Clinical manifestations and treatment outcomes of rare genera fungal keratitis in China

Background: Fungal keratitis is a common cause of blindness, and although major fungal genera in China have been widely reported, little is known regarding rare genera of fungal keratitis. This study is aimed at investigating the clinical manifestations and treatment outcomes of rare genera of fungal keratitis and improving clinicians’ understanding of these genera in China. Methods: A retrospective analysis was performed at Shandong Eye Hospital from October 2008 to December 2017. The clinical manifestations, risk factors, confocal microscopic images, histopathological features, in vitro drug sensitivity tests, and treatment outcomes of 16 patients with 8 rare fungal genera were evaluated. Results: Slit lamps were used to observe Rhizopus, Pythium insidiosum, and Purpureocillium lilacinum, which presented with endothelial plaque and hypopyon. Curvularia crescentulum keratitis mainly presents with feathered edges. Cases of Colletotrichum , Exserohilum, and Bipolaris also showed these feathered edges extending to the periphery, while Sarocladium keratitis only showed obvious edema and infiltration. The mean age was 54.75±12.70 years with an increase in male preponderance. Plant trauma and foreign body scratching caused ten cases (62.5%), and six cases (37.5%) were caused without inducement. In 2/3 cases, Curvularia crescentulum keratitis was cured with antifungal medication, while in 14/16 cases (87.5%), surgery was required. An exception occurred in three cases of Pythium insidiosum , where patients with fungal recurrence and final evisceration was performed; no recurrence of infection occurred in the rest of the patients. 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 found to be statistically significant [(95 % CI, 0.33–1.43) ( P =0.003)]. Conclusions: We report on the rare fungal genera collected in China in order to improve

the knowledge of clinicians. In our study, Curvularia crescentulum seems to respond well to antifungal drugs. Patients affected by Pythium insidiosum often need long-term recovery and multiple keratoplasty, and postoperative fungal recurrence rate is high. For fungal keratitis, the combination of early sensitive antifungal drugs and necessary surgical treatment may still be a beneficial factor in treatment.

Background
Fungal keratitis can result in serious visual impairment and mainly concentrated in tropical and subtropical regions in developing countries, such as India and Brazil [1][2][3], as it accounts for 40% to 50% of all isolated keratitis cases [4,5]. Currently, there are more than 70 pathogens that can cause fungal keratitis [6], but the clinical isolates of these pathogens are mainly concentrated in a few genera. The fungal isolates vary from country to country due to climate and environmental impacts. Fusarium and Candida are the most common pathogens in developed countries (such as the United States and the United Kingdom) [7]. Rare pathogens, such as Rhizopus, Sarocladium, Colletotrichum, Exserohilum, Bipolaris, Curvularia crescentulum,Purpureocillium lilacinum, and Pythium insidiosum have been reported in dozens of cases among both white and black populations. Fusarium, Aspergillus, and Alternaria are the main pathogens in China [8], but the rare fungal genera mentioned above have not been reported in China.
Different clinical manifestations of fungal keratitis and different sensitivities to antifungal drugs makes it very difficult to treat fungal keratitis. The clinical features and drug sensitivity of common fungal genera have been widely reported, but treatment guidelines for rare genera are lacking. Therefore, the purpose of this study is to gather cases of rare fungal genera treated in Shandong Eye Hospital and analyse the relevant factors predisposing their occurrence, the clinical manifestations, the confocal microscopic images, the drug sensitivity tests, and the treatment outcomes.

Subjects
The study isolated and identified fungal pathogens of 1608 patients with fungal keratitis from Shandong Eye Hospital between October 2008 and December 2017. Sixteen cases caused by eight rare fungal genera were analysed from these patients. This study was approved by the Ethics Committee of Shandong Eye Hospital. The tenets of the Declaration of Helsinki were adhered to in the conduct of this study, and all surgical patients signed written informed consent forms.

Diagnostic methods
Confocal microscopy, anterior segment coherence optical tomography, corneal scraping cultures, and slit lamp microscopy examinations were performed on all patients. After admission, corneal lesions were examined using cytology smearing, and patients' scraped tissue was inoculated into Sabouraud dextrose agar (SDA) and blood agar plates and were cultured in incubators. The growth characteristics of the colonies were recorded. Internal Transcribed Spacer (ITS) gene sequencing was used to identify those whose morphology could not be identified.
Amphotericin B, fluconazole, itraconazole, and voriconazole (provided by Shandong Boke Biological Co., Ltd.) were tested for vitro antifungal susceptibility, and the minimum inhibitory concentration (MIC) values were reported. Corneal buttons excised from surgical patients were submitted for histopathology evaluation.

