Subcutaneous Phaeohyphomycosis Caused By New Species Of The Genus Scopulariopsis:Clinical And Laboratory Research

Background: Phaeohyphomycosis is the dematiaceous fungi induced infection that involves superficial tissues, skin, cornea, subcutaneous tissues, and sometimes the whole body system. The pathogenic dematiaceous fungi are soil saprophytes and plant pathogenic fungi, and their spores are widespread in the air. Many of the dematiaceous fungi are contaminant microorganism among experiments and can stay on the body surface for a short period of time. In the cases of the compromised immune system, diabetes, leukocythemia, and long-term use of immunosuppressive drug and glucocorticoids, the pathogenic dematiaceous fungi may travel into the body and cause infections through lungs and skin lesions; moreover, they may lead to systematic infections via blood circulations. Some of the pathogenic dematiaceous fungi have neurotropism, they may affect the brain tissues and meninges, causing central nervous system infections. This study starts with the separating identification of a new Scopulariopsis species that caused subcutaneous Phaeohyphomycosis, followed by the studies on the phenotype, in vitro antifungal susceptibility, and molecular identification.Part 1: A case of phaeohyphomycosis case that caused by a new Scopulariopsis infection, and the isolation and identification and clinical research that focus on the new Scopulariopsis. A 52-year-old male patient presented with a verrucous hyperplastic plaque on the lateral margin of left hand over seven years. The patient had experienced a trauma on the same site before the onset of the lesion. A small verrucous nodule was observed at the beginning, and the lesion gradually enlarged centrifugally. He was treated by 2 times cryotherapy which yield to poor outcome. Dermatological examination showed a 7×7 cm2 sized, welldemarcated verrucous hyperplastic plaque with the central area healing on the lateral margin of the left hand. The plaque had a cauliflower like and rough surface with tiny blood crusts. Histopathological examination of biopsied lesion tissue showedhyperkeratosis, pseudo-epitheliomatous hyperplasia in the epidermis, while infectiou granuloma formation in the dermis with predominately mixed inflammatory cells infiltration. Direct microscopic examination demonstrated long septate hyphae withou sclerotic bodies. Fungal culture isolated a slow-growing, dark brown, woolly-like colony with radial wrinkled surface and cracking medium. Phylogenetic analysis based on ITS and LSU sequencing revealed that the isolate was clustered in a separate clade which wa grouped in the genus Scopulariopsis. The final diagnosis was confirmed a phaeohyphomycosis caused by a new species of the genus Scopulariopsis. The patien had been treated by itraconazole 400 mg/day and acitretin 20mg/day. The lesion was completely faded after three months treatment and no recurrence was observed during the following six-month follow up.Part 2: The new Scopulariopsis was isolate from the skin lesion of the patient and cultured on different culture mediums, observed colonial morphology and growth rate. The in vitro antifungal susceptibility was performed in terms of the Clinical and Laboratory Standards Institute(CLSI) M38-A2 standard on Sporulation of filamentous fungi. Results showed that the new Scopulariopsis’ morphology varied among different mediums, and achieved the fast growth rate on GC medium, they produce more spores on PDA,SDA,MEA medium than others. Slide microculture of PDA showed light brown mycelium, branched separation, conidiophore upright. There were one to three phialide with spontaneous distribution. the conidiospore chain were sporadically present on top of the phialides with uneven length.. In vitro antifungal susceptibility showed good sensitivity on itraconazole and terbinafine. Scanning electron microscopy showed the phialide with inflated tip and clusters of conidiospore chain and penicillus. Temperature tolerance test showed the ability for the growth of the new Scopulariopsis at 37℃.Part 3: Molecular identification includes the abstraction, amplification, and sequencing of the DNA. The amplified PCR segments had been sent to purification and bidirectional sequencing at Sangon Biotech(Shanghai)Co., Ltd. The phylogenic analysis on the resultsof the sequencing had been performed, the ITS and LSU sequencing showed 567 bp and 548 bp segments; after uploading the results to Gen Bank, the accession number of KP764411 and KR006348 were matched. Phylogenetic tree construction result showed the new Scopulariopsis was located at an independent branch.Conclusion: 1. This case was phaeohyphomycosis caused by a new species of the genus Scopulariopsis. The patient had been treated by itraconazole 400 mg/day and acitretin 20mg/day. The lesion was completely faded and no recurrence was observed during the following six-month follow up.2. Results showed that the new Scopulariopsis’ morphology varied among different mediums. In vitro antifungal susceptibility showed good sensitivity on itraconazole and terbinafine. Temperature tolerance test showed the ability for the growth of the new Scopulariopsis at 37℃. 3. Phylogenetic tree construction result showed the new Scopulariopsis was located at an independent branch.