Lubricating drops for contact lens discomfort in adults.

BACKGROUND: Contact lens discomfort is a symptom-based clinical diagnosis that affects 13% to 75% of contact lens wearers. The Tear Film and Ocular Surface Society defines contact lens discomfort as "a condition characterized by episodic or persistent adverse ocular sensations related to lens wear either with or without visual disturbance, resulting from reduced compatibility between the lens and ocular environment, which can lead to decreased wearing time and discontinuation from lens wear." Signs of the condition include conjunctival hyperemia, corneal and conjunctival staining, altered blinking patterns, lid wiper epitheliopathy, and meibomian gland dysfunction. Eye care specialists often treat contact lens discomfort with lubricating drops, including saline, although there is no clear evidence showing this treatment is effective and safe. OBJECTIVES: To evaluate the efficacy and safety of lubricating drops for ocular discomfort associated with contact lens wear in adults. SEARCH METHODS: We searched CENTRAL (which contains the Cochrane Eyes and Vision Trials Register), MEDLINE, Embase.com, two other databases, and two trials registries to May 2024, without date or language restrictions. SELECTION CRITERIA: We included parallel-group randomized controlled trials (RCTs) that evaluated lubricating drops, including saline, versus no treatment, or that evaluated lubricating drops versus saline, in adult contact lens wearers. We included studies regardless of publication status, language, or year of publication. DATA COLLECTION AND ANALYSIS: We applied standard Cochrane methodology. The critical outcome was contact lens discomfort. Important outcomes were corneal fluorescein staining and conjunctival redness. Adverse outcomes were incident microbial keratitis, inflammatory corneal infiltrates, and participant discontinuation. We assessed risk of bias for outcomes reported in the summary of findings table using the Cochrane risk of bias tool RoB 2, and we rated the certainty of the evidence using GRADE. MAIN RESULTS: We included seven RCTs conducted in the USA, Canada, Italy, and France. They randomized a total of 463 participants to lubricating drops, saline, or no treatment. Four trials evaluated lubricating drops and saline versus no treatment, but one of them provided no usable outcome data. Three trials evaluated lubricating drops versus saline. Study characteristics All trial participants were adults, and the mean age ranged from 25.7 years to 36.7 years. The proportion of women varied from 15% to 82%. The trials lasted between one and four weeks. Of the five trials that reported contact lens discomfort, we judged three at high risk of bias, and considered the other two had some risk of bias concerns. Lubricating drops (including saline) versus no treatment Lubricating drops compared with no treatment may reduce contact lens discomfort, measured on a 37-point scale (lower is better), but the evidence is very uncertain (mean difference [MD] -5.9 points, 95% confidence interval [CI] -3.74 to -8.05; 2 RCTs; 119 participants). One trial found no difference between lubricating drops and no treatment in "end-of-day" comfort. The trial that compared saline with no treatment provided no results for the control group. Two studies measured corneal fluorescein staining on a scale of 0 to 20 (lower is better). We found low-certainty evidence of little to no difference between lubricating drops and no treatment in changes in the extent (MD -0.15 points, 95% CI -0.86 to 0.56; 2 RCTs; 119 participants), depth (MD -0.01 points, 95% CI -0.44 to 0.42; 2 RCTs; 119 participants), or type (MD 0.04 points, 95% CI -0.38 to 0.46; 2 RCTs; 119 participants) of corneal fluorescein staining scores. Regarding conjunctival redness, measured on a scale of 0 to 4 (lower is better), there was low-certainty evidence of little to no difference between lubricating drops and no treatment in nasal region scores (MD 0.10, 95% CI -0.29 to 0.49; 1 RCT; 73 participants) and temporal region scores (MD 0.00, 95% CI -0.39 to 0.39; 1 RCT; 73 participants). No studies reported microbial keratitis or inflammatory corneal infiltrates, and no trials reported vision-threatening adverse events up to four weeks of treatment. All trials reported the proportion of participants who discontinued participation. In two trials, no participants left any treatment group. Our meta-analysis of another two studies suggests little difference in the number of people who dropped out of the lubricating treatment group versus the no treatment group (risk ratio [RR] 1.42, 95% CI 0.19 to 10.94; 138 participants; low-certainty evidence). Lubricating drops versus saline Lubricating drops may have little to no effect compared with saline on contact lens discomfort measured on a visual analog scale of 0 to 100 (lower is better), but the evidence is very uncertain (MD 9.5 points, 95% CI -4.65 to 23.65; 1 RCT; 39 participants). No studies reported corneal fluorescein staining or conjunctival redness. No studies reported microbial keratitis or inflammatory corneal infiltrates, and no trials reported vision-threatening adverse events up to four weeks of treatment. Our meta-analysis of three studies suggests little difference in the number of people who dropped out of the lubricating treatment group versus the saline group (RR 1.56, 95% CI 0.47 to 5.12; 269 participants; low-certainty evidence). AUTHORS' CONCLUSIONS: Very low-certainty evidence suggests that lubricating drops may improve contact lens discomfort compared with no treatment, but may have little or no effect on contact lens discomfort compared with saline. Low-certainty evidence also suggests that lubricating drops may have no unwanted effects that would lead to discontinuation over one to four weeks. Current evidence suggests that prescribing lubricating drops (including saline) to people with contact lens discomfort is a viable option. However, most studies did not assess patient-reported contact lens (dis)comfort using a validated instrument. Therefore, further well-designed trials are needed to generate high-certainty evidence on patient-reported outcomes as well as on longer-term safety outcomes.

