Hormographiella aspergillata: an emerging basidiomycete in the clinical setting? A case report and literature review

Background Filamentous basidiomycetes are mainly considered to be respiratory tract colonizers but the clinical significance of their isolation in a specimen is debatable. Hormographiella aspergillata was first reported as a human pathogen in 1971. We discuss the role of this mold as a pathogen or colonizer and give an update on diagnostic tools and in vitro antifungal susceptibility. Case presentation We identified three cases of H. aspergillata with respiratory symptoms in a short period of time. One invasive infection and two colonizations were diagnosed. Culture supernatants showed that H. aspergillata can produce galactomannan and β-D-glucan but not glucuronoxylomannan. For the first time, isavuconazole susceptibility was determined and high minimum inhibitory concentrations (MICs) were found. Liposomal amphotericin B and voriconazole have the lowest MICs. Conclusion To date, 22 invasive infections involving H. aspergillata have been reported. On isolation of H. aspergillata, its pathogenic potential in clinical settings can be tricky. Molecular identification and antifungal susceptibility testing are essential considering high resistance against several antifungal therapies.

neutropenia and anorexia. He had a history of acute myeloid leukemia (AML) and hematopoietic stem cell transplantation (HSCT). His C-reactive protein (CRP, positivity threshold value: 3 mg/L) was 135 mg/L and empirical antibiotic therapy (ceftriaxone) was started at day 210 (D210, 7th month) post-HSCT. Chest computed tomography (CT) scan showed right lower lobe opacification ( Fig. 1a) that had increased 1 week later (Fig. 1b). Invasive fungal infection (IFI) was suspected, and liposomal amphotericin B (lAmB 5 mg/kg/day) was started on D232 (7th month). Microscopic examination of a bronchoalveolar lavage (BAL) sampled at D237 (7th month) showed septate hyphae (Fig. 2) but cultures on Sabouraud media incubated at 25°C and 35°C were sterile after 7 days. H. aspergillata was identified by sequencing the internal transcribed spacer (ITS) region of fungi directly from the BAL. Interestingly, serum galactomannan monitoring was negative (< 0.1 on repeated samples; Platelia® Aspergillus assay, Bio-Rad; positivity threshold index: > 0.5) and β-Dglucan (Fungitell®, Cape Cod; positivity threshold value: 80 pg/mL) was weakly positive on D237 (7th month; 98 pg/mL) but negative on D248 (8th month; 46 pg/mL). In accordance with the 2008 European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, the patient was classified as having probable IFI [23]. His condition worsened following pulmonary Stenotrophomonas maltophilia infection and so it was decided to initiate palliative care. lAmB was stopped on D253 (8th month), 3 weeks after its introduction. The patient died on D298 (9th month).
The second patient (HA2) was a 49-year-old man admitted to the intensive care unit for pneumopathy with acute respiratory failure. He had a history of psychiatric disorders, diabetes mellitus, asthma, smoking and middle cerebral artery stroke with persistent sequelae. CRP was negative on admission. The following day, it was positive at 108.0 mg/L but procalcitonin remained negative. Mechanical ventilation and empirical antibiotic therapy (ceftazidime) were initiated. A mucous plug containing purulent secretions in the left lung was removed by fibroscopy and transmitted to Bacteriology and Mycology Laboratories. Microscopy examinations of samples were negative but cultures identified oropharyngeal microbiota associated with a white mold on Sabouraud media at 25°C and 35°C after 7 days. Subcultures of mold grew with white to slightly cream-colored velvety colonies ( Fig. 3a and b) on potato dextrose agar media. Microscopy examination of cultures showed hyaline septate hyphae with conidiophores producing cylindrical arthroconidia ( Fig. 3c and d). H. aspergillata identification was confirmed by sequencing the ITS region. In vitro antifungal susceptibility testing was performed via broth microdilution technique according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [24]. Minimum inhibitory concentrations (MICs) are given in Table 1. The chest CT scan was unremarkable and there was no risk factor for IFI and so no antifungal  therapy was initiated. The inflammatory syndrome decreased rapidly 3 days later, and the patient's condition improved. A putative diagnosis of bacterial aspiration pneumonia with fungal colonization was established.
The third patient (HA3) was a 28-year-old woman admitted for investigation of an inflammatory disease affecting the central nervous system treated by methylprednisolone for 3 days (1 g/day). Bronchial fibroscopy was performed along with other investigations. Initial microscopy examination of the sample was negative but H. aspergillata grew after 3 weeks on Lowenstein-Jensen medium at 35°C because of mycobacterial suspicion (identification confirmed by ITS sequencing). Antifungal susceptibility testing was performed as described above ( Table 1). The patient was asymptomatic and her chest CT scan normal, suggesting colonization, and so no antifungal treatment was initiated.

Literature review
We reviewed the literature since 1971 to date using the terms "Hormographiella aspergillata" or "Coprinus cinereus" and "infection" in MEDLINE database (Tables 1   and 2). For each strain, antifungals MIC with the method used were reported in Table 1 when available. According to the 2008 EORTC/MSG criteria, all probable or proven IFI due to H. aspergillata were reported in Table 2 with significant clinical details.
H. aspergillata can also be a colonizer of the respiratory tract, as illustrated in our three patients, all of whom had an underlying respiratory condition. The weak clinical significance of the isolation of basidiomycetes in healthy subjects, in contrast with their lifethreatening potential in immunocompromised patients, has already been described with Schizophyllum commune or Ceriporia lacerata, for example [27,28]. These fungi are widely present in the environment, and their spores are easily inhaled and can grow in pulmonary alveoli in cases of local or systemic impaired function of alveolar macrophages.
As yet there are no EUCAST nor Clinical and Laboratory Standards Institute (CLSI) breakpoints to interpret the antifungal MICs for H. aspergillata. However, previous articles have reported in vitro resistance to echinocandins, fluconazole along with high MIC for flucytosine (Table 1). We found higher MICs for isavuconazole (4 and 16 mg/L) than what is usually observed for basidiomycetes [28,29]. In the light of our findings and data from the literature, lAmB and voriconazole have the lowest MICs. However, H. aspergillata infections have a poor prognosis even when surgical debridement is performed.
In conclusion, on isolation of H. aspergillata, its pathogenic potential in clinical samples should be interpreted together with the patient's history. Formal identification of the fungus can be tricky and usually requires molecular tools in addition to culture. Basidiomycetes can also be contaminants or colonizers and so microscopy examination of samples and/or histology in combination with biomarkers are crucial for diagnosis. Respiratory tract colonization is probably not uncommon given that the fungus is widespread in the environment but seems to be restricted to patients with underlying respiratory diseases. lAmB and voriconazole seem to be the antifungals of choice.  Table 3 Galactomannan (GM), β-D-glucan and glucuronoxylomannan antigen assays on culture supernatant. For each strain, 5 to 10 colonies incubated at 35°C for 4 days on Sabouraud media were suspended in 1 ml distilled water. After vigorous agitation, the suspensions were centrifuged for 5 min at 10,000 g.