Profiling ocular surface responses to preserved and non-preserved topical glaucoma medications: A 2-year randomized evaluation study.

BACKGROUND: Use of topical glaucoma medications has been reported to cause ocular surface (OS) discomfort and inflammation. This study explores the profile of inflammatory cytokines and OS symptoms induced in response to preserved and non-preserved drops. METHODS: Prospective, randomized evaluation on 36 treatment-naïve patients over 24 months of three differently preserved glaucoma drop preparations: Preservative-free (PF), polyquad (PQ) and benzalkonium chloride (BAK). Study participants were evaluated at baseline and then at 1, 3, 6, 12 and 24 months while on medication. At each visit, participants completed the OS disease index (OSDI) questionnaire, had basal tear sampling and impression cytology (IC) of the conjunctival epithelium. Quantitative polymerase chain reaction was performed to measure the gene expression of inflammatory cytokines [interleukin (IL)-6, IL-8, IL-10, IL-12A, IL-12B, IL-17A, IL-1ß and tumour necrosis factor-α] in the IC samples. Corresponding protein expression of cytokines in tear samples was assessed by the Becton-Dickinson cytometric bead arrays. RESULTS: Compared to PF and PQ groups, mRNA and protein expression of IL-6, IL-8 and IL-1ß increased in samples from the BAK group in a time-dependent fashion, whereas all other cytokines showed a non-significant increase. In the BAK group, there was a strong correlation between OSDI and the levels of IC/IL-1ß (r = .832, R2 = .692 and P = .040); IC/IL-10 (r = .925, R2 = .856 and P = .008) and tear/IL-1ß (r = .899, R2 = .808 and P = .014). CONCLUSIONS: BAK-preserved topical drops stimulate a sterile inflammatory response on the OS within 3 months which is maintained thereafter, whereas PF-drops and PQ-preserved drops showed no significant OS inflammation.

Fate of endothelial cells after intrastromal implantation of Descemet's membrane-endothelial cell tissue.

In non-Descemet Stripping Automated Endothelial Keratoplasty (nDSAEK), the host DM and endothelium are not removed surgically before the introduction of the posterior lamellar graft; the result is that the patient has both the healthy donor endothelium and the diseased or residual host endothelium. Conversely, DSAEK tissues, that are inserted with inverted polarity (upside down), do not survive and the graft fails. While the mechanism of endothelial cell transplantation is clear, the fate of the endothelial cells retained between two stromal interfaces and their physiological role, if any, is not well understood. The aim of our study was therefore to evaluate the viability of a healthy endothelial-Descemet's membrane (EDM) graft after the insertion into a stromal pocket of a recipient donor cornea. Research corneas (n = 52) were divided into three groups: Group A, where an EDM (obtained from another cornea) with good endothelium was inserted in a stromal pocket endothelium side down; Group B, consisting of control corneas with a stromal pocket but without EDM insertion; and Group C, pre-stripped membranes resting on their stroma (not in a stromal pocket). The tissues were preserved in tissue culture medium for 21 days at 31 °C. Parameters including viability of endothelial cells, expression of tight junctions (ZO-1) and thickness were evaluated. After 21 days, all the membranes inserted within the stromal pocket of Group A survived, although an average endothelial cell loss of 30.1% (± 18.10) and a mortality of 10.2% (± 22.86) were recorded. Qualitative analysis using triple staining with Hoechst, ethidium homodimer and calcein AM confirmed the mortality. ZO-1 was expressed where the cells were present, showing good integrity of tight junctions. Group C showed an average endothelial cell loss of 1.9% (± 3.38), a mortality of 0.02% (± 0.07) and a higher expression of ZO-1. An EDM graft with endothelium facing downwards can survive in a stromal pocket for at least 3 weeks, with an overall cell mortality of 30%. Further studies are needed to evaluate the possible outcomes of the insertion of a healthy intrastromal EDMs with reverse polarity and in edematous corneas.

Efficacy of topical microemulsion of fatty acids of the ω-3 series on the sub-epithelial corneal nerves regeneration after epithelium-off corneal collagen cross-linking for keratoconus.

