Invasive Fungal Infection by Hormographyella aspergillata: A Tricky Diagnosis Triggered by (1,3)-Beta-D-Glucan Assay

Raffaella Koncan1*, Gianpaolo Nadali2, Vincenza Favuzzi3, Marco Ligozzi1, Annarita Sorrentino4 and Giuliana Lo Cascio4 1Microbiology and Virology Unit, Department of Diagnostic and Public Health, University of Verona, Verona, Italy 2Hematology Section, Department of Clinical and Experimental Medicine, University of Verona, Italy 3Pathological Anatomy Unit, Department of Diagnostic and Public Health, University of Verona, Verona, Italy 4Microbiology and Virology Unit, Department of Pathology, Integrated University Hospitals of Verona, Italy

Hormographiella aspergillata is a Basidiomycete; it belongs to the Psathyrellaceae family. It is the asexual form of Coprinopsis cinerea which is normally found in compost and sewage. The potential of common environmental filamentous basidiomycetes (BM) as human pathogens in infections has not yet been examined under laboratory conditions; instead, BM from clinical specimens usually present non-sporulating mold that grow as cottony white colonies [1]. It has been rarely found in humans as pulmonary and skin infections, endocarditis, endophthalmitis, chronical sinusitis [2] but the incidence of human disease caused by environmental fungi has been on rise. This is primarily attributed to risk factors such as wide use of immunosuppressive drugs, invasive medical instrumentation, large-scale usage of azole antifungals in agriculture [1].
The most of reported cases of H. aspergillata infection occurred in patients with hematologic malignancies. The first case was documented in 1971 and the second twenty six years later. Subsequently, the number of cases grew up to a total of fourteen reported in the following fifteen years [1][2][3]. This increasing incidence might be due to the higher number of immunocompromised patients, but lack of proper identification is probably the main reason for the low number of observations. This is the first case described in Italy.

Case Presentation
On May 2014, 26 years old male of south Asian ethnic origin, was diagnosed with Mixed Phenotype Acute Leukemia (MPAL). MPAL is a rare disease, representing only 3-5% of acute leukemia of all age groups, and 2.4-3.7% in children [4]. It affects adults more frequently than children, with a slight male preference. The prognosis for MPAL is poor comparing to other acute leukemia, with an overall survival of eighteen months [5].
The patient was admitted in the Hematology Unit of the University Hospital of Verona and treated accordingly to an acute myeloid leukemia chemotherapy induction program with Fludarabine, Cytarabine and Idarubicin, (FLAI regimen).

Abstract
We report on a pulmonary infection caused by the basidiomycete Hormographiella aspergillata. For the first time, (1,3)-beta-D-Glucan serum levels were monitored and they progressively increased during the development of clinical symptoms of infection. This assay and subsequent molecular analysis resulted essential for accurate identification and indication of proper antifungal therapy.
On August 2014, the patient received a second FLAI chemotherapy course. Since Quantiferon test resulted positive, isoniazid prophylaxis was provided. Treatment was complicated by septic shock, sustained by Escherichia coli and multidrug resistant Morganella morganii, completely solved by carbapenems and aminoglicosides. During hospitalization, the patient received antifungal prophylaxis using posaconazole oral solution. However, during the second course of chemotherapy, posaconazole was interrupted because of nausea and vomiting.
Fifteen days after chemotherapy, the patient developed intense spontaneous pain in the left scapular area. Chest X-Rays were persistently negative up to day nineteen. At day twenty, thoracic computed tomography (CT) scan revealed two lesions compatible with invasive fungal infection (IFI), with a possible abscess involving the soft tissues of the thoracic wall ( Figure 1).
The patient underwent thoracotomy with atypical resection of the left upper lobe and drainage of the abscess cavity [6]. The purulent material was analyzed. After 72 h on Sabouraud Dextrose agar (SDA) plates (Oxoid, Basingstoke, England), incubated at 30°C, cottony white colonies grew. Identification was reached by microscopic examination of mycelium, stained with lactophenol-blue ( Figure 2) but the precise diagnosis of Hormographiella aspergillata was confirmed by sequencing of ITS and D1-D2 region of ribosomal sub-unites [7,8].
Spectrophotometric identification by MALDI-TOF (bioMerieux, SA, Marcy l'Etoile, France), did not lead to any result using SARAMIS software package (AnagnosTec). It is noteworthy that the first BG test was positive seven days before CT imaging, thus suggesting IFI, even forty days before microbiological identification of the causative agent.
Conventional chest X-Rays were repeatedly negative but the rapid raise of BG serum level directed further investigation in the hypothesis of IFI. GM test was always negative, reaching a value of 0.4, close to the cut-off of 0.5, when BG was >523 pg/mL.
Intravenous voriconazole was the therapeutic choice. The pharmacological treatment was successful: BG showed progressively decreasing values and the patient did not show any clinical symptoms of IFI.
A bone marrow aspirate showed persistence of residual disease (8%).
The patient decided to receive further treatment at his home town. He died of leukemia progression few weeks later.

Discussion and Conclusion
Aspergillus spp. represent the most common pathogens causing pulmonary infection in immunocompromised patients and is  usually revealed by the GM assay; in twelve cases of infection by Hormographiella GM was always negative, resulting of no diagnostic utility [1].
Identification of filamentous Basidiomycetes is difficult because key diagnostic features are often lacking, usually only arthroconidia are formed [9].
In Hormographiella aspergillata, also the shape of arthroconidia is non-diagnostics, therefore definitive identification requires sequencing of the ribosomal DNA [1]. The MALDI-TOF is still not useful: filamentous fungi are difficult to be treated by conventional protocols with formic acid and the database is still poor. This is the first report on the use of a pan-fungal biomarker like BG to monitor the trend of infection by this rare pathogen: in our experience it has been useful to prompt diagnostic efforts in the identification of IFI.
It is known that Basidiomycetes are not susceptible to caspofungin, whereas Aspergillus spp. is susceptible to this antifungal drug: in one reported case, treatment for Aspergillus spp. was not effective for Hormographiella and the patient died [10]. Since very few documented cases have been observed, there is no established treatment for H. aspergillata; however the detection of the causative agent should be always pursued to avoid wrong therapy. In our opinion, BG should be considered a reliable diagnostic help in infections caused by Basidiomycetes.