**Methods:** An UHPLC/PDA method was first developed and validated to specifically characterize both compounds. Chromatographic separation was achieved using an ACQUITY UPLC-H Class apparatus. The analytical conditions were selected after testing different parameters such as extraction procedures, column type, mobile phase composition, etc. We applied the developed method to define temporal kinetics of GT and bmGT production by using a fungal infection in two different cell line models, A549 (ATCC CCL-185, alveolar basal epithelial cell of human adenocarcinoma), and RAW 264.7 (ATCC TIB-71, murine macrophages) and an *Asperillus fumigatus* clinical isolate, GT producer. After the establishment of a specific infection model (ratio1:1, inoculum size 5x10t5 spores), we harvested each supernatant from cell line cultures at fixed time points, for assessment of GT/bmGT production. Galactomannan (GM) antigen was also quantified in order to establish the fungal burden at each time. In addition, a microscopic evaluation was performed to prove spores progression in each cell line. Specific stains (Sytox, calcofluor) were used to better define the characteristics of infection

**Results:** The best overall selectivity and peak shape were achieved by using a silica column (ACQUITY UPLC® HSS, C18, 2,1 x 75 mm 1,8µm particle size) combined with a mobile phase consisting of a gradient of water and acetonitrile and a flow rate of 0.1 mL/min. The developed method was applied to analyze the extracted supernatant (using chloroform) obtained from each condition. In the laboratory condition established, GT was firstly detected after 30 h of incubation. The kinetic profile showed a maximum production after 48 h and then a continuous decrease. Enrichment of GT in infected cell lines did not correlate with GM. Additionally, the level of bmGT, the inactive derivate of GT, was detected later. Contrary to GT, the levels of bmGT showed a continuous increase after 48 h of incubation. No great differences were found in GM, GT and bmGT amounts by cell lines studied. In both models, GT showed a concentration-dependent cytotoxic effect. Of note, bmGT did not show any toxic effect and appear to be more stable than GT.

**Conclusion:** 1) GT and bmGT may be excreted to extracellular media during the course of *Aspergillus fumigatus* infection, although these components showed different kinetics during the *Aspergillus fumigatus* growth. 2) The chromatographic method described was successfully applied to characterize these secondary metabolites, having potential as a diagnostic technique for aspergillosis.

P181

In vitro comparison between two commercial kits for the detection of Aspergillus galactomannan.
Preliminary results.
D.-A. Giannakopoulou, E. Zachrou, E. Zarkada, T.-A. Vyzantiadis

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

**Objectives:** Galactomannan is an important component of fungal cell wall, mainly measured for the early diagnosis and monitoring of invasive Aspergillosis. Several commercial kits have been released and the aim of this study was to compare the recently available kit of Dynamiker Aspergillus Galactomannan Assay (Dynamiker Biotechnology Co, Ltd, China) to the internationally used and broadly evaluated Platelia™ Aspergillus Ag (Bio-Rad, France).

**Methods:** Both kits are immunoenzymatic assays, based on the same principle with slight technical differences. An advantage of the Dynamiker method is the use of breakable strips, while both produce results after the calculation of an index value. The interpretation of positivity is based on the cut-off index of 0.5 for serum and 1.0 for BAL, defined after years of use and evaluation of results produced by the Bio-Rad kit. Forty four (39 serums, 4 BALs, 1 pleural fluid) specimens were tested by both methods (18 positive by the Bio-Rad kit). The positive and negative controls of the Bio-Rad kit were also tested by the Dynamiker one. Further on, in order to test the precision of the Dynamiker kit, twelve specimens were tested for their inter-day variation between two or three sequential measurements and their intra-day variation concerning double measurements. Alongside, two positive specimens (one serum and one BAL) were further diluted in three sequential two-fold sub dilutions (1:2, 1:4 and 1:8) in order to test the detection ability of the kit.

**Results:** The values (mean±SEM) were 1.12±0.24 by Bio-Rad and 0.68±0.12 by Dynamiker. Although, the strict numeric values were quite different, they didn't differ statistically (p = 0.35) and they were positively correlated (r = 0.65, p = 0.000001). The precision of the Dynamiker kit was good with an intra-run CV (coefficient of variation) of 13.01% and an inter-run CV of 13.25%. However, while all negative (26 specimens) by Bio-Rad were found again negative by Dynamiker, this was not the case with the positive specimens, where there was a disagreement in six out of eighteen (33.3%, positive by Bio-Rad and negative by Dynamiker) and the strict numeric values differed statistically (p = 0.016). Both Bio-Rad positive and negative controls were found at the same level with the Dynamiker and all dilutions of the tested two positive samples provided positive results.

**Conclusion:** If the long used Bio-Rad kit is going to be considered the reference kit for the comparison of other recently available commercial kits, then it is probable that the here-tested Dynamiker kit is quite efficient and could probably offer a reliable alternative. However, these preliminary results show that there is a possible issue of sensitivity by not being able to detect a percentage of positive samples, while the kit is highly specific. Further in vitro studies with larger number of samples in combination to clinical information and outcome would provide a more documented and evaluated use of the kit.

P182

**External Validation of a bedside prediction rule for candidemic patients**

K. Alobaid¹, A. Chadha², A. Alansari³

¹Mycology Reference Lab, Mubarak Alkabeer hospital, Jabryia, Kuwait, ²Microbiology Unit, Mubarak Alkabeer hospital, Jabryia, Kuwait, ³Medical Department, Mubarak Alkabeer hospital, Jabryia, Kuwait

**Objectives:** The aim of this study is to examine Ostrosky-Zeichner's rule (CPR) on predicting cases of candidemia among hospitalized patients in ICU and non ICU settings.

**Methods:** This is a prospective study on cases of candidemia from March 2017 till May 2018 in a secondary hospital. All candidemic cases were analyzed and tested by applying Ostrosky-Zeichner's rule (CPR). Another group of patients who developed bacteremia were also tested as a control. The inclusion criteria for cases and controls are (1) positive blood culture in a patient with signs of infection, (2) hospital acquired infections. CPR was modified to include ICU and non ICU patients. The rule included presence of central line or broad spectrum antibiotic plus two of the following: steroids, immune suppressive agents, pancreatitis, major surgery, TPN, and major surgery.
**Results:** In the period from March 2017 till May 2018, 50 cases of candidemia were found. Two cases were excluded, as they represented contamination. Control group involved 60 patients. Majority of cases were hospitalized in medical wards (27), seven patients were surgical, 12 patients were hospitalized in ICU, and two patients were pediatric cases. **The sensitivity, specificity, PPV, and NPV of modified Ostrosky-Zeichner's rule were 40.4%, 98.3%, 67%, and 95% respectively. Area under the curve (AUC ROC) is 0.69 and 95% CI is (0.62-0.76).** Although 29 cases (59.6%) were not predicted by the rule, 6 patients (21%) had femoral catheters.

**Conclusion:** Clinical prediction rules are helpful adjunctive tool to rationalize the use of antifungals. Modified Ostrosky-Zeichner's rule, in particular, appears to have good specificity for candidemic patients. The results also suggest that around 40% of patients can be treated empirically before culture results and this may improve survival. The possibility of femoral catheters as a risk factor for candidemia needs further evaluation. As shown in other studies, the high negative predictor value helps avoiding or stopping unnecessary use of antifungals.

P183

**Real Time PCR and galactomannan test for detection of Aspergillus spp. in bronchoalveolar lavage fluid of patients with aspergillosis**

S. Ignatyeva¹, V. Spiridonova¹, T. Bogomolova², O. Shadrivova³, Y. Borzova¹, E. Desyatik², N. Vasilyeva², N. Klimko⁴

¹Kaschkin Research Institute Of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ²Kaschkin Research Institute Of Medical Mycology. Department Of Medical Microbiology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ³Department Of Clinical Mycology, Allergy And Immunology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ⁴Department Of Clinical Mycology, Allergology And Immunology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation

**Objectives:** The success of treatment of fungal infection depends on early etiological diagnostics. The aim of the study was to test a real time PCR with High Resolution Melt analysis (mHRM-RT-PCR) and galactomannan (GM) for detection of *Aspergillus* spp. in bronchoalveolar lavage fluid (BAL) of patients with aspergillosis.

**Methods:** We tested 33 BAL from 33 patients with aspergillosis in Saint-Petersburg between 2015 and 2018 yy. As controls, 50 BAL were collected from patients without mycoses. BAL was examined by direct microscopy with calcofluor white and culture. Fungal DNA was extracted from clinical samples by a chloroform-isoamyl extraction method. DNA amplification was performed using aspergillus - specific primers and EvaGreen based mHRM-RT-PCR on Rotor-Gene 6000 cycler. Detection of GM was performed using "Platelia Aspergillus Ag" (Bio-Rad). The test was considered as positive with a cut-off of ≥0,5. We calculated the sensitivity and specificity for the PCR assay and GM with the level of statistical significance <0,05.

