Donor related corneal graft infection: a review of literature and preventive strategies.

PURPOSE: Donor-related infections are a serious threat to patient safety after corneal transplantation. We provide a concise review of literature from the last decade on donor-related graft infections, sources of contamination and means to reduce the contamination of donor tissue and preservation media. METHODS: We reviewed 50 papers from year 2005 to 2021 related to donor-related graft infections. We included 14 studies related to the risk factors associated with post-keratoplasty infection and preventive methods. RESULTS: Incidence of post-keratoplasty infections has been reported to be approximately 0.2%-0.77% for endophthalmitis and 6.5%-10.5% for microbial keratitis. We analyzed six important studies regarding the risk factors related to donor contamination. It was observed that younger donor age, increased death to retrieval time, warming cycles and increased eye bank processing time and positive corneo-scleral rim cultures were important risk factors for donor-related infections post keratoplasty. Eye banks have adapted newer protocols over the time period for prevention of donor-related contamination. Recommended preventive strategies were published in about eight important studies over the past decade. In addition to meticulous donor screening, rapid warming cycles, double contact with povidone iodine during retrieval and addition of antifungals like amphotericin B, Voriconazole and cycloheximide have been suggested over the last decade although their use is still in debate with regard to the efficacy, toxicity and cost-effectiveness. CONCLUSION: The last decade has witnessed a relative rise of fungal infections and multidrug resistant bacterial infections post-keratoplasty. Eye bank prepared corneas for lamellar surgeries are at increased risk for donor contamination due to increased exposure to the higher temperatures during their processing. Addition of antifungals and broad spectrum antibiotics to the hypothermic preservation media needs to be considered in the new era of increasing trends of lamellar keratoplasty.

Resolvin D1 protects against Aspergillus fumigatus keratitis in diabetes by blocking the MAPK-NF-κB pathway.

Fungal keratitis (FK) is one of the main causes of blindness in China. People with diabetes are susceptible to corneal epithelial disease, even fungal keratitis. At present, there are few studies on this disease. Resolvins (Rv) has been reported as a mediators that exert crucial anti-inflammatory and immune regulation roles in serval diseases. In order to investigate the roles and underlying mechanism of Resolvins D1 (RvD1) on the Aspergillus fumigatus (A. fumigatus) keratitis in diabetes, we established in vivo and in vitro models of A. fumigatus keratitis, which were then exposed to high glucose. The expression levels of RvD1, 5-lipoxygenase (5-LOX), and 15-lipoxygenase (15-LOX) in A. fumigatus keratitis patients with diabetes were determined through Enzyme Linked Immunosorbent Assay (ELISA), Western blot and immunohistochemistry. Reactive Oxygen Species (ROS) production, ELISA, flow cytometry, Hematoxylin-Eosin (HE) staining and fungal loading determination were conducted to evaluate the severity of A. fumigatus infection. Lymphangiogenesis and angiogenesis were examined by immunofluorescence assay. Western blot was applied to detect the proteins of the MAPK-NF-κB pathway. The results showed that RvD1 diminished the high glucose-induced oxidative stress and inflammatory response, as evidenced by the reduction of ROS production, Interleukin-6 (IL-6), Interleukin-8 (IL-8), Heme Oxygenase-1 (HMOX-1), and the elevation of Cyclooxygenase-2 (COX2), Superoxide Dismutase (SOD-1), and Glutathione Peroxidase-2 (GPX2) levels in A. fumigatus-infected Human Corneal Endothelial Cells (HCECs). Additionally, lymphangiogenesis and angiogenesis prominently decreased after intervention with RvD1. Furthermore, RvD1 significantly reduced the levels of p-MEK1/2 and p-ERK1/2, and restrained the NF-κB and GPR32 activation. The above results showed that RvD1 protects against A. fumigatus keratitis in diabetes by suppressing oxidative stress, inflammatory response, fungal growth, and immunoreaction via modulating MAPK-NF-κB pathway. RvD1 provides clues for the therapeutic targets of Fungal keratitis complicated with diabetes.

