Antifungal Efficacy of Luliconazole in an Experimental Rabbit Model of Fungal Keratitis Caused by Fusarium solani.

Fungal keratitis is a corneal fungal infection that potentially leads to blindness and is mainly caused by filamentous fungi, such as Fusarium, with limited drug options available, such as natamycin and voriconazole. Therefore, this study aimed to evaluate the therapeutic effects of the imidazole antifungal drug-luliconazole-using a rabbit experimental model of fungal keratitis caused by Fusarium solani, which is the dominant causative agent of fungal keratitis. F. solani was inoculated into rabbit corneas. luliconazole 1% suspension or natamycin 5% eye drops were administered four times a day (N = 6 for each group) 3 days after inoculation. Signs were scored up to 14 days after inoculation to evaluate the efficacy of the drugs. Compared with the peak mean sign scores of the placebo control group, there was a significant decrease in the mean sign scores of both the treatment groups (P < 0.05). Sign score trends were similar between the two treatment groups. In conclusion, luliconazole demonstrated therapeutic efficacy comparable to that of natamycin in treating experimental fungal keratitis. This suggests that luliconazole can be a novel therapeutic agent for human fungal keratitis.

Fungal Keratitis Caused by Talaromyces coalescens: A Case Report.

Fungal keratitis is a severe corneal infection, and the causative fungi include various rare fungal species. Fungal keratitis caused by Talaromyces species has yet to be reported, and there is no information about this fungus as a cause of keratitis. A 77-year-old man developed fungal keratitis while waiting for a donor cornea due to bullous keratopathy in his left eye. Fungal culture of a corneal scraping grew filamentous fungi, which were morphologically identified as Paecilomyces species. The corneal infection did not improve after topical administration of 1% voriconazole, and ribosomal DNA sequencing definitively verified the fungus to be Talaromyces coalescens. The lesion gradually improved after switching to topical 5% natamycin. Antifungal susceptibility tests determined the high minimum inhibitory concentrations of voriconazole to be > 8 µg/mL. This is the first report of Talaromyces fungal keratitis. Clinicians, especially those in ophthalmology, need to be aware of this rare fungus.

Successful Management of Fungal Keratitis by Alternaria alternata Complicating Mooren's Ulcer.

Mooren's ulcer is an idiopathic peripheral ulcerative keratitis whose pathogenesis is thought to be due to an autoimmune reaction. The first-line treatment for Mooren's ulcer is the use of topical steroids, which can be difficult to discontinue. The 76-year-old patient in this case was receiving topical steroids for bilateral Mooren's ulcer and developed a feathery corneal infiltration and perforation in the left eye. On suspicion of a fungal keratitis complication, we started topical voriconazole treatment and performed lamellar keratoplasty. Topical betamethasone was continued twice a day. The identified causative fungus was Alternaria alternata, which is known to be susceptible to voriconazole. The minimum inhibitory concentration of voriconazole was later proven to be 0.5 µg/mL. After 3 months of treatment, the residual feathery infiltration disappeared and the left vision recovered to 0.7. In this case, topical voriconazole was effective, and the eye was successfully treated with continuing topical steroids. Fungal species identification and antifungal susceptibility test proved helpful for symptom management.

Analysis of treatment protocols using azithromycin eye drops for bacterial blepharitis: second report-bacteriological investigation.

PURPOSE: To gain new insights into the etiology of blepharitis, we investigated the causative bacteria in patients with blepharitis and the effects of 1% azithromycin ophthalmic solution. STUDY DESIGN: A multicenter, prospective observational study. METHODS: After the subjects were diagnosed as having blepharitis they were administered 1% azithromycin ophthalmic solution for up to 14 days. Bacterial cultures and smear microscopic examinations of the eyelid margin were conducted at the initial visit, after administering eye drops, and 1 month after the end of eye drop administration. The minimum inhibitory concentrations (MICs) of azithromycin were measured. RESULTS: At the initial visit, the bacterial morphology determined by smear microscopic examinations coincided with that of strains isolated by culture taken from 22 of 45 patients. All detected bacteria were gram-positive, and Corynebacterium spp., Cutibacterium acnes, Staphylococcus epidermidis, Streptococcus spp., and Enterococcus faecalis were isolated most commonly. Compared with the initial visit the number of isolated strains per eye decreased significantly at 7 days after the start of eye drop administration and 1 month after the end of eye drop administration. The azithromycin MICs were temporarily increased after the start of eye drops but then decreased. CONCLUSION: Our study suggests that in blepharitis pathogenicity is characterized by increased strain numbers and amounts of indigenous bacteria. Administering a 1% azithromycin ophthalmic solution suppresses the number of bacterial strains within 1 month after the end of eye drop administration without increasing drug resistance.

