Publications by authors named "Stephen B Kaye"

147 Publications

Functional and morphological outcomes of the inverted internal limiting membrane flap technique in small and medium sized macular holes <400µm.

Retina 2021 Feb 25. Epub 2021 Feb 25.

Hospital rechts der Isar, Technical University of Munich (TUM), Ismaninger Str. 22, 81675 Munich, Germany Royal Liverpool University Hospital, Prescot Street, Liverpool L1 8XP, United Kingdom.

Purpose: To assess the effect of an internal limiting membrane (ILM) flap (IF) in MH surgery on best-corrected visual acuity (BCVA) and integrity of the ellipsoid zone (EZ) and external limiting membrane (ELM).

Methods: Patients were included who had successful surgery for MH <400µm with or without an IF. Main outcome measures were BCVA and restoration of the ELM and EZ at 12 months.

Results: 60 patients were included, 36 with conventional peeling and 24 with an IF. BCVA improved from 0.74 (±0.30) logMAR (20/110 Snellen) to 0.26 (±0.20) (20/36 Snellen) in patients without and from 0.77 (±0.32) logMAR (20/118 Snellen) to 0.18 (±0.12) (20/30 Snellen) in patients with an IF, respectively. There was no difference in the integrity of the EZ and ELM in patients with or without an IF at either 3 (p=0.58, p=0.20), 6 (p=0.81, p=0.10) or 12 months (p=0.60, p=0.20) or in BCVA at 3 (p=0.24), 6 (p=0.18) and 12 months (p=0.11). In the multivariable model, only pre-operative BCVA (p<0.01), EZ integrity (p=0.001) and age (p<0.01) were associated with post-operative BCVA.

Conclusions: In patients undergoing surgery for MH <400µm the use of an IF did not affect BCVA or the integrity of the EZ and ELM.
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http://dx.doi.org/10.1097/IAE.0000000000003160DOI Listing
February 2021

Intrastromal voriconazole for refractory infectious crystalline keratopathy associated with Candida pararugosa.

Can J Ophthalmol 2021 Mar 18. Epub 2021 Mar 18.

St Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom.

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http://dx.doi.org/10.1016/j.jcjo.2021.02.023DOI Listing
March 2021

Impact of COVID-19 on keratoconus patients waiting for corneal cross linking.

Eur J Ophthalmol 2021 Mar 15:11206721211001315. Epub 2021 Mar 15.

Department of Ophthalmology, Royal Liverpool University Hospital, Liverpool, UK.

Purpose: Royal College of Ophthalmologist recent guidance recommended delaying cross-linking services during the COVID-19 pandemic. This study investigates the effects of such delays in the delivery of cross-linking services in patients with keratoconus progression.

Methods: Retrospective observational study of 46 patients with keratoconus progression, whose cross-linking was delayed due to the COVID-19 pandemic. Demographic and clinical details were obtained from assessments on the day of listing, and subsequent review on the day of the procedure. Topographic indices included keratometry of the posterior and anterior corneal surface, maximum keratometry (), thinnest corneal thickness, ABCD progression and progression based on standard criteria recommendations (1.5 D & 20 microns thinning).

Results: A total of 46 eyes were analysed with an average time between being listed for CXL and having the procedure done was 182 ± 65 days. The delay due to COVID-19 was of 3 months. In this time period they had a significant worsening of all keratometric indices and lost almost one line of visual acuity (0.19 ± 0.19 to 0.26 ± 0.18 LogMAR, : 0.03). Thirty two eyes (70%) demonstrated progression in accordance with the ABCD progression criteria, while 18 eyes (39%) showed either an increase in of more than 1.5D or a thinning in corneal thickness of at least 20 μm.

Conclusions: The treatment delay for the keratoconus patients caused further progression and vision worsening. We recommend that corneal collagen crosslinking needs to be considered as a high priority intervention.
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http://dx.doi.org/10.1177/11206721211001315DOI Listing
March 2021

Keratitis: Incidence, Pathophysiology, Risk Factors and Novel Strategies for Treatment.

J Clin Med 2021 Feb 13;10(4). Epub 2021 Feb 13.

Department of Eye and Vision Science, University of Liverpool, Liverpool L7 8TX, UK.

Bacterial keratitis is a devastating condition that can rapidly progress to serious complications if not treated promptly. Certain causative microorganisms such as and are notorious for their resistance to antibiotics. Resistant bacterial keratitis results in poorer outcomes such as scarring and the need for surgical intervention. Thorough understanding of the causative pathogen and its virulence factors is vital for the discovery of novel treatments to avoid further antibiotic resistance. While much has been previously reported on , has been less extensively studied. This review aims to give a brief overview of epidemiology, pathophysiology and clinical characteristics as well as summarise the current evidence for potential novel therapies.
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http://dx.doi.org/10.3390/jcm10040758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918096PMC
February 2021

Biomaterials for corneal endothelial cell culture and tissue engineering.

J Tissue Eng 2021 Jan-Dec;12:2041731421990536. Epub 2021 Feb 16.

Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK.

