Rare Fungal Keratitis Caused by Coprinellus Radians

A case of fungal keratitis due to Coprinellus radians is reported. To our knowledge, fungal keratitis caused by this species was rare. Fungal hyphae were detected in corneal scrapings, and isolates were identified by morphology and by sequencing the internal transcribed spacer region of ribosomal DNA. The patient was treated with systemic and local antifungal therapy for 5 days, and lamellar keratoplasty was performed after no obvious improvement in symptoms. The in vitro antifungal susceptibilities of the case strain were tested for six antifungal agents. The results showed that 5-fluorouracil was resistant, fluconazole was moderately sensitive, and the other drugs assayed (amphotericin B, posaconazole, itraconazole and voriconazole) were highly effective against this fungus.


Introduction
At present, more than 100 species that belong to 50 genera of fungi pathogens have been reported which cause human corneal infection [1]. The pathogens of fungal keratitis vary in different climatic regions. Filamentous fungi are the main pathogens throughout the world, especially in tropical and subtropical areas, and the most common pathogens are Fusarium and Aspergillus [1][2][3][4][5]. In China, the most common pathogenic agents are Fusarium, Aspergillus and Alternaria [6]. As a basidio-mycotic fungus, C. radians which is commonly known as a contaminant has not been reported as the cause of this infection. Therefore, we describe a case of keratitis due to C. radians.

Case Report
A 48-year-old female farm worker, her left eye was scratched by corn leaves 40 days ago, complained of foreign body sensation, with vision loss, lacrimation, photophobia and no other obvious discomfort. She was diagnosed with a corneal ulcer at local hospital and was given topical antibiotic eyedrops (specific medication is unknown), significant improvement in symptoms of self-discomfort. Two weeks later, her condition deteriorated, the symptoms such as eye abrasion and vision loss were significantly aggravated, accompanied by difficulty in opening eyes, so she went to the local hospital again and received systemic and topical anti-infection treatment, but because of no effect, she was referred to our hospital.
On first examination, the patient's vital signs were normal and her existing spectacle correction visual acuity was FC/20 cm in the left eye, which showed diffuse conjunctival hyperemia. There was a 5 mm 9 mm gray-white infiltration with irregular borders and edema in the center cornea, invasion of shallow and middle stroma (Fig. 1a). No abnormality was observed in the right eye.
On image logical examinations, anterior segment coherence optical tomography (AS-OCT) (Optovue, USA) showed that the central cornea infiltration depth was about 100 lm and in vivo confocal microscopy (IVCM) (Heidelberg HRT3, RCM) showed that the accumulation of medium amount of necrotic material and the invasion depth of hyphae structure reached 200 lm, with a large number of chlamydospore-like structures clustered (Fig. 2). The endothelial cell count was about 2269 /mm 2 .
Corneal scraping was performed at the bottom and edge of corneal ulcer, and placed on a microscope slide which drip 10% potassium hydroxide; it showed abundant thick hyaline septate and branched hyphae, with abundant chlamydospore in the middle or terminal of the hyphae (Fig. 3a), while other scrapings were directly inoculated into 5% sheep blood agar and Sabouraud glucose agar (SDA), and incubated at 28°C and 35°C, respectively. After 2-3 days, small white colonies, apparently of a single fungus species, appeared on all the culture media.
After hyphae were detected, the patient was given voriconazole 10 mg/ml eyedrops four times per hour,  amphotericin B 0.25% eyedrops once an hour, levofloxacin 0.5% eyedrops four times a day, pranoprofen 0.1% eyedrops four times a day and ofloxacin 0.3% eye ointment once a night. Five days later, the inflammation did not subside. In order to avoid missing the operation opportunity, partial lamellar keratoplasty (glycerin preserved corneal tissue) was performed under peribulbar anesthesia after the patient signed the informed consent. After surgery, there was therapy with voriconazole 10 mg/ ml eyedrops once an hour, amphotericin B 0.25% eyedrops every two hours, ofloxacin 0.3% eye ointment four times a day and recombinant bovine basic fibroblast growth factor eye gel four times a day. Five days later, the voriconazole and amphotericin B 0.25% eyedrops were decreased to four times daily; at the same time, levofloxacin and pranoprofen eye drops were given four times a day. Eight days after the surgery, the corneal ulcer healed and the patient was discharged from hospital (Fig. 1b), and her visual acuity with spectacle correction improved to 20/200. The topical antifungals were tapered and discontinued, and outpatient follow-up was conducted.