A Rare Case of Fungal Keratitis Caused by Plectosphaerella cucumerina Diagnosed With Repeated Corneal Scrapings: A Case Report.

Plectosphaerella cucumerina is a filamentous fungus that infects plants and crops, but there are few previous reports of human infections. The current case was an 82-year-old woman who was referred to us for corneal infection in her left eye that did not improve with antibacterial and anti-inflammatory treatment. The best-corrected visual acuity (BCVA) of the eye at the first visit to us was hand motion. Slit-lamp examination revealed extensive white infiltration and ulceration in the anterior corneal stroma of the left eye. Intensive antibacterial and antiviral treatment for one month did not improve the condition of the cornea. Although initial culture testing and polymerase chain reaction (PCR) testing of corneal scraping samples did not reveal the causative microorganism, repeated culture testing identified P. cucumerina. The corneal infection eventually subsided after topical and systemic treatment with voriconazole (VRCZ). The final BCVA in the left eye was 1.3 logMAR. This was a rare case of fungal keratitis due to P. cucumerina. Our case suggests that it is important to perform repeated examinations with corneal scrapings, especially when the treatment response is poor.

Lacrimal Duct Obstruction and Infection Associated with Non-Traumatic Corneal Perforation: A Case Series.

Purpose: To report a case series of lacrimal duct obstruction and infection associated with non-traumatic corneal perforation. Case Series: This study included 6 eyes in 6 patients with non-traumatic corneal perforation treated between April 2019 and March 2021. All 6 cases were associated with lacrimal duct obstruction and infection. Purulent discharge caused by lacrimal duct infection was observed in all 6 patients (100%). However, three of the 6 patients (50%) did not show purulent discharge at initial examination and lacrimal duct obstruction was therefore not initially recognized. Dry eye was observed in five of the 6 patients (83%) and may have caused corneal deterioration, increasing susceptibility to perforation. Further, dry eye masks symptoms of lacrimal duct obstruction and infections, such as epiphora and regurgitation of purulent discharge, making the association with lacrimal duct obstruction and infection difficult to determine. All patients were treated for both corneal perforation and lacrimal duct disease, and conditions improved, with no recurrence of either corneal perforation or lacrimal duct disease. Conclusion: In patients with a combination of lacrimal duct disease and corneal perforation, treatment of both diseases resulted in stabilization of patient condition. Dry eyes may mask symptoms of lacrimal duct diseases, such as epiphora and purulent discharge, and lacrimal duct disease may thus be underdiagnosed.

Retinal ganglion cell analysis in patients with sellar and suprasellar tumors with sagittal bending of the optic nerve.

The study investigated clinical features of sellar and suprasellar tumors with optic nerve bending. Twenty-five patients (13 men/12 women; age, 59.0 ± 12.9 years) with optic nerve bending in one eye who underwent tumor resection for sellar and suprasellar tumors were included. The other eye, without optic nerve bending, was the control. The pre- and postoperative best-corrected visual acuity (BCVA) and ganglion cell layer (GCL) + inner plexiform layer (IPL) thickness were studied retrospectively using optical coherence tomography. Preoperative BCVA in the eye with optic nerve bending was significantly poor and improved significantly after tumor resection. Eyes with optic nerve bending had significantly less GCL + IPL thickness on the temporal side than eyes without optic nerve bending. Preoperative GCL + IPL thickness of the entire macula was reduced in eyes with optic nerve bending and poor postoperative BCVA compared to those with good postoperative BCVA. There was no significant difference in GCL + IPL thickness of eyes with optic nerve bending before and after tumor resection. Optic nerve bending caused by sellar and suprasellar tumors resulted in visual impairment and decreased retinal ganglion cells. Eyes with optic nerve bending and severely reduced GCL + IPL thickness may have less BCVA improvement after tumor resection.

