gold-standard method for dermatophyte identification is microscopy and culture. Microscopy has
low specificity and culture has low

**Methods:** (1) The gold-standard methods routinely used in the laboratory were microscopy using 30% KOH and culture using DTM (dermatophyte test medium) agar incubated at 28°C for up to 4 weeks. For 12 months, from 1.1.2016 until 31.12.2016 samples from skin, nail and hair from patients with suspected tinea were analyzed using the gold-standard methods. (2) A new Israeli commercial molecular kit called DERMADYN Dermatophytes detection test V.01 (Dyn R&D) kit that identifies 7 dermatophytes; *T. rubrum* complex, *T. tonsurans*, *T. violaceum*, *M. gypseum*, *E. floccosum*, *M. canis* and *T. mentagrophytes* complex was evaluated compared to the gold-standard on 103 samples. (3) From 1.8.18 until 31.12.18, 1179 samples were analyzed for the presence of Dermatophytes using the molecular kit only.

**Results:** Using the gold-standard only 480 (23.6%) samples were positive for dermatophytes with a distribution of 462 (23%), 12 (0.6%), 2 (0.1%), 2 (0.1%) 1 (0.05%), 1 (0.05%) positive for *T. rubrum* complex, *T. mentagrophytes* complex, *M. canis*, *Scopulariopsis brevicaulis*, *T. tonsurans*, and *M. gypseum*, respectively. Discrepancies between microscopy and culture were found in 27% of the samples. (2) There was an agreement between the gold-standard and the molecular kit in 60 out of the 103 samples tested (58%), a complete disagreement in 9 (9%) and a partial disagreement in 34 (33%) of them. (3) Using the molecular approach 578 (49%) samples were positive for dermatophytes with the distribution of 551 (46.7%), 2 (0.2%), 8 (0.7%), 6 (0.5%), 1 (0.08%) and 10 (0.85%) positive for *T. rubrum* complex, *T. mentagrophytes* complex, *M. canis*, *T. tonsurans*, *M. gypseum* and *E. floccosum*, respectively.

**Conclusion:** Twice the number of dermatophytes were identified using the molecular approach compared to the gold-standard with a similar distribution of species having *T. rubrum* complex the main pathogen. The molecular assay has a much shorter training period, a high capacity of performance and high sensitivity. Although the kit contains only 7 dermatophytes, these are the most prevalent species in Israel. As discrepancies between microscopy and culture is a common problem and as culture has low sensitivity Labs should consider evaluating a molecular based diagnostic method for dermatophyte identification to increase detection rates.

P483

In the *Aspergillus* section *Nigri*, *Aspergillus welwitschiae* is more often responsible for otomycosis than *Aspergillus tubingensis* and *Aspergillus niger*

M. Gits-Muselli¹,², S. Hamane², B. Verillaud³, E. Cherpin², N. Guigue², D. Garcia-Hermoso¹, A. Alanio⁴, S. Bretagne⁴

¹Molecular Mycology, Institut Pasteur, PARIS, France, ²Parasitology-mycology Laboratory, Lariboisière Saint-Louis Fernand Widal Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), PARIS, France, ³Ent Department, Lariboisière Saint-Louis Fernand Widal Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), PARIS, France, ⁴Paris-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:** Fifteen percent of external otitis are otomycosis (Ninkovic et al. 2008). *Aspergillus* section *Nigri* are the most frequent molds isolated from external ear conduct (EEC) samples followed by *Aspergillus* section *Flavi* (R. Sarvan et al. 2012). *Aspergillus* section *Nigri* include several cryptic species difficult to differentiate using phenotypic features. We wondered whether some species of the *Nigri* section could be more frequently observed in some specific anatomical sites.

**Methods:** We identified at the species level all the isolates of *Aspergillus* section *Nigri* consecutively collected between 2010 and 2017 in our laboratory and stored frozen at -20 C°. After thawing and subculture on Sabouraud medium, DNA was extracted using a MagnaPure DNA extractor and species identification was performed upon the partial sequence of the calmodulin gene. The two primers used for amplification of the targeted gene were CMD5 (CCGAGTACAAGGARGCCTTC) and CMD6 (CCGATRGAGGTCATRACGTGG) (Hong et al. 2005). The sequences were compared to
the Mycobank database. The sequences obtained were aligned to build a phylogenic tree using
Geneious software.

**Results:** A total of 186 strains were sequenced: 75 EEC (64 patients), 78 respiratory samples (78 patients), and 33 air environmental isolates. When considering the first strain per patient, we identified by decreasing frequency: *Aspergillus tubingensis* (78/175; 45 %), *Aspergillus welwitschiae* (59/175; 34 %), *Aspergillus niger* (34/175; 19 %), *Aspergillus luchuensis* (2/175; 1 %), and *Aspergillus japonicus* (2/175; 1 %). When several isolates were available for a given patients, the following isolates belonged to the same species. The species repartition was statistically different when comparing EEC samples to respiratory and air samples with *A. welwitschiae* overrepresented in otomycosis (53% 34/64) (p<0.001). In contrast, the distribution of the species was not different between respiratory and air samples, with 46% and 61% *A. tubingensis* respectively (Figure 1).

<figure><img src="image_107.png" /><figcaption>Figure 1: Species repartition within the *Aspergillus Nigri* section by site of sampling</figcaption></figure>

Inside each species, the sequence identity was high (96.5% for *A. tubingensis*, 99.3% *A. welwitschiae*, and 99.4% *A. niger* isolates) with no difference according to the isolation site. The phylogenetic tree based on the calmodulin gene partial sequences showed a stackable organization to the tree generated using whole genome sequencing (Vesth et al. (2018)), with *Aspergillus luchuensis* included
within the *A. tubingensis* clade, and *A. welwitschiae* closed to the *A. niger* clade (Figure 2).

<figure><img src="image_108.png" /><figcaption>**Figure 2:** Phylogenetic tree obtained with the *Aspergillus* section *Nigri* isolates included in the study using calmodulin gene partial sequences

The sequence of *Aspergillus japonicus* was used as the out-group.</figcaption></figure>

**Conclusion:** Among the 175 Aspergillus species belonging to the Nigri section, *A. welwitschiae* was found significantly associated with otomycosis whereas the species distribution was similar between respiratory and environmental isolates. This finding suggests specific physical properties of *A. welwitschiae* to thrive in the external ear. One hypothesis could be a better fitness to infect EEC containing different lipids linked to the ear-wax.