**Results:** Direct microscopic examination of BAL from patients with aspergillosis was positive in 42% cases, *Aspergillus* spp. were isolated in 91% cases. Cultures of 33 BAL samples revealed various *Aspergillus* spp.: A. fumigatus (63%), A. niger (19%), A. flavus (6%), A. versicolor (3%). From 3 patients (9%) two different species were isolated: A. fumigatus + A. niger (6%) and A. fumigatus + A. flavus (3%). GM test was positive in 23 BAL samples from patients with confirmed aspergillosis. Sensitivity of PCR assay in BAL was higher, than sensitivity of GM test (84% vs. 70%, p = 0,013). At the same time, specificity of GM test was higher than of PCR assay in patients without aspergillosis (87% vs. 71%, p = 0,018). The positive results of PCR assay in patients with aspergillosis correlated with positive results of microscopy and cultural investigations from BAL in 75% and 78%, positive GM test - in 55% and 95%, respectively. Complex investigation of biological materials by immunological and molecular methods increased the diagnostic effectiveness to 91 - 92%.
**Conclusion:** This study indicated that the mHRM-RT-PCR may be a very useful tool for detection of *Aspergillus* spp. in bronchoalveolar lavage fluid of patients with aspergillosis. Real Time PCR assay together with GM test increase the diagnostic effectiveness of biological materials investigations.

P184

Detection and identification of mucormycosis etiologic agents in biological samples of
oncohematological patients using multiplex real time PCR

S. Ignatyeva¹, V. Spiridonova¹, T. Bogomolova², Y. Avdeenko¹, O. Shadrivova³, I. Zyuzgin⁴, Y. Chudinovskikh⁵, M. Popova⁶, O. Uspenskaya⁷, N. Vasilyeva², N. Klimko⁸

¹Kaschkin Research Institute Of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ²Kaschkin Research Institute Of Medical Mycology. Department Of Medical Microbiology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ³Department Of Clinical Mycology, Allergy And Immunology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, ⁴N.N. Petrov National Medical Research Centre of Oncology, Saint Petersburg, Russian Federation, ⁵N.N. Petrov National Medical Research Centre of Oncology, Ministry of Health of Russian Federation, St. Petersburg, Russian Federation, ⁶I.Pavlov First Saint Petersburg State Medical University, St. Petersburg, Russian Federation, ⁷Leningrad Regional Clinical Hospital, St. Petersburg, Russian Federation, ⁸Department Of Clinical Mycology, Allergology And Immunology, North-Western State Medical University n.a. I.I. Mechnikov, Saint-Petersburg, Russian Federation

**Objectives:** Mucormycosis is an opportunistic infection characterized by rapid progression, high morbidity and mortality. The success of treatment of fungal infection depends on early etiological diagnostics. The aim of the study was to test a multiplex real time PCR with High Resolution Melt analysis (mHRM-RT-PCR) on clinical samples for simultaneous detection and identification of Mucormycetes in biological samples.

**Methods:** We tested 12 clinical samples from 10 oncohematological patients with mucormycosis in Saint-Petersburg between 2014 and 2018. All samples were collected from patients without mycoses. Fungal DNA was extracted from clinical samples by a chloroform-isoeamyl extraction method. DNA amplification was performed using mucormycetes - specific primers and EvaGreen based mHRM-RT-PCR on Rotor-Gene 6000 cycler.

**Results:** The mHRM-RT-PCR allows to identify 7 clinical samples from patients with aspergillosis and mucormycosis the representatives of *Aspergillus* to the genus and Mucormycetes to the species level: *Rhizopus arrhizus*, *Mucor racemosus*, *Rhizomucor pusillus*, *Lichtheimia corymbifera*. We tested 3 native tissue, 7 formalin-fixed paraffin-embedded tissue samples and 2 BAL of patients with mucormycosis. In 1 of 3 native tissue and 2 BAL with positive direct microscopic examinations *Lichtheimia corymbifera* was isolated. In 7 formalin-fixed paraffin-embedded tissue non-septate mycelium of mucormycetes was detected. mHRM-RT-PCR was positive in native, formalin-fixed paraffin-embedded tissue samples and BAL of all patients with mucormycosis. PCR assay allowed to identify the representatives of Mucormycetes: *Lichtheimia corymbifera* in 10 and *Rhizomucor pusillus* in 2 from 12 samples. In biological specimens of 2 patients the PCR assay detected a mixed infection by *Aspergillus* and *Mucormycetes* spp. The results of mHRM-RT-PCR in patients with mucormycosis correlated with traditional methods. In 21 controls tissue samples and 20 BAL PCR test was negative.

**Conclusion:** The multiplex RT-PCR has high sensitivity and specificity in patients with mucormycosis. This study indicated that the mHRM-RT-PCR may be a very useful tool for detection of etiologic agents of mycoses, particularly in the case of a mixed infection caused by *Aspergillus* and *Mucormycetes* spp.

P185

Clinical application of free circulating microRNAs in patients with hematologic malignancies as
promising biomarkers in monitoring invasive aspergillosis
E. Tolnai, K. Okechukwu Nwozor, G. Fidler, R. Szasz, L. Rejto, S. Biro, M. Paholcsek

Faculty Of Medicine, Department Of Human Genetics, University of Debrecen, Debrecen, Hungary
**Objectives:** MicroRNAs (miRNAs) are small 19- to 23- nucleotide long, noncoding single stranded RNA molecules. They play an important role in regulating various biological processes through complementary base pairing with 3'- untranslated regions (3' UTR) of the corresponding target mRNAs. MiRNAs are key regulators of various physiological and pathophysiological processes like cell proliferation, differentiation, apoptosis, inflammation and cancer. Altered expression levels of miRNAs have been associated with many diseases. Increasing number of evidences demonstrate that specific miRNAs can be a great promise as novel biomarkers for clinical diagnosis of many types of diseases due to their stability in various body fluids. However, little is known about the potential use of circulating miRNAs as biomarkers in fungal infections. Invasive aspergillosis (IA) is a life-threatening infection caused by *Aspergillus* especially in immunocompromised patients. With this pilot study our aim was to verify the different expression profiles of 21 pre-selected target miRNAs in hematologic malignant patients with (proven, probable IA) and without (possible IA and healthy controls) IA. We assessed the association of IA disease characteristics with the abundance of the free circulating microRNAs in the liquid biopsy samples of these patients to identify potential diagnostic markers for further studies.

**Methods:** We obtained whole blood (WB) samples from 40 patients at the onset of fevered neutropenia (FN) and from 5 healthy volunteers. Total RNA was isolated from the WB samples using the MagMax™ mirVana™ Total RNA Isolation Kit (Thermo Fisher Scientific). MiRNAs were reverse transcribed to cDNA with TaqMan® Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific). TaqMan® quantitative real-time PCR (miRNA assay) was used for detecting miRNA expression profiles. Relative miRNA expressions were calculated with the ΔΔCt method.

**Results:** We found that 7 out of the 21 miRNAs were strongly associated with IA by being significantly (*p*<0.05; *p*<0.01) upregulated in our patient group compared to the control group. Increase in miRNA expressions were also observed in the case of further 7 miRNAs which expression differences did not prove to be statistically significant.

**Conclusion:** Based on the result of this pilot study, we detected significant expression differences in the case of seven miRNAs. We think that these may be promising and effective biomarker targets to support the diagnosis of IA. We also believe that the diagnostic strategy relying on the combination of multiple circulating miRNAs promises better positive predictive values in monitoring of IA.

P190

**Indication of opportunistic micromycetes in mycobiota of municipal solid waste compost by metagenomic approach**

G. Kozlov¹, N. Vasilyeva², I. Riabinin²

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

**Objectives:** Objective of the study is to perform the genotaxonomic analysis of mycobiota of thermal-treated compost from municipal solid waste (CMSW) to identify the microfungi of medical interest.

**Methods:** The metagenomic study of samples using an internal transcribed spacer (ITS2) as a ticosomic marker was performed on the Illumina MiSeq instrument (Illumina, USA) according to the Manufacturer's protocol. DNA-extraction, post-PCR purification of apmplicons and DNA concentration measurement were made by utilization PowerSoil DNA Isolation (Qiagen, Germany), AMpure XP magnetic beads (Beckman Coulter, USA) commercial kits and Qubit 4 Fluorometer (ThermoFisher Scientific, USA) respectively. Bioinformatic data processing was performed using QIIME 1.9.1 software, Silva and Unite databases. Classification algorithm of open-reference operational taxonomic units was utilized.