Inhibiting miR-129-5p alleviates inflammation and modulates autophagy by targeting ATG14 in fungal keratitis.

To investigate the role of miR-129-5p in inflammation and autophagy in fungal keratitis, we established a keratitis mouse model infected with Fusarium solani (F. solani) and conducted experiments on corneal stromal cells infected with F. solani. The expression of miR-129-5p was detected via quantitative real-time polymerase chain reaction (PCR). The miR-129-5p antagomir was used to transfect cells and mice to study the regulatory role of miR-129-5p in autophagy and inflammation after fungal infection. The expression of Beclin1 and LC3B and colocalization of LC3B with lysosomes were detected via Western blotting and immunofluorescence. CCK-8 was used to determine the viability of corneal stromal cells. The expression of IL-1ß were detected by ELISA. Bioinformatics software was used to predict the potential targets of miR-129-5p, which were verified by a luciferase reporter gene assay. RT-PCR showed that miR-129-5p expression in mouse corneas was significantly increased after infection with F. solani. Subconjunctival injection of the miR-129-5p antagomir significantly enhanced the proteins Beclin-1 and LC3B. At the same time, inhibiting miR-129-5p expression could reduce the inflammatory response in FK and significantly increase the viability of corneal stromal cells infected with F. solan. Moreover, the dual luciferase reporter assay indicated that Atg14 was a direct target of miR-129-5p. Our study shows that miR-129-5p is a novel small molecule that regulates autophagy by targeting Atg14, indicating that it may be a proinflammatory and therapeutic target for fungal keratitis.

The Role of Corneal Donor Rim Fungal Cultures: Pro Side.

ABSTRACT: Fungal infection after corneal transplantation is a rare but potentially devastating complication. It is of paramount concern for transplant surgeons and the eye banking community. The value of universal corneal rim cultures for keratoplasty remains controversial. In 2016, The Eye Bank Association for America reported an increasing trend in the incidence of post keratoplasty fungal infections and a higher incidence of post keratoplasty [penetrating keratoplasty and endothelial keratoplasty (EK)] fungal endophthalmitis cases. This increasing trend in rate over time from previous Eye Bank Association for America reports was disproportionately associated with EK and Candida species. Additionally, several studies confirmed a high correlation between positive corneoscleral donor rim fungal cultures and postoperative infections, and a higher risk to the mate eye of a cornea that had the positive fungal corneal rim culture and developed an infection. Positive fungal donor rim cultures-especially in the setting of interface keratitis after EK surgery-can raise the index of suspicion for a fungal cause and may help direct therapy, especially in the early stages, where the symptoms and signs of spread may not be obvious and obtaining direct cultures is inherently difficult without surgical intervention.

Role of IL-36γ/IL-36R Signaling in Corneal Innate Defense Against Candida albicans Keratitis.

Purpose: Interleukin (IL)-36 cytokines have been shown to play either beneficial or detrimental roles in the infection of mucosal tissues in a pathogen-dependent manner, but their involvement in fungal keratitis remains elusive. We herein investigated their expression and function in mediating corneal innate immunity against Candida albicans infection. Methods: Gene expression in mouse corneas with or without C. albicans infection was determined by regular RT- and real-time (q)-PCR, Western blot analysis, ELISA or proteome profile assay. The severity of C. albicans keratitis was assessed using clinical scoring, bacterial counting, and myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration. IL36R knockout mice and IL-33-specific siRNA were used to assess the involvement IL-33 signaling in C. albicans-infected corneas. B6 CD11c-DTR mice and clodronate liposomes were used to define the involvement of dendritic cells (DCs) and macrophages in IL-36R signaling and C. albicans keratitis, respectively. Results: IL-36γ were up-regulated in C57BL6 mouse corneas in response to C. albicans infection. IL-36 receptor-deficient mice display increased severity of keratitis, with a higher fungal load, MPO, and IL-1ß levels, and lower soluble sIL-1Ra and calprotectin levels. Exogenous IL-36γ prevented fungal keratitis pathogenesis with lower fungal load and MPO activity, higher expression of sIL-1Ra and calprotectin, and lower expression of IL-1ß, at mRNA or protein levels. Protein array analysis revealed that the expression of IL-33 and REG3G were related to IL-36/IL36R signaling, and siRNA downregulation of IL-33 increased the severity of C. albicans keratitis. Depletion of dendritic cells or macrophages resulted in severe C. albicans keratitis and yet exhibited minimal effects on exogenous IL-36γ-induced protection against C. albicans infection in B6 mouse corneas. Conclusions: IL-36/IL36R signaling plays a protective role in fungal keratitis by promoting AMP expression and by suppressing fungal infection-induced expression of proinflammatory cytokines in a dendritic cell- and macrophage-independent manner.