Drugs and surgical procedures
Sixteen patients were hospitalized and received topically polyene (5% natamycin eye drops or 0.25% amphotericin eye drops) and imidazole (0.5% fluconazole eye drops or 10mg/ml voriconazole eye drops). When their corneal ulcers became worse, that is, increased infiltrate size or increasing hypopyon, a 0.2 mg/ml intravenous drip was utilized once a day, or 200 mg itraconazole orally once a day were added to the therapy.
Surgical treatment was used when drug therapy was shown to be ineffective after approximately one week. keratectomy was performed when the lesion was located in the paracentral or periphery where the infection size was less than 5 mm with a depth of less than 1/2 the corneal stroma, when the depth reaches 1/2, conjunctival flap can be combined. Lamellar keratoplasty (LKP) was performed when the lesion was located in the optic axis and reached the deep corneal stroma but not the corneal endothelium.
Therapeutic penetrating keratoplasty (TPK) was performed when the fungal infection had reached the corneal endothelium, and evisceration was performed when the fungal recurrence after keratoplasty was uncontrollable.

Statistical Analysis Method
Microsoft Excel 2010 was used for data input and management. To analyze the changes of best-corrected visual acuity (BCVA) before and after operation with the Wilcoxon signedrank test. All statistical analyses were performed using Predictive Analytics Software (PASW) version 18.0.

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) (

Discussion
There are different growth patterns among fungal species, so the clinical manifestations, pathogenic invasiveness, and microbial characteristics also differ. There is a challenge presented in the clinical diagnosis of infectious keratitis based on slit lamp examination, and microbial detection is still the gold standard [9]. However, early empirical treatment based on lesion manifestations is also necessary. Many studies have reported that Fusarium keratitis more commonly shows feathery edges due to horizontal growth, while Aspergillus keratitis more commonly shows immune rings and hypopyon due to vertical growth [10,11]. In this study, we observed the clinical manifestations of Rhizopus keratitis showing endothelial plaque and hypopyon. Warner et al. [12], and Azari et al. [13] reported that Rhizopus belongs to the Mucorales order of fungi, and is rarely seen in human eyes. Such an infection can rapidly destroy the tissue structure and accelerate the progression of ocular diseases because of its strong pathogenic invasiveness [14]. Fewer than five cases have been reported, and there are still no reports in China.
Pythium insidiosum classified as an oomycete, which is a fungal-like organism that is seen as branching, sparsely, septate or aseptate filaments; its host animals are mammals, but eye infections are rare in China. The main predisposing factors are water exposure, wearing contact lenses, and trauma. Although this study discussed Pythium insidiosum with fungi, its microbial species still requires further discussion. Its clinical manifestations are mainly endothelial plaque, hypopyon, and peripheral reticular infiltration, which indicate that the genus causes strong enzymatic hydrolysis in surrounding and deep tissues, probably because of the lack of ergosterol drug targets in the cytoplasmic membrane leading to insensitivity to antifungal drugs and difficulty in controlling diseases [15,16].
Curvularia crescentulum belongs to the family of dematiaceous fungi. Its clinical manifestations are raised lesions and feathered edges; hypopyon rarely occurs, and the prognosis is quite good. The medication rate of Curvularia crescentulum keratitis in this study was 2/3 cases (66.67%). As such, Curvularia crescentulum keratitis seems to respond well to antifungal drug. Exserohilum, Bipolaris, and Colletotrichum are also dematiaceous fungi where foreign bodies or plant trauma are predisposing factors [17].
Human infections mainly invade the skin and respiratory tract, rarely infecting the cornea [18][19][20][21][22]. Moreover, no cases of these three genera infections have been reported in China.
In this study, the most common manifestation of the three fungal genera was feathered edges, which invaded the surrounding corneal tissues in a carpet-like manner. The main reason for this was the melanin in the cell wall, which can affect the host's immune response to infection and reduce the toxicity of pathogens to deep invasion. In addition, the unique temperature sensitivity of Colletotrichum also inhibited its progression to deeper levels (≥35°C growth restriction), although the low virulence of these three genera still needs to be verified in future cases.
Chidambaram et al. [23] reported that the average branching angle of Fusarium and Aspergillus was 59.7° and 63.3°, respectively. In our study, confocal microscopy images showed an average branching angle of 39.03° 46.85° for the eight rare fungal genera, which was close to that of Fusarium. However, Colletotrichum's unique cluster-like manifestations and Pythium insidiosum's bead-like manifestations were different from those of Fusarium and Aspergillus's manifestations [24]. The positive results of histopathology also showed that most hyphae grew horizontally. However, the direction of hyphae growth should be judged by clinical manifestations, confocal microscopy, and pathological findings together, which can provide a reference for the choice of surgical methods in the future.
Polyene and imidazole are still the main antifungal therapies used in China. Natamycin is considered to be the first-line treatment of filamentous fungal keratitis , such as Fusarium spp and Aspergillus spp [11,25]. There was no zoospore in the culture of Pythium insidiosum in our study, and no results were obtained from drug susceptibility tests. The other seven genera were susceptible to amphotericin B, voriconazole, and itraconazole; among them, voriconazole seemed more effective against fungi, especially for dematiaceous species. It is impossible to test the susceptibility of natamycin due to a lack of test reagents.
Seven patients (43.75%) with Colletotrichum, Exserohilum, Bipolaris, Purpureocillium lilacinum, and Sarocladium keratitis were treated with keratectomy (one case of Sarocladium combined with conjunctival flap). The average healing time of the corneal epithelium was 5.17±0.79 days postoperatively, and there was no evidence of fungal recurrence which effectively shortened the course of the disease. This indicated that if the above genera are ineffective in drug treatment, surgery without delay could be beneficial to control illness. Most of the foreign reports of Purpureocillium lilacinum keratitis had a poor prognosis because the role of hydrolase progressed deeper and with more ease [26][27][28]. Todokoro et al. [29]reported that two patients were cured completely using only antifungal drugs. In our study, the two patients with Purpureocillium lilacinum keratitis underwent surgical treatment, and had a good clinical effect. This might be considered as being related to the combination of preoperative antifungal-sensitive drugs and the total eradication of lesions. At present, there are no effective treatment for Pythium insidiosum keratitis, antifungal therapy or surgery is still the main treatment method used in other countries, but afflicted patients face prolonged recovery often requiring multiple keratoplasty, and the fungal recurrence rate after surgery is high [24,30,31]. In our study, four cases of Pythium insidiosum keratitis were ineffective in antifungal therapy, and three cases (75%) showed fungal recurrence after corneal transplantation. For