**Results:** Among a variety of microfungi from CMSW-mycobiota representatives of 20 taxa to varying degrees associated with pathologies and the indigenous human microbiota were revealed. They can
be divided into three groups. Group A consist of micromycetes, which do not colonize the human body normally (or occasional contaminants of the body's open loci) but can cause opportunistic mycoses: *Aspergillus niger*, *Cryptococcus uniguttulatus*, *Rhodotorula glutinis*. Group B including micromycetes, sometimes occurring in biomaterials of human body, as well as colonizing environmental objects: *Candida zeylanoides#*, *C. inconspicua*#, *C. membranifaciens#*, *Pichia norvegensis*#, *Meyerozyma guilliermondii#*, *Kluyveromyces marxianus*, *Lodderomyces elongisporus*, *Issathenkia orientalis*#, *Trichosporon asahii* and *Cutaneotrichosporon cutaneum*. Group C: micromycetes that live mainly in the human body (sometimes in animals) and are not common inhabitants of abiotic objects: *C. albicans*, *C. parapsilosis#*, *C. metapsilosis*, *C. glabrata*#, *C. tropicalis#*, *Malassezia sp*.

**Conclusion:** CMSW is expected to be a habitat for microorganisms that are interesting in two aspects: (1) cause of their capability of carrying out unique metabolic pathways associated with the biodegradation of plant polymers, synthetic plastics and metallic materials and, on the other hand, (2) cause of representation by them the «microbiome imprint» of human population of a settlement area. Some of the detected yeast organisms are known to be resistant to antimycotics: the lists of groups B and C contain species with intrinsic resistance to fluconazole (*) as well as containing strains with secondary resistance mechanisms (#). The most unusual representative among the identified microbiota was *Malassezia sp*. Despite the deep adaptation of malasseziae to the humans and animals skin and numerous unsuccessful attempts to find their free-living related yeast, the application of molecular genetics technologies in the 21st century made it possible to identify the genetic material of these fungi in the most unexpected habitats (deep-sea sediments, invertebrates, some freshwater fishes, soil of arctic zones etc.). Ways to supply the high nutritional needs of malasseziae in the environment are not yet clear, theoretically, *Malassezia spp*. in CMSW can borrow the polyunsaturated fatty acids necessary for them directly from the remnants of dietary fats, or through symbiotic interaction with other members of the microbial community. Conducted research made it possible to consider the CMSW-mycobiota as a potential niche associated with the circulation of opportunistic micromycetes in the anthropogenic ecosystem of the city.

P191

Hunt for *Cryptococcus* : two years survey of the wild avian fauna of Southern France

P. Drakulovski<fnref n="1" />, T. Gouveia<fnref n="1" />, J. Pierru<fnref n="2" />, D. Krasteva<fnref n="1" />, C. Pottier<fnref n="1" />, E. Arianiello<fnref n="2" />, V. Bellet<fnref n="1" />, F. Roger<fnref n="1" />, S. Bertout<fnref n="1" />

**Objectives:** *Cryptococcus* sp. are environmental yeast whose natural biotope is still poorly understood. Indeed, *C. neoformans* has been isolated mostly from pigeon faeces but also from vegetable surfaces and several trees. *C. gattii* has been mostly isolated from decaying wood of various tree species, like *Eucalyptus*, *Pinus*, *Olea*, and *Ceratonia* trees but also from animals. *C. laurentii*, *C. albidus* have been isolated from soil, water but also from animal faeces. A few other species like *C. terrestris* or *C. aerius* are considered environmental only. In order to clarify the biotope of these yeasts, we are conducting a two year survey of the wild avian fauna in Southern France.

**Methods:** Six hundred and four birds from 70 genera and 86 species, randomly selected from the animals arriving for care at the Center for Protection of the Wild Fauna of Herault were sampled. Sampling was performed during care protocols either by direct cloacal swab or, when impossible, by swabbing of faecal drops present in cages. The samples were treated as per the standardized protocol used by the ISHAM Cryptococcal Working Group i.e transferred onto Niger Agar seed medium and grown at 28°C and 37°C during 7 days minimum. Each suspected *Cryptococcus* colony was checked for the presence of a capsule by India ink staining, for its urease activity and identified by API ID32C and ITS sequencing.

**Results:** Identification allowed to find 39 *C. albidus*, 30 *C. magnus*, 13 *C. laurentii*, 2 *Cutaneotrichosporon curvatus*, 2 *C. oeirensis*, 2 *C. diffluens*, 2 *C. carnescens*, 2 *Filobasidiella elegans*, 1 *C. wiriengae*, 1 *C. terrestris* and 1 *C. aerius* coming from 33 different bird species. No *C. neoformans* nor *C. gatti* were found so far. Global cryptococcal prevalence was of 15.7%.

<footnote n="1">¹Laboratoire De Parasitologie Et Mycologie Médicale, University of Montpellier, Montpellier, France,</footnote>

<footnote n="2">²Centre De Sauvegarde De La Faune Sauvage, LPO, Villeveyrac, France</footnote>
**Conclusion:** Several *Cryptococcus* were found from bird wherein they were not described until now. As such, this study brings new data on the avian species potentially carrying various *Cryptococcus* sp yeast.

P192

Microbial monitoring and environmental influence before and after hospital architectural design

F. Bonadio¹, M. Caetano², J.P. Siqueira³, M. Almeida¹

¹Infectious Disease, Medical School, sao josé do rio preto, Brazil, ²UNESP, São José do Rio Preto, Brazil, ³Infeciosas Disease, FAMERP, São José do Rio Preto, Brazil

**Objectives:** To assess the microbial contamination and the environmental impact of reform in a hemodynamic service hospital ward, in a Hospital of the Interior of the State of São Paulo, Brazil.

**Methods:** Environmental samples were collected from 10 surfaces (floor, walls, sinks, and counters), in two different moment, in rooms of medical procedures. RODAC® plates were imprinted in the different surfaces. Considering fungi and bacteria, plates were incubated at 35°C for up to 24 hours to allow all organisms to grow. Colonies were isolates in agar BHI (DIFCO®) with identification of bacterial species conducted by automated system VITEK®, and fungi by slide culture in Potato Agar (DIFCO®) and biochemical characteristic. After first sampling, the rooms of the Hemodynamics Service in the hospital ward was reformed, using all principles and materials commercially available to control microorganisms. Innovative material and procedures of civil construction were adopted, including paint, mortar, and coating following normative criteria according to Brazilian legislation. Then, 10 new surface samples were obtained from the same points of the previous collection, at the same incubation, isolation, and identification criteria.

**Results:** In the first sampling, it was detected contaminated surfaces with high microbial load: 100% for bacteria, and 80% for fungi, ranging from 1 to 400 CFU, according to the collection point. Considering the microbial diversity, there was prevalence for Gram positive bacteria from Staphylococcus spp, and filamentous fungi, especially Aspergillus spp., Acremoniumspp., and Scopulariopsis spp. After the reform, microbiological analysis showed a significant reduction of microbial counts, as well as reduction in the diversity of bacterial and fungal species. It was noted that 80% of the cultures were negative. No fungi were observed and the bacterial isolates are still being identified.

**Conclusion:** The hospital environment should be constantly monitored for its competence in non-proliferation of infectious agents. The presence of bacteria and fungi on surfaces may constitute a risk factor for dissemination and occurrence of infectious diseases, with great impact on Public Health. Old constructions may favor the dissemination of these organisms. This study confirmed the high microbial contamination in the hospital environment before reform and showed that a well-designed architectural design, with appropriate material and adoption of normative procedures, can minimize the risks of contamination and dissemination of potentially pathogenic microorganisms.

P193

Study of fungal environmental contamination in nests of the penguin enclosure of a large French animal zoo park.

N. Cartier¹, R. Le Sénéchal¹, M. Becerra¹, E. Bailly¹, E. Cateau¹, J. Chandenier²,³, B. Mulot⁴, A. Leclerc⁴, G. Desoubeaux²,³

¹Parasitology - Mycology - Tropical Medicine, Hôpital Bretonneau, Tours, France, ²Parasitology - Mycology - Tropical Medicine, Hôpital Bretonneau, TOURS, France, ³Cepr Inserm U1100, Université de Tours, Tours, France, ⁴Clinique Vétérinaire, ZooParc de Beauval & Beauval Nature, Beauval, France

**Objectives:** *Aspergillus fumigatus* is an environmental mold with opportunistic pathogenicity. It is responsible for aspergillosis, a disease particularly common in penguins that are kept in a confined or insufficiently ventilated environment. At ZooParc de Beauval (Saint-Aignan, France), the incidence of aspergillosis was estimated by autopsy at 4% in Humboldt penguins (*Spheniscus humboldti*) during the period [April 2018 - April 2019]. Antifungal treatment is based on the
administration of azole drugs. This study assessed the environmental contamination in the penguin enclosure and searched for azole resistances in *A. fumigatus* isolates.

**Methods:** Two sets of environmental samples were carried out in the spring and fall 2018, by swabbing the artificial nests of the penguin enclosure in ZooParc de Beauval. The collected specimens were inoculated onto malt extract agar at 35°C. In case of fungal growth, a count of the colony number, as well as a phenotypic and molecular identification were achieved. Each DNA of all the *A. fumigatus* isolates was systematically sequenced to look for azole resistance mutations in *CYP51* gene.