The corneal endothelium is the posterior monolayer of cells that are responsible for maintaining overall transparency of the avascular corneal tissue via pump function. These cells are non-regenerative in vivo and therefore, approximately 40% of corneal transplants undertaken worldwide are a result of damage or dysfunction of endothelial cells. The number of available corneal donor tissues is limited worldwide, hence, cultivation of human corneal endothelial cells (hCECs) in vitro has been attempted in order to produce tissue engineered corneal endothelial grafts. Researchers have attempted to recreate the current gold standard treatment of replacing the endothelial layer with accompanying Descemet's membrane or a small portion of stroma as support with tissue engineering strategies using various substrates of both biologically derived and synthetic origin. Here we review the potential biomaterials that are currently in development to support the transplantation of a cultured monolayer of hCECs.
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http://dx.doi.org/10.1177/2041731421990536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894589PMC
February 2021

Indocyanine Green Angiographic Assessment of Conjunctival Melanocytic Disorders.

Cornea 2021 Feb 15. Epub 2021 Feb 15.

Department Ophthalmology, Medical University of Innsbruck, Austria; and Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom.

Purpose: To investigate whether transvascular indocyanine green (ICG) dye leakage is associated with conjunctival malignancy.

Methods: This is a prospective interventional study. Patients presenting with circumscribed conjunctival melanocytic disorders (CMDs) were included and examined using color photography, anterior segment optical coherence tomography to measure lesion size, and fluorescein and ICG angiography to measure vascular pattern and leakage. Time to vascular leakage was measured by 2 independent observers. Lesions were characterized as benign or malignant based on histopathological features.

Results: Thirty patients with CMD were included: 22 lesions were benign (conjunctival nevus, n = 20; conjunctival melanocytic intraepithelial neoplasia without atypia, n = 2) and 8 were malignant (in situ conjunctival melanoma n = 2; invasive conjunctival melanoma, n = 6). Malignant lesions had larger mean maximal diameters (11.0 ± 4.5 vs. 4.2 ± 2.5 mm, P = 0.003) and more frequently showed intrinsic tumor vasculature (8 of 8 vs. 10 of 22, P = 0.007). The mean time to ICG leakage was 350.9 ± 165.9 seconds in benign and 59.6 ± 22.1 seconds (P = 0.002) in malignant lesions and was inversely correlated with lesion size and thickness.

Conclusions: Time to angiographic ICG dye leakage is significantly shorter in malignant versus benign CMD.
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http://dx.doi.org/10.1097/ICO.0000000000002681DOI Listing
February 2021

A pipeline to evaluate inhibitors of the Pseudomonas aeruginosa exotoxin U.

Biochem J 2021 Feb;478(3):647-668

Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, U.K.

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. In severe P. aeruginosa infections, the phospholipase activity of the type 3 secretion system toxin, ExoU, induces lysis of target host cells and results in the poorest clinical outcomes. We have developed an integrated pipeline to evaluate small molecule inhibitors of ExoU in vitro and in cultured cell models, including a disease-relevant corneal epithelial (HCE-T) scratch and infection model using florescence microscopy and cell viability assays. Compounds Pseudolipasin A, compound A and compound B were effective in vitro inhibitors of ExoU and mitigated P. aeruginosa ExoU-dependent cytotoxicity after infection of HCE-T cells at concentrations as low as 0.5 µM. Addition of the antimicrobial moxifloxacin controlled bacterial load, allowing these assays to be extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration for 24 h, but, when used in combination with either Pseudolipasin A, compound A or compound B, a greater amount of viable cells and scratch healing were observed. Thus, our pipeline provides evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat infections due to ExoU producing P. aeruginosa, as well as the means to identify more potent ExoU inhibitors for future therapeutics.
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http://dx.doi.org/10.1042/BCJ20200780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886320PMC
February 2021

Tomographic and aberrometric assessment of first-time diagnosed paediatric keratoconus based on age ranges: a multicentre study.

Acta Ophthalmol 2020 Dec 30. Epub 2020 Dec 30.

Department of Ophthalmology, Regional University Hospital of Malaga, Malaga, Spain.

Purpose: To describe paediatric keratoconus (KC) patients by tomographic and aberrometric characteristics at first diagnosis, in a multicentre study.

Methods: We included 278 eyes from 139 paediatric patients, with a first tomographic diagnosis (Pentacam ) of KC prior to 18 years old. KC classification was based on the KC Index (≥ 1.07) and Topographic Keratoconus Classification (TKC ≥ 1). Patients were divided based on age ranges (14 and under and over 14 years) and gender. Statistical analysis was performed with SPSS statistics 25.0. ANOVA factor was carried out comparing to compare groups.

Results: 278 eyes were screened, and 230 eyes were diagnosed with paediatric KC. Mean age was 15.48 ± 2.33 (6 to 18) years. We found differences in terms of TKC (2.08 ± 0.89 and 2.38 ± 0.82, p < 0.05) and spherical aberration (-0.71 ± 0.97 and -1.07 ± 1.36, p < 0.05) among the 14 years old or under and above 14 years old groups, respectively. Overall, female paediatric KC patients presented a more severe TKC, Belin Ambrosio Display, maximum keratometry, asphericity and primary and secondary coma aberrations compared to male KC patients. We observed a correlation between CDVA and asphericity (r = 0.71, p < 0.01), as well as between CDVA and spherical aberration (r = 0.69, p < 0.01).