PURPOSE: To evaluate efficacy of a microemulsion of fatty acids of the ω-3 series on the regeneration of the sub-epithelial corneal nerve plexus in patients with keratoconus after epi-off cross-linking. METHODS: In this prospective study, we recruited 40 patients, 18 females, mean age 28 years (range 22-37), who were randomly divided in two groups. Group A, 20 patients, after cross-linking were treated with a microemulsion of fatty acids of the ω-3 series. Group B were treated with hyaluronic acid (0.15%)-based tear substitute. Nerve tortuosity, reflectivity and density were examined with in vivo confocal microscopy. Ocular surface disease index (OSDI) questionnaire at the preoperative and at each follow-up visit (1, 3 and 6 months) after treatment was completed. RESULTS: No significant difference between the two groups was noted at 1 month in terms of nerve density and OSDI. A statistically significant difference between the two groups was detected at 3 months in terms of nerve fibers density (6 ± 0.82 in Group A and 1 ± 0.51 in Group B, P = 0.0001). Reflectivity and tortuosity of the fibers did not show significant differences between the two groups at any time point. At 1 month, OSDI average value in group A and in group B was 31.5 ± 1.94 and 30 ± 1.96, at 3 months 13 ± 1.71 and 28 ± 1.83, and at 6 months 10.5 ± 1.87 and 9.0 ± 1.81, respectively. CONCLUSION: The use of a microemulsion of fatty acids appears to ensure a faster regeneration of nerve fibers in patients undergoing epi-off cross-linking.

Natural course of adult-onset vitelliform lesions in eyes with and without comorbid subretinal drusenoid deposits.

PURPOSE: Adult vitelliform lesions (AVL) are associated with age related macular degeneration (AMD) and subretinal drusenoid deposits (SRDD). We evaluated the natural course of AVL, assessing the influence of SRDD on disease progression, visual function and incidence of macular atrophy (MA) and choroidal neovascular membranes (CNVM). METHODS: A retrospective cohort study was conducted between January 2011 and March 2016. Demographic, clinical and imaging data from 26 consecutive AVL patients were analysed following case note review. Optical coherence tomography images were graded for SRDD and patients divided into those with/without SRDD. Outcomes included presenting/changes in best corrected visual acuity (BCVA) and incidence of MA/CNVM. RESULTS: Mean age was 78.6 ± 7.6 years. Mean follow-up was 51.5 ± 25.6 months. Twelve patients (46.2%) had SRDD at presentation with 3 more (11.5%) developing them. Subjects with SRDD were older (mean 81.7 ± 6.1 years vs 74.3 ± 7.6 years, p = 0.010). Mean presenting BCVA was worse in SRDD eyes (0.39 ± 0.31 logMAR vs 0.19 ± 0.18 logMAR, p = 0.017). Eight of 15 patients with SRDD (53.3%) developed incident MA or CNVM; higher than those with no SRDD (1/11, 9.1%; p = 0.036). Two patients (7.7%) developed full thickness macular holes. CONCLUSIONS: Patients with AVL and SRDD likely represent an advanced pathological stage or phenotype with worse visual outcome and higher risk of MA/CNVM. Possible overlap with AMD exists. Follow-up, counselling and provisions for early detection/treatment of complications should be made. Better classification including improved understanding of phenotypic and genetic variations with reference to comorbid diseases including AMD is required. Presence of SRDD in AVL offers a dichotomous classification, indicating risk of future MA/CNVM formation.

In vivo microscopic and optical coherence tomography classification of neurotrophic keratopathy.

