Phialemoniopsis limonesiae sp. nov. causing cutaneous phaeohyphomycosis in an immunosuppressed woman

ABSTRACT Rare or opportunistic fungal infections are mostly described in immunosuppressed patients. We present a case of a cutaneous phaeohyphomycosis that developed on the dorsal foot in an immunosuppressed woman suffering from AIDS, caused by a novel Phialemoniopsis species. It clinically presented as an indurated violaceous plaque, surmounted by nodules exuding a sero-purulent discharge. A filamentous fungus was isolated from pus and cutaneous biopsy. ITS and LSU sequences phylogenetically resolved the fungus as an unknown species of Phialemoniopsis, which is an unresolved family within Sordariomycetes. In this study we describe the new species as Phialemoniopsis limonesiae, which clusters on a single branch clearly separated from its closest phylogenetic neighbours. This new strain showed low MIC to itraconazole, voriconazole and posaconazole.


Introduction
Up to 2000 fungal species are being newly described each year [1], several of which are associated with human infections. The genus Phialemoniopsis (Sordariomycetes) presently contains six species, four of which have been isolated from human fluids, skin or nails [2]. Since the introduction of Phialemoniopsis in 2013 [2], a further two species have been added, namely P. hongkonensis, from a nodule on a human forearm [3], and P. endophytica, the only species thus far isolated from plants [4].
Phialemoniopsis, which is a genus of dematiaceous hyphomycetes, can be distinguished from Phialemonium by having phialides and adelophialides with collarettes, and the development of sporodochium-or pycnidium-like conidiomata, which are both absent in Phialemonium s. str.
In the present communication, we report a case of cutaneous phaeohyphomycosis in an immunosupressed woman caused by a novel Phialemoniopsis species described here as P. limonesiae.

Case report
A 56-year-old woman was referred at our consultation for a persistent wound on her foot. She was followed for AIDS (CD4+ T lymphocytes 38/µl and controlled HIV viremia), disseminated histoplasmosis in 2015, and metastatic pulmonary adenocarcinoma since 2017. Her usual treatment included abacavir/dolutegravir/lamivudine (Triumeq ® ), cotrimoxazole prophylaxis, itraconazole as secondary prophylaxis (200 mg oral capsules bid) since her histoplasmosis and osimertinib 40 mg/kg, respectively. The patient's history included a trip to Ecuador prior to the onset of the skin lesions. It initially erupted as a unique papule, progressing to multiple violaceous papulo-pustules and local pain (Figure 1(a)). There was no history of skin trauma.
Her general condition was poor with asthenia and anorexia. She had no fever. Skin examination showed an indurated purplish painful plaque surmounted by papules and nodules on the dorsum of the right foot, sometimes ulcerated with a sero-purulent discharge with grains ( Figure 1(b)). No peripheral adenopathy was noted. Clinical diagnosis suspected a deep mycosis (including a relapse of histoplasmosis with cutaneous location), bacterial actinomycosis, or mycobacterial infection.
Histological examination (H&E) revealed a dense dermal, predominantly neutrophilic infiltrate, with abscess formation. The overlying epidermis was slightly acanthotic with parakeratosis and slight spongiosis.
Yeast-like structures were seen within the abscess ( Figure  2(a,b)). The use of special stains (PAS) confirmed the fungal nature of these structures ( Figure 3). The X-ray did not reveal any signs of osteitis. Bacterial and mycobacterial cultures from swabs and cutaneous biopsy were negative. Two repeated swabs from the pus and one cutaneous biopsy showed, on direct examination, the presence of hyphae which grew on brain heart infusion (BHI) agar with chloramphenicol and gentamicin after 5 days incubation at 30°C , later to be identified as a species of Phialemoniopsis by ITS sequencing. The identification by MALDI-TOF MS (Bruker Daltonics) using two different data bases showed a discrepancy. The results obtained with the Bruker database (MBT Compass Library 8431 MSP) and the MSI database were Phialemoniopsis curvata (score: 2.03) and Phialemoniopsis cornearis (score: 21.29), respectively [3,5]. Cultures from the swab and skin biopsy were sent to the Westerdijk Fungal Biodiversity Institute, in the Netherlands, for species identification. The strain was suspected to be a new species of Phialemoniopsis, and is described below.
The clinico-microbiological presentation was consistent with the diagnosis of a deep cutaneous phaeohyphomycosis of the foot in a context of immunosuppression.
We treated the patient with povidone iodine and after culturing the fungus, itraconazole capsules were replaced by an oral solution of itraconazole 200 mg b.i.d. in order to increase the biodisponibility in this HIV+ patient [6]. The lesion improved with plaque regression and diminution of nodule infiltration under itraconazole solution. A relapse was noted when we changed the compounded formulation (solution to capsule) due to adverse effects (nausea). At that time low itraconazole blood level (0.81 mg/ml)   was confirmed under capsules intake. In addition, we topically applied a compress soaked with itraconazole solution, twice a day for 10 min. After 5 weeks of oral and topical itraconazole, the cutaneous lesions were stable and asymptomatic. The new itraconazole blood level was 1.40 mg/ml.
The patient died of her oncologic issue after 7 months of treatment. The cutaneous lesions regressed but had not completely healed at the time of her death.