**Objectives:** Malignant otitis externa is an uncommon, but potentially fatal, complication of otitis externa affecting immunocompromised and elderly patients, particularly those with diabetes. Fungal infection is rare, severe and the clinical presentations pose significant problems in diagnosis and treatment. The purpose of our study is to report the diagnostic and therapeutic difficulties of fungal necrotizing external otitis.

**Methods:** It is a retrospective study spread over 5 years conducted between January 2012 and December 2017. It included patients with documented diagnosis of fungal necrotizing external otitis.

**Results:** There were eight men and three women, all diabetic with a mean age of 64 years. The reason for the consultation was treatment-resistant otalgia and otorrhea. The clinical examination and the TDM (tomodensitometry) were in favor of a necrotizing external ear infection. All patients were treated with antipyocyanic therapy antibiotic (ceftazidime and ciprofloxacin) for an average duration
of one month. A swab of the infected ear was taken for mycological examination and a mycosic agent was revealed within an average of 45 days. *Candida albicans* was isolated in four cases and *Candida parapsilosis* in one case. Concerning molds, *Aspergillus flavus* was isolated in four cases, *Aspergillus fumigatus* and *Rhizopus spp* in one case respectively. Treatment was based on Amphotericin B in two cases and on Amphotericin B followed by Voriconazole in the other cases. The average duration of treatment was four months. Seven patients evolved well, two were lost to follow-up and two died. The average follow-up period was 11 months.

**Conclusion:** When a necrotizing external ear infection, occurs in an immunocompromised patient, with no response to antibiotic therapy, a fungal infection should be suspected and treated early and effectively to ensure a better prognosis.

P485

Chromagars for differentiation between Trichophyton benhamiae, Microsporum canis and M. audouinii

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

**Objectives:** **Objective:** *Trichophyton benhamiae* has increasingly been isolated in Europe during the last years. In culture, this dermatophyte is difficult to differentiate macroscopically from immature colonies of *Microsporum canis* and sometimes from non-sporulating form of *M. audouinii*. Here we have used different culture conditions aiming to establish the macroscopic differences between these species, which could be useful for identification.

**Methods:** **Method:** *T. benhamiae* (6 isolates), *M. canis* (8 isolates), and *M. audouinii* (5 isolates) were cultured at 28 °C on Trichophyton agars for nutritional requirements (5 d), urease (5 d), Chromagar CandiSelect™ (BioRad) (7-10 d) and CHROMAgar™ Candida (CHROMAgar) (7-10 d). Identification of *T. benhamiae*, *M. canis* and non-sporulating form of *M. audouinii* were confirmed by ITS sequencing.

**Results:** **Results:** Five of 6 *T. benhamiae* isolates had requirement for vitamins (inositol and thiamine) and had positive urease test after 5 days incubation at 28 °C. *M. canis* and *M. audouinii* isolates showed no vitamin requirements. *M. audouinii* showed weak or negative urease test. The tested dermatophyte species could be differentiated by colony colour on Chromagar CandiSelect™: *T. benhamiae* presented turquoise diffuse pigment, meanwhile *M. canis* had light yellow and *M. audouinii* purple pigment around the colony. On CHROMAgar™ Candida the colonies showed macroscopical differences: *T. benhamiae* had colonies with white centre and yellow margin, and the other two dermatophytes had brownish colonies.

**Conclusion:** **Conclusion:** Identification and differentiation between *T. benhamiae*, *M. canis* (immature colonies) and *M. audouinii* (non-sporulating form) by conventional methods is difficult. Using biochemical tests like vitamin requirements, urease and Chromagars, may be helpful for identification.

P487

First results in the study of mycetoma in Turkana (Kenya)

M.F. Colom¹, C. Ferrer¹, S. Leting², L. Ramírez¹, J. Ekai², C. Hernández³

¹Medical Mycology Lab, University Miguel Hernandez, Sant Joan d'Alacant, Spain, ²Medical Diagnosis Laboratory, Lodwar County Referral Hospital, Lodwar, Kenya, ³General Surgery, Hospital Clínico San Carlos, Madrid, Spain

**Objectives:** The aim of the study was to assess the prevalence of mycetoma in the county of Turkana (Northwest Kenya), as well as to describe the main causative agents involved in the disease and their possible environmental origin, using simple methods that can be applied locally.

**Methods:** Based on the data collected by the team of cooperative medicine *Cirugia en Turkana*, a specific study for mycetoma was started during the 16th campaign of that team in February 2019. Patients with suspected mycetoma were studied at the Lodwar County Referral Hospital (LCRH). After informing the patient and getting their consent, the lesions were examined and sampled (mainly by biopsy) and clinical data were recorded. Samples were washed in sterile saline solution
and cut in fragments. Some of these were inoculated in Sabouraud Dextrose Agar and Malt Extract Agar plates. One fragment of each sample was used for DNA extraction. The DNA and the rest of the fragments of samples were kept at -20°C. Environmental samples were also taken from soils, trees and shrubs in different locations of the county. These samples were also fragmented and inoculated in the same culture media. All cultures were incubated at room temperature at the LCRH laboratory. The DNA obtained from clinical samples was submitted to PCR amplification of the ITS-5.8S and the V4-V5 16S rRNA gene region, for the detection and identification of fungi and bacteria respectively.

**Results:** In this 2019 campaign 11 patients were studied. All were men between 13 and 70 Yo (mean age 34). Ten of them were shepherds and one worked as a builder. Lesions were mainly located at the feet (91%). Three of them referred black grains in the exudate and 6 yellow or cream coloured. Culture of clinical and environmental samples yield 5 and 24 fungal colonies respectively. For clinical samples, most of cultures were negative. From environmental samples, fungi morphologically compatible with *Madurella mycetomatis* and *Trematosphaeria grisea* were obtained as well as other mycetoma-related genera (*Alternaria* and *Aspergillus*). Molecular identification of showed *Aspergillus terreus*, *Aspergillus flavus*, and *Paecilomyces formosus* in the environment. *Cellulosimicrobium cellulans*, an actinobacteria that has not been previously reported as causative agent of mycetoma, was detected in two of the clinical samples, *Alternaria* sp. in one and *Penicillium spp* in 5 (*P. glabrum*, *P. spinulosum*, *P. corylophilus*). After DNA amplification, three samples remained negative.