Neurotrophic keratopathy (NK) is a rare degenerative corneal disorder characterized by instability of epithelial integrity with consequent epithelial defects that can worsen up to persistent epithelial defects with stromal melting and ulceration. The pathogenesis of NK springs from a variable degree of damage to the trigeminal nerve plexus, leading to a reduction or total loss of corneal sensitivity. Mackie classification (1995) distinguishes three stages of NK, based on the severity of clinical presentation. The technological innovations in corneal diagnostic imaging allow clinicians to accurately study the morphometry and morphology of corneal structure with microscopic resolution. In this study, 45 patients affected by NK at different stages underwent in vivo confocal microscopy (IVCM) and anterior segment optical coherence tomography (AS-OCT) with particular attention to analyze subbasal nerve plexus fibers and the stromal structure. At the light of IVCM and AS-OCT observations, we propose a different staging of NK with respect to the Mackie's classification that takes into account the severity of subbasal nerve fibers damage and the extension in depth of stromal ulceration; this classification better defines, at the time of diagnosis, the cellular and structural alterations in the affected corneas, with possible prognostic and therapeutic values in the management of NK.

Early results on the use of chitosan-N-acetylcysteine (Lacrimera®) in the management of dry eye disease of varied etiology.

PURPOSE: To evaluate the effect of once daily administration of chitosan-N-acetylcysteine (Lacrimera®) in the management of dry eye disease (DED). METHODS: Eighteen patients (3 male, 15 female) aged 25-86 years (mean 61.1) and suffering from moderate to severe DED with superficial punctate keratitis (SPK) were retrospectively evaluated after a trial of Lacrimera® drops (1 drop in the morning for 5 days only). All the patients were using other artificial tears before the treatment. All lubricants were stopped, and Lacrimera® was started instead. Slit-lamp examination and images were taken before and at 1 and 3 weeks follow-up after the treatment. The subjective (Ocular Surface Disease Index, OSDI) and objective (Oxford Grading System, OGS) evaluation was recorded. A paired student's t test was performed to analyse the data. RESULTS: At baseline, the SPK grade was I to IV (OGS) and the OSDI ranged from 25 to 71.4. Fifteen patients showed a statistically significant (p < 0.0001) improvement in OGS and the OSDI at 3 weeks post-treatment. Three patients showed no improvement. CONCLUSIONS: A single-dose instillation of chitosan-N-acetylcysteine for five consecutive days improved signs and symptoms in patients affected from DED from a variety of causes, who were refractory to standard treatment with lubricants. Given its posology, the absence of side effects and the results obtained Lacrimera® should be taken into consideration as a viable option in patients with moderate to severe DED.

Cultivation and characterisation of the surface markers and carbohydrate profile of human corneal endothelial cells.

BACKGROUND: The study aims to characterise human corneal endothelial cell (HCEnC) cultures generated by the peel-and-digest method based on their surface protein/carbohydrate expression pattern. METHODS: Quantitative polymerase chain reaction was used to compare expression of vimentin, CD90, Cytokeratin-19, ZO-1 and Claudin 14 in cultured HCEnC and cell line B4G12 versus stromal cells. Fluorescence-activated cell sorting was used to assess surface protein distribution of cultured and uncultured HCEnC. Distribution of surface proteins/carbohydrates was visualised by immunofluorescent and lectin staining. RESULTS: Human corneal endothelial cell and B4G12 showed lower expression level for vimentin, CD90, Cytokeratin-19 compared with stromal cells; while ZO-1 was expressed in endothelial cells, Claudin 14 was detected in B4G12 only. Fluorescence-activated cell sorting analyses revealed CD166, CD47, CD44, CD54, CD73, CD90, CD105, CD106, CD112, CD146 and CD325 to be present, with CD34 to be absent from cultured HCEnC. Freshly isolated, non-cultivated HCEnCs were CD90, CD73, CD146 and CD325 positive. Carbohydrates were detected by lectins LCA, PHA E, PHA L, PSA, sWGA, Con A, RCA 120 and WGA, but cultured HCEnC showed negative for GSL I, SBA, DBA, PNA and UEA I. CONCLUSION: Cultures established by the peel-and-digest method are probably not prone to stromal contamination, but the cells are likely to undergo endothelial-to mesenchymal transition as suggested by apparent morphological changes.

Creeping posterior synechiae following hyperopic iris-fixated phakic implants.