Conclusions
In this study, the clinical manifestations, auxiliary examination and treatment outcomes of rare fungal genera in China are discussed. Analysis should be carried out in order to improve clinicians' understanding of these genera and to promote early diagnosis and improve target treatment. Whether it's common or rare fungal genera, the combination of early sensitive antifungal drugs and the necessary surgical treatments may still beneficial factors in treatment.However, the deficiency of this study is that there are few cases of rare genera, and more information is needed to study their clinical characteristics and treatment outcomes.

Acknowledgments
The author thank the imaging department and clinical laboratories of Shandong Eye Hospital for their help in collecting information of this manuscript.

Authors' contributions
XW is responsible for the acquisition of clinical data and the writing of manuscripts. XHL participates in the preparation of culture medium and identification of fungal pathogens.
SXL, TW and YNJ guided the data analysis. STW, JTW and CXD performed data collection.
WYS reviewed and revised the manuscript. All authors read and approved the final manuscript.

Funding
This work was partially supported by the National Natural Science Foundation of China

Availability of data and materials
The datasets used and/or analyzed to support the findings of this study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This study followed the tenets of the Helsinki Declaration on ethical principles for medical research involving human subjects and was approved by the ethics committee of Shandong Eye Hospital, Shandong Provincial Key Laboratory of Ophthalmology, China. Written informed consents were obtained from all subjects.

Consent for publication
Not applicable.
The authors declare that they have no competing interests.     The treatment process of three evisceration cases of Pythium insidiosum keratitis. A1,B1,C1: Slit lamp biomicroscopy photography. A2: Fungal recurrence was observed four days after LKP, and the whole corneal graft was infiltrated. A3: Fungal recurrence was observed two days after TKP, accompanied by hypopyon.
B2: Hypopyon occurred five days after TPK. B3: Hypopyon increased half a month after surgery. C2: Corneal graft, implant bed infiltration, and hypopyon were observed three days after TKP. C3: Whole corneal graft infiltration was observed three days after keratectomy.

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