Conclusion: Our findings revealed that the debut of KC is usually in a moderate to advanced stage in the paediatric population at first diagnosis, particularly in female patients. Corneal tomography should be systematically performed in children with recent onset of corneal astigmatism.
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http://dx.doi.org/10.1111/aos.14715DOI Listing
December 2020

Changes in pupillometry associated with dissipated energy during phacoemulsification.

Eur J Ophthalmol 2020 Dec 29:1120672120984377. Epub 2020 Dec 29.

St Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, UK.

Purpose: To investigate the effect of ultrasound level during phacoemulsification on pupil dynamics.

Methods: Comparative retrospective study on patients who underwent routine cataract surgery at the Royal Liverpool University Hospital. Clinical parameters, anterior chamber measurements, axial length, surgeon grade, time of surgery, level of ultrasound used (cumulative dissipated energy, CDE), intra- and post-operative complications were collected. Pupil diameters were collected before and 4 ± 1 weeks after surgery in static scotopic, mesopic, photopic pupil conditions. Also, pupil dynamic measurements after luminous stimulus were recorded. Changes in static pupil diameters, relative dilation at 3.5 s after luminous stimulus, and time to reach 75% and 95% of maximum dilation were measured.

Results: Forty-eight eyes of 24 patients (13 males) were included with a mean age of 73.1 ± 14.6 years. Mean CDE value was 18.11 ± 10.56. Mean scotopic pupil diameters decreased by 0.24 ± 0.48 mm ( = 0.021) in the operated eye. Significant correlation was found between reduction in scotopic pupil diameter and CDE ( = 0.05). A generalized linear model confirmed that the level of CDE was significantly associated with reduction in scotopic pupil diameter ( = 0.026). Patients who underwent surgeries with lower CDE (0 < CDE ⩽ 10 and 10 < CDE ⩽ 20) did not experience significant changes in scotopic pupil diameter after surgery ( = 0.28 and  = 0.79, respectively) as opposed to those with higher CDE (CDE > 20;  = 0.03).

Conclusion: Phacoemulsification cataract surgery and the cumulative dissipated energy may be associated with changes in pupil behaviour.
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http://dx.doi.org/10.1177/1120672120984377DOI Listing
December 2020

16S Ribosomal RNA PCR Versus Conventional Diagnostic Culture in the Investigation of Suspected Bacterial Keratitis.

Transl Vis Sci Technol 2020 12 1;9(13). Epub 2020 Dec 1.

Department of Eye and Vision Sciences, University of Liverpool, Liverpool, UK.

Purpose: The purpose of this study was to compare conventional diagnostic culture (CDC) to 16S ribosomal RNA polymerase chain reaction (PCR) analysis for diagnosing bacterial keratitis.

Methods: Samples collected from 100 consecutive patients presenting to the Royal Liverpool University Hospital with bacterial keratitis were processed using CDC and 16S PCR analysis.

Results: The overall detection rate using both methods was 36%. Of these, 72.2% (26/36) were detected by PCR and 63.9% (23/36) isolated by CDC ( = 0.62). Using a combination of both PCR and CDC increased the detection rate for pathogenic bacteria by 13% compared to using CDC alone ( = 0.04). In CDC negative samples, 16S PCR identified more pathogens than CDC in 16S PCR negative samples. Neither order of sample collection nor prior antimicrobial use affected the detection rate.

Conclusions: 16S rRNA gene PCR performed in addition to CDC on corneal samples from patients with clinically suspected bacterial keratitis led to additional pathogen detection.

Translational Relevance: 16S rRNA gene PCR should be developed to become an additional part of clinical service for patients with bacterial keratitis rather than used in isolation.
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http://dx.doi.org/10.1167/tvst.9.13.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718820PMC
December 2020

DSAEK and STC-6 Needle.

Cornea 2021 Mar;40(3):e5

Department of Corneal Diseases, St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, UK.

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http://dx.doi.org/10.1097/ICO.0000000000002600DOI Listing
March 2021

Eye bank versus surgeon prepared DMEK tissues: influence on adhesion and re-bubbling rate.

Br J Ophthalmol 2020 Oct 30. Epub 2020 Oct 30.

UCL, London, UK.

Aim: To investigate the difference in adhesion and rebubbling rate between eye bank and surgeon prepared Descemet membrane endothelial keratoplasty (DMEK) tissues.

Methods: Laboratory and clinical retrospective comparative interventional case series. Research corneal tissues were obtained for laboratory investigation. The clinical study involved patients with endothelial dysfunction who underwent DMEK surgery and tamponade with air. Tissues were stripped using a standard DMEK stripping technique (SCUBA) and shipped as prestripped or loaded in a 2.2 intra-ocular lens cartridge with endothelium facing inwards (preloaded) before transporting from the eye bank to the surgeon. For surgeon prepared tissues, all the grafts were stripped in the theatre and transplanted or stripped in the laboratory and tested immediately. Adhesion force and elastic modulus were measured in the centre and mid-periphery in a laboratory ex vivo investigation using atomic force microscopy, while rebubbling rates were recorded in the clinical study.