The purpose of this study was to report on the occurrence and management of posterior synechiae (PS) in three hyperopic eyes after iris-fixated anterior chamber phakic IOL implantation. This is a case report of a 55-year-old man and a 55-year-old lady who had undergone iris-fixated anterior chamber phakic IOL implantation to correct hyperopic astigmatism (one eye) and hyperopia (both eyes), respectively. The three eyes developed creeping PS and pigment dispersion within 4-6 weeks of surgery. Synechiolysis and pupil stretching in one eye did not work as the synechiae promptly recurred. Definitive management in the form of removal of the phakic implant and phacoemulsification with lens implant was successful in restoring normal vision in all three eyes. Although implantation of phakic intraocular lenses has been reported to be very effective for the correction of moderate to high degrees of ametropia, this complication can occur in hyperopic eyes. Pigment dispersion has been reported in myopic eyes also, but PS seem to be more an issue with hyperopic eyes. Age of the patient may also be a factor. This information is important in the selection and counselling of patients.

Concise review: evidence for CD34 as a common marker for diverse progenitors.

CD34 is a transmembrane phosphoglycoprotein, first identified on hematopoietic stem and progenitor cells. Clinically, it is associated with the selection and enrichment of hematopoietic stem cells for bone marrow transplants. Due to these historical and clinical associations, CD34 expression is almost ubiquitously related to hematopoietic cells, and it is a common misconception that CD34-positive (CD34(+) ) cells in nonhematopoietic samples represent hematopoietic contamination. The prevailing school of thought states that multipotent mesenchymal stromal cells (MSC) do not express CD34. However, strong evidence demonstrates CD34 is expressed not only by MSC but by a multitude of other nonhematopoietic cell types including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors. In many cases, the CD34(+) cells represent a small proportion of the total cell population and also indicate a distinct subset of cells with enhanced progenitor activity. Herein, we explore common traits between cells that express CD34, including associated markers, morphology and differentiation potential. We endeavor to highlight key similarities between CD34(+) cells, with a focus on progenitor activity. A common function of CD34 has yet to be elucidated, but by analyzing and understanding links between CD34(+) cells, we hope to be able to offer an insight into the overlapping properties of cells that express CD34.

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

BACKGROUND AIMS: The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. METHODS: Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. RESULTS: All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. CONCLUSIONS: Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy.

Collagen cross-linking with photoactivated riboflavin (PACK-CXL) for the treatment of advanced infectious keratitis with corneal melting.

PURPOSE: To investigate the efficacy and safety of corneal collagen cross-linking (CXL) with photoactivated riboflavin (photoactivated chromophore for infectious keratitis [PACK]-CXL) in the management of infectious keratitis with corneal melting. DESIGN: Prospective clinical trial. PARTICIPANTS: Forty eyes from 40 patients with advanced infectious keratitis and coexisting corneal melting. METHODS: Twenty-one patients (21 eyes) underwent PACK-CXL treatment in addition to antimicrobial therapy. The control group consisted of 19 patients (19 eyes) who received only antimicrobial therapy. MAIN OUTCOME MEASURES: The slit-lamp characteristics of the corneal ulceration, corrected distance visual acuity, duration until healing, and complications were documented in each group. The Mann-Whitney U test was used for statistical analysis. P values less than 0.05 were considered statistically significant. RESULTS: The average time until healing was 39.76 ± 18.22 days in the PACK-CXL group and 46.05 ± 27.44 days in the control group (P = 0.68). After treatment and healing, corrected distance visual acuity was 1.64 ± 0.62 in the PACK-CXL group and 1.67 ± 0.48 in the control group (P = 0.68). The corneal ulceration's width and length was significantly bigger in the PACK-CXL group (P = 0.004 and P = 0.007). Three patients in the control group demonstrated corneal perforation; infection recurred in 1 of them. No serious complications occurred in the PACK-CXL group. CONCLUSIONS: Corneal CXL with photoactivated riboflavin did not shorten the time to corneal healing; however, the complication rate was 21% in the control group, whereas there was no incidence of corneal perforation or recurrence of the infection in the PACK-CXL group. These results indicate that PACK-CXL may be an effective adjuvant therapy in the management of severe infectious keratitis associated with corneal melting.