Effective diagnosis by real-time PCR of herpes simplex diffuse endotheliitis that is similar in appearance to fungal keratitis: case series.

PURPOSE: Herpes simplex diffuse endotheliitis with accompanying feathery infiltration is difficult to diagnose due to corneal findings that are similar to fungal keratitis. This case series reports on the effectiveness of using real-time polymerase chain reaction (PCR) to diagnose herpes simplex diffuse endotheliitis that is similar in appearance to fungal keratitis. METHODS: After extracting corneal smear sample DNA, samples were then applied to two independent PCR assays, a qualitative multiplex 24-pathogen strip PCR assay, and a quantitative real-time PCR assay of herpes simplex virus type 1 (HSV-1). RESULTS: All 3 cases showed ciliary injection, feathery infiltration in the corneal stroma and hypopyon, which are corneal findings similar to that observed for fungal keratitis. Retrocorneal plaques, which showed clear boundaries between the corneal endothelial surfaces and retrocorneal plaques in anterior segment optical coherence tomography, were observed in 2 out of 3 cases. Corneal scraping was performed in all cases, followed by initiation of antifungal treatment. However, real-time PCR of the corneal scraping detected 6.0 × 106, 1.0 × 105 and 5.0 × 105 copies/µg glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of HSV-1 DNA per each microgram of the samples. Fungi were not cultured in any of the cases. After switching the medication from antifungal to antiviral, the feathery corneal infiltration was cured with only mild scarring. CONCLUSIONS: Real-time PCR was an effective tool in diagnosing HSV diffuse endotheliitis with feathery infiltration. Topical corticosteroids in conjunction with oral and topical antivirals were an effective treatment.

Intraocular penetration of liposomal amphotericin B after intravenous injection in inflamed human eyes.

PURPOSE: To determine the intraocular penetration of amphotericin B (AMPH-B) after an intravenously injection of liposomal amphotericin B (L-AMB) in inflamed human eyes. METHODS: Seven eyes of 5 patients with fungal eye diseases (endophthalmitis in 6 eyes and keratitis in 1 eye) were treated with intravenous injections of 100-250 mg/day of L-AMB. Samples of blood, corneal button, aqueous humor, and vitreous humor were collected and assessed for AMPH-B. RESULTS: The AMPH-B level in the cornea (604.0 µg/g) of the case with fungal keratitis exceeded the minimum inhibitory concentration. However, the levels in the aqueous and vitreous humors of the cases with fungal endophthalmitis were lower, e.g., 0.02 ± 0.01 µg/ml (0.09% of serum level) in the aqueous humor and 0.05 ± 0.08 µg/ml (0.17% of serum level) in the vitreous humor. CONCLUSIONS: The AMPH-B levels administered intravenously were very low in the aqueous and vitreous humors. Our findings indicate that intravenous L-AMB can be considered only for patients with mild endogenous fungal endophthalmitis, e.g., isolated chorioretinitis without vitreous extensions.

In Vitro and In Vivo Evaluation of Three Newly Isolated Bacteriophage Candidates, phiEF7H, phiEF14H1, phiEF19G, for Treatment of Enterococcus faecalis Endophthalmitis.

Post-operative endophthalmitis caused by Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Therefore, novel alternative treatments that are effective against enterococcal endophthalmitis are required. Bacteriophage therapy has the potential to be an optional therapy for infectious diseases. Therefore, we investigated the therapeutic potential of three newly isolated enterococcal phages, phiEF7H, phiEF14H1, and phiEF19G, in E. faecalis-induced endophthalmitis. These phages could lyse the broad-range E. faecalis, including strains derived from endophthalmitis and vancomycin-resistant E. faecalis in vitro, as determined by the streak test. Morphological and genomic analyses revealed that these phages were classified into the Herelleviridae genus Kochikohdavirus. The whole genomes of these phages contained 143,399, 143,280, and 143,400 bp, respectively. Endophthalmitis was induced in mice by injection of three strains of E. faecalis derived from post-operative endophthalmitis or vancomycin-resistant strains into the vitreous body. The number of viable bacteria and infiltration of neutrophils in the eye were both decreased by intravitreous injection of phiEF7H, phiEF14H1, and phiEF19G 6 h after injection of all E. faecalis strains. Thus, these results suggest that these newly isolated phages may serve as promising candidates for phage therapy against endophthalmitis.