Results: There was no difference in endothelial cell viability between surgeon or eye bank prepared tissue. Surgeon-stripped DMEK grafts in the laboratory investigation showed significantly higher elastic modulus and adhesion force compared to prestripped and preloaded tissues (p<0.0001). In the clinical data, rebubbling rates of 48%, 40% and 15% were observed in preloaded, prestripped and surgeon-stripped DMEK grafts, respectively. Rebubbling rates were significantly associated with combined cataract surgery (p=0.009) and with time from harvesting the graft to the surgery (p=0.02).

Conclusions: Decreased adhesion forces and elastic modulus in eye bank prepared tissues may contribute to increased rebubbling rates.
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http://dx.doi.org/10.1136/bjophthalmol-2020-317608DOI Listing
October 2020

Recurrent microbial keratitis and endogenous site Staphylococcus aureus colonisation.

Sci Rep 2020 10 29;10(1):18559. Epub 2020 Oct 29.

Department of Eye and Vision Sciences, University of Liverpool, 6 West Derby Street, Liverpool, L7 8TX, UK.

This study investigated Staphylococcus aureus carriage in patients with microbial keratitis (MK). 215 patients with MK, 60 healthy controls and 35 patients with rheumatoid arthritis (RA) were included. Corneal scrapes were collected from patients with MK. Conjunctival, nasal and throat swabs were collected from the non-MK groups on a single occasion and from the MK group at presentation and then at 6 and 12 weeks. Samples were processed using conventional diagnostic culture. 68 (31.6%) episodes of clinically suspected MK were classed as recurrent. Patients with recurrent MK had a higher isolation rate of S. aureus from their cornea than those with a single episode (p < 0.01) and a higher isolation rate of S. aureus from their conjunctiva compared to control participants, 20.6% (14/68) versus 3% (5/60) respectively (p = 0.01). Significantly more patients with recurrent MK (12/68, 17.6%) were found to have S. aureus isolated from both their conjunctiva and nose than those with a single episode of MK (7/147, 4.8% p = 0.002) and compared to patients in the control group (3/60, 5.0% p = 0.03). The results indicate that patients with recurrent MK have higher rates of carriage of S. aureus suggesting endogenous site colonisation as a possible source of recurrent infection.
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http://dx.doi.org/10.1038/s41598-020-75821-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596706PMC
October 2020

En-face analysis of the human limbal lymphatic vasculature.

Exp Eye Res 2020 12 28;201:108278. Epub 2020 Sep 28.

Department Ophthalmology, Medical University of Innsbruck, Austria. Electronic address:

Purpose: To describe the location and morphometric characteristics of the human limbal lymphatic vasculature and its relation to the marginal corneal vascular arcades (MCA).

Methods: Ex vivo confocal microscopic (CM) imaging and immunofluorescence double staining for CD-31 and D2-40 of histological en-face sections using 12 preserved human cadaveric corneoscleral discs were performed, followed by a semi-automated morphometric analysis of the two-dimensional vascular network architecture.

Results: Ex vivo CM confirmed the presence of 2 distinct vascular networks. The haematic limbal vascular complex (HLVC) extended further into the cornea, forming typical MCAs. The lymphatic limbal vascular complex (LLVC) was peripheral from the termination of Bowman's layer and was also found to be peripheral to and deeper than the HLVC. LLVC and HLVC were significantly different with respect to vessel diameter, segment length and wall thickness.

Conclusion: The lymphatic vasculature of the human corneoscleral limbal region displays specific morphometric features that allow its differentiation from haematic vessels using CM.
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http://dx.doi.org/10.1016/j.exer.2020.108278DOI Listing
December 2020

Eye Banking: One Cornea for Multiple Recipients.

Cornea 2020 Dec;39(12):1599-1603

St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom.

Purpose: Corneal transplantation is the most frequently performed transplant procedure. In much of the world, the demand for donor tissue heavily outstrips supply. With developments within lamellar corneal graft surgery, the use of split corneal donor tissue to increase donor tissue supply seems a pragmatic solution to reduce the supply and demand mismatch. This is especially important with tissue supply expected to be affected by the COVID-19 pandemic.

Methods: A literature review of techniques was performed, enabling multiple transplants to be derived from a single donor and simulation of a model to quantify the number of corneas potentially saved.

Results: Studies on splitting corneal donor tissue have demonstrated that up to 5 recipients may benefit from 1 donor scleral button. The impact of splitting donor tissue may provide a saving of up to 25.3% of donor graft tissue.

Conclusions: Splitting and preparing the donor tissue within an eye bank will improve tissue validation and donor tissue availability and may increase surgeon efficiency.
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http://dx.doi.org/10.1097/ICO.0000000000002476DOI Listing
December 2020

BMJ Open Ophthalmology.

Authors:
Stephen B Kaye

BMJ Open Ophthalmol 2020 1;5(1):e000530. Epub 2020 Sep 1.

Department of Eye and Vision Science, University of Liverpool, Liverpool, UK.