**Conclusion:** The detection of *Cellulosimicrobium cellulans* could be a significative result of this study. Nevertheless, it should be confirmed in more samples and patients. For *Alternaria* sp, and *Aspergillus* sp, they had been reported as mycetoma agents previously. The lack of results from some of the yellow grain mycetomas could be since some patients were under antibiotic treatment and, for the black grain ones, this is probably due to the low sensitivity of the method in the detection of these fungi. The isolation of mycetoma agents from the environmental samples confirms that not only Acacia thorns, but also other vegetal products, can be the origin and vehicle of inoculation of the pathogens

P488

Examining the efficacy of Origanum heracleoticum essential oil against fungal pathogens of the nails: preliminary results.

K. Samaras, A.-M. Markantonatou, T.-A. Vyzantiadis

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

**Objectives:** Fungal infections of the nails (onychomycoses) are very common and often need long term treatments, not always effective. In addition to the available chemical antifungal agents, natural products have been used as well. Scope of this study was to test the possible efficacy of commercial antifungal regimens based on *Origanum heracleoticum* essential oil as well as the efficacy of their main antifungal substance, carvacrol, against common fungal pathogens of the nails.

**Methods:** Three different regimens were tested. Regimen (A), contains *Origanum heracleoticum* essential oil (purity>99%, carvacrol >86%), whereas regimen (B) contains the essential oil (28% v/v) in combination with other natural essential oils. Both formulations are suitable for external use. The antifungal activity of pure carvacrol was also tested. Possible indication of the aforementioned regimens (A and B) is the treatment of microbial pathogens of the nails. The proposed dosage for regimen (A) is 4-6 drops (20 µL per drop) oregano essential oil diluted in a teaspoon (5 mL) of another oil (for example olive oil), which is equivalent to carvacrol concentration of approximately 13.76-20.64 mg/ml. Regimen (B) is proposed to be used undiluted (carvacrol concentration approximately 240 mg/ml). In order to demonstrate their possible antifungal activity, serial dilutions of regimens (A), (B) and pure carvacrol from 1,024 mg/L to 0.031 mg/L were tested against dermatophytic (*Trichophyton rubrum* and *Trichophyton interdigitale*) and non dermatophytic isolates (*Candida albicans*, *Candida parapsilosis*, *Candida glabrata* and *Scopulariopsis brevicaulis*) which are all common causes of onychomycoses. The fungal strains were examined also for their sensitivity in fluconazole and
itraconazole. The susceptibility testing was performed according to the proposed EUCAST methodology (microdilution method).

**Results:** All tested regimens exhibited antifungal activity in high concentrations. Depending on the fungal strain the range of MICs was 64-256 mg/L for regimen (A), 64-512 mg/L for regimen (B) and 64-512 mg/L for pure carvacrol. The ranges of MICs were 0.063-4 mg/L for itraconazole and 0.5-64 mg/L for fluconazole.

**Conclusion:** The results of the study demonstrate that the regimens tested may have antifungal activity against both dermatophytic and non dermatophytic isolates, although in much higher concentrations than the commonly used antifungal drugs. However, being substances of only local use on the fungal lesions, there is the probability that the above mentioned concentrations could be achieved on the nail application. Further experimental data and studies of clinical use are required in order to specify potential benefit, duration of treatment and possible toxicity.

P489

Cutaneous ulcer due to *Candida parapsilosis* associated with skin trauma in an adult patient with psoriasis vulgaris

G. Ge¹, D. Shi²

¹Jining Medical University, Jining, China, ²Jining No. 1 People's Hospital, Jining, China

**Case Report:** The *Candida parapsilosis* (*C. parapsilosis*) family has emerged as a major opportunistic and nosocomial pathogen. It causes multifaceted pathology in immuno-compromised and normal hosts. Common symptoms of *Candida* skin infections include thickening of the skin, hyperkeratosis, and erythema. *C. parapsilosis* rarely induced the skin ulcer without assistance from other pathogens. Here, we present 27-year-old female with cutaneous ulcer on her left foot due to *C. parapsilosis* after trauma. She has a 6-month history of psoriasis vulgaris without treatment. *C. parapsilosis* was isolated from skin lesion for several times and was confirmed by morphological and cultural characteristics as well as by DNA molecular analysis. Patient was successfully treated with oral itraconazole in 200 mg daily for seven weeks. To our knowledge, it was the first report that *C. parapsilosis* induced cutaneous ulcer in a patient with psoriasis; Cutaneous candidiasis maybe associated with the skin barrier damage (the trauma) or psoriasis.

P490

Two genetic lines of *Trichophyton rubrum* population in Russia

I. Pchelin, Y. Mochalov, G. Chilina, N. Vasilyeva, A. Taraskina

Kashkin Research Institute Of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint Petersburg, Russian Federation

**Objectives:** *Trichophyton rubrum* is the most prevalent agent of onychomycosis and tinea pedis in most countries, including Russia. However, data on its population structure remain controversial, with some studies reporting the absence of the structure and others reporting bipartite structure. We aimed at studying *T. rubrum* population by microsatellite analysis and typing of TERG_02941 locus.

**Methods:** The study included 59 clinical isolates of *T. rubrum* from Saint Petersburg and Yekaterinburg, Russia. Species identification was done by ITS rDNA region sequencing. Typing was performed by real time PCR of the locus TERG_02941 and microsatellite analysis of 12 loci, including 4 original ones. The data were processed in the R software using the polysat and poppr packages. Genetic distances were calculated by Bruvo method.