Material and methods
The new species of Phialemoniopsis was described by standard morphological examination of the fungal culture and by molecular phylogenetic analysis.
Strains isolated from both pus and cutaneous biopsy from the patient were inoculated on brain heart infusion (BHI) agar (BD Difco TM ) with chloramphenicol (50 µg/ml) and gentamicin (50 µg/ml) and on Candida CHROMagar plates (CHROMagar Candida, France). First growth was seen after 5 days of incubation at 30°C.
Phylogenetic analyses were based on a concatenated alignment of act, ITS, LSU, and tub2. The Maximum Likelihood (ML) analyses were performed on the CIPRES Science Gateway portal [16] using RAxML v. 8.2.9. For ML analyses, the default parameters were used, and bootstrap support (MLBS) was carried out using the rapid bootstrapping algorithm with the automatic halt option. A MLBS value >50% was considered as statistically significant. Bayesian analyses (BI) were performed using MrBayes v. 3.2.6 [17]. Markov Chain Monte Carlo sampling (MCMC) analyses of four chains were started in parallel from a random tree topology. Four simultaneous Markov chains were run for 10 M generations with a sampling frequency set to the 1000th generation (resulting in 10,000 total trees per parallel run). The first 25% trees represented the burn-in phase of the analyses and were discarded, and the remaining trees were used to calculate posterior probabilities (BPP). FigTree v. 1.4 was used to visualize the final tree.
Morphological features were determined on oatmeal agar (OA), 2% potato dextrose agar (PDA), 2% malt extract agar (MEA) and synthetic nutrient-poor agar (SNA) [18]. Cultures were incubated at 25°C in the dark for 2 weeks. Macroscopic characters and diameters were measured after 14 d of incubation, and the colony colour (surface and reverse) rated after Rayner (1970) [19]. Slide preparations were mounted in clear lactic acid to study the micromorphological structures. Micromorphological observations were processed with a Nikon Eclipse 80i compound microscope with differential interference contrast (DIC) optics and a Nikon AZ100 dissecting microscope, both equipped with a Nikon DS-Ri2  Figure 5). The strain isolated in this study clustered within the genus Phialemoniopsis, but proved to be distinct from any known species ( Figure 5). Phialemoniopsis limonesiae A. Riat, L.W. Hou & Crous, sp. nov. MycoBank MB837525.
Etymology: Named after the patient's name, "Limones," from whom this fungus was isolated. An informed consent was obtained from the patient in order to present her case and name this novel fungus after her.

Discussion
In this study we described a new opportunistic human pathogen, named Phialemoniopsis limonesiae.
Dematiaceous fungi cause opportunistic cutaneous infections most often in solid organ transplant recipients than in AIDS patients [21,22]. In AIDS patients, cryptococcosis, histoplasmosis and Talaromyces marneffei infections are more common [23] The genus Phialemoniopsis is closely allied to Phialemonium, and is widely distributed in air, soil, and plant materials [2]. Different species of Phialemoniopsis are associated with infections in humans like endocarditis, endovascular infections, endophthalmitis, meningitis and skin and soft tissue infections [3]. In our case, its most probable origin was Ecuador where our patient travelled prior to symptom development. One possible hypothesis of its emergence could be the use of prolonged antifungal prophylaxis [24].
In our patient, phaeohyphomycosis developed under a treatment of itraconazole capsules at a therapeutic dosage, although the susceptibility testing of the strain showed a low MIC (= 0.06) for itraconazole. However, a low itraconazole blood level (0.81 mg/ ml) was confirmed under capsules intake. The lesions improved after the switch to an oral itraconazole solution 200 mg b.i.d. and topical formula consisting of applying an empirical oral itraconazole solution on a dressing during 10 min in addition to standard antiseptics. A higher blood level of oral itraconazole solution compared to the capsules was already described in HIV positive patients treated for oral candidiasis [6]. Our case confirms that the oral solution improves the poor bioavailability due to capsule intake [24]. Standard treatment of known Phialemoniopsis spp. such as P. curvata and P. ocularis relies on oral itraconazole therapy 100-200 mg daily. Amphotericin B, voriconazole and surgical excision in the case of solitary lesions have also been used [21].
Identification of microbial species as human pathogens is challenging. Particularly moulds and yeasts are commonly isolated from skin on superficial samples. Clear clinical and laboratory criteria need to be used to confirm pathogenicity. We documented each step: repeated samples, biopsy and multiple microbiological analyses. Among these analyses the MALDI-TOF system used in many clinical laboratories for common fungal identification proved inconclusive for this case. To identify rare fungi, the MALDI-TOF system has shown its limitation and its dependency on the quality and robustness of the database used [25].
To conclude, Phialemoniopsis limonesiae is a new opportunistic fungus to be added to the growing number of dematiaceous hyphomycete human pathogens, affecting a deeply immunocompromised patient. The clinical presentation as subcutaneous abscesses and nodules on the skin is similar to known Phialemoniopsis species in humans.