Atypical hydrops in keratoconus.

To report the clinical presentation, progress and management of atypical acute hydrops. A retrospective case study of three patients with keratoconus, two of whom had previously undergone penetrating keratoplasty. The patients underwent full ophthalmological examination and digital slit-lamp imaging of the cornea throughout the course of the condition. The two patients who had previously undergone keratoplasty had spontaneous hydrops primarily affecting the host bed but in one case extended to the graft inferiorly; however, in the third patient it was traumatic in origin. The Descemet's tear affected the host rim in only one patient, which resolved spontaneously. In another patient, the hydrops was related to an internal dehiscence of the graft-host junction and had to be managed by an endothelial transplant covering the dehisced graft-host junction. In the third patient, hydrops secondary to trauma was also associated with acute haemops. Progression of keratoconus post keratoplasty can occur exclusively in the recipient bed leading to acute hydrops in the host sparing the transplanted cornea. The progressive thinning and ectasia of the recipient bed can also result in internal graft-host dehiscence leading to chronic oedema. Rapid entry of aqueous or blood cells into the corneal stroma following acute rupture of the Descemet's membrane suggests that the abnormal stroma of the eye with keratoconus may have an important role to play in the pathogenesis of acute hydrops/haemops.

Amniotic membrane graft (AMG) for persistent epithelial defects following infective corneal ulcers and keratitis - A systematic review.

ABSTRACT: Management of nonhealing corneal ulcers is challenging as it can lead to complications such as melting, descemetocele, and perforation, which can be prevented by amniotic membrane graft (AMG). The absence of any review (English language) on AMG for corneal ulcers prompted us to conduct this review. We looked for clinical trials (randomized and controlled) on the effect of AMG combined with other treatments, through various search engines utilizing the terms amniotic membrane and corneal ulcer. All patients who received AMG and/or conventional medication were included. Risk of bias tools derived from Cochrane collaboration were used to ascertain the standard and quality of the trials. Visual acuity and epithelial healing time were the main outcome measures studied. Three trials were finally selected for analysis. AMG was noted to be associated with better uncorrected visual acuity and reduction in neovascularization. There was no difference in best corrected visual acuity, epithelial healing time, and corneal opacity. There is evidence that the use of AMG is associated with improved uncorrected visual acuity as well as reduction in neovascularization of the cornea among patients with corneal ulcers, but there was no improvement in epithelial healing time or corneal opacity. Further multicenter randomized controlled trials are needed to elicit the beneficial effects of the intervention and augment the validity of this review.

Increasing incidence of contact-lens-related Acanthamoeba keratitis in a tertiary ophthalmology department in an Italian population.

PURPOSE: The purpose of this paper is to report the increasing incidence of contact-lens related Acanthamoeba keratitis (AK) in a tertiary ophthalmology department in Umbria, central Italy. METHODS: Observational and retrospective case series were carried out. A total of nine eyes with a diagnosis of AK were examined. All patients underwent a full slit lamp examination, in vivo confocal microscopy (IVCM) and corneal scraping. The IVCM was repeated at one and two-week and at one, three and six-month intervals. Samples of domestic tap water were also examined for PCR analysis. Patients were treated with levofloxacin0,5%, Polyhexamethylene biguanide 0.02%, and Propamidine Isetionate0,1%. RESULTS: All patients were contact lens wearers. The average patient age was 27.75 (range 18-45), with three men and five women. The main clinical features were ciliary congestion, diffuse epitheliopathy with punctuated keratitis, multiple, small sub-epithelial, greyish, corneal infiltrates with epithelial defect, pseudodendritic corneal lesions, perineural infiltrates, corneal stromal cellularity, and stromal infiltrates. IVCM was indicative of Acanthamoeba in seven out of the nine eyes. All the positive IVCM images were section images showing double walled, bright-spot cysts with a clear chain-like arrangement of five or more cysts identified in three of the patients. PCR analysis of the water was negative in all cases. CONCLUSION: Although PCR is the most common method used, the increased incidence of AK could mainly be related to a proper IVCM interpretation. A broad-spectrum antibiotic, such as levofloxacin might play a role in the early treatment of AK reducing the virulence of the amoeba.