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http://dx.doi.org/10.1136/bmjophth-2020-000530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467524PMC
September 2020

Bilateral Keratoconus Progression: Immediate Versus Delayed Sequential Bilateral Corneal Cross-linking.

J Refract Surg 2020 Aug;36(8):552-556

Purpose: To compare immediate sequential bilateral corneal cross-linking (CXL), wherein both eyes are treated on the same day, to delayed sequential bilateral CXL, where each eye is treated on different days for bilateral progressive keratoconus.

Methods: This was a retrospective case note review of all patients who underwent sequential CXL or delayed CXL for keratoconus at Royal Liverpool University Hospital, United Kingdom. Parameters assessed were the change in maximum keratometry and minimum central corneal thickness on corneal tomography scan, corrected distance visual acuity, and cost estimates of treatment.

Results: A total of 38 patients (31 men and 7 women) with keratoconus with a mean age of 25.3 ± 7.0 years were included. Twenty patients received sequential CXL and 18 received delayed CXL treatment. In both the sequential CXL and delayed CXL groups, the treated eye(s) showed no evidence of progression after the corneal CXL treatment at last follow-up visit after 358 ± 158 days for sequential CXL and 451 ± 205 days for delayed CXL. There were no complications from the treatment in either group. In the delayed CXL group, the mean time interval between the two CXL procedures was 146 ± 129 days. Five of 18 patients (27%) showed progression of keratoconus in their second eye during the waiting time. Economical evaluation showed that four visits were saved for each sequential CXL treatment compared to delayed CXL.

Conclusions: Delayed CXL carries a risk of progression in the second eye and is associated with a higher economic burden. [J Refract Surg. 2020;36(8):552-556.].
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http://dx.doi.org/10.3928/1081597X-20200629-01DOI Listing
August 2020

Antimicrobial Activity of Poly-epsilon-lysine Peptide Hydrogels Against Pseudomonas aeruginosa.

Invest Ophthalmol Vis Sci 2020 08;61(10):18

Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, United Kingdom.

Purpose: To determine the antimicrobial activity of poly-epsilon-lysine (pɛK) functionalization of hydrogels against Pseudomonas aeruginosa.

Methods: Antimicrobial activities of pɛK and pɛK+ hydrogels were tested against both keratitis and a laboratory strain of Paeruginosa at a range of inocula sizes, over 4 and 24 hours. The number of viable CFU on pɛK and pɛK+ hydrogels or commercial contact lenses (CL) was investigated. Ex vivo porcine corneas were inoculated with Paeruginosa PAO1 (103 CFU) and incubated with pɛK+ hydrogels or commercial hydrogel CL for 24 hours and the effects of infection determined.

Results: PɛK+ hydrogels showed log reductions in viable CFU compared with pɛK hydrogels for all Paeruginosa strains, depending on inocula sizes and incubation time. After 24 hours pɛK+ hydrogels showed >5 and >7.5 log reduction in CFU compared with commercial hydrogel CL at 103 and 106 CFU, respectively. In an ex vivo porcine corneal infection model, pɛK+ hydrogels led to a significant decrease in viable PAO1 CFU and histologic analysis indicated a decreased infiltration of PAO1 into the stroma.

Conclusions: PɛK+ hydrogels demonstrated enhanced antimicrobial activity versus nonfunctionalized pɛK hydrogels against clinically relevant Paeruginosa strains. PɛK+ hydrogels have the potential to be used as a bandage CL with innate antimicrobial characteristics to minimize the risk of microbial keratitis.
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http://dx.doi.org/10.1167/iovs.61.10.18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441358PMC
August 2020

DSAEK Centration and Interface Folds: Surgical Management.

Cornea 2020 Nov;39(11):1457-1459

St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom; and.

Purpose: To describe a method to center a Descemet stripping automated endothelial keratoplasty lamellar graft and address interface folds.

Methods: A novel technique to center a Descemet stripping automated endothelial keratoplasty graft without undue manipulation of endothelial surface using a STC-6 needle passed obliquely through the anterior corneal surface to engage and progress the lenticule along the posterior surface after injection of air.

Result: Centration of graft achieved and macroscopic folds resolved.

Conclusions: Here, we describe an atraumatic and reproducible method for graft centration.
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http://dx.doi.org/10.1097/ICO.0000000000002411DOI Listing
November 2020

Free-Floating DMEK in the Host Anterior Chamber: Surgical Management.

Cornea 2020 Nov;39(11):1453-1456

St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom; and.

Purpose: To describe a method to visualize and manage a completely detached Descemet membrane endothelial keratoplasty (DMEK) tissue scroll in the anterior chamber.

Methods: A 56-year-old male patient with pseudophakic bullous keratopathy, who underwent uncomplicated DMEK surgery, had a complete graft detachment diagnosed at 1-week follow-up. The graft was reattached using a new technique, that is, the free-floating graft was stained in the anterior chamber with trypan blue, immediately followed by air injection to separate the host stroma from the dye. The stained DMEK graft was opened by gentle tapping and attached to the host stroma by air tamponade.

Result: This technique allowed sufficient staining of DMEK tissue to further evaluate and correct the graft orientation inside the anterior chamber without compromising the stroma. The DMEK graft was attached 1 week after the reattachment procedure. The cornea cleared confirming the functionality of the endothelial cells.