**Results:** Overall in spring and fall sampling, 0.8 and 2.6 mean fungal CFU *per* sample of penguin nest were counted, *i.e.* 83% and 100% nests were contaminated with at least one fungal species, respectively. Moreover, *Aspergillus fumigati* section was isolated in 67% and 75% of them. Thus, the concordance rate was 67% between the two sets of sampling. Through to DNA sequencing, all but one isolate (*Aspergillus nishimurae*) were eventually identified as *A. fumigatus stricto sensu*. To date, only one resistance mutation for azole drugs was detected once in the *CYP51* gene (G138A).

**Conclusion:** Despite the geographical location of ZooParc de Beauval in a rural environment, intensive adjacent agricultural practices do not exhibit any impact on the selection of resistant strains. However, the very high contamination rates herein encourage great caution and regular checks of the fungal populations.

P200

**Specific Identification and Antifungal Susceptibility Pattern of Fungal Species Isolated from Patients with Onychomycosis**

I. Haghani¹, M. Shams-Ghahfarokhi², T. Shokohi³, M.T. Hedayati³, A. Dalimi Asl⁴, M. Fathi⁴

¹Medical Mycology, Mazandaran university of Medical sciences, Sari, Iran, ²Department Of Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran, ³Department Of Medical Mycology, Invasive Fungi Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, Sari, Iran, ⁴Department Of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

**Objectives:** Onychomycosis is a common nail problem, accounting for up to 50% of all nail diseases. The aim of the present study was to determine the species distribution based on the restriction fragment length polymorphism and susceptibility patterns of the causative agents of onychomycosis.

**Methods:** This cross-sectional study was conducted on nail samples collected from 257 patients suspected of onychomycosis within a year. Fungal isolates was identified by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) with the enzymes *Msp I*, *Mva I*, *Alw I* and sequencing.

**Results:** According to the results, Out of the 257 patients participating in the study, onychomycosis was diagnosed in 180 (70.03 %) cases, among which 51.1% were caused by non-dermatophyte molds (NDMs), 34.4% by yeasts, and 10.6% by dermatophytes. Numerous cryptic species recovered from onychomycosis for the first time. In the majority of cases, novel triazoles and imidazoles (i.e., efinaconazole, luliconazole, and lanoconazole) showed potent activity in comparison to other antifungal agents. The minimum inhibitory concentration (MIC) of luliconazole and lanoconazole ranged within 0.001 to >1µg/ml and their geometric mean MICs were 0.0154 and 0.0309 µg/ml against all isolates, respectively.

**Conclusion:** It seems that obtained data will be useful to improve the knowledge of researchers, clinicians, and dermatologists about onychomycosis distribution, species diversity and adoption of appropriate treatment.

P201

**Ibrexafungerp (formerly SCY-078) Demonstrates Activity Against Candida auris: In Vitro, In Vivo and Clinical Case Studies of Candidemia**

S. Barat, D. Angulo

SCYNEXIS, Inc., Jersey City, United States of America
**Objectives:** *Candida auris* is a growing global threat; a pathogen associated with high mortality (up to 60%), multi-drug resistance, the ability to spread from person-to-person and surface-to-person, presenting high risk for outbreaks in healthcare facilities. Ibrexafungerp is a novel IV/oral glucan synthase inhibitor (triterpenoid) antifungal with activity against *Candida*, *Aspergillus* and *Pneumocystis* spp, in Phase 3 development. Given the potent activity of ibrexafungerp against *Candida* spp., Scynexis has embarked on a development program to understand the activity and effectiveness against *Candida auris*. We will present the current preclinical and clinical data sets of ibrexafungerp against *Candida auris*.

**Methods:** *In vitro* studies tested ibrexafungerp against >100 clinical isolates of *C. auris*. Other *in vitro* studies evaluated the effects of ibrexafungerp against *C. auris* biofilms. *In vivo* activity against *C. auris* was evaluated using a disseminated murine model and a cutaneous infection guinea pig model. In humans, an ongoing open-label trial of ibrexafungerp for treatment of patients with infections caused by *C. auris* (the CARES study) has been initiated in the USA and India.

**Results:** *In vitro* and *in vivo* studies demonstrated that ibrexafungerp is active against *C. auris*, including MDR strains. The MIC mode for ibrexafungerp was 1ug/ml and the MIC₅₀ and MIC₉₀ were 0.5 and 1 ug/ml, respectively. Many echinocandin resistant *C. auris* isolates have shown susceptibility to ibrexafungerp. Further, ibrexafungerp has been shown to reduce biofilm thickness. In animal models of *C. auris* infection, treatment with ibrexafungerp resulted in improved survival and reduced fungal burden in both the murine model of disseminated infection and the guinea pig model of cutaneous infection as compared to untreated controls. In humans, two patients with difficult to treat *C. auris* candidemias were enrolled in the CARES study and responded positively to oral ibrexafungerp with eradication of the infection.

**Conclusion:** This data demonstrate that ibrexafungerp possess potent *in vitro* and *in vivo* activity as well as promising clinical activity. Therefore, continued clinical evaluation of ibrexafungerp as an option to treat *C. auris* infections is warranted.

P202

**Microsporum ferrugineum in Germany – an anthropophilic dermatophyte on the rise**

S. Uhrlass¹, C. Sitaru², C. Scholz², M. Gebhardt³, A. Baunacke⁴, C. Friedrichs⁵, I. Ranke-Greve⁶, U. Cleffmann⁶, D. Koch¹, H. Muetze¹, C. Krueger¹, N. Rahmig⁷, U.-C. Hipler⁷, P. Nenoff¹

¹Partnership Dr. C. Krueger & Prof. P. Nenoff, Laboratory of medical microbiology, Roetha OT Moelbis, Germany, ²Klinik Für Dermatologie Und Venerologie- Mykologisches Labor, Universitätsklinikum, Freiburg, Germany, ³Hautarztpraxis, Zwickau, Germany, ⁴Hautarztpraxis, Radeberg, Germany, ⁵Medizinisches Labor Ostsachsen, Görlitz, Germany, ⁶Dr. Stephan Bartels, Hautarztpraxis, Göttingen, Germany, ⁷Klinik Für Hautkrankheiten, Universitätsklinikum, Jena, Germany

**Objectives:** In Germany, the anthropophilic dermatophyte *Microsporum (M.) ferrugineum* was nearly never isolated during the last 50 years. Currently, starting with the year 2016, single strains were detected, and small outbreaks of infections due to this difficult to differentiate fungus have been observed in Germany. From July 2016 until April 2019, altogether 19 *M. ferrugineum* strains were isolated. The dermatophytes originated from patients all over Germany.

**Methods:** Cultural investigation revealed slowly growing colonies with white thallus and peripheral yellow brownish submerged hyphae bundles. The reverse side of the furrowed colonies showed cream coloured to yellow staining. Microscopically, large spherical and oval double-walled intercalary localized chlamydospores, typical ‘bamboo’ hyphae, and acute-angled branched hyphae developed. Fungal culture material from all isolates was identified by PCR and genomic Sanger sequencing of the internal transcribed spacer (ITS) region and/or the translation elongation factor (TEF)-1α gene. Reference strain for comparative identification was *M. ferrugineum* CBS 497.48 (Centraalbureau voor Schimmelcultures CBS, Utrecht, The Netherlands, www.westerdijkinstitute.nl). Patients were children and adolescents under 18 years, mainly males. Suggested source of infection was martial sports, e.g. wrestling, judo, and boxing. Surprisingly, a significant part of affected patients counted to Russian Germans. A migrant 3 years old boy from
Afghanistan suffering from tinea capitis was also among the patients. Another strain was isolated from a 10 years old wrestler with suspicion of tinea corporis. There was no background of migration or contact to foreigners, the boy did not stay abroad. The mycological challenge is the cultural identification of *M. ferrugineum*. Confusion with the morphological similar species *M. canis*, but also *M. audouinii*, and *Trichophyton verrucosum* is possible.

**Results:** The comparison of the found ITS-2 sequence with known sequences deposited at the database of the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, U.S., confirmed the species identification *M. ferrugineum*. The phylogenetic analysis of the dermatophytes – the dendrogram of fungal strains – demonstrated the genetic differences between *M. ferrugineum* strains and *M. audouinii* or *M. canis*. The three species could clear distinguished from each other. In particular, sequencing of the TEF-1α gene allowed discrimination between *M. ferrugineum* and *M. audouinii* or *M. canis* better than sequencing the ITS-2-region. Meanwhile, the *M. ferrugineum* strain 208361/2016 is deposited at the „Deutsche Sammlung von Mikroorganismen und Zellkulturen" (DSMZ, German Collection of Micro organisms and Cell cultures) in Braunschweig, Germany (DSM no. 103785). The DNA sequences of the *M. ferrugineum* strains are currently deposited as MF173061-ITS - and as MF173060 - TEF-1α gene - at the NCBI database.