Ocular surface immune transcriptome and tear cytokines in corneal infection patients.

Background: Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears. Methods: Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1ß, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay. Results: A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance. Conclusions: During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight.

BCLA CLEAR Presbyopia: Management with contact lenses and spectacles.

This paper seeks to outline the history, market situation, clinical management and product performance related to the correction of presbyopia with both contact lenses and spectacles. The history of the development of various optical forms of presbyopic correction are reviewed, and an overview is presented of the current market status of contact lenses and spectacles. Clinical considerations in the fitting and aftercare of presbyopic contact lens and spectacle lens wearers are presented, with general recommendations for best practice. Current options for contact lens correction of presbyopia include soft simultaneous, rigid translating and rigid simultaneous designs, in addition to monovision. Spectacle options include single vision lenses, bifocal lenses and a range of progressive addition lenses. The comparative performance of both contact lens and spectacle lens options is presented. With a significant proportion of the global population now being presbyopic, this overview is particularly timely and is designed to act as a guide for researchers, industry and eyecare practitioners alike.

Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates.

Acanthamoeba, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as Acanthamoeba keratitis. In its natural environment, Acanthamoeba performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct Acanthamoeba spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by in situ hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; Pseudomonas species was abundant in Australian corneal isolates (P < 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates (P < 0.017), and Bacteroidota was abundant in Australian water isolates (P < 0.019). The bacterial beta diversity of Acanthamoeba isolates from keratitis patients in India and Australia significantly differed (P < 0.05), while alpha diversity did not vary based on the country of origin or source of isolation (P > 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within Acanthamoeba, thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens.

Epidemiology of and Genetic Factors Associated with Acanthamoeba Keratitis.

Acanthamoeba keratitis (AK) is a severe, rare protozoal infection of the cornea. Acanthamoeba can survive in diverse habitats and at extreme temperatures. AK is mostly seen in contact lens wearers whose lenses have become contaminated or who have a history of water exposure, and in those without contact lens wear who have experienced recent eye trauma involving contaminated soil or water. Infection usually results in severe eye pain, photophobia, inflammation, and corneal epithelial defects. The pathophysiology of this infection is multifactorial, including the production of cytotoxic proteases by Acanthamoeba that degrades the corneal epithelial basement membrane and induces the death of ocular surface cells, resulting in degradation of the collagen-rich corneal stroma. AK can be prevented by avoiding risk factors, which includes avoiding water contact, such as swimming or showering in contact lenses, and wearing protective goggles when working on the land. AK is mostly treated with an antimicrobial therapy of biguanides alone or in combination with diaminidines, although the commercial availability of these medicines is variable. Other than anti-amoeba therapies, targeting host immune pathways in Acanthamoeba disease may lead to the development of vaccines or antibody therapeutics which could transform the management of AK.