**Results:** Ribosomal ITS region sequencing revealed nucleotide sequence, identical to that one, deposited under KT285224 accession in all but two isolates. Real-time PCR of the TERG_02941 locus revealed one SNP at position 793. At this position, adenine was found in 59% of isolates and guanine was found in 34% of isolates. The scatterplot, based on the results of the analysis of the lengths of microsatellite loci by the method of principal components, contained two separate clouds of points. One of the clouds was formed by isolates with TERG_02941 793A genotype, the other — by 793G isolates (Fig. 1). The value of the Simpson index, calculated for equal samples, was not significantly different for TERG_02941 genotypes. The association index, $r^2 = 0.02483$, was calculated using the
Multilocus Style Permutation method after deleting one non-informative locus and performing clonal correction. The value laid beyond the right boundary of the distribution of expected frequencies. The probability $r_d$ was estimated as 0.002, which made it possible to reject the null hypothesis of the absence of connections between the markers. The $\chi^2$ test showed no connection between the TERG_02941 isolate genotype and the localization of the lesion on the patient's body.

**Conclusion:** Microsatellite analysis with an extended set of loci confirms the presence of two genetic lines of the fungus *T. rubrum* in the territory of the Russian Federation, with the same diversity. In the population of the fungus there is a significant deviation from the Hardy-Weinberg equilibrium.

P491

**Comparison of three skin sampling methods and two media for culturing *Malassezia* yeast**

A. Abdillah¹, S. Khelaifia², D. Raoult², F. Bittar³, S. Ranque¹

¹Vitrome, Aix Marseille Univ, IRD, AP-HM, SSA, Marseille, France, ²Culturomics, IHU Méditerranée Infection, Marseille, France, ³Mephi, Aix Marseille Univ, IRD, AP-HM, SSA, Marseille, France

**Objectives:** *Malassezia* species are lipid-dependent basidiomycetous yeast. They are resident commensals of the human skin but also cause common skin diseases, such as pityriasis versicolor, seborrheic dermatitis, *etc*. Whether they are associated with other human diseases remains an open question, partly because of the lack of efficient medium to culture these fastidious yeast. Besides, the absence of a standardized skin sampling technique introduces a major bias in culture-based descriptive studies. The objectives of this study were: 1) to compare three methods of skin sampling for the detection of *Malassezia* yeast and 2) to evaluate the performances of « FastFung » a new culture medium based on the Schædler agar, for the growth of *Malassezia* spp. by culture compared to the reference Dixon medium.

**Methods:** A total of 10 healthy volunteers were included in this study. Fifteen skin samples were collected at 5 different body sites for each participant using three sampling methods: a) dry cotton swabs, b) swabs in a liquid transport medium « Transwab®», and c) a sterile gauze. The three methods were applied successively in a random order, which was noted, at each sampling site. Each skin sample was then plated in parallel, in a random order on both Dixon and FastFung media. All plates were incubated aerobically at 30°C and cultures were examined each day for one week for *Malassezia* spp. growth.

**Results:** *Malassezia* yeast grew in 93/300 (31%) culture plates, corresponding to 150 samples. A total of 1 082 *Malassezia* spp. colonies were isolated and identified by MALDI-TOF Mass Spectrometry, of which 455 (42%) were *M. globosa*, 424 (40%) *M. sympodialis*, and 203 (18%) *M. restricta*. Multivariate statistical analysis found a significant independent effect ($P<10^{-3}$) of the sampling methods. Of the 93 positive culture, 62 (67%) were sampled using a sterile gauze, 17 (18%) with Transwab®, and 14 (15%) with the dry cotton swab. We also found a statistically significant independent effect ($P = 0.003$) of the culture medium, with the FastFung and Dixon media yielding 58 (62%) and 35 (38%) positive culture, respectively. Interestingly, the majority of the fastidious *M. globosa* and *M. restricta* species were isolated on the FastFung medium.

**Conclusion:** Our findings demonstrate that sterile gauze is the most efficient and reliable skin sampling technique to culture *Malassezia* yeast; and that the novel FastFung culture medium is suitable, and more efficient than the Dixon medium for *Malassezia* spp. culture. We propose implementing sterile gauze skin sampling and the FastFung culture medium in the routine *Malassezia* spp. cultivation procedures.

P495

**Morphological and molecular characterization of the *T. benhamiae* isolates.**

A. Packeu¹, F. Baert¹, K. Goens¹, S. Roesems¹, D. Stubbe², M. Hendrickx³

¹Mycology & Aerobiology, Sciensano, Brussels, Belgium, ²Mycology & Aerobiology, Bccm/ihem Collection, Sciensano, Brussels, Belgium, ³Mycology, Sciensano, BCCM/IHEM, Brussels, Belgium

**Objectives:** Dermatophytes are keratinophilic fungi responsible for causing dermatophytosis in both man and animals. Recently, several genera were (re)introduced in addition to *Epidermophyton*,
*Microsporum*, and *Trichophyton* and depending on their primary habitat, divided into geophilic, zoophilic and anthropophilic species. Due to the fact that they are affecting more than 20-25% of the world's population, able to affect healthy individuals and in some cases are communicable makes this specific group of fungi of great importance for public health. Recent taxonomical changes (de Hoog *et al.*, 2017) proposed an increase in the number of genera, simplifying identification of dermatophytes in routine diagnostics. The implementation of these changes necessitated a taxonomic re-evaluation of the dermatophytes within the Belgian BCCM/IHEM culture collection. And furthermore, an evaluation of such a large dataset leads to further insights in the taxonomy of this specific group of dermatophytes.

**Methods:** In the present study, the members of the *T. benhamiae* and *T. bullosum* clade were evaluated, using different molecular methods (sequencing and MALDI-TOF MS). The obtained results were coupled with conventional physiological, morphological, clinical, and geographical data of the strains in order to obtain a correct nomenclature for each analysed isolate. As proposed by de Hoog *et al.*, *T. benhamiae* strains can be categorized into two subclades: the *T. bullosum* clade and the *T. benhamiae* clade. The *T. bullosum* clade comprises the type strain of this zoophilic species isolated from cutaneous lesion of a horse described for the first time in 1933 by Lebasque. The *T. benhamiae* clade comprises isolates from the zoophilic species *T. benhamiae*, *T. erinacei*, *T. verrucosum* and *T. eriotrephon* and the anthropophilic *T. concentricum* species. However, in both clades delineation of the different species still remains unclear. Especially for *T. benhamiae* a lot of confusion still exists, where 2 different phenotypes (white and yellow) have been described.