**Conclusion:** In Germany, in the last decades, *M. ferrugineum* was not isolated and described so far. Currently, with this forgotten dermatophyte must be expected in particular in migrants and asylum seekers. The species identification of *M. ferrugineum* represents a challenge for the dermatologist. If there are morphological and microscopic features suspicious for such a rare *Microsporum*-species, molecular identification of the causative pathogen should be performed. For oral therapy of tinea capitis due to *M. ferrugineum* griseofulvin should be given, alternatively itraconazole or fluconazole.

P203

*Arthroderma chiloniense* - a new geophilic dermatophyte - isolated from human and hedgehog

S. Uhrlass¹, D. Rimek², V. Hubka³, W. Schroedl⁴, M. Reuschel⁵, J. Brasch⁶, H. Muetze¹, D. Koch¹, C. Krueger¹, Y. Graeser⁷, P. Nenoff¹

¹Partnership Dr. C. Krueger & Prof. P. Nenoff, Laboratory of medical microbiology, Roetha OT Moelbis, Germany, ²Gesundheitsschutz, Thueringer Landesamt für Verbraucherschutz, Bad Langensalza, Germany, ³Laboratory Of Fungal Genetics And Metabolism, Institute of Microbiology ASCR, Prague, Czech Republic, ⁴Ag Mykologie, Institut für Bakteriologie und Mykologie, Leipzig, Germany, ⁵Klinik Für Heimtiere, Reptilien, Zier- Und Wildvögel, Stiftung Tierärztliche Hochschule, Hannover, Germany, ⁶Klinik Für Dermatologie, Venerologie Und Allergologie, Universitätsklinikum Schleswig-Holstein, Kiel, Germany, ⁷Institut Für Mikrobiologie Und Hygiene, Universitätsmedizin – Charité, Berlin, Germany

**Objectives:** Due to the new taxonomy of the dermatophytes, geophilic fungal species are summarized within the genus *Arthroderma* (A.). Until now known *Arthroderma* species are e.g. *A. insingulare*, *A. gertleri*, *A. uncinatum*, *A. thuringiensis*, besides, there are the three relatively new species *A. amazonicum*, *A. crocatum*, and *A. vespertilio*. Altogether four strains of an until now not known geophilic fungus - suggested as mould or dermatophyte - were isolated between 2011 and 2017 from different patient's materials. Underlying dermatoses were a hand eczema or suspected tinea manus, nail changes (nail plate pigmentation) to exclude an onychomycosis, and skin scrapings from the lower leg and body trunk to exclude a tinea corporis. An additional strain of *A. chiloniense* was found in clinical material originating from the skin and stings of a hedgehog with alopecia diffusa and scaly skin.

**Methods:** Conventional and molecular mycological diagnostic was performed. Kind of growth of the fungal colonies on common fungal media was assessed. Fungal culture material from all five isolates was identified by PCR and genomic Sanger sequencing of the internal transcribed spacer (ITS: 18S rRNA, ITS1, 5.8S rRNA, ITS2, and 28S rRNA) region, the translation elongation factor (TEF)-1α gene, and β-tubulin. Human strains 216228/16- DSM106573, 211495/17- DSM106575, 211496/17- DSM106574 and 215488/17- DSM108904 are deposited at the „Deutsche Sammlung von Mikroorganismen und Zellkulturen" (DSMZ, German Collection of Micro organisms and Cell
cultures) in Braunschweig, Germany. The from hedgehog isolated strain with laboratory number 250173/2018 has already been submitted to the DSMZ, too.

**Results:** The white, flat, fast growing colonies presented with yellow reverse sides. Microconidia are small, elongated, sessile lateral directly at the hyphae or the conidiophores, also in dense aggregates. Later arthroconidia and multiple chlamydospores have developed, however, no macroconidia. Urea splitting of all isolates was positive. By sequencing of the gene regions ITS-1 and ITS-2, TEF-1α and β-tubulin showed, that the new species showed the highest genetic similarity to the species *A. crocatum* and *A. amazonicum*. Ultimately, the sequences of the here isolated unknown fungi could be assigned to the new described geophilic dermatophyte *Arthroderma chiloniense* sp. nov.

**Conclusion:** *A. chiloniense* presents a new geophilic dermatophyte, first described in Kiel, Germany, in 2018, and published 2019. This species is obviously widely prevalent in Germany. Isolated from human beings under the suspicion of a dermatophytosis of the skin or nails, seems to indicate that *A. chiloniense* possesses pathogenic potential. Here, *A. chiloniense*, for the first time was isolated from an animal, an European hedgehog.

P204

## The First Case of *Candida auris* Fungemia in Thailand

M. Chayakulkeeree¹, P. Ungulkraiwit², P. Chongtrakool³, P. Ngamskulrungroj³, T. De Groot⁴, J. Meis⁵
¹Medicine, Division Of Infectious Diseases And Tropical Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, ²Department Of Medicine, Vejthani Hospital, Bangkok, Thailand, ³Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, ⁴Medical Microbiology And Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, Netherlands, ⁵Department Of Medical Microbiology And Infectious Diseases, Excellence Center For Medical Mycology (ecmm), Canisius Wilhelmina Hospital, Nijmegen, Netherlands

### Case Report:

**Objectives:** *Candida auris* is an emerging fungal species resistant to several antifungal agents. There is an increasing worldwide awareness of *C. auris*. We therefore aim to report the first case of *C. auris* infection in Thailand.

**Methods:** We present the first case of *C. auris* fungemia in an elderly woman from Thailand.

**Results:** A 81-year-old woman was admitted in a private hospital in January 2018 due to status epilepticus which required ICU support with prolonged mechanical ventilation. In addition, she had chronic kidney disease requiring hemodialysis through her right subclavian central venous catheter. During 4 months in the hospital, she experienced catheter-related bacterial blood stream infection, nosocomial pneumonia, urinary tract infection and tracheobronchitis which was treated with several regimens of broad-spectrum antibiotics. On day 110 of hospitalization, she had hypotension with low-grade fever during hemodialysis. Blood cultures from her central venous catheter grew *Candida* spp. While peripheral line cultures remained negative. The central catheter was removed and she was treated with micafungin. The *Candida* isolate was subsequently identified as *Candida auris* by MALDI-TOF and sequencing. Swab cultures from the central catheter exit site also grew *C. auris*. She denied any travel outside of Thailand prior to admission. Despite intravascular catheter removal, she had again candidemia from *C. auris* three weeks later and micafungin was re-introduced. However, the patient died 6 days after restart of treatment. After diagnosis, strict contact precautions were installed with no further intra-hospital transmission. CLSI microbroth dilution antifungal susceptibility testing of all isolates showed resistance to fluconazole (32 mg/L) and voriconazole (0.25 mg/L), higher MICs of amphotericin B (1 mg/L) and low MICs to echinocandins (0.063 mg/L). Isavuconazole had low MICs of < 0.016 mg/L. By using AFLP and MLVA genotyping the isolates had the same genotype and were shown to belong to the south-Asian *C. auris* clade.

**Conclusion:** We report the first case of *C. auris* (south-Asian clade I) fungemia from Thailand. The index case for this *C. auris* introduction into Thailand remained unclear and no secondary cases were discovered. Active surveillance for this emerging antifungal-resistant *Candida* species should be brought into attention to prevent healthcare-associated outbreaks.
P205

Disseminated phaeohyphomycotic lymphadenitis with Cladophialophora carrionii

J. Farooqi¹, S. Moin¹, S.F. Mahmood², R. Idress¹, F. Naqvi¹, A. Zafar¹

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

Case Report:

**Objective:** This study describes a case of disseminated phaeohyphomycotic lymphadenitis in a young woman with delayed diagnosis and good clinical response after appropriate treatment.

**Methods:** A 32 year old lady presented with erythematous to violaceous papules, with oozing discharge bilaterally in her inguinal region, for a few months. The history revealed that she had a past history of tuberculous meningitis, treated with first line antituberculous therapy for eighteen months, and two years back she developed pigmented discharging lymph nodes bilaterally in her axillae. The histopathology of the biopsy showed chronic granulomatous inflammation with multiple branching septate fungal hyphae. She received amphotericin B for 21 days but without improvement. Now, sample from these inguinal lesions was sent for histopathology and culture.

**Results:** Histopathology of the biopsy material showed dense inflammation and well-formed granuloma comprising of epithelioid cells and multinucleate giant cells. Multiple branching septate fungal hyphae noted in the inflammatory debri and in and around granuloma. PAS stain highlighted septate fungal hyphae. Gram stain revealed moderate septate hyphae with numerous pus cells. Culture on Sabouraud dextrose agar yielded velvety olive-black colony in the fourth week. Microscopic slide examination of culture material was suggestive of *Cladophialophora carrionii*. The patient was started on voriconazole and was continued this for 6 months and the patient improved. On discontinuing voriconazole after six months, there was a relapse in the disease. Voriconazole was restarted and the patient improved.