Inhibition of miR-665-3p Enhances Autophagy and Alleviates Inflammation in Fusarium solani-Induced Keratitis.

Purpose: Accumulated evidence has shown that microRNAs (miRNAs) are closely related with the regulation of autophagy, which plays vital roles in fungal keratitis (FK). Microarray data showed elevated expression of miR-665-3p in mouse corneal tissues after infection with Fusarium solani (F. solani). Here, we investigated the effect of miR-665-3p in regulating autophagy in experimental F. solani keratitis and determined the potential molecular mechanisms involved. Methods: In this article, we established an in vivo mouse model of FK and an in vitro model of corneal stromal cells by inoculating with F. solani. We divided them into the following six groups: control, chloroquine (CQ), rapamycin (Rapa), miR-665-3p antagomir (ant-665), miR-665-3p agomir (miR-665), and the negative control group (miR-NC). The levels of autophagy were detected by electron microscopy, Western blotting, and immunofluorescence. Then, we used a dual-luciferase reporter assay to determine the binding of miR-665-3p to the autophagy-related gene (ATG)5 3'UTR. Detection of IL-1ß protein levels and hematoxylin and eosin (H&E) staining of corneal tissues were used to observe the effect of miR-665-3p on inflammation in FK. Results: Here, we showed that inhibition of miR-665-3p expression in FK upregulated autophagy and alleviated inflammation. Nevertheless, the opposite results were found by overexpressing miR-665-3p. Additionally, ATG5 was a direct target gene for miR-665-3p. Conclusions: Together, our data demonstrated that miR-665-3p might be involved in F. solani keratitis of mice by regulating autophagic pathways and inflammation.

The Role of Donor Rim Fungal Cultures.

ABSTRACT: Culturing all donor rims for fungus makes no sense. Only 1% of all cultures will be positive, and of those positive cultures, only 6% will also have a clinical infection. Prophylactically treating all positive cultures means 94% of patients will be treated unnecessarily. Fungal cultures do not reliably direct specific medication choice, and fungal infections of the interface in endothelial keratoplasty and deep anterior lamellar keratoplasty are nearly impervious to medical therapy. Suspected fungal infections of the deep stromal interface should be treated expeditiously with penetrating keratoplasty before peripheral spread or endophthalmitis occur.

The Search for Antifungal Prophylaxis After Artificial Corneal Surgery-An In Vitro Study.

PURPOSE: To evaluate the antifungal properties of topical antibiotics (already being used successfully to prevent bacterial endophthalmitis) and some promising antiseptics for antifungal prophylaxis in the setting of artificial corneal implantation. METHODS: Several commonly used antibiotics for antimicrobial prophylaxis after artificial corneal implantation, in addition to antiseptics [benzalkonium chloride (BAK), povidone-iodine (PI), and some ionic liquids (ILs)], were tested in vitro against Candida albicans, Fusarium solani, and Aspergillus fumigatus. The time-kill activity was determined. Toxicity was assayed in vitro on human corneal epithelial cultures using trypan blue. Adhesion and tissue invasion experiments were also carried out on porcine corneas and commonly used contact lenses, with or without gamma irradiation, and by analysis with fluorescence microscopy. RESULTS: Polymyxin B (PMB)/trimethoprim/BAK (Polytrim), PMB alone, gatifloxacin with BAK (Zymaxid), and same-concentration BAK alone exhibited antifungal activity in vitro. Moxifloxacin (MOX) or gatifloxacin without BAK-as well as trimethoprim, vancomycin, and chloramphenicol-had no effect. 1% PI and ILs had the highest efficacy/toxicity ratios (>1), and Polytrim was species dependent. Subfungicidal concentrations of Polytrim reduced adhesion of C. albicans to Kontur contact lenses. Gamma-irradiated corneas showed enhanced resistance to fungal invasion. CONCLUSIONS: Of antibiotic preparations already in use for bacterial prophylaxis after KPro surgery, Polytrim is a commonly used antibiotic with antifungal effects mediated by both PMB and BAK and may be sufficient for prophylaxis. PI as a 1% solution seems to be promising as a long-term antifungal agent. Choline-undecanoate IL is effective and virtually nontoxic and warrants further development.