**Results:** Preliminary molecular results (ITS and BT), obtained with the BCCM-IHEM isolates, point to the existence of only one *T. benhamiae* clade with well supported clades for the species *T. erinacei*, *T. verrucosum* and *T. eriotrephon*. The remaining species are, however, not well resolved. While most *T. benhamiae* strains clustered together with *T. concentricum* as a subclade in the ITS+BT phylogeny, some strains clustered with *T. bullosum* forming a highly supported clade.

**Conclusion:** In order to better delineate the different taxonomic entities and to define criteria for their identification, a combining of all results obtained via the proposed polyphasic approach will be primordial.

P496

**Phenotypic and phylogenetic characterization of malbranchea-like isolates from clinical specimens in United States**

J.F. Cano-Lira¹, E. Rodriguez-Andrade¹, N. Wiederhold², A. Stchigel¹

¹Mycology, Universitat Rovira i Virgili, Reus, Spain, ²Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, United States of America

**Objectives:** The main objective of this work was to identify phenotypically and molecularly twenty-two strains from clinical specimens in USA, preliminary classified as *Malbranchea* spp.

**Methods:** For cultural characterization, the strains were grown onto potatoes extract agar (PDA), phytone yeast extract agar and oatmeal agar, and the colonies measured and described after two weeks at 25°C. Vegetative and reproductive structures were observed and documented from slide cultures mounted on water and 60% lactic acid by a BH1 Olympus microscope. Photo micrographs were taken using a Zeiss Axio-Imager M1 light microscope with a DeltaPix Infinity X digital camera using Nomarski differential interference contrast. For phylogenetic analysis, total DNA was extracted from colonies after 7-10 days of growth on PDA at 25 °C. DNA was used to amplify and to sequence a fragment of the 28S nrRNA gene (LSU) using the primer pair LR0R and LR5, and the ribosomal internal transcribed spacers (ITS) by using the primer pair ITS5 and ITS4. A preliminary molecular identification was performed with ITS and LSU sequences using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi). A maximum level of identity of ≥ 98% respect to the sequences of type or reliable reference strains deposited in the GenBank/EMBL database, and the phenotypic characteristics, were used for identification at species-level. LSU sequences generated by us were aligned with other from known taxa belonging to the order Onygenales by ClustalW application of MEGA v. 6.06. The phylogenetic tree was built
using maximum-likelihood (ML) and Bayesian-inference (BI) methods with RAxML v. 8.2.10 and MrBayes v3.2.6 computer programs, respectively. *Oidiodendron truncatum* and *Myxotrichum deflexum* (*Myxotrichaceae* family) were used as outgroup.

**Results:** Hitherto, taking into account the phylogenetic data and the phenotypic features, eleven strains were identified at species level. We report the finding of *Auxarthron alboluteum* (two strains), *A. conjugatum* (two strains), *A. umbrinum* (three strains) and *A. zuffianum* (one strain); and of *Malbranchea aurantiaca* (two strains) and *M. flocciformis* (one strain). One strain of *Arachnomyces*, two of *Auxarthron*, one of *Malbranchea* and two of *Spiromastigoides* could not be identified at species-level. In addition, two strains did not matched at genus-level.

**Conclusion:** The phenotypic characterization and the phylogenetic analysis carried out by us only allowed us to identify at species-level 11/19 strains previously identified as *Malbranchea* spp. Most of these strains finally resulted known species of the genus *Auxarthron*, and only three of them could be identified as *Malbranchea* species. This indicates that the presumptive morphological identification is unreliable, and molecular data are needed for the correct identification of this sort of fungi. Surprisingly, eight of nineteen strains were identified at genus-level or not identified, which suggests that these malbranchea-like fungi could be putatively new taxa

P497

**Fast mold identification with MALDI-TOF MS: The importance of the spectra acquisition method**

K. Mucek, T. Maier, M. Timke, M. Kostrzewa

Research And Development, Microbiology And Diagnostics, Bruker Daltonik GmbH, Bremen,
Germany

**Objectives:** Invasive fungal infections are a major cause for morbidity and mortality in ill, immunocompromised and paediatric patients [1, 2]. Clinical diagnosis of mold infections and identification of fungal contamination is often based on morphological criteria which encounter issues like robustness of phenotypic procedures, often sporulation as a prerequisite for identification and very few skilled mycologists that are capable of differentiating molds in the medical environment. In order to solve this situation one of the most promising alternative to identify clinically and environmental relevant molds is MALDI-TOF MS. Through the generation of protein spectra and the comparison with a reference library, molds can be identified fast and reliable. However, in some cases the acquisition of mass spectra particular from filamentous fungi is challenging since the frond mycelium often results in reduced spectra quality.

**Methods:** A minimum about 56 filamentous fungi was measured with MALDI-TOF in duplicates with at least two different operators. Each strain was cultivated on Sabouraud agar for 24 – 48 h, front mycel was smeared on a MALDI target plate. One of two spots was pre-treated with formic acid before all spots were overlaid with HCCA matrix and the measurement started. Two different acquisition methods were applied. The Bruker ‘standard’ method and the ‘filamentous fungi’ method was compared. Subsequently all spectra were matched against the Filamentous Fungi Library 3.0.

**Results:** After cultivation of filamentous fungi biological material could be transferred successfully to a MALDI-TOF target plate in duplicates for each strain. The results showed that the rate of spectra acquisition by using the filamentous fungi method was significant higher (about 25%) compared to the standard acquisition method. It could be shown additionally that the acquired spectra resulted in an increased identification rate.