Conclusions: The technique described may be useful in the cases of complete detachment of DMEK tissue and poor visualization of the DMEK tissue orientation. Staining with trypan blue under a "protective" air bubble can provide sufficient visualization to ensure the unfolding of DMEK tissue and reduce the risk of host stromal staining.
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http://dx.doi.org/10.1097/ICO.0000000000002380DOI Listing
November 2020

Shotgun sequencing to determine corneal infection.

Am J Ophthalmol Case Rep 2020 Sep 8;19:100737. Epub 2020 May 8.

International Center for Ocular Physiopathology, The Veneto Eye Bank Foundation, Venice, Italy.

Purpose: To investigate if shotgun-sequencing method could be useful in detailed diagnosis of herpes simplex virus (HSV) infection and compare it with the conventional diagnostic method.

Observations: Using a sterile scraper, the infectious part of the ocular surface was scraped gently and placed on a glass slide for conventional diagnosis using PCR and histology and in RNA stabilizing reagent for shotgun sequencing respectively. Concentration of the DNA was determined using a sensitive fluorescence dye-based Qubit dsDNA HS Assay Kit. Shotgun-sequencing libraries were generated using the NEBNext DNA ultra II protocol. The samples were sequenced on the Illumina NextSeq 500 in high output mode with 2X150 bp paired-end sequencing. Taxonomic and functional profiles were generated.Conventional diagnostic method suspected herpetic keratitis. The results indicated presence of an amplified product of 92 bp positive HSV-DNA. Conventional diagnostic method detected the presence of Herpes Simplex Virus DNA (type 1). Shotgun sequencing confirmed the diagnosis of HSV along with the taxonomical profiling of the virus. These results were achieved using 1.9 ng/μL of DNA concentration (114 ng in 60 μL) of the total sample volume.

Conclusions And Importance: Shotgun sequencing is a hypothesis-free approach that identifies full taxonomic and functional profile of an organism. This technology is advantageous as it requires smaller sample size compared to conventional diagnostic methods.
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http://dx.doi.org/10.1016/j.ajoc.2020.100737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229483PMC
September 2020

Mass Spectrometry Reveals α-2-HS-Glycoprotein as a Key Early Extracellular Matrix Protein for Conjunctival Cells.

Invest Ophthalmol Vis Sci 2020 03;61(3):44

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Purpose: To determine the composition of extracellular matrix (ECM) proteins secreted by a conjunctival epithelial cell line and to identify components that aid conjunctival epithelial cell culture.

Methods: Human conjunctival epithelial cell line (HCjE-Gi) cells were cultured in serum-free media and their ECM isolated using ammonium hydroxide. Growth characteristics were evaluated for fresh HCjE-Gi cells plated onto ECMs obtained from 3- to 28-day cell cultures. Mass spectrometry was used to characterize the ECM composition over 42 culture days. Cell adhesion and growth on pre-adsorbed fibronectin and α-2-HS-glycoprotein (α-2-HS-GP) were investigated.

Results: Day 3 ECM provided the best substrate for cell growth compared to ECM obtained from 5- to 28-day cell cultures. Mass spectrometry identified a predominantly laminin 332 matrix throughout the time course, with progressive changes to matrix composition over time: proportional decreases in matrix-bound growth factors and increases in proteases. Fibronectin and α-2-HS-GP were 5- and 200-fold enriched as a proportion of the early ECM relative to the late ECM, respectively. Experiments on these proteins in isolation demonstrated that fibronectin supported rapid cell adhesion, whereas fibronectin and α-2-HS-GP both supported enhanced cell growth compared to tissue culture polystyrene.

Conclusions: These data reveal α-2-HS-GP as a candidate protein to enhance the growth of conjunctival epithelial cells and raise the possibility of exploiting these findings for targeted improvement to synthetic tissue engineered conjunctival substrates.
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http://dx.doi.org/10.1167/iovs.61.3.44DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401837PMC
March 2020

Conjunctival Biopsy Site in Mucous Membrane Pemphigoid.

Am J Ophthalmol 2020 08 15;216:1-6. Epub 2020 Feb 15.

Department of Ophthalmology, Royal Liverpool University Hospital, Liverpool, United Kingdom; Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom.

Purpose: To investigate if there is an association between the location of the conjunctival biopsy site (lesional, perilesional, or nonaffected) and the result of the direct immunofluorescence (DIF) test in patients with suspected mucous membrane pemphigoid (MMP) involving the ocular surface.

Design: Retrospective case series.

Methods: Records of patients with clinically suspected ocular MMP were reviewed to determine the location of the conjunctival biopsy. Conjunctival biopsy locations were defined as "lesional," "perilesional," and "nonaffected" conjunctiva. The DIF was considered positive when there was deposition of at least 1 of either IgM, IgG, IgA, or C3 at the basement membrane of the specimen; nondiagnostic when only fibrinogen was found at the same location; and negative when none of these features were present.