The role of naturally acquired intracellular Pseudomonas aeruginosa in the development of Acanthamoeba keratitis in an animal model.

BACKGROUND: Acanthamoeba is an environmental host for various microorganisms. Acanthamoeba is also becoming an increasingly important pathogen as a cause of keratitis. In Acanthamoeba keratitis (AK), coinfections involving pathogenic bacteria have been reported, potentially attributed to the carriage of microbes by Acanthamoeba. This study assessed the presence of intracellular bacteria in Acanthamoeba species recovered from domestic tap water and corneas of two different AK patients and examined the impact of naturally occurring intracellular bacteria within Acanthamoeba on the severity of corneal infections in rats. METHODOLOGY/PRINCIPAL FINDINGS: Household water and corneal swabs were collected from AK patients. Acanthamoeba strains and genotypes were confirmed by sequencing. Acanthamoeba isolates were assessed for the presence of intracellular bacteria using sequencing, fluorescence in situ hybridization (FISH), and electron microscopy. The viability of the bacteria in Acanthamoeba was assessed by labelling with alkyne-functionalized D-alanine (alkDala). Primary human macrophages were used to compare the intracellular survival and replication of the endosymbiotic Pseudomonas aeruginosa and a wild type strain. Eyes of rats were challenged intrastromally with Acanthamoeba containing or devoid of P. aeruginosa and evaluated for the clinical response. Domestic water and corneal swabs were positive for Acanthamoeba. Both strains belonged to genotype T4F. One of the Acanthamoeba isolates harboured P. aeruginosa which was seen throughout the Acanthamoeba's cytoplasm. It was metabolically active and could be seen undergoing binary fission. This motile strain was able to replicate in macrophage to a greater degree than strain PAO1 (p<0.05). Inoculation of Acanthamoeba containing the intracellular P. aeruginosa in rats eyes resulted in a severe keratitis with increased neutrophil response. Acanthamoeba alone induced milder keratitis. CONCLUSIONS/SIGNIFICANCE: Our findings indicate the presence of live intracellular bacteria in Acanthamoeba can increase the severity of acute keratitis in vivo. As P. aeruginosa is a common cause of keratitis, this may indicate the potential for these intracellular bacteria in Acanthamoeba to lead to severe polymicrobial keratitis.

Identification and quantification of Acanthamoeba spp. within seawater at four coastal lagoons on the east coast of Australia.