**Conclusion:** The MALDI Biotyper Filamentous Fungi Module with adapted acquisition settings for filamentous fungi improves the identification rate for molds. MALDI-TOF based differentiation is less error prone and faster compared to classical identification. Results can be easily obtained in one hour from cultivation plate. Moreover, the adapted filamentous fungi method compared to the standard spectra acquisition method is a vast advantage. Time consuming repetition of target preparation and re-measurement is reduced as well as previous protein extraction for MALDI-TOF measurement. Thus, mold infections like aspergillosis or mucormycosis or fungal contaminations are identified more reliable and faster. [1] Prattes, J., Heldt, S., Eigl, S. et al. Curr Fungal Infect Rep (2016)
10: 43. https://doi.org/10.1007/s12281-016-0254-5 [2] Gullo, A. Drugs (2009) 69(Suppl 1): 65. https://doi.org/10.2165/11315530-000000000-00000

P498

**Trichophyton mentagrophytes – a new genotype in Cambodia**

S. Uhrlass¹, M. Sithach², D. Koch¹, F. Wittig¹, H. Muetze¹, C. Krueger¹, P. Nenoff¹

¹Partnership Dr. C. Krueger & Prof. P. Nenoff, Laboratory of medical microbiology, Roetha OT Moelbis, Germany, ²Department Of Dermatology, Preah Kossamak Hospital, Phnom Penh, Cambodia

**Objectives:** According to the currently suggested classification and new taxonomy of the dermatophytes, the former *Trichophyton mentagrophytes* (*TM*)-complex is now differentiated in *TM* (zoophilic strains) and *Trichophyton interdigitale* (*TI*, anthropophilic strains).

**Methods:** Mycological diagnostics was done during a survey of superficial dermatophytoses in Cambodia.

**Results:** 1) The new genotype „Cambodia“ of *TM* (strain 201341/18) was found in a 8 years old male child with skin lesions starting already 3 months ago, prior the dermatology consultation. Inflammatory papules on the right part of the scalp were to be seen. 2) A 31 years old male suffered from a tinea cruris. The dermatophyte isolate was found to be the new *TM* genotype Cambodia (strain 218292/17). 3) A 26 years old male farmer showed lesions appearing since one year, started with scaly pruriginous plaques on both hips. Clinical diagnosis was tinea corporis. By mycological diagnostics, from skin scrapings, a dermatophyte was isolated. By sequencing of the DNA it was identified as *TM* genotype VIII India (strain 218294/17). 4) A one-year-old male was diagnosed as tinea corporis. The dermatophyte which was growing from skin scrapings of the baby’s cutaneous lesions was identified as *T. interdigitale* (*TI* strain 250037/18 genotype II* - mixed type between *TI* and *TM*).

**Conclusion:** Today, due to sequencing of the ITS region of the rDNA, at least 10 genotypes of *TM* and *TI* are known. These genotypes are mostly associated to the geographic origin or to the source of infection. Currently, two genotypes of *TI* (ITS Type I [Europe] and ITS Type II [Cosmopolite]), were described. *TM*, however, comprises the common zoophilic "German" ITS Type III (Europe) and III* (Cosmopolite), genotype IV (UK, USA, South Africa and Feancde), V (Asia, Egypt, Iran, Iraq and Japan), VI (Europe, Russia and Finland), VII (from Thailand originating isolates in Switzerland and Germany, USA, Australia, Georgia, Russia, and Vietnam), VIII (Asia, India, Iran, Oman, Australia), and last but not least the Australian genotype (ITS Type IX). Based on results of sequencing preferably of the ITS region of the rDNA, we are able to demonstrate that 2 out of 3 *TM* strains in Cambodia represent a new, until now not described, ITS genotype which is called now the "Cambodian" genotype of *TM*. Surprisingly, we found one additional *TM* strain of the Indian genotype VIII. An association between this dermatophytosis in Cambodia and the current Indian outbreak of superficial dermatophytosis can only be suspected. The fourth here isolated dermatophyte belongs to the until now very rarely described genotype *TI* II*. Phylogenetic, this genotype is the so called mixed type between anthropophilic *TI* and zoophilic *TM*. Each genotype of the *TM/TI* complex seems to be specific for e.g. a geographic area, for a country, or for a distinct animal which represents the source of infection, speaking for a special way of transmission of the infection.

P499

**Novel *Cunninghamella* species causing mucormycosis in an apparently immunocompetent individual**

V. Hallur¹, M. Sable², P. C³, S. M Rudramurthy⁴, P. Sharma⁵, H. Prakash⁴, A. Chakrabarti⁴

¹Microbiology, All India Institute of Medical Sciences, Bhubaneswar, Bhubaneswar, India,
²Pathology, All India Institute of Medical Sciences, Bhubaneswar, Bhubaneswar, India, ³Ent, All India
Institute of Medical Sciences, Bhubaneswar, Bhubaneswar, India, ⁴Medical Microbiology,
Postgraduate Institute of Medical Education and Research, Chandigarh, India, ⁵Ent, All India
Institute of Medical Sciences Bhubaneswar, Bhubaneswar, India
**Case Report: Title:** Novel *Cunninghamella* species causing mucormycosis in an apparently immunocompetent individual Mucormycosis due to *Cunninghamella* spp. is extremely rare clinical entity. We report here a case of mucormycosis due to *Cunninghamella* sp. in an apparently immunocompetent individual. **Material & Methods: Patient & microbiological methods:** Tissue biopsy from the lesion was subjected to histopathology, and culture. Formalin fixed paraffin embedded tissue (FFPE) and culture were sent to Mycology Reference Laboratory at PGIMER Chandigarh for species identification & susceptibility testing. **Molecular identification:** DNA was extracted from FFPE tissue and cultures as described previously by Rickerts *et. al* (1) & Prakash *et.al*. (2) DNA from FFPE tissue was PCR amplified using mucoromycetes specific primers. The ITS and 26s genes of ribosomal DNA of the isolate were amplified using pan fungal primers. Consensus sequences obtained from amplified products were analyzed using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST). **Phylogenetic characterization:** The 26S rDNA and ITS sequences of different *Cunninghamella* were retrieved from NCBI nucleotide database. Phylogenetic analysis was performed as per Prakash *et.al*. (2)