Exophiala lecanii-corni keratitis presenting as a serpiginous pigmented superficial lesion: a case report.

RATIONALE: Patients with bullous keratopathy (BK) treated by Descemet stripping automated endothelial keratoplasty (DSAEK) have a compromised cornea, due to the administration of topical steroid, postsurgical use of contact lenses, and impaired barrier function of the corneal epithelium by BK. We report a case of Exophiala lecanii-corni (E lecanii-corni) keratitis presenting as a serpiginous pigmented superficial lesion after DSAEK. PATIENT CONCERNS: An 81-year-old woman who had undergone cataract surgeries, suffered from decreased vision in the left eye. She was diagnosed BK and she underwent DSAEK. Two months after DSAEK, a pigmented superficial lesion developed on the left cornea. The lesion migrated and recurred repeatedly and she was referred to our department. Best corrected vision was 20/220. DIAGNOSES: Light microscopy of a corneal scraping revealed branching fungal hyphae. Fungal culture showed growth of a black colony, identified as E lecanii-corni by ribosomal DNA sequencing. INTERVENTIONS: We started topical treatment with 1% voriconazole and 1.5% levofloxacin. Antifungal susceptibility testing showed that the minimum inhibitory concentration of voriconazole was 0.06 µg/mL. OUTCOMES: The lesion scarred after treatment for 3 months, and left best corrected vision improved to 20/40. LESSONS: Genus Exophiala is known as 1 of the "black molds" and a cause of chromomycosis. This is the first description of E lecanii-corni keratitis, and pigmented corneal epithelial lesions may be characteristic of this fungal genus.

Corynebacterium Species of the Conjunctiva and Nose: Dominant Species and Species-Related Differences of Antibiotic Susceptibility Profiles.

PURPOSE: Nondiphtherial Corynebacterium species are normal residents of human skin and mucosa, including the conjunctiva and nose, but can cause conjunctivitis and keratitis. Recently, resistance against various classes of antibiotics has been reported in Corynebacterium. The present study investigated the type of species and antibiotic susceptibilities of the conjunctival and nasal Corynebacterium species. METHODS: This study examined 183 strains of Corynebacterium species that were isolated from patients undergoing preoperative examinations for cataract surgery. Species were identified by RNA polymerase ß-subunit-encoding gene (rpoB) sequencing. Antibiotic susceptibility tests were performed by the microdilution method according to the Clinical and Laboratory Standards Institute standard method M45. RESULTS: Corynebacterium macginleyi was the most predominant species (84%; 46 of 55) in the conjunctiva. The 2 major species in the nasal cavity were Corynebacterium accolens and Corynebacterium propinquum (44% and 31%, respectively), followed by Corynebacterium pseudodiphtheriticum (8%), Corynebacterium jeikeium (7%), and C. macginleyi (3%). In contrast to other nasal Corynebacterium species, only C. macginleyi showed a high susceptibility to macrolides. However, among nonconjunctival Corynebacterium species, C. propinquum, was unique in having a high resistance rate to levofloxacin (29%), comparable with that observed in C. macginleyi (36%). Penicillin G and tobramycin showed good susceptibility in almost all strains. CONCLUSIONS: Drug resistance against fluoroquinolones and macrolides was observed in Corynebacterium species, with the antibiotic susceptibility profiles correlating with differences of the species and niche. Nasal and conjunctival Corynebacterium profiles of drug resistance suggest habitat segregation strictly at the species level.

Therapeutic Effects of Intravitreously Administered Bacteriophage in a Mouse Model of Endophthalmitis Caused by Vancomycin-Sensitive or -Resistant Enterococcus faecalis.