**Conclusion:** Incomplete investigation of infectious lesions may delay diagnosis. Both histopathological and microbiological assessments are equally important for making a conclusive diagnosis. Antifungal therapy may delay growth of fungi that normally grow within a week, hence a longer incubation time may be warranted for fungal smear positive samples. In phaeohyphomycosis, the infection is usually introduced by traumatic inoculation or inhalation of the etiologic agent; and clinical presentations are greatly influenced by the immune status of the host. In our case there was no known immune deficiency and no trauma that the patient could recall.

P206

Genomic sequencing of *Candida auris* cases in Canada, 2012 - Feb 2019

A. Bharat¹, D. Alexander², T.C. Dingle³, P.J. Dufresne⁴, L.M. Hoang⁵, J.V. Kus⁶, I.S. Schwartz⁷, L. Gade⁸, A.P. Litvintseva⁸, A. Mcgeer⁹, M.R. Mulvey¹

¹Antimicrobial Resistance And Nosocomial Infections, National Microbiology Laboratory Canada, Winnipeg, Canada, ²Cadham Provincial Laboratory, Winnipeg, Canada, ³Alberta Public Laboratories, Edmonton, Canada, ⁴Laboratoire de santé publique du Québec, Sainte-Anne-de-Bellevue, Canada, ⁵BC Centres for Disease Control, Vancouver, Canada, ⁶Public Health Ontario, Toronto, Canada, ⁷University of Alberta, Edmonton, Canada, ⁸Mycotic Diseases Branch, US Centers for Disease Control and Prevention, Atlanta, United States of America, ⁹Mount Sinai Hospital, Toronto, Canada

**Objectives:** *Candida auris* is an emerging yeast that is associated with high rates of antifungal resistance and healthcare-associated outbreaks that can be difficult to control. Our objective was to carry out genomic characterization of all known *C. auris* cases in Canada to monitor the emergence of this species.

**Methods:** Nineteen isolates of *C. auris* identified from 2012 - Feb 2019 were submitted to the Canadian Mycology Reference Centre which is in development at Canada's National Microbiology Laboratory (NML). Isolates from all 19 cases were subjected to whole genome sequencing (WGS) on the Illumina Nextseq platform. Phylogenetic analysis based on nucleotide differences in the core genome was carried out with the SNVPhyl v1.1 pipeline and assemblies were performed with SPAdes v1.6.
**Results:** The isolates were obtained from axilla/groin (n = 7), blood (n = 4), ear (n = 3), and other sites (n = 5). All Canadian isolates of *C. auris* fell within the four known genomic lineages (clades), which were initially named after the continents where they were identified and then renamed Clades I - IV. Canadian cases were linked to the Clade I (South Asian clade, n = 10), Clade IV (South American clade, n = 4), Clade III (African clade, n = 3), and Clade II (East Asian clade, n = 1). The four geographical clades were highly divergent with approximately 15,000 - 60,000 single nucleotide variants (SNV) between clades. Canadian isolates within a clade were often clonal, for example, isolates from Clade IV (South American clade) differed by only 14-26 SNVs. Canadian isolates from Clade I (South Asian clade) differed by an average of 79 SNVs, however, epidemiologically-linked isolates within this clade differed by only 1-19 SNVs. We identified mutations associated with fluconazole resistance (ERG11 Y143R) and voriconazole resistance (ERG11 Y132F) but not echinocandin resistance (FKS1 hotspot mutations).

**Conclusion:** The number of known cases of *C. auris* in Canada remains low and all cases were susceptible to echinocandins. Whole genome sequencing confirmed the global origins of Canadian cases. Isolates tended to be clonal within each clade but high resolution WGS may be helpful in discriminating between patient transmission and separate introductions into a healthcare facility.

P207

Misidentification of *Candida duobushaemulonii* associated to superficial infection and reduced susceptibility to azoles

I. Jurado¹, E. Varela-Echegaray¹, C. Marcos-Arias¹, P. De Groot², G. Quindós¹, *E. Eraso*¹

¹Inmunology, Microbiology, Parasitology, University of the Basque Country (UVP/EHU), Bilbao, Spain, ²Regional Center For Biomedical Research, Castilla-la Mancha Science & Technology Park, University of Castilla-La Mancha, Albacete, Spain

**Objectives:** Candidiasis by *Candida haemulonii* complex (*Candida haemulonii* and *Candida duobushaemulonii*) and closely related species, such as *Candida auris* and *Candida pseudohaemulonii* that show reduced susceptibility to antifungal drugs are increasing. Conventional phenotypic diagnostic methods are unable to identify these species. Therefore, their prevalence may have been underestimated. The aim was to use novel molecular techniques to identify isolates from a stock culture collection with dubious identities, which could belong to emerging and multi-resistant species related to *C. haemulonii*.

**Methods:** The study included 150 clinical isolates from the yeast stock collection of the Medical Mycology Laboratory at the University of the Basque Country (UPV/EHU) collected between 1993 and 2017 that could be inaccurately identified by API®ID 32C (bioMérieux, France). Three clinical confirmed isolates (two *C. auris* and one *C. duobushaemulonii*) and one *C. pseudohaemulonii* reference strain (CBS 10004) were included as positive controls. All isolates were analysed by two different PCR techniques: a *C. auris*-specific PCR¹ and a *C. haemulonii* complex-tetraplex PCR². Species identities were confirmed by sequencing the ITS1-5.8S-ITS2 region. Finally, *in vitro* susceptibility to nine antifungal agents (anidulafungin, micafungin, caspofungin, posaconazole, voriconazole, itraconazole, fluconazole, amphotericin B and 5-fluorocytosine) was tested using Sensititre™ YeastOneYO10 (Thermo Scientific, USA).

**Results:** *C. auris*-specific PCR accurately identified both confirmed isolates of *C. auris* and no bands were detected for the other isolates. Eight isolates previously identified as *C. haemulonii* were confirmed by tetraplex PCR. Neither *C. auris* nor *C. pseudohaemulonii* were detected in the collection. One isolate from a toenail previously identified in 1996 by API®ID 32C panel as *Candida intermedia* produced a 115 bp sized band in the tetraplex PCR, corresponding to *C. duobushaemulonii*. All identifications were confirmed by ITS sequencing. The *C. duobushaemulonii* isolate was re-analysed by the current API®ID 32C panel, which identified it as *Candida sake*. The *C. duobushaemulonii* isolate was susceptible dose-dependent to itraconazole (MIC 0.5 µg/mL) and fluconazole (MIC 32 µg/mL). All *C. haemulonii* were susceptible dose-dependent to itraconazole (MIC 0.5 µg/mL) and fluconazole (MIC 16 µg/mL) except two, who were resistant (MIC 1 and 64 µg/mL, respectively), and their susceptibility to amphotericin B was reduced (MIC 2->8 µg/mL). The *C. pseudohaemulonii* reference
strain CBS 10004 was susceptible to all antifungal drugs. Both *C. auris* isolates showed resistance to fluconazole (MIC >256 µg/mL), dose-dependent susceptibility to itraconazole (MIC 0.25 µg/mL), and one of them was also susceptible dose-dependent to voriconazole (MIC 2 µg/mL).

**Conclusion:** *Candida haemulonii* complex and closely related species may have been associated to superficial infections prior to its emergence related to invasive infections, as indicated by the identification of a *C. duobushaemulonii* isolate from before 2012, when current classification of the complex was established. This complex shows reduced susceptibility to azoles and amphotericin B.

**Funding:** GIC15/78 IT-990-16 (Gobierno Vasco) and SAF2017-86188-P (MINECO, Spain). 1. Ruiz-Gaitán et al. *Molecular identification of Candida auris by PCR amplification of species-specific GPI protein-encoding genes*. Int J Med Microbiol. 2018;308:812-818. 2. Arastehfar et al. *Low-cost tetraplex PCR for the global spreading multi-drug resistant fungus, Candida auris and its phylogenetic relatives*. Front Microbiol. 2018;29:91119.

P208

**Acute fungal obstructive pyelonephritis, echinocandin is a possible treatment? A case report**

J. Testa¹,², A. Calori³, L.A. Nicolini⁴, M.G. Pizzi², C.G. Zeroli², R. Martegani², B. Menzaghi²

¹Biotechnologies And Life Sciences Department, Center for Clinical Ethics, University of Insubria, Varese, Italy, ²Busto Arsizio Hospital, Asst-valleolona, Infectious Diseases Unit, Busto Arsizio (VA), Italy, ³Busto Arsizio Hospital, Asst-valleolona, Urology Unit, Busto Arsizio, Italy, ⁴Department Of Health Science (dissal), Infectious Diseases Unit, Genova, Italy

**Case Report:**

**Objectives/Introduction:** Urinary tract candidiasis is the most frequent nosocomial fungal infection worldwide; ascending acute pyelonephritis (AP) due to Candida species are uncommon complications. Nowadays we observe a change in the distribution of *Candida* species, with an increasing number of non-albicans Candidaisolations. In this setting Fluconazole is the treatment of choice; the role of echinocandin is unknown, because of the limited repository of antifungals that achieve adequate urinary concentrations.