Acanthamoeba is an opportunistic free-living heterotrophic protist that is the most predominant amoeba in diverse ecological habitats. Acanthamoeba causes amoebic keratitis (AK), a painful and potentially blinding corneal infection. Major risk factors for AK have been linked to non-optimal contact lens hygiene practices and Acanthamoeba contamination of domestic and recreational water. This study investigated the incidence and seasonal variation of Acanthamoeba spp. within coastal lagoons located on the eastern coast of Australia and then examined the association between Acanthamoeba and water abiotic factors and bacterial species within the water. Water samples were collected from four intermittently closed and open lagoons (ICOLLs) (Wamberal, Terrigal, Avoca and Cockrone) every month between August 2019 to July 2020 except March and April. qPCR was used to target the Acanthamoeba 18S rRNA gene, validated by Sanger sequencing. Water abiotic factors were measured in situ using a multiprobe metre and 16S rRNA sequencing (V3-V4) was performed to characterise bacterial community composition. Network analysis was used to gauge putative associations between Acanthamoeba incidence and bacterial amplicon sequence variants (ASVs). Among 206 water samples analysed, 79 (38.3%) were Acanthamoeba positive and Acanthamoeba level was significantly higher in summer compared with winter, spring, or autumn (p = 0.008). More than 50% (23/45) water samples of Terrigal were positive for Acanthamoeba which is a highly urbanised area with extensive recreational activities while about 32% (16/49) samples were positive from Cockrone that is the least impacted lagoon by urban development. All sequenced strains belonged to the pathogenic genotype T4 clade except two which were of genotype clades T2 and T5. Water turbidity, temperature, intl1 gene concentration, and dissolved O2 were significantly associated with Acanthamoeba incidence (p < 0.05). The ASVs level of cyanobacteria, Pseudomonas spp., Candidatus spp., and marine bacteria of the Actinobacteria phylum and Acanthamoeba 18S rRNA genes were positively correlated (Pearson's r ≥ 0.14). The presence of Acanthamoeba spp. in all lagoons, except Wamberal, was associated with significant differences in the composition of bacterial communities (beta diversity). The results of this study suggest that coastal lagoons, particularly those in urbanised regions with extensive water recreational activities, may pose an elevated risk to human health due to the relatively high incidence of pathogenic Acanthamoeba in the summer. These findings underscore the importance of educating the public about the rare yet devastating impact of AK on vision and quality of life, highlighting the need for collaborative efforts between public health officials and educators to promote awareness and preventive measures, especially focusing lagoons residents and travellers.

Redefining the human corneal immune compartment using dynamic intravital imaging.

The healthy human cornea is a uniquely transparent sensory tissue where immune responses are tightly controlled to preserve vision. The cornea contains immune cells that are widely presumed to be intraepithelial dendritic cells (DCs). Corneal immune cells have diverse cellular morphologies and morphological alterations are used as a marker of inflammation and injury. Based on our imaging of corneal T cells in mice, we hypothesized that many human corneal immune cells commonly defined as DCs are intraepithelial lymphocytes (IELs). To investigate this, we developed functional in vivo confocal microscopy (Fun-IVCM) to investigate cell dynamics in the human corneal epithelium and stroma. We show that many immune cells resident in the healthy human cornea are T cells. These corneal IELs are characterized by rapid, persistent motility and interact with corneal DCs and sensory nerves. Imaging deeper into the corneal stroma, we show that crawling macrophages and rare motile T cells patrol the tissue. Furthermore, we identify altered immune cell behaviors in response to short-term contact lens wear (acute inflammatory stimulus), as well as in individuals with allergy (chronic inflammatory stimulus) that was modulated by therapeutic intervention. These findings redefine current understanding of immune cell subsets in the human cornea and reveal how resident corneal immune cells respond and adapt to chronic and acute stimuli.

Correction: Peguda et al. The Activity of Polyhomoarginine against Acanthamoeba castellanii. Biology 2022, 11, 1726.

In the original publication [...].

Human tear metabolites associated with nucleoside-signalling pathways in bacterial keratitis.

OBJECTIVE: The study aimed to profile and quantify tear metabolites associated with bacterial keratitis using both untargeted and targeted metabolomic platforms. METHODS: Untargeted metabolomic analysis using liquid-chromatography-Q Exactive-HF mass-spectrometry explored tear metabolites significantly associated with bacterial keratitis (n = 6) compared to healthy participants (n = 6). Differential statistics and principal component analysis determined meaningful metabolite differences between cases and controls. Purines and nucleosides were further quantified and compared between 15 cases and 15 controls in the targeted metabolomic platform using TSQ quantum access triple quadrupole mass spectrometry. Compound quantification was done by plotting the calibration curves and the difference in the compound levels was evaluated using the Wilcoxon rank-sum test. RESULTS: In the untargeted analysis, 49 tear metabolites (27 upregulated and 22 downregulated) were differentially expressed between cases and controls. The untargeted analysis indicated that the purine metabolism pathway was the most affected by bacterial keratitis. Metabolite quantification in the targeted analysis further confirmed the upregulation of xanthine (P = 0.02) and downregulation of adenine (P < 0.0001), adenosine (P < 0.0001) and cytidine (P < 0.0001) in the tears of participants with bacterial keratitis compared to that of healthy participants. CONCLUSIONS: Bacterial keratitis significantly changes the tear metabolite profile, including five major compound classes such as indoles, amino acids, nucleosides, carbohydrates, and steroids. This study also indicates that tear fluids can be used to map the metabolic pathways and uncover metabolic markers associated with bacterial keratitis. Conceivably, the inhibition of nucleoside synthesis may contribute to the pathophysiology of bacterial keratitis because nucleosides are required for maintaining cellular energy homeostasis and immune adaptability.