Endophthalmitis due to infection with Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Given that the frequency of this condition caused by vancomycin-resistant Enterococcus faecalis has been increasing, the development of novel therapeutics is urgently required. We have demonstrated the therapeutic potential of bacteriophage ΦEF24C-P2 in a mouse model of endophthalmitis caused by vancomycin-sensitive (EF24) or vancomycin-resistant (VRE2) strains of E. faecalis Phage ΦEF24C-P2 induced rapid and pronounced bacterial lysis in turbidity reduction assays with EF24, VRE2, and clinical isolates derived from patients with E. faecalis-related postoperative endophthalmitis. Endophthalmitis was induced in mice by injection of EF24 or VRE2 (1 × 104 cells) into the vitreous. The number of viable bacteria in the eye increased to >1 × 107 CFU, and neutrophil infiltration into the eye was detected as an increase in myeloperoxidase activity at 24 h after infection. A clinical score based on loss of visibility of the fundus as well as the number of viable bacteria and the level of myeloperoxidase activity in the eye were all significantly decreased by intravitreous injection of ΦEF24C-P2 6 h after injection of EF24 or VRE2. Whereas histopathologic analysis revealed massive infiltration of inflammatory cells and retinal detachment in vehicle-treated eyes, the number of these cells was greatly reduced and retinal structural integrity was preserved in phage-treated eyes. Our results thus suggest that intravitreous phage therapy is a potential treatment for endophthalmitis caused by vancomycin-sensitive or -resistant strains of E. faecalis.

Bilateral Necrotizing Herpes Simplex Keratitis in an Immunocompetent Patient With Genetic Analysis of Herpes Simplex Virus 1.

PURPOSE: To report a case of severe bilateral necrotizing herpes simplex keratitis (HSK) in an immunocompetent patient, with genotyping of the underlying herpes simplex virus 1 (HSV-1). METHODS: Genetic analyses of HSV-1 in tear samples were performed with polymerase chain reaction-based restriction fragment length polymorphism, targeting the viral genes unique short (US)2, US4 (glycoprotein G), and US7 (glycoprotein I). RESULTS: A 64-year-old woman with no history of atopy or immune disorders manifested bilateral keratitis with geographic ulcer. Her initial visual acuity was 20/1000 (OD) and 20/20 (OS). Polymerase chain reaction testing of a tear sample revealed the presence of HSV-1 in both eyes, and the patient was diagnosed with bilateral HSK. Both eyes progressed to necrotizing keratitis during the treatment course. Continuous intensive treatment, at first with acyclovir ointment and oral valacyclovir and later with steroid eye drops for stromal keratitis, finally improved the patient's condition. However, after 2 years, her visual acuity was limited to 20/250 (OD) and 20/60 (OS) because of corneal opacity from scarring. We found that the strain in the current case had a genotype combination of C/A/B (for US2/US4/US7), a known pattern in Japan, in both eyes. CONCLUSIONS: We successfully performed an unprecedented genetic analysis of an HSV-1 strain isolated from a case of bilateral necrotizing HSK in an immunocompetent patient. The association of the HSV-1 genotype with the clinical manifestation remains unclear, calling for more data from new cases, especially from different geographic regions.

Acute retinal necrosis following herpes simplex encephalitis: a nationwide survey in Japan.

PURPOSE: Acute retinal necrosis (ARN) is a severe necrotizing retinitis caused by varicella-zoster virus or herpes simplex virus (HSV) that often results in blindness. Occasionally, HSV-caused ARN develops after herpes simplex encephalitis (HSE). It remains unknown, however, when and how often ARN develops after HSE. To investigate the actual conditions of patients with ARN following HSE and the interval period between the prior HSE and the onset of ARN, a retrospective nationwide survey of the Japanese population was performed. STUDY DESIGN: Retrospective. METHODS: Questionnaires were sent out to the neurology and ophthalmology departments of teaching hospitals in Japan. They inquired about HSE patients in neurology departments and ARN patients diagnosed with HSV in ophthalmology departments. The proportion of the HSV-ARN patients with a history of HSE and their interval periods were obtained from the questionnaires returned by the ophthalmology departments. RESULTS: Neurology departments of 324 hospitals responded to the questionnaires (response proportion: 40.9%), and 53 HSE cases were reported. Ophthalmology departments of 535 hospitals responded the questionnaires (response proportion: 54.3%), and 67 HSV-ARN cases were reported. Among the 67 HSV-ARN cases, 16 (23.9%) had histories of prior HSE. Although the interval periods from the prior HSE to the onset of HSV-ARN varied among cases, nearly half developed HSV-ARN within 2 years. CONCLUSIONS: This nationwide survey of the Japanese population showed that HSV-ARN develops after HSE in higher frequency within 2 years. Neurologists and ophthalmologists should be aware that HSE survivors have a risk of contracting HSV-ARN.