Cost-Effectiveness of Antifungal Supplementation of Corneal Cold Storage Media.

PURPOSE: To evaluate the cost-effectiveness of supplementing hypothermic cold storage media (CSM) with antifungal therapy. DESIGN: Cost-effectiveness analysis (CEA). PARTICIPANT: Base case of a patient with Fuch's endothelial dystrophy undergoing a first eye keratoplasty. METHODS: Cost-effective analysis of the base case with corneal tissue stored in CSM or CSM supplemented with antifungal therapy over a 16-year time horizon. Multiple clinical scenarios were considered, including endothelial keratoplasty (EK) and penetrating keratoplasty (PK); amphotericin B, voriconazole, caspofungin, and combination therapy; and third-party payer and societal perspectives. The incidences were derived from PubMed literature searches and average wholesale prices of medications; all costs were discounted 3% per annum and adjusted for inflation to 2019 US dollars. MAIN OUTCOME MEASURES: Incremental cost-effectiveness ratios (ICERs). RESULTS: In the reference case, a corneal endothelial graft stored in amphotericin B-supplemented CSM was the most cost-effective approach from a third-party payer and societal perspective. Probability sensitivity analysis (PSA) of the societal model for the EK was robust, with 93.5% being below an arbitrary willingness-to-pay threshold (WTP) of $20 000 per fungal infection averted. Voriconazole, caspofungin, and combination antifungals were less cost-effective than amphotericin B. The main factors influencing the CEA were the incidences of postkeratoplasty fungal infections, potential increases in graft failures, and antifungal costs. For grafts intended for PKs, antifungal supplementation was less cost-effective than for EKs. CONCLUSIONS: Antifungal supplementation with amphotericin B for EK grafts was the most cost-effective approach of the studied antifungals; however, the CEA was sensitive to potential changes in graft failure rates, underlining the importance of long-term safety studies. For full-thickness corneal grafts, antifungal supplementation was less cost-effective.

Efficacy and Safety of Various Amphotericin B Concentrations on Candida albicans in Cold Storage Conditions.

PURPOSE: To determine the concentration of amphotericin B that would be both effective against Candida albicans contamination and safe for corneal endothelial cells (CECs) in cold storage conditions. METHODS: Triplicate media cultures were inoculated with 10 colony-forming units (CFUs)/mL of C. albicans (American Type Culture Collection 10231), supplemented with amphotericin B (0-20 µg/mL), stored in cold conditions (2°C-8°C) for 72 hours, and analyzed quantitatively for CFUs. C. albicans concentration in each sample was determined initially and after 6, 24, 48, and 72 hours of storage. CEC mitochondrial function (oxygen consumption rate), apoptosis, and necrosis were examined in donor corneas after 7 days of amphotericin B exposure and compared with untreated controls. CEC viability was also examined by calcein-AM staining and Fiji segmentation after 72 hours or 2 weeks of amphotericin B exposure to mimic potential eye bank practices. RESULTS: Amphotericin B concentrations of 1.25, 2.5, and 5.0 µg/mL resulted in 0.47, 1.11, and 1.21 log10 CFU reduction after only 6 hours of cold storage and continued to decrease to 3.50, 3.86, and 4.49 log10 reductions after 72 hours, respectively. By contrast, amphotericin B 0.255 µg/mL showed only 1.01 log10 CFU reduction after 72 hours of incubation. CEC mitochondrial function and viability did not differ in donor corneas exposed to amphotericin B ≤2.59 µg/mL compared with the controls. CONCLUSIONS: Optimal efficacy of amphotericin B against C. albicans is achieved in cold storage conditions at concentrations ≥1.25 µg/mL, and 2.5 µg/mL reduces Candida contamination by >90% after 6 hours of cold storage without sacrificing CEC health.