**Methods:** We report a *Candida tropicalis* complicated urinary tract infection (cUTI) in a patient with diabetes mellitus who successfully treated by high dosage of Caspofungin and oral fluconazole (FLC). The infection was triggered by the placement of a ureteral double J stent and bladder catheter, arranged for kidney stones.

**Results:** A 67-year-old Italian woman affected by diabetes mellitus, metabolic syndrome and recent breast cancer, was transferred in the autumn of 2018 from an outside institution after double J stent positioning for left ureteral stone complicated by grade III hydronephrosis. At the admission fever and increase inflammatory values were present, so blood and urine cultures were performed. Empiric antibiotic therapy with carbapenems was started, without clinic improvement in 5-6 days, so we interrupted this therapy. We isolated only pan-sensitive *Candida tropicalis* in urine sample but with few colonies, so we considered as colonization. She therefore underwent to CT-scan with urological and thoracic studies: we found left AP complicated by micro abscesses and pulmonary thrombo-embolism. We decided to introduce high dosage of FLC and to remove ureteral catheter double J and bladder catheter; we isolated from them another time the same type of *Candida tropicalis*. Considering a diffuse disease, trans-thoracic echocardiography and fundus oculi exam were performed, excluding fungal manifestations. After few days she remained febrile and blood inflammation biomarkers not decreased very well, so we decided to switch therapy with an alternative one, suspending FLC, we started Caspofungin at the dosage of 70 mg/die. Immediately after initiation of treatment we observed a rapid improvement with disappearance of the fever. After two weeks, abdomen CT-scan showed an inflammation reduction and she was discharged home with maintenance high dosage FLC (800 mg/die), concluding antifungal therapy after 3 overall months. Finally, after therapy, CT-scan and urine culture were prescribed with no evidence of recurrence. The patient is currently alive, without urinary problems.

**Conclusion:** *Candida tropicalis* is a rare cause of AP. Echinocandins have been traditionally avoided in the treatment of cUTI because of their poor urinary concentrations; their use with high dosage,
owes an achieved high renal tissue concentration. This case shows that in fungal cUTI we can use
high dosage echinocandin if fluconazole is no possible for any reasons. We want also to stress that is
necessary a prolonged therapy in fungal AP.

P209

The first two cases of *Candida auris* in The Netherlands

E. Vogelzang¹, A. Weersink², J. Meis³, K. Van Dijk¹

¹Medical Microbiology And Infection Control, AUmc, location VUmc, Amsterdam, Netherlands,
²Medical Microbiology And Immunology, Meander Medical Center, Amersfoort, Netherlands,
³Department Of Medical Microbiology And Infectious Diseases, Excellence Center For Medical Mycology (ecmm), Canisius Wilhelmina Hospital, Nijmegen, Netherlands

**Objectives:** *Candida auris* is a rapidly emerging, multidrug resistant pathogenic yeast. In recent years an increasing number of *Candida auris* invasive infections and colonized patients have been reported from several continents, including the USA, Africa, Europe and Asia. Here, we describe the first two cases of *Candida auris* in The Netherlands.

**Methods:** Case 1 was a middle aged patient repatriated from an Indian hospital to a Dutch intensive care unit (ICU). He had been hospitalized for five weeks in an Indian hospital for treatment of sepsis with multiple organ failure due to pneumonia. He was mechanically ventilated and repatriated with a tracheostomy tube and continuous renal replacement therapy for which he had a central venous catheter. Routine admission screening cultures for MRSA and multidrug-resistant gram-negative bacteria showed yeast growth from Continuous renal replacement therapya removed central venous catheter which the Bruker MALDI-TOF MS identified as *Candida auris*. Case 2 was a middle aged patient, referred by the Amsterdam airport medical services to the hospital due to shortness of breath and inability to stand without assistance. He was in Amsterdam airport for a layover flying from India to the USA. Due to renal insufficiency, he had received hemodialysis in an Indian healthcare facility up to two days before presentation in our hospital. Due to subfebrile temperature a urine sample was cultured which grew 10^4-5 CFU yeasts. The Bruker MALDI-TOF MS identified the yeast as *Candida auris*.

**Results:** In both cases contact precautions were instituted since patients had been previously admitted to an Indian healthcare facility. Routine screening cultures were taken from nose, throat and perineum to identify potential multidrug-resistant gram-negative bacteria and MRSA. At present no routine screening for *Candida auris* is performed in the Netherlands. Screening failed to detect *Candida auris*, but groins and axilla screens were not included. Molecular typing with AFLP and MLVA showed that both isolates had closely similar genotypes and both belonged to the South Asian *Candida auris* Clade I. Broth microdilution showed that both *Candida auris* isolates had high fluconazole (>64 mg/L) and voriconazole (4 mg/L) MICs. Amphotericin B (0.5-1 mg/L), anidulafungin (<0.016-0.063 mg/L) and micafungin (0.063 mg/L) had low MICs. In both cases antifungal therapy was not indicated since there were no signs of an infection. Due to installment of contact precautions no transmission of *Candida auris* was observed.

**Conclusion:** We report the first two cases of *Candida auris* in The Netherlands in patients who were recently admitted to an Indian healthcare facility. The patients were immediately placed in contact isolation after admission and no transmission of *Candida auris* occurred. Concern is raised regarding surveillance for multidrug-resistant organisms, since routine screening of patients from foreign hospitals is still focused on multidrug-resistant gram-negative bacteria and MRSA and not yet on (multi)drug resistant yeast, such as *Candida auris*.

P210

Evaluation of the disinfectant activity of a portable ultraviolet C equipment

A. López-Saiz, *E. Sevillano*, A. Guridi, I. De-La-Fuente, E. Mateo, E. Eraso, G. Quindós Immunology, Microbiology And Parasitology, University of the Basque Country (UPV/EHU), Bilbao, Spain
**Objectives:** Healthcare-associated infections are caused by different microorganisms, including bacteria, viruses and fungi. In the recent years, *Candida auris* has emerged as a multidrug-resistant fungal pathogen difficult to eradicate from hospital environment. It has been isolated from medical equipment, and from different objects that hardly ever are disinfected and can act as fomites (bed hand-controllers or mobile phones). In some cases, routine chemical and physical decontamination approaches are used but not all the above-mentioned objects can be treated with them due to possible deterioration. Ultraviolet C radiation could represent an alternative, which does not cause aesthetic or functional alterations in inanimate objects. Therefore, the objective of this study was to evaluate the disinfection capacity of a portable ultraviolet C (UV Sanitizer Corvent®) equipment.

**Methods:** The disinfectant effect of the UV sanitizer equipment was evaluated using different exposure times (30 s, 1 min, 2 min, 3 min and 5 min), against five *Candida* isolates: three clinical strains of *C. auris*, and two reference strains, *Candida albicans* SC5314 and *Candida parapsilosis* ATCC 22019. These microorganisms were inoculated onto slides, starting from a suspension of 1.5-5.0 x 10⁹ ufc/mL, that was mixed with an interference solution to analyze the disinfecting effect in both clean and dirty conditions (presence of organic matter). Slides were exposed to ultraviolet C radiation inside the equipment. The results were compared with those obtained after the disinfection of the slides by immersion in 70% ethanol following the protocol described in EN 14562:2006 standard.

**Results:** Disinfection, considered as a logarithmic reduction equal to or greater than 5 from the initial inoculum, was achieved at a minimum exposition time of 3 min, in both clean and dirty conditions. The presence of organic matter interfered in the disinfection process to some extent, since the final values were slightly higher after the treatment of the samples in dirty conditions. The effectivity of 3 min immersion in 70% ethanol was very high against all microorganisms, obtaining a total reduction in all cases.

**Conclusion:** The ultraviolet C radiation applied for 3 minutes using the portable ultraviolet C UV Sanitizer Corvent® equipment was effective in the disinfection of slides inoculated with *C. auris*, *C. albicans* and *C. parapsilosis*. This equipment is a promising alternative for implementing disinfection protocols in hospitals and laboratories with inanimate objects of common use (mobile phones, TV or bed controllers) reducing risk of infection transmission.

P211

Emergence of ERG11 Y132F azole resistant *Candida parapsilosis* blood isolates in a Turkish hospital: A 10 year experience (2009-2019)

A. Arastehfar¹, F. Daneshnia¹, S. Hilmioğlu-Polat², W. Fang³, M. Yaşar², F. Polat², D. Yeşim Metin², K. Zomorodian⁴, T. Coenye⁵, M. Ilkit⁶, W. Pan³, W. Liao³, F. Hagen¹, M. Kostrzewa⁷, C. Lass-Flörl⁸, T. Boekhout¹

¹Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands, ²Department of Microbiology, Faculty of Medicine, University of Ege, Izmir, Turkey, ³Medical Mycology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China, ⁴Basic Sciences In Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, ⁵UGent, Ggent, Belgium, ⁶Division of Mycology, Department of Microbiology, Faculty of Medicine, University of Çukurova, Adana, Turkey, ⁷Research And Development, Microbiology And Diagnostics, Bruker Daltonik GmbH, Bremen, Germany, ⁸Div Hygiene & Med. Microbiology, Med. Univ. Innsbruck, Innsbruck, Austria

**Objectives:** The recent emergence of azole resistant *C. parapsilosis* isolates carrying Y132F mutation in *ERG11* is a matter of concern in India, Kuwait, South Korea, and USA. Given the horizontal transfer of *C. parapsilosis* and the propensity of Y132F mutants to colonize the hospital environments, their prompt identification followed by application of appropriate infection control strategies are of importance.

