Among the 1608 fungal keratitis cases, the isolates of Fusarium were the most common (53.3%), followed by Aspergillus (23.4%), Alternaria (12.6%), and other genera (10.7%) such as Candida, Scedosporium, Acremonium, Trichosporon, and rare genera. The 16 patients of rare fungal keratitis consisted of 1 case of Rhizopus (0.06%), 2 cases of Sarocladium (0.12%), 4 cases of Pythium insidiosum (0.25%), 1 case of Exserohilum (0.06%), 1 case of Bipolaris (0.06%), 2 cases of Colletotrichum (0.12%), 2 cases of Purpureocillium lilacinum (0.12%), and 3 of cases Curvularia crescentulum (0.19%).
Demographics and clinical presentation
Among the 16 patients, 13 were males and 3 were females; most occupations are farmers. The average age was 54.75±12.70 years. Three cases (18.75%) were caused by plant trauma, seven cases (43.75%) were due to foreign body scratching, and six cases (37.5%) were without inducement. None of the 16 cases had a history of topical steroid use. The onset time was 13.81 ±7.20 days. Pythium insidiosum keratitis developed quickly, and the onset time was 10.5±5.20 days. The duration of hospitalization was 17.81 ± 8.83 days, Exserohilum keratitis was the shortest (<15 days) and Pythium insidiosum keratitis was the longest (>30 days).
When medicated for about one week, Rhizopus, Pythium insidiosum, and Purpureocillium lilacinum had endothelial plaques and hypopyon was observed via slit lamp. Curvularia crescentulum keratitis was mainly presented with feathered edges. Cases of Colletotrichum, Exserohilum, and Bipolaris cases also showed feathered edges extending to the periphery, but were not associated with endothelial plaque, satellite lesions, or hypopyon in our study. Sarocladium cases only showed obvious edema and infiltration without typical fungal clinical characteristics (Figure 1, and Table 1).
Confocal microscopy images
Hyphae were found in 16 cases (85.25%) upon examination using confocal microscopy, and generally changed as follows: the hyphae were highly reflective; they were partially branched and segregated; their diameter was about 2.3 – 4.4 μm; their shape was dendritic, linear, or short and rod-like; they had irregular hyphal morphology; the average branching angle was 39.03 – 46.85 degrees; and the spore detection rate is very low. The confocal manifestations of different genera are shown in Figure 2.
Histopathological section results
In the 16 patients, 14 cases had corneal pathological sections, and hyphae structures were found in 8 cases (57.14%). A small number of hyphae and inflammatory cells were observed in Sarocladium and Exserohilum keratitis. When conducting periodic acid–Schiff (PAS) staining, Pythium insidiosum keratitis was not stained, only vacuolar hyphae-like structures were found in the whole stroma, and the inflammatory cells were not obvious. Furthermore, immunofluorescence staining yielded positive results. More hyphae and a few spores were observed in Purpureocillium lilacinum and Curvularia crescentulum keratitis (Figure 3).
In vitro drug sensitivity tests
In addition to Pythium insidiosum, the MIC values of amphotericin B, voriconazole, and itraconazole in seven genera were low (0.016 to >16), indicating that they were all susceptible to the three antifungal agents. Among them, Rhizopus was found to be more sensitive to amphotericin B. Sarocladium was shown to be equally sensitive to three antifungal agents. Colletotrichum, Exserohilum, Bipolaris, and Purpureocillium lilacinum were more sensitive to voriconazole, while Curvularia crescentulum was more sensitive to itraconazole and fluconazole was highly resistant in all genera studied (MIC 4 to>256) (Table 2).
Among the collected patients, 111 cases (13.07%) of Fusarium keratitis were cured by antifungal medication，and surgery was performed on 746 (86.93%) cases, including 362 cases (48.53%) of LKP, 237 cases (31.77%) of TPK, 137 cases (18.36%) of keratectomy, and 9 cases (1.21%) of evisceration. The postoperative fungal recurrence rate was 7.5%, and all patients were successfully cured by TPK after recurrence. The rare fungal genera patients’ ulcers gradually healed in 2/3 cases (66.67%) of Curvularia crescentulum keratitis after antifungal drug treatment. After one week of medication, 14/16 cases (87.5%) underwent surgical treatment after persistent progression or a lack of improvement in symptoms. Among the 14 cases , keratectomies were performed in 7 (50%) cases , the average healing time of the corneal epithelium was 5.17±0.79 days post-operation. Two cases (14.29%) were treated with LKP, one case of Pythium insidiosum keratitis recurred after operation, and fungal recurrence was detected by confocal microscopy after corneal transplantation. Five cases (35.71%) were treated with TPK, and two cases of Pythium insidiosum keratitis recurred after operation. Evisceration was performed in three recurrent cases with an uncontrollable infection, and the average time of first recurrence was 3.33 days (Figure 4, Table 1).
The best-corrected visual acuity (BCVA, LogMAR) of 13 cases was 1.48 ±0.85 before treatment and 0.60 ±0.45 after treatment, and the difference was statistically significant [(95 % CI, 0.33–1.43) (P=0.003)], with an average increase of 4.55±2.62 lines.