Fungal infection after keratoplasty and the role of antifungal supplementation to storage solution: a review.

Fungal infection after corneal transplantation is a rare, yet potentially devastating, postoperative complication and has become a growing concern for the transplant surgeon and eye banking community. The Eye Bank Association of America (EBAA) has reported an increasing trend in the rate of postkeratoplasty fungal infections and a reversal in the previously documented predominance of bacterial over fungal infections. Additionally, several studies have confirmed a high correlation between positive corneoscleral donor rim fungal cultures and postoperative infections. Optisol GS (Bausch & Lomb, Irvine, California, USA), the most extensively used corneal storage solution in US eye banks, does not currently contain any antifungal supplementation. Although large randomised control trials evaluating the efficacy and safety of routine antifungal supplementation to corneal storage solution are lacking, several investigative studies have assessed the role of antifungal agents in reducing fungal contamination of donor corneas without causing undue corneal toxicity. This review will present the current epidemiology of postkeratoplasty fungal infections and evidence for obtaining routine fungal rim cultures and antifungal supplementation of storage solution.

Fungal keratitis: Pathogenesis, diagnosis and prevention.

As a kind of serious, potentially sight-threatening corneal infections with poor prognosis, fungal keratitis can bring a heavy economic burden to patients and seriously affect the quality of life, especially those in developing countries where fungal keratitis is more prevalent. Typical clinical features include immune rings, satellite lesions, pseudopods, hypha moss, hypopyon and endothelial plaques. The ideal therapeutic effects could not be achieved by current treatments for many reasons. Therefore, under the current status, understanding the pathogenesis, early diagnosis and prevention strategies might be of great importance. Here, in this review, we discuss the recent progresses that may advance our understanding of pathogenesis, early diagnosis and prevention of fungal keratitis.

Increasing Povidone-Iodine Exposure in Endothelial Keratoplasty Tissue Processing and Fungal Infection Impact.

PURPOSE: To evaluate the effect on donor rim cultures and postoperative infections of doubling the povidone-iodine exposure time during corneal tissue recovery before its use in keratoplasty. METHODS: Consecutive donor cornea recoveries were evaluated for positive donor corneal rim cultures and postoperative infections before and after a protocol change of doubling the exposure time of povidone-iodine during donor preparation. RESULTS: In 631 consecutive cornea donor recoveries, 18 (2.9%) had positive fungal rim cultures and 41 (6.5%) had positive bacterial rim cultures. Three (0.48%) developed postoperative fungal infections, and no bacterial infections occurred. After doubling the povidone-iodine exposure time during the recovery process, 725 consecutive corneas were reviewed. Four (0.6%) had positive fungal rim cultures, and 29 (4.0%) had positive bacterial rim cultures. No postoperative fungal or bacterial infections occurred. No noticeable increase in epithelial toxicity developed between the 2 groups. CONCLUSIONS: Increasing the povidone-iodine exposure time during the donor cornea recovery process decreased the rate of positive donor corneal rim fungal cultures (P = 0.001), positive donor corneal rim bacterial cultures (P = 0.04), and postoperative fungal infections (P = 0.06).

Effect of Optisol Supplementation With 0.255 µg/mL Amphotericin B on Elimination of Yeast at 5°C.