Relationship of posterior peripheral corneal layers and the trabecular meshwork: an immunohistological and anatomical study.

BACKGROUND/AIM: With the popularity of endothelial keratoplasty (EK) procedures, Descemet membrane (DM) EK and pre-Descemet EK, considerable work has been done on understanding the posterior corneal anatomy. Most of the information available relates to the central cornea. We evaluated the peripheral cornea to explore the immunohistological and anatomical relationship between the pre-Descemet layer (PDL), DM and trabecular meshwork (TM). METHODS: Six donor human sclerocorneal discs were studied. PDL, DM and TM were examined by light microscopy, transmission electron microscopy (TEM) and immunohistology. The DM was peeled from the centre to the limit of its peripheral attachment, to reach the transition zone (TZ) between TM and peripheral cornea. Ten-micron sections were stained with antibodies against collagens 1, 2, 3, 4, 5, 6, 12, elastin, myocilin, wnt-1, aquaporin, tenascin C, laminin and integrin alpha 3. RESULTS: Collagens 2, 3, 4, laminin and myocilin were predominantly seen in the TZ between TM and peripheral cornea. Wnt-1, integrin alpha 3 and tenascin C were highly concentrated in TM. Collagen 1 was present predominantly in the corneal stroma. On TEM; DM was thinner with a denser banded structure spread throughout its thickness in the periphery compared with the central cornea where it presents as the distinct anterior banded layer. CONCLUSION: The TZ between DM, PDL and TM shows a unique histological structure at the periphery. The collagen and elastin fibres of the TM are continuous with the PDL. The structures are firmly attached to each other. These findings provide structural information that is relevant to the preparation of DMEK donor tissue.

A Biosynthetic Alternative to Human Amniotic Membrane for Use in Ocular Surface Surgery.

Purpose: The biosynthetic Symatix membrane (SM) was developed to replace fresh human amniotic membrane (hAM) in ocular surgical applications. The purpose of this study was to test the biocompatibility of the SM with human limbus-derived epithelial cells with regard to their physical and biological properties. Methods: Different physical properties of SM were tested ex vivo by simulation on human corneas. In vitro, primary limbal epithelial cells from limbal explants were used to test biological properties such as cell migration, proliferation, metabolic activity, and limbal epithelial cell markers on the SM, hAM, and freeze-dried amniotic membrane (FDAM). Results: The surgical handleability of the SM was equivalent to that of the hAM. Ultrastructural and histological studies demonstrated that epithelial cells on the SM had the typical tightly apposed, polygonal, corneal epithelial cell morphology. The epithelial cells were well stratified on the SM, unlike on the hAM and FDAM. Rapid wound healing occurred on the SM within 3 days. Immunofluorescence studies showed positive expression of CK-19, Col-1, laminin, ZO-1, FN, and p-63 on the SM, plastic, and FDAM compared to positive expression of ZO-1, Col-1, laminin, FN, and p63 and negative expression of CK-19 in the hAM. Conclusions: These results indicate that the SM is a better substrate for limbal epithelial cell migration, proliferation, and tight junction formation. Altogether, the SM can provide a suitable alternative to the hAM for surgical application in sight-restoring operations. Translational Relevance: The hAM, currently widely used in ocular surface surgery, has numerous variations and limitations. The biocompatibility of corneal epithelial cells with the SM demonstrated in this study suggests that it can be a viable substitute for the hAM.

Clinical features, risk factors and outcomes of contact lens-related bacterial keratitis in Nottingham, UK: a 7-year study.