Results: The records of 41 patients were analyzed. Of these, 32 were eligible to be included in the study. Biopsies were lesional in 22% of cases (7/32), perilesional in 22% (7/32), and from nonaffected conjunctiva in 56% (18/32). DIF results were positive in 14% of lesional biopsies, in 86% of perilesional biopsies, and in 17% of those from nonaffected conjunctiva (P = .003). Perilesional biopsies gave higher positive DIF than lesional biopsies (P = .029).

Conclusions: Perilesional conjunctival biopsies are associated with an increase in positive DIF results. These results support the need to sample perilesional conjunctival tissue in patients with suspected MMP.
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http://dx.doi.org/10.1016/j.ajo.2020.01.033DOI Listing
August 2020

Toxin ExoU as a Therapeutic Target in the Treatment of Bacterial Infections.

Microorganisms 2019 Dec 16;7(12). Epub 2019 Dec 16.

Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool L7 8TX, UK.

The opportunistic pathogen employs the type III secretion system (T3SS) and four effector proteins, ExoS, ExoT, ExoU, and ExoY, to disrupt cellular physiology and subvert the host's innate immune response. Of the effector proteins delivered by the T3SS, ExoU is the most toxic. In infections, where the ExoU gene is expressed, disease severity is increased with poorer prognoses. This is considered to be due to the rapid and irreversible damage exerted by the phospholipase activity of ExoU, which cannot be halted before conventional antibiotics can successfully eliminate the pathogen. This review will discuss what is currently known about ExoU and explore its potential as a therapeutic target, highlighting some of the small molecule ExoU inhibitors that have been discovered from screening approaches.
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http://dx.doi.org/10.3390/microorganisms7120707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955817PMC
December 2019

Antimicrobial Nitric Oxide Releasing Contact Lens Gels for the Treatment of Microbial Keratitis.

ACS Appl Mater Interfaces 2019 Oct 2;11(41):37491-37501. Epub 2019 Oct 2.

School of Engineering , University of Liverpool , Brownlow Hill , Liverpool L69 3GH , United Kingdom.

Microbial keratitis is a serious sight threatening infection affecting approximately two million individuals worldwide annually. While antibiotic eye drops remain the gold standard treatment for these infections, the significant problems associated with eye drop drug delivery and the alarming rise in antimicrobial resistance has meant that there is an urgent need to develop alternative treatments. In this work, a nitric oxide releasing contact lens gel displaying broad spectrum antimicrobial activity against two of the most common causative pathogens of microbial keratitis is described. The contact lens gel is composed of poly-ε-lysine (pεK) functionalized with nitric oxide (NO) releasing diazeniumdiolate moieties which enables the controlled and sustained release of bactericidal concentrations of NO at physiological pH over a period of 15 h. Diazeniumdiolate functionalization was confirmed by Fourier transform infrared (FTIR), and the concentration of NO released from the gels was determined by chemiluminescence. The bactericidal efficacy of the gels against and was ascertained, and between 1 and 4 log reductions in bacterial populations were observed over 24 h. Additional cell cytotoxicity studies with human corneal epithelial cells (hCE-T) also demonstrated that the contact lens gels were not cytotoxic, suggesting that the developed technology could be a viable alternative treatment for microbial  keratitis.
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http://dx.doi.org/10.1021/acsami.9b13958DOI Listing
October 2019

Differential effects of primary disease and corneal vascularisation on corneal transplant rejection and survival.

Br J Ophthalmol 2020 05 28;104(5):729-734. Epub 2019 Aug 28.

Moorfields Eye Hospital NHS Foundation Trust, London, UK

Aims: To investigate the relative risk of pretransplant corneal vascularisation on rate of rejection and graft failure within 5 years of surgery when categorised by indication for transplantation.We analysed all adults recorded in the UK transplant registry who had a first cornea transplant for keratoconus (KC), pseudophakic bullous keratopathy (PBK) or previous infection (viral/bacterial/fungal/protozoan) between 1999 and 2017. We analysed the number of quadrants of the recipient cornea vascularised before transplant and type of vascularisation, the interval post-transplant to rejection, if any, and the outcome at 5 years post-transplant. Risk factors for rejection and transplant failure were modelled by multivariable risk-adjusted Cox regression.

Results: Corneal vascularisation was recorded in 10%, 25% and 67% of patients with KC, PBK and infection, respectively. Individuals with PBK had an increased hazard of transplant rejection only when there were more than two quadrants of vascularisation (HR 1.5, p=0.004) when either superficial and/or deep vascularisation was present (HR 1.3 and 1.4, respectively, p=0.004). Individuals who had a transplant for previous infection had an increased hazard of rejection with four quadrants of vascularisation (HR 1.6, p=0.003). There was no risk-adjusted increase in transplant failure associated with vascularisation in any group. There was weak evidence of reduction in risk of rejection and/or failure associated with lamellar compared with penetrating transplantation in KC and PBK in vascularised recipient corneas.

Conclusion: Vascularisation is a risk factor for corneal allograft rejection within 5 years. The indication for transplantation has a clinically significant effect on the magnitude of this risk.
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http://dx.doi.org/10.1136/bjophthalmol-2019-314200DOI Listing
May 2020

Increasing Donor Endothelial Cell Pool by Culturing Cells from Discarded Pieces of Human Donor Corneas for Regenerative Treatments.