**Methods:** During 10 years (2009-2019), 228 non-duplicate isolates of *C. parapsilosis* were recovered from the blood samples of patients admitted to Ege University Hospital, Izmir, Turkey. All isolates were primarily identified by API 20C AUX and re-identified via MALDI-TOF MS. Regardless of fluconazole MIC values, *ERG11* sequencing was performed for all 228 isolates. In the next step, *MRR1*,
TAC1, and UPC2 sequencing was performed for 90 isolates containing all resistant isolates with or without ERG11 Y132F mutation and randomly selected susceptible dose-dependent and susceptible isolates.

**Results:** Surprisingly, Y132F was the most predominant mutation in *ERG11* (*n* = 46, 20.2%), followed by K143R (*n* = 21, 9.2%), and G458S (*n* = 6, 2.6%), all implicated in fluconazole resistance. Moreover, Q250K and G307A were two newly suggestive mutations exclusively occurred in fluconazole resistant isolates. Among isolates harboring *ERG11* Y132F, 43.5%, 17.3%, and 10.8% of isolates carried nonsynonymous mutations in *UPC2*, *TAC1*, and *MRR1*, which is inconsistent to what was previously reported from a South Korean study. The first isolate carrying Y132F mutation emerged in 2012 and isolates carrying this mutation persistently recovered from the blood samples onwards (2012-2019). Strikingly, the number of isolates harboring *ERG11* Y132F, doubled and increased 5 fold between 2009-2015 and 2009-2019, respectively (Figure 1). Future studies are ongoing to determine the corresponding genotype of each isolate using microsatellite typing and the antifungal susceptibility data for voriconazole, micafungin, and anidulafungin remain to be completed.

**Conclusion:** We observed a significant and persistent isolation of emerging *ERG11* Y132F fluconazole-resistant *C. parapsilosis* isolates, which is a worrisome complication in our hospital requiring implementation of typing and sequencing of target genes followed by establishing appropriate infection control strategies to confine further spread of such isolates. The widespread use of azoles in clinical settings, especially developing countries, highlights the importance of worldwide surveillance studies to evaluate the prevalence and spread of *ERG11* Y132F isolates. Figure 1. Accumulation of *ERG11* Y132F mutation in *C. parapsilosis* isolates over 10 years of study from the blood samples of patients admitted to Ege University Hospital, Izmir, Turkey.

P212

*Candida auris:* Epidemiology, Risk Factor Analysis and Management of Invasive Infections in Tertiary Care Hepatobiliary Centre

P. Kale¹, V. Khillan¹, S.K. Sarin²

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

**Objectives:** *Candida auris* involvement in candidemia and other deep-seated invasive infections is associated with very high mortality. The inability of several available commercial identification systems to quickly diagnose *C. auris* remains a challenge to early therapy. The data is limited especially in patients with hepatobiliary diseases, in whom the risk factors and management differs due to the risk of antifungal toxicity. We aimed to study the epidemiology, risk factors and therapeutic management of invasive infections caused by *C. auris* in patients with hepatobiliary diseases.

**Methods:** Single-center, prospective study of patients with suspected invasive fungal infections between January 2017-December 2018. The patients with hepatobiliary diseases were included in the study based on clinical diagnosis and radiological features. Demographics, comorbidities, and laboratory variables were recorded. The samples received in the microbiology laboratory as a part of clinical diagnostic protocols were processed accordingly. The positive yeast cultures were subjected to identification by Vitek 2(Biomérieux, India) with updated software using YST cards, and antifungal sensitivity confirmed by Broth microdilution in accordance with CLSI guidelines. The final outcome considered was mortality within one month after diagnosis or discharge of the patient with stable parameters.

**Results:** Total of 109 isolates of *C. auris* from 55 patients, blood 14(12.8%), abdominal fluids 14(12.8%), urine 58(53.21%), respiratory 4(3.6%), liver abscess 2 (1.83%), pancreatic abscess 1(0.9%) and wound infections 3 (2.75%). Underlying disease was chronic liver disease 37 (68%), 10 (20%) post liver transplant patients, acute on chronic liver failure 4 (7%), acute liver failure 2 (3%), acute pancreatitis 1 (1.8%) and pancreatic neuroendocrine tumor 1 (1.8%). 14 (25%) patients were discharged, mortality was 41 (74.04%). Risk factors were MELD 40 (p0.04), Child C (p 0.05) and CTP score above 12. Prior use of steroids (p 0.02), neutropenia (p 0.03), prolonged hospital stay (0.029), use of broad spectrum antibiotics more than 7 days (p 0.05) were the risk factors significantly associated with development
of *Candida auris* infections and higher mortality. Co-morbidities, acute renal failure, diabetes, hepatitis
infection were not significantly associated with mortality. The antifungal resistance: fluconazole
68.42%, voriconazole 14.03%, flucytosine 59.64%, amphotericin B 28 % with no resistance to
caspofungin and micafungin.

**Conclusion:** *C. auris* infections are associated with misidentification, intrahospital transmission, poor treatment outcomes and higher mortalities. Prompt detection, earlier initiation of therapy and effective surveillance can curtail *C. auris* in hospitals. Our study depicts the spectrum of invasive infections caused by *C. auris*, its prevalence, risk factors and therapeutic options. The presence of risk factors like steroid use, neutropenia, broad spectrum antibiotic use and hospital stay of more than 7 days should prompt towards escalating diagnostic measures for rapid identification of *C. auris* for early initiation of therapy. Active screening of patients with risk factors can also reduce mortality. The study results also help to guide empiric therapy with echinocandins as azoles and amphotericin B show high resistance in these isolates. This study will guide prophylactic and therapeutic options in hepatobiliary disease patients with underlying risk factors to reduce mortality.

P213

Lignin degradation pathway by *Scedosporium* species

W. Poirier, S. Giraud, J.-P. Bouchara

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

**Objectives:** *Scedosporium* are usually soil saprophytes. Among the ten species recognized within the genus, three have been regularly reported as causing human infections, particularly in patients with cystic fibrosis (CF): *Scedosporium apiospermum*, *Scedosporium aurantiacum* and *Scedosporium boydii*. Because of their low sensitivity to almost all available antifungal drugs, a better understanding of the pathogenic mechanisms of these fungi is required in order to identify new therapeutic targets. Likewise, identification of the origin of the contamination of patients may be helpful to propose prophylactic measures. In this aim, environmental studies were conducted on the ecology of these opportunistic molds. *Scedosporium* species are abundant in human-made environments, in relation with specific traits of the fungi: ability to survive at low oxygen pressure and to degrade hydrocarbons. Recent studies have demonstrated the correlation between the lignocellulolytic properties of filamentous fungi and their ability to degrade environmental organic pollutants such as aromatic hydrocarbons. Among the lignocellulosic compounds, the particular properties of lignin make it an interesting study molecule. In this context, we attempted to elucidate the mechanisms developed by *Scedosporium* species to degrade this polyphenolic molecule, with a special emphasis on the opening of aromatic rings.

**Methods:** Culture in specific media and biochemical analyses were performed to characterize *Scedosporium* properties. Orthologue enzymes from the literature were used to screen *Scedosporium* genomic data by tBLASTn (Basic Local Alignment Search Tool) searches and real-time PCR experiments were then conducted to analyze the relative expression of these candidate genes.

**Results:** The lignolytic properties of *Scedosporium* species were first confirmed by cultivation of the fungi on agar media containing the different components of lignocellulose as carbon source. According to literature, the early steps in the lignin degradation pathway involve peroxidases and oxidases. Biochemical analyses allowed us to detect these enzyme activities in the culture supernatant of these fungi grown in lignin-containing media. Fifteen orthologue genes were identified by tBLASTn searches, four encoding members of the peroxidase family and the others encoding oxidase. However real-time PCR experiments revealed that only four of these genes encoding peroxidases (2) and oxidases (2) were over-expressed in the presence of lignin. Based on an exhaustive bibliography, at least three different pathways, *i.e* the β-ketoadipate, the oxoadipate and the gentisate pathways, were identified as potential routes for the degradation of molecules resulting from the early oxidation steps and the opening of the aromatic rings. In all *Scedosporium* species, enzymes involved in the gentisate pathway are encoded by genes organized into a six membered gene cluster as revealed by