**Results: Patient & microbiological methods:** A 26-year-old healthy man presented with slowly progressive swelling on the left cheek for 2 years and ulceration over the swelling for two months. Laboratory investigations revealed a normal blood sugar, hemogram, CD4 counts, liver and renal function tests. He was non-reactive for HIV. Contrast enhanced computed tomography scan of the head and neck showed mild enhancing lesions on left side of the face along with thickening of floor of maxilla. Biopsy from the lesion showed focal areas of chronic inflammation with multinucleated giant cells containing broad aseptate fungal hyphae. A rapidly growing white mould with aseptate hyphae & erect sporangiophores ending in a swollen vesicle, with 1-spored sporangiola suggestive of *Cunninghamella* was isolated. The isolate had minimum inhibitory concentrations (MICs) of 4µg/ml for amphotericin B, itraconazole, and posaconazole; and 3µg/ml for Terbinafine as per CLSI M38A2. He was managed with repeated debridement over 7.5 months, amphotericin B and posaconazole for 1.5 months & 6 months respectively. As there was no improvement, he was finally treated with oral Itraconazole. At present the ulcer has healed, but the swelling is still persisting. **Molecular identification:** The fungus was identified as *Cunnighamella* sp. by analysis of DNA sequence of the FFPE tissue. ITS sequences of the isolate showed 97.54% base identity with uncultured *Cunninghamella* spp. (MG571234.1) isolated from Thailand. **Phylogenetic analysis:** Phylogenetic analysis of ITS and 26s regions of all known *Cunninghamella* species with the present isolate suggested that the isolate may be a new or undefined species under genus *Cunninghamella*. **Conclusion:** We report recalcitrant mucormycosis due to a *Cunninghamella* sp., which is phylogenetically different from other described species in this genus. Further molecular characterization is essential to identify the species. References: Eur J Clin Microbiol Infect Dis. 2006 Jan;25(1):8-13. Med Mycol. 2016 Aug 1;54(6):567-75. doi: 10.1093
Development, Microbiology And Diagnostics, Bruker Daltonik GmbH, Bremen, Germany, ¹⁶First Faculty Of Medicine, Charles University in Prague, Prague, Czech Republic, ¹⁷Department Of Botany, Charles University, Prague, Czech Republic

**Objectives:** Species of *Trichophyton benhamiae* complex have a worldwide distribution and their host range includes many pet and livestock animals. Nowadays, this complex has become very important due to epidemic spread of *T. benhamiae* complex species in children and their pet animals in Europe. No causal mechanism has been found that would explain this increase. Moreover, the incidence of several other species from this complex is also increasing in Europe due to higher interest of people in pet hedgehogs (host of *T. erinacei*), insufficient prophylactic vaccination (*T. verrucosum* ) or increasing use of molecular methods in the dermatological diagnostics leading to more accurate identification (*T. bullosum*, *T. eriotrephon*). A considerable genetic and phenotypic variability has been revealed in these emerging pathogens, but the species limits are not always clearly defined. Therefore, the aim of this study was to elucidate species boundaries and phylogenetic relationships between species belonging to the *T. benhamiae* complex.

**Methods:** A total of 352 clinical isolates from *T. benhamiae* complex associated with human and animal dermatophytoses were analysed using molecular markers (DNA sequence data and microsatellites), and morphological and physiological methods.

**Results:** Members of the *T. benhamiae* complex were resolved into three major clades (designated *T. benhamiae*, *T. erinacei* and *T. africanum* clades). All analyses supported delimitation of four species in the *T. benhamiae* clade, i.e., *T. benhamiae*, *T. concentricum*, and two novel species, *T. europaeum* and *T. japonicum*. In contrast to DNA sequence and based phylogeny, the population genetic and phenotypic data distinguished North American strains of *T. benhamiae* (mostly isolated from dogs) and emerging European strains associated with quinea pigs. The status of new variety, *T. benhamiae var. luteum*, is proposed for these strains. The *T. erinacei* clade consisted of three species, namely *T. erinacei*, *T. verrucosum* and *T. eriotrephon*. *Trichophyton bullosum* and one new species, *T. africanum* (previously African race of *Arthroderma benhamiae*) together with *T. bullosum* formed the *T. bullosum* clade

**Conclusion:** Species boundaries in the *T. benhamiae* clade were resolved by using polyphasic approach. This approach supported recognition of nine species, including three new zoophilic species. *Trichophyton benhamiae* was split into two varieties; *T. benhamiae var. luteum* is currently responsible for the European outbreak of zoonotic infections.

## Video abstracts

### V01

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

S. Colman, L. Cattoir, A. Boel

Microbiology, OLV Hospital, Aalst, Belgium

**Objectives:** *Phaeohyphomycosis* (PHM) is a chronic infection caused by dematiaceous fungi which usually involves the skin and subcutaneous tissue. As a result of the use of molecular diagnostic techniques, previously unrecognized causative agents of *phaeohyphomycosis* have been discovered. Here we report the first case of phaeohyphomycosis caused by *Phaeoacremonium fuscum* in an immunocompetent patient.

**Methods:** Case presentation A 60 year old Filipino male presented with rotator cuff pathology to the orthopedics consultation. On physical examination a painless, soft, subcutaneous mass above the right patella was noted, which didn't cause the patient any complaints. The suprapatellar nodule had been growing slowly for 3 years. The patient denied any apparent antecedent trauma, arthropod bite, family history or contact with a person with similar lesions. The man still visits the Philippines on an infrequent basis. He had no fever, weight loss or other symptoms. Detailed physical examination further revealed a 4x3 cm, immobile, non-pulsatile light red mass. There were no other signs of inflammation of the skin. A serous effusion was aspirated from the mass.

**Results:** Fungal culture of the aspirated fluid showed growth of a filamentous fungus on chocolate blood agar and Sabouraud's dextrose agar. Molecular identification was performed by sequence
analysis of the internal transcribed spacer region 2 (ITS2). BLAST search revealed a similarity of 100% with *Phaeoacremonium fuscum*. The antifungal susceptibility profile was established by EUCAST broth microdilution: itraconazole >16 mg/L, posaconazole 0.25 mg/L, amphotericin B 0.50 mg/L, voriconazole 0.50 mg/L. This susceptibility profile is similar to those previously published. A wide local excision of the lesion was performed a couple of weeks later. The patient had an uneventful postoperative recovery. No antifungal therapy was started postoperatively since the patient was not immunocompromised and there was lack of reimbursement. Histopathological examination of the specimen showed fibrous tissue with a mixed cellular infiltrate including lymphocytes, plasma cells, macrophages and neutrophils. Periodic acid-schiff stain and Grocott stain showed evidence of branching septate hyphae.