Acanthamoeba Keratitis Risk Factors for Daily Wear Contact Lens Users: A Case-Control Study.

PURPOSE: This study was designed to establish risk factors for the development of Acanthamoeba keratitis (AK) for daily disposable (DD) contact lens (CL) users compared with daily wear (DW) reusable lens users and for risks unique to DD users. This is important because, in many major economies, CL use is the principal cause of microbial keratitis, of which AK accounts for approximately 50% of cases with sight loss. Determining these AK risks informs practitioner advice and consumer behavior. DESIGN: Case-control study. PARTICIPANTS: Cases and controls were recruited from an Accident and Emergency Department serving South-East England. Cases were new CL users with AK recruited retrospectively from January 2011 to February 2013 and prospectively thereafter until August 2014. Controls were recruited prospectively from February 2014 to June 2015. METHODS: Analysis of a self-administered questionnaire. MAIN OUTCOME MEASURES: Independent risk factors and population attributable risk percentage (PAR%) for AK. RESULTS: A total of 83 AK cases and 122 controls were recruited; DD use was reported by 20 (24%) cases and 66 (54%) controls. In multivariable analyses adjusted for potential confounders, the odds of AK was higher for DW reusable soft lenses (odds ratio [OR], 3.84; 95% confidence interval [CI], 1.75-8.43) and rigid lenses (OR, 4.56; 95% CI, 1.03-20.19) than for DD lenses. Within the DD-using subset, AK was associated with the following modifiable risk factors: less frequent professional follow-up visits (OR, 10.12; 95% CI, 5.01-20.46); showering in lenses (OR, 3.29, 95% CI, 1.17-9.23); lens reuse (OR, 5.41; 95% CI, 1.55-18.89); and overnight wear (OR, 3.93; 95% CI, 1.15-13.46). The PAR% estimated that 30% to 62% of cases could be prevented by switching from reusable soft lens to DD lens use. CONCLUSIONS: Acanthamoeba keratitis risks are increased > threefold in DW reusable lens users versus DD lens use. Acanthamoeba keratitis risks for DD lens users can be minimized by adherence to safe use guidelines (no reuse, overnight wear, or contamination by water). Safe CL use can be improved by increasing the prominence of risk avoidance information from manufacturers and regulators. Because AK accounts for half of severe keratitis in CL users, these measures can be expected to have public health benefits.

The Activity of Polyhomoarginine against Acanthamoeba castellanii.

Arginine-rich peptides can have broad-spectrum anti-bacterial and anti-fungal activities. Polyhomoarginine consists of highly cationic residues which can act on the negatively charged microbial cell membranes. Acanthamoeba is a free-living protozoan known to cause a rare corneal infection which is difficult to diagnose and treat. This study evaluated the activity of the polyhomoarginines against Acanthamoeba castellanii. Acanthamoeba amoebicidal, amoebistatic, encystation and excystment assays were performed using protocols described in the literature. The activity of polyhomoarginines (PHAs) of different lengths (10 to 400 residues) was measured against the trophozoites and cysts of Acanthamoeba castellanii ATCC30868 in concentrations ranging from 0.93 µM to 15 µM. Data were represented as mean ± SE and analysed using one-way ANOVA. Overall, PHAs demonstrated good anti-acanthamoeba activity against both trophozoites and cysts. PHA 30 reduced the number of viable trophozoites by 99%, inhibited the formation of cysts by 96% and the emergence of trophozoites from cysts by 67% at 3.75 µM. PHA 10 was similarly active, but at a slightly higher concentration of 15 µM, reducing the numbers of viable trophozoites by 98%, inhibiting cyst formation by 84% and preventing the emergence of trophozoites from cysts by 99%. At their greatest anti-amoeba concentrations, PHA 10 gave only 8% haemolysis at 15 µM while PHA 30 gave <40 % haemolysis at 3.75 µM. Polyhomoarginine 10 showed excellent anti-amoebic activity against both forms of Acanthamoeba castellanii and was non-toxic at its most active concentrations. This implies that polyhomoarginines can be developed into a potential therapeutic agent for Acanthamoeba keratitis. However, there is a need to carry out further pre-clinical and then in vivo experiments in the AK animal model.

