A Case of Beauveria Bassiana Keratitis Confirmed by Gene Sequencing Sung-Dong Chang, M.D., Jong-Hwa Jun, M.D. Department of Ophthalmology, School of Medicine, Dongsan Medical Center, Keimyung University, Daegu, Korea

INTRODUCTION Beauveria genus is composed by the insect pathogens, soil fungus, and Beauveria bassiana is distributes worldwide. 8 cases of human infection, and 4 cases of ocular infection have been reported. Ocular infection induces very severe keratitis, and surgical removal such as deep lamellar dissection or penetrating keratoplasty of infected tissues is required. As the diagnosis, in most cases, early diagnosis is difficult resulting in the administration of antifungal agents under the condition that keratomycosis is progressed substantially, and thus early appropriate treatment is delayed and poor prognosis is shown. Among cases reported until now, none of cases was definitely diagnosed by gene sequencing for more early diagnosis, and it is considered that for cases suspicious to be fungal keratomycosis, by performing gene sequencing, it could be definitely diagnosed early and aggressive treatments could be administered, hence, the prognosis of patient could be improved

A 70 years old male patient visited our clinic for the chief complaint of the progressive deterioration of the visual acuity after injury with a splint in the left eye 1 week ago. In the past history, he had a history of chemotherapy due to nasopharyngeal and gastric cancer, and the past history of ocular surgery was not shown. The visual acuity of the left eye was 0.06, and the intraocular pressure of the left eye was 8 mmHg. In the slit lamp examination, severe conjunctival injection and follicular hypertrophy were shown, and after fluorescein staining, in the slightly nasal side of the corneal center of the left eye, linear epithelial defect 0.7*1.0 mm in size was detected(Figure A,B) CASE A B

In the vicinity, infiltration of immune cells, Desmets’ membrane folds, and edema findings in the stroma were detected, and in the border of the epithelial defect, a pattern of the slight elevation of the healed epithelium was shown. In the anterior chamber, weak inflammation findings and flare were detected. Smear and culture test were performed by corneal scraping, and in the smear test, special findings were not detected (Figure A) During the follow up observation while administering antibiotic eye drops, approximately after 2 weeks, the size of epithelial defect was increased to 3.7*2.7 mm, and the pattern of the augmentation of inflammation findings in the anterior area was shown (Figure B) CASE A B

At the 5 weeks of the treatment, smear and culture were repeated, and several hyphae were detected, and thus the sample was cultured in Sabouraud dextrose agar, it was suspected to be Beauveria spp, and gene sequencing was performed by the use of the MicroSeq D2 LSU r DNA fungal sequencing kit, and it was identified to be Beauveria bassiana. Natamycin eye drops were administered every hour, and at the 7 weeks of treatment, the epithelial defect showed a pattern to be slightly decreased, however, due to the thinning of the stroma caused by ongoing destruction, Desmatocele was developed and thus amniotic membrane transplantation was performed (Figure A) After amniotic membrane transplantation, amphotericin B eye drops were administered additionally, and at the 1 week of treatment, the epithelial defect was healed completely, and noticeable reduction of inflammation in the anterior chamber could be observed (Figure B) CASE A B

DICUSSION Recently, the incidence of keratomycosis shows a trend to be increased continuously due to the expanded use of antibiotics, steroid, and immunosuppressant, and consequently, interests on it are on the rise. Among them, regarding Beuaveria bassiana, prior to the infection in the cornea in 1985 by Sachs et al. for the first time, it has been known to be a fungus with weak pathogenicity that could not induce keratomycosis. After the report in 1985 by Sachs et al. infection cases such as keratomycosis, deep tissue infection, fungal empyema, disseminated infection, etc. have been reported, and particularly, it has been reported that upon the development of keratomycosis, it is detected to be negative in fungus culture by corneal scraping in many cases, and early appropriate fungus treatment is delayed in many cases, hence, even in cases definitely diagnosed to be fungus keratomycosis, surgical treatments such as penetrating keratoplasty are required in most cases

In keratomycosis cases, 4 cases have been reported, and for 3 cases required surgical treatments, penetrating keratoplasty was performed in 2 cases, and 1 case was treated by deep lamellar dissection, and regarding cases treated with medical management, Kisla et al. have reported 1 case, and thus for cases developed keratomycosis, surgical treatments were required in most cases. In our case, in the second corneal scraping test, several hyphae were detected, and thus medical agents were switched to natamycin eye drop, and afterward, it showed a pattern to be improved, nonetheless, because of the progressive destruction of the stroma, permanent amniotic membrane transplantation was required. Afterward, amphotericin B eye drops were administered additionally, and the epithelial defect and cell infiltration findings in the vicinity were improved rapidly, and without penetrating keratoplasty, his ocular intergrity could be maintained. DICUSSION

Conclusion Different from previously reported 8 cases of human infection, in our case, by fungal culture, Beauveria spp. was suspected, and thus for its definite diagnosis, gene sequencing was performed, and it could be ultimately diagnosed as Beauveria bassiana, and with the aggressive use of antifungal agents, the rapid improvement pattern of keratitis could be observed. In our case also, the initial diagnosis by corneal scraping and culture failed, the disease course was prolonged, the level of corneal destruction was severe, and amnion membrane transplantation was required, and it is considered that from now, for cases suspected to be fungal keratomycosis, by the application of aggressive diagnosis methods such as gene sequencing, and by the administration of of appropriate antifungal agents, conservation of the eye could be achieved.