PURPOSE: Fungal infections in lamellar keratoplasty are a growing concern. Optisol-GS does not contain an antifungal agent and supplementation with 0.255 µg/mL Amphotericin B (AmpB) has been considered. This study tested the ability of 0.255 µg/mL AmpB in Optisol-GS to eliminate yeast contamination of corneal tissue. METHODS: Three isolates of Candida albicans, 1 of Candida parapsilosis, and 1 of Candida glabrata were tested in Optisol with and without AmpB. Corneoscleral rims stored at -80°C were thawed and placed in 10 multiwell plates (4 per plate). The rims were inoculated with 4 respective loads of yeast: 0, 10, 10, and 10 colony-forming units in 2 sets of 5 for 5 yeasts. One set was filled with Optisol plus AmpB and the other with Optisol only. All 10 plates were incubated at cold storage (2°C-8°C) for 48 hours. After 48 hours, all corneal rims were placed into 10 mL of yeast extract peptone dextrose medium; a swab culture of each well was plated onto Sabouraud plates; and all plates with the remaining Optisol were incubated at 30°C. Yeast growth was monitored for 10 days. Minimum inhibitory concentration and minimum fungicidal concentration were determined. RESULTS: All corneoscleral specimens were positive regardless of fungal load or presence of AmpB. All controls remained negative. Minimum inhibitory concentrations and minimum fungicidal concentrations were equivalent and ranged between 0.5 and 2.0 µg/mL. CONCLUSIONS: AmpB at a concentration of 0.255 µg/mL in Optisol-GS at cold storage (2°C-8°C) over 48 hours did not eliminate yeast from corneal tissue.

Phomopsidione nanoparticles coated contact lenses reduce microbial keratitis causing pathogens.

Microbial keratitis is the infection caused by pathogenic microorganisms that commonly occurs among the contact lens users. Various antimicrobial compounds were coated on contact lenses to kill keratitis causing microorganisms, however these compounds caused several adverse side effects. Hence, the aim of this study is to develop a silicone hydrogel contact lens coated with phomopsidione nanoparticle that inhibit keratitis causing clinical isolates. Phomopsidione nanoparticles were synthesized using polyvinyl alcohol as encapsulant. The nanoparticles showed an average size of 77.45 nm, with neutral surface charge. Two drug release patterns were observed in the drug release profile, which are the initial slow release phase with extended drug release (release rate 46.65 µg/h), and the burst release phase observed on Day 2 (release rate 2224.49 µg/h). This well-regulated drug delivery system enables the control of drug release to meet the therapeutic requirements. On agar diffusion assay, 3 out of 5 test microorganisms were inhibited by phomopsidione nanoparticle coated contact lenses, including two Gram negative bacteria. Besides, all test microorganisms showed at least 99% of growth reduction, with the treatment of the contact lens model. The drug loaded onto the nanoparticles is sufficient to prevent the bacterial growth. In conclusion, this study provides an effective alternative to combat keratitis-causing microorganisms among contact wearers.

Efficacy of Amphotericin B Against Fusarium and Aspergillus in Corneal Storage Medium.

PURPOSE: The incidence of postkeratoplasty fungal infection is increasing in the United States, and our most commonly used corneal storage medium, Optisol-GS, contains antibiotics but no antifungal agents. We previously demonstrated the efficacy of amphotericin B additives in eliminating Candida albicans contaminants in Optisol-GS. The purpose of this study was to determine whether amphotericin B would also be efficacious against Fusarium solani and Aspergillus fumigatus. METHODS: Vials of Optisol-GS were supplemented with 0.255 µg/mL of amphotericin B. Half of the vials were inoculated with F. solani and half with A. fumigatus. Positive control vials were inoculated with the fungi but no amphotericin B. The vials were refrigerated, sampled, and plated at different time points. The plates were then incubated at 36°C for 48 hr after which fungal colony counts were performed. RESULTS: There was an average reduction in the growth of F. solani in the amphotericin B-supplemented vials of 44% on day 2, 79% on day 7, and 80% on day 14 when compared with the positive control vials. There was an average reduction in the growth of A. fumigatus in the amphotericin B-supplemented vials of 40% on day 2 and 14% on day 7 when compared with the positive control vials. Both amphotericin B-supplemented and control vials grew less than 2 colonies of A. fumigatus on day 14. CONCLUSIONS: This study suggests that amphotericin B additives in Optisol-GS reduce the growth of F. solani and A. fumigatus.