The Anti-Amoebic Activity of a Peptidomimetic against Acanthamoeba castellanii.

Acanthamoeba is a free-living protozoan known to cause keratitis most commonly, especially among contact lens wearers. Treatment of Acanthamoeba keratitis is challenging as Acanthamoeba can encyst from the active form, a trophozoite, into a hibernating cyst that is refractory to antibiotics and difficult to kill; therefore, there is a need for more effective anti-amoebic strategies. In this study, we have evaluated the anti-amoebic activity of the antimicrobial peptide mimic RK-758 against Acanthamoeba castellanii. RK-758 peptidomimetic was subjected to biological assays to investigate its amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects on A. castellanii. The anti-amoebic activity of the peptide mimic RK-758 was compared with chlorhexidine against the Acanthamoeba castellanii ATCC30868 and Acanthamoeba castellanii 044 (a clinical strain) with the concentrations of both ranging from 125 µM down to 7.81 µM. All experiments were performed in duplicate with three independent replicates. The data were represented as mean ± SE and analysed using a two-sample t-test and two-tailed distributions. A p < 0.05 was considered statistically significant. The peptidomimetic RK-758 had anti-Acanthamoeba activity against both trophozoites and cysts in a dose-dependent manner. The RK-758 had amoebicidal and growth inhibitory activities of ≥50% at a concentration between 125 µM and 15.6 µM against the trophozoites of both Acanthamoeba strains. Inhibitory effects on the cyst formation and trophozoite re-emergence from cysts were noted at similar concentrations. Chlorhexidine had 50% activity at 7.81 µM and above against the trophozoites and cysts of both strains. In the haemolysis assay, the RK-758 lysed horse RBCs at concentrations greater than 50 µM whereas lysis occurred at concentrations greater than 125 µM for the chlorhexidine. The peptidomimetic RK-758, therefore, has activity against both the trophozoite and cyst forms of Acanthamoeba and has the potential to be further developed as an anti-microbial agent against Acanthamoeba. RK-758 may also have use as an anti-amoebic disinfectant in contact lens solutions.

Assessment of genotypes, endosymbionts and clinical characteristics of Acanthamoeba recovered from ocular infection.

INTRODUCTION: Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified. AIMS: This retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations. METHODOLOGY: Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit (rns) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH). RESULTS: The mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis. Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively. CONCLUSIONS: Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK.

Acanthamoeba, an environmental phagocyte enhancing survival and transmission of human pathogens.

The opportunistic protist Acanthamoeba, which interacts with other microbes such as bacteria, fungi, and viruses, shows significant similarity in cellular and functional aspects to human macrophages. Intracellular survival of microbes in this microbivorous amoebal host may be a crucial step for initiation of infection in higher eukaryotic cells. Therefore, Acanthamoeba-microbe adaptations are considered an evolutionary model of macrophage-pathogen interactions. This paper reviews Acanthamoeba as an emerging human pathogen and different ecological interactions between Acanthamoeba and microbes that may serve as environmental training grounds and a genetic melting pot for the evolution, persistence, and transmission of potential human pathogens.