Fungal keratitis caused by Didymella gardeniae (formerly Phoma gardeniae) successfully treated with topical voriconazole and miconazole.

Fungal keratitis by Phoma species is rare, and little information has been accumulated. We report a case of keratitis caused by Didymella gardeniae, formerly known as P. gardeniae. The patient had a history of stromal herpetic keratitis and had been treated with long-term topical betamethasone. He developed infectious keratitis in the left eye, with the causative fungus identified by ribosomal DNA sequencing. Combination treatment with topical voriconazole and miconazole proved effective.

Efficacy of Luliconazole Against Broad-Range Filamentous Fungi Including Fusarium solani Species Complex Causing Fungal Keratitis.

PURPOSE: Fungal keratitis can be difficult to medically treat. Topical antifungals are usually applied empirically as the initial option in treating fungal keratitis. Natamycin (NAT) and/or voriconazole (VRCZ) have been widely used in the treatment of fungal keratitis. However, Fusarium solani species complex (FSSC), which are the dominant species of fungal keratitis, are resistant to VRCZ. This study investigated in vitro efficacy of luliconazole (LLCZ), a new imidazole antifungal, against FSSC and other filamentous fungi. METHODS: A total of 18 Fusarium isolates and 7 others were grown on potato dextrose agar at 30 and 37°C. For Fusarium, species identification and phylogenetic tree analysis were performed based on elongation factor-1α (EF-1α) DNA sequencing. The broth microdilution method was used for antifungal susceptibility testing of 11 antifungal drugs including LLCZ. RESULTS: The 18 identified Fusarium isolates belonged to FSSC (n = 13), Fusarium oxysporum species complex (FOSC; n = 2), Fusarium chlamydosporum species complex (FCSC; n = 1), Fusarium incarnatum-equiseti species complex (FIESC; n = 1), and Fusarium fujikuroi species complex (FFSC; n = 1). We further divided 13 FSSC isolates into 3 clades, FSSC5 (n = 8), FSSC3 + 4 (n = 4), and FSSC9-a (n = 1), with 8 FSSC strains growing at 37°C. LLCZ showed lowest minimum inhibitory concentrations (MICs) against all tested filamentous fungi, with a MIC90 against the Fusarium species of 0.06 µg/mL, whereas MIC90 for NAT and VRCZ were 4 and 8 µg/mL, respectively. CONCLUSIONS: LLCZ has the strongest in vitro antifungal activity among all drugs used against broad-range filamentous fungi including FSSC. LLCZ may potentially be a new medical treatment option for fungal keratitis.

Genetic diversity and persistent colonization of Enterococcus faecalis on ocular surfaces.

PURPOSE: Enterococcus faecalis causes severe acute endophthalmitis and often leads to poor visual outcomes. Conjunctival bacterial cultures occasionally grow atypical bacteria including E. faecalis, which can potentially contribute to the development of postoperative endophthalmitis. However, the characteristics of these ocular E. faecalis strains are unknown. This study is the first attempt to determine the population characteristics of E. faecalis clinical isolates from eye infections and ocular commensals. STUDY DESIGN: Retrospective METHODS: Twenty-eight E. faecalis ocular isolates were collected from 23 patients at 3 referring hospitals. The multilocus sequence typing (MLST) data were analyzed using the eBURST program. Phenotypes of cytolysin and gelatinase, antibiotic susceptibility, and mutations of the quinolone resistance-determining regions (QRDRs) of gyrA and parC were also examined. Pulsed-field gel electrophoresis (PFGE) was performed for strains from the same patients. RESULTS: PFGE revealed that 3 patients retained identical strains for 10 months to 2 and a half years. MLST identified 12 sequence types (STs), which were clustered into 3 clonal complexes (CCs) and 8 singletons, with ST179 the largest. Thirteen of the 23 isolates (56.5%) belonged to CC58, CC8, or CC2, which have previously been reported to be major CCs. Six of the 23 strains (26.0%) exhibited high-level quinolone resistance derived from mutations of the QRDRs in both gyrA and parC. CONCLUSIONS: The sequence types of E. faecalis ocular isolates were divergent, with no eye-specific lineages observed. Persistent colonization of E. faecalis on the ocular surface was demonstrated in patients with chronic ocular surface diseases.