J Ophthalmol 2019 21;2019:2525384. Epub 2019 Jul 21.

International Center for Ocular Physiopathology, Fondazione Banca degli Occhi del Veneto Onlus, Venice, Italy.

Purpose: To investigate if the peripheral corneal endothelium that is usually discarded after a corneal transplant could be used for endothelial cell culture.

Methods: Donor corneas ( = 19) with a mean age of 72 years, male : female ratio of 15 : 4, and death-to-preservation time of 10 hours were assessed for endothelial cell density (ECD) and number of dead cells before isolation. Alizarin red staining ( = 3) was performed to check the morphology of cells in the center and periphery. Descemet's membrane-endothelial complex was peeled from the center (8.25 mm) and the periphery (2.75 mm) and plated in two different wells of an 8-well chamber slide with media refreshed every alternate day. The confluence rate was monitored by microscopy. Live/dead analysis was performed ( = 3) at confluence. Tag-2A12 as a monoclonal antibody against peroxiredoxin-6 (Prdx-6) ( = 4), ZO-1 (zonula occludens-1) as a tight junction protein ( = 4), and Ki-67 as a proliferative cell marker ( = 4) were used to characterize the cells at confluence.

Results: At confluence, 8.25% average increase in the number of cells was observed from the central zone compared with 16.5% from the peripheral zone. Proliferation rate, hexagonality, Ki-67 positivity, and the cell area did not significantly differ between the groups ( > 0.05). All the proteins corresponding to the biomarkers tested were expressed in both the groups.

Conclusions: Although there are significantly fewer amounts of peripheral cells available after graft preparation for keratoplasty, these cells can still be used for endothelial cell culture due to their proliferative capability. The peripheral cells that are discarded after graft preparation can thus be utilized to increase the donor endothelial cell pool for regenerative treatments.
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http://dx.doi.org/10.1155/2019/2525384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679880PMC
July 2019

Use of a corneal impression membrane and PCR for the detection of herpes simplex virus type-1.

J Med Microbiol 2019 Sep 24;68(9):1324-1329. Epub 2019 Jul 24.

Department of Eye and Vision Science, University of Liverpool, Liverpool, UK.

. To investigate the use of a corneal impression membrane (CIM) for the detection of herpes simplex virus type 1 (HSV-1) in suspected herpes simplex keratitis (HSK).. In the laboratory study, swabs and CIMs made from polytetrafluoroethylene were spiked with different concentrations of HSV-1. DNA was extracted and real-time PCR undertaken using two sets of primers. In the clinical study, consecutive patients presenting with suspected HSK were included. For each patient, samples were collected from corneal lesions with a swab and a CIM in random order. Clinical details were collected using a standardized clinical form and patients were categorized into probable, presumed and possible HSK.. There was no difference in the performance of both primer sets for all HSV-1 dilutions (=0.83) using a CIM or between a CIM and a swab (=0.18). In total, 110 patients were included. Overall, 73 patients (66.4 %) had probable, 20 patients (18.2 %) presumed and 17 patients (15.5 %) possible HSV-1 keratitis. The HSV-1 detection rate was significantly higher using a CIM (40/110, 36.4 %) than a swab (28/110, 25.5 %) (=0.004). In the probable HSV keratitis group, the detection rate using a CIM was 43.8 % compared to 27.4 % for a swab (=0.004). The cycle threshold values obtained for the conjunctival swabs were higher than those obtained for the CIMs (<0.001).. In suspected HSK, a CIM is a useful alternative to a swab and more likely to detect the presence of HSV-1.
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http://dx.doi.org/10.1099/jmm.0.001041DOI Listing
September 2019

Metagenomics in ophthalmology: current findings and future prospectives.

BMJ Open Ophthalmol 2019 4;4(1):e000248. Epub 2019 Jun 4.

Fondazione Banca Degli Occhi Del Veneto Onlus, Zelarino, Venezia, Italy.

Less than 1% of all microorganisms of the available environmental microbiota can be cultured with the currently available techniques. Metagenomics is a new methodology of high-throughput DNA sequencing, able to provide taxonomic and functional profiles of microbial communities without the necessity to culture microbes in the laboratory. Metagenomics opens to a 'hypothesis-free' approach, giving important details for future research and treatment of ocular diseases in ophthalmology, such as ocular infection and ocular surface diseases.
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http://dx.doi.org/10.1136/bmjophth-2018-000248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557081PMC
June 2019

Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium.

BMJ Open Ophthalmol 2019 20;4(1):e000246. Epub 2019 Apr 20.

International Center for Ocular Physiopathology, Fondazione Banca degli Occhi del Veneto Onlus, Venice, Italy.

Objective: To detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method.

Methods: Seven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16 S and 18 S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments.

Results: In both, OC and hypothermic storage and control samples, the most abundant genera were and and were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus which was found in both the storage conditions. CDM was negative for microorganisms in all the samples.

Conclusion: Metagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turn-around time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.
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http://dx.doi.org/10.1136/bmjophth-2018-000246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6528759PMC
April 2019