**Conclusion:** PHM is caused by brown pigmented fungi containing melanin in their cell wall. Phaeoacremonium species are known as vascular plant pathogens, responsible for wilting and dieback of woody plants. They are also opportunistic pathogens in humans, mainly causing cutaneous or subcutaneous PHM. However, sporadic cases of respiratory tract infection, septic arthritis and disseminated infection are described. The true prevalence of Phaeoacremonium species is probably underestimated due to the asymptomatic nature of most subcutaneous lesions and the difficult identification. There is no standard antifungal regimen or evidence regarding the necessity for antifungal treatment as concomitant with surgery for localized infections. Long courses of antifungals, such as voriconazole treatment, are often employed to reduce the risk of recurrence. Case reports suggest that surgery can be curative if the infection is localized and adequately removed, as was the case in our patient. Regardless of the immune status of the patient, clinicians should consider the possibility of a fungal infection when they encounter chronic subcutaneous nodules without major symptoms.

## V02

### *Trichosporon diagnosis- The Right path*

T. Premamalini<fnref n="1" />, P. Kennedy Kumar<fnref n="1" />, N. Anish Kumaran<fnref n="1" />, A.J. Kindo<fnref n="2" />

**Objectives:** To discuss about the correct path in identifying *Trichosporon* sp. To emphasize the importance of performing conventional laboratory diagnostic methods like microscopy, along with molecular methods in the diagnosis of *Trichosporon* sp.

**Methods:** Yeast-like colonies were observed for microscopic characteristics by Gram staining. The isolates which showed the presence of budding yeast cells, pseudohyphae and arthroconidia were tested for production of the enzyme urease. Urease positive test isolates were provisionally identified as belonging to the genus *Trichosporon*. To confirm the identity of all phenotypically identified *Trichosporon* isolates, *Trichosporon* genus specific colony PCR was performed. This pair of primer is *Trichosporon* specific and it amplified part of the nucleotide sequences of the rDNA small subunit (18S).

**Results:** The study of colony morphology of the *Trichosporon* isolates on SDA at 25°C after 10 days of incubation revealed varied colony morphology. Most commonly, they may appear as white to cream, farinose or cerebriform colonies, with furrows. The important characteristic feature of this yeast is the production of arthroconidia and enzyme urease. Performing Gram stain or Dalmau technique to search for arthroconidia is a very useful tool for screening of *Trichosporon* spp. Finally, the accurate identification of this yeast was done using DNA based methods like *Trichosporon* genus specific colony PCR. The members of the genus *Trichosporon* produced DNA bands of approximately 170 bp.

**Conclusion:** *Trichosporon* sp. has been so far reported as the second most common cause of disseminated yeast infections in humans, next to the genus *Candida*. They are emerging opportunistic pathogens causing disseminated infections in immunocompromised patients. Such infections are usually difficult to diagnose, do not respond to treatment with routinely used antifungal agents, and are associated with high mortality rates. Hence, correct identification of *Trichosporon* sp. helps the clinician in providing appropriate treatment. This also applies for other emerging yeast infections,

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

<footnote n="2">²Microbiology, Sri Ramachandra Medical College and Research Institute, chennai, India</footnote>
where combination of conventional and molecular methods may help in timely and accurate
diagnosis.

V03

Disseminated rhinosporidiosis with different morphological lesions involving various anatomical sites.

J. Chander¹, R.P.S. Punia², A. Dass³, A.K. Attri⁴

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

**Objectives:** Rhinosporidiosis is a chronic granulomatous disease caused by *Rhinosporidium seeberi*, which is a taxonomically debated endosporulating aquatic eukaryotic organism since long. Rhinosporidiosis predominantly involves the mucous membranes of the nose and eyes mainly conjunctiva. In recent past, giant lesions and disseminated type of disease, has also been reported in some of the clinical settings. This disease is hyperendemic in the Indian subcontinent, particularly in Sri Lanka and southern India. However, sporadic cases have occasionally been reported from other continents.

**Methods:** A case report of rare disseminated rhinosporidiosis with varied morphological lesions involving simultaneously three anatomical sites i.e. nasal, cutaneous and bones in a hepatitis B virus surface antigen (HBsAg) positive patient. A 35-years old male, resident of district Madhubani, Bihar, fisherman by occupation presented with complaints of left sided nasal obstruction, insidious in onset, gradually progressive and associated with change in voice since the last 6 years. Also, there was slowly progressive swelling over medial aspect of left ankle for the last 1 year and a similar looking swelling, smaller in size also appeared in the left post-auricular region four months back and has gradually increased in size.On examination, a polypoidal fungating mass was present over left medial aspect of foot which was non-tender, highly vascular, large, hemi-spherical in shape and bled on touch. On anterior rhinoscopy, a warty, irregular, non-tender mass, not bleeding on touch, occupying whole of left nasal cavity was seen. Oral cavity showed bulging soft palate and a bilobed pale polypoidal mass with smooth surface hanging from nasopharynx to oropharynx. There was a solitary ovoid growth measuring 2 x 2 cm with normal appearing overlying skin in the left upper post-auricular region. Serum of the patient on testing for HBsAg was positive. The patient was known chronic alcoholic and smoker.

**Results:** A wedge biopsy of the lesion when examined in normal saline showed sporangia of *R. seeberi* in different stages of development. Routine haematoxylin and eosin (H & E) staining, periodic acid-Schiff (PAS) staining as well as mucicarmine staining of the tissue also revealed findings of rhinosporidiosis with many sporangia and scattered endospores seen in a background of inflammatory infiltrate composed of polymorphs, lymphocytes and plasma cells including the bony involvement. A final diagnosis of disseminated rhinosporidiosis presenting simultaneously with three different kinds of lesions; nasal, cutaneous and osteomyelitis of medial malleolus and distal end of tibia was made. The patient was treated surgically by performing below knee amputation; transnasal and transoral excision with electrocoagulation as well as excision of postauricular mass was also performed. Histopathology of the excised bone and other growths was consistent with the preoperative diagnosis. The patient was discharged and advised dapsone therapy 100 mg bd to prevent recurrence. Presently, the patient is on follow up and there is no recurrence of the disease.

**Conclusion:** This is prudent for clinicians, microbiologists and pathologists to keep rhinosporidiosis as a differential diagnosis when managing patients with polypoidal growths even in non-endemic areas. In such a background high index of suspecion is the key to reach the final diagnosis.

<figure><img src="image_109.png" /></figure>

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).