BACKGROUND/OBJECTIVES: To examine the clinical characteristics, risk factors and outcomes of contact lens-related bacterial keratitis (CLBK) in a large UK tertiary referral centre. SUBJECTS/METHODS: A retrospective analysis of all patients who presented to the Queen's Medical Centre, Nottingham, UK, with suspected CLBK between October 2015 to September 2022 (a 7-year period) was performed. Relevant data on demographic factors, CL wear behaviour, causes, clinical characteristics, and outcomes were analysed. RESULTS: We included 138 patients with CLBK; the mean age was 42.0 ± 17.8 years and 74 (53.6%) patients were male. Most CLBK were related to soft CL wear (94.5%), particularly monthly disposable (42.5%) and daily disposable (24.4%) CLs. Poor CL wear behaviour/hygiene was documented in 57.1% cases. Among the 64 (46.4%) microbiological-positive cases (n = 73 organisms), Pseudomonas aeruginosa (36, 49.3%) and Staphylococcus spp. (16, 21.9%) were most commonly identified. Six (4.3%) cases were polymicrobial. Most (97.0%) patients were successfully treated with topical antibiotics alone, with 80.6% achieving good final corrected-distance-visual-acuity (CDVA) of ≥ 0.30 logMAR. Poor visual outcome (final CDVA < 0.30 logMAR) was significantly associated with presenting CDVA < 0.6 logMAR (p = 0.002) and central ulcer (p = 0.004). Poor corneal healing (complete healing of > 30 days from initial presentation) was significantly associated with age > 50 years (p = 0.028), female gender (p = 0.020), and infiltrate size >3 mm (p = 0.031). CONCLUSIONS: Poor CL wear behaviour/hygiene is commonly observed in CLBK, highlighting the importance of improved counselling and awareness regarding CL use and hygiene. When presented early and managed appropriately, most patients are able to achieve good clinical outcomes with medical treatment alone.

Microbiological culture versus 16S/18S rRNA gene PCR-sanger sequencing for infectious keratitis: a three-arm, diagnostic cross-sectional study.

Background: To compare the diagnostic performance of microbiological culture and 16S/18S rRNA gene polymerase chain reaction (PCR)-Sanger sequencing for infectious keratitis (IK) and to analyse the effect of clinical disease severity on test performance and inter-test concordance. Methods: This was a three-arm, diagnostic cross-sectional study. We included all eligible patients who presented with presumed bacterial/fungal keratitis to the Queen's Medical Centre, Nottingham, UK, between June 2021 and September 2022. All patients underwent simultaneous culture (either direct or indirect culture, or both) and 16S (pan-bacterial)/18S (pan-fungal) ribosomal RNA (rRNA) PCR-Sanger sequencing. The bacterial/fungal genus and species identified on culture were confirmed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Relevant clinical data were also collected to analyze for any potential clinico-microbiological correlation. Main outcome measures included the diagnostic yield, test accuracy (including sensitivity and specificity), and inter-test agreement [including percent agreement and Cohen's kappa (k)]. Results: A total of 81 patients (86 episodes of IK) were included in this study. All organisms identified were of bacterial origin. Diagnostic yields were similar among direct culture (52.3%), indirect culture (50.8%), and PCR (43.1%; p = 0.13). The addition of PCR enabled a positive diagnostic yield in 3 (9.7%) direct culture-negative cases. Based on composite reference standard, direct culture had the highest sensitivity (87.5%; 95% CI, 72.4-95.3%), followed by indirect culture (85.4%; 95% CI, 71.6-93.5%) and PCR (73.5%; 95% CI, 59.0-84.6%), with 100% specificity noted in all tests. Pairwise comparisons showed substantial agreement among the three tests (percent agreement = 81.8-86.2%, Cohen's k = 0.67-0.72). Clinico-microbiological correlation demonstrated higher culture-PCR concordance in cases with greater infection severity. Conclusions: This study highlights a similar diagnostic performance of direct culture, indirect culture and 16S rRNA PCR for bacterial keratitis, with substantial inter-test concordance. PCR serves as a useful diagnostic adjuvant to culture, particularly in culture-negative cases or those with lesser disease severity (where culture-PCR concordance is lower).