Mycology
We identified fungi by morphology and molecular methods. The clinical isolate was subcultured on SDA and incubated in 28°C. After 8 days, the colonies on SDA were floccose, with dark yellow fluffy aerial mycelia in the middle, light yellow around and the  123 reverse was yellow and yellow-brown (Fig. 4). Microscopically, the hyphae were transparent, septated and branched. Conidiophore on both sides and tip of hyphae were short and thick. The conidia were single-celled, the ends were blunt, sausage-like, S-shaped and C-shaped and clustered in slimy head (Fig. 5).
According to its morphological and microbial characteristics, it was preliminarily identified as Coprinellus spp.; however, the genus could not be ascribed to which species. To confirm the identification, the internal transcribed spacer (ITS) region was sequenced and DNA sequences were determined at Kingdom clinical trial center (Guangzhou, China). The resulting sequences were deposited in the GenBank database. Species identification was performed by searching databases using the BLAST sequence analysis tool (https://www.ncbi.nlm.nih. gov/BLAST/). The ITS sequence of the isolate was 99.69% similar to that of strain C. Radians.
Although the relevant information of C. radians was available in the previous literature, most of them were isolated from sputum specimens, diabetic feet, abdominal drainage fluid and skin ulcer wounds, which could not exclude the possibility of contamination, and there were few clinical reports of eye infection caused by this genus. The patient's left eye was scratched by corn leaf and had a long course of disease. Slit-lamp examination showed a gray-white ulcer lesion of about 5 mm 9 5 mm in the cornea central pupil area. Combining with corneal scrape histology, IVCM and culture results, it conformed to the predisposing causes, clinical manifestations and etiological diagnosis of ocular fungal infection.
Coprinellus radians has low nutritional requirements and can grow on a variety of media, the most suitable medium is SDA and PDA, and the culture time is about 10 days. The colony characteristics were obvious and grew rapidly on SDA medium. Its color is bright and like cotton flocculent, the middle is fluffy, dark yellow, and the surrounding is light yellow as well as yellow or yellow-brown on the back. The color gradually disappeared after many generations. The spores are single-celled, the ends are blunt, sausagelike, S-shaped and C-shaped and clustered in slimy head. Although the colonies and spores of the genus are distinctive and easy to identify, they should be distinguished from those of some Fusarium species in order to avoid confusion ( Table 2).
In vitro drug sensitivity test showed that the MIC values of amphotericin B, voriconazole, posaconazole and itraconazole were lower, while the MIC values of fluconazole and 5-fluorocytosine were higher. Due to the lack of a unified drug sensitivity break point, it is not yet possible to determine whether drug resistance exists. However, according to the MIC values, the rare genus was highly sensitive to amphotericin B, itraconazole, posaconazole and voriconazole, and resistant to fluconazole. Whether there is intrinsic drug resistance has not been reported. The patient was first treated in a primary hospitals and its specific medication was not clear; when she came to our hospital, the lesion had invaded the stroma, and sensitive antifungal agents and timely lamellar keratoplasty are effective. Therefore, fungal keratitis needs early diagnosis to identify different fungi genera and symptomatic treatment.
In summary, to our knowledge, this case of mycotic keratitis represents the first known reported infection caused by C. Radians, as well as being the first known reported in humans caused by this rare species.
We provided the checklist according to the request of the magazine [11].

Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict of interest and no ethical issue.
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