The Co-Creation of a Patient Information Leaflet for Patients With the Rare Eye Infection Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a rare but severe eye disease. A research engagement event, "The Cornea Day," in London, UK in 2013, identified the lack of credible information about AK and a need for practical day to day management strategies. Experiences of 15 AK patients attending The Cornea Day were distilled into a survey that was administered to a wider group of 76 patients, carers, researchers, and clinicians. A Patient Information Leaflet was cocreated and then represented to additional patients for final modification. The AK Patient Leaflet (revised 2019) is available in several languages and used globally.

Tissue resident memory T cells inhabit the deep human conjunctiva.

Mucosal linings of the body, including the conjunctiva, are enriched in tissue-resident memory T cells (TRMs) whose defining feature is their continual tissue protection that does not rely on migration to lymphoid organs to elicit immune responses. Hitherto, conjunctival TRMs have only been identified in the superficial epithelium. This work aims to develop a more complete understanding of the conjunctival immunological capacity by investigating the presence of TRMs within the deeper, more stable layers of the healthy human conjunctiva. Using immunofluorescence microscopy and antibodies against CD3, CD4, CD69 and HLA-DR on bulbar conjunctival biopsies obtained from 7 healthy adults (age range = 32-77 years; females = 4), we identified CD69+TRM subsets in all layers of the human conjunctiva: the superficial epithelium, the basal epithelium, the adenoid, and the fibrous layers. Interestingly, the adenoid layer showed significantly higher densities of both CD4 and CD8 TRMs when compared to the fibrous layer and conjunctival epithelia. Additionally, CD4 TRMs predominated significantly over CD8 TRMs in the adenoid layer. The abundance of deep conjunctival CD69+TRMs within the healthy human may suggest the presence of defence mechanisms capable of inducing long-term immunogenic memory. Understanding this spatial distribution of conjunctival CD69+TRMs is essential to improving mucosal vaccine design.

Ocular Distribution of the Renin-Angiotensin-Aldosterone System in the Context of the SARS-CoV-2 Pandemic.

The COVID-19 pandemic has resulted in an unprecedented impact on global health, economy, and way of life. SARS-CoV-2, the virus responsible for the disease, utilizes the ACE2 receptor found on host cells to mediate entry, replication, and infection. Numerous studies have elucidated the presence of many components of the renin-angiotensin-aldosterone system (RAAS) in the eye, including the ACE2 receptor. Considering this, and the anatomical vulnerability that the exposed ocular surface offers with its interconnectedness to the respiratory system, there is a theoretical risk of pathogen entry from the ocular route as well as the development of COVID-19-associated eye disease. Despite this, the actual epidemiological data demonstrates low ocular symptoms, possibly due to differing ACE2 receptor expression across age, ethnicity, and sex coupled with the protective properties of tears. We summarize the current literature on ocular RAAS with specific focus on the ACE2 receptor and its interplay with the SARS-CoV-2 virus.

Epidemiology, Microbiology, and Genetics of Contact Lens-Related and Non-Contact Lens-Related Infectious Keratitis.

ABSTRACT: Infectious keratitis is a rare but severe condition associated with a range of ocular and systemic predisposing conditions, including ocular trauma, prior surgery, surface disease, and contact lens (CL) wear. This review explores the epidemiology of infectious keratitis, specifically the differences in disease incidence and risk factors, causative organism profile and virulence characteristics and host microbiome, genetics, gene expression, proteomics, and metabolomic characteristics in CL-related and non-CL-related diseases. Differences exist in the epidemiology, demographics, causative organisms, and their virulence characteristics in CL-related and non-CL-related diseases, and there is less evidence to support differences between these groups of individuals in the ocular surface microbiome, genetics, and pathways of disease. Genetic variations, however, in the host immune profile are implicated in both the onset and severity of infectious keratitis in CL and non-CL wearers. As technologies in metabolomics, proteomics, and genomics improved to be better able to process small-volume samples from the ocular surface, there will be improved understanding of the interplay between the CL, ocular surface, host immune profile, and the microbial environment. This may result in a more personalized approach in the management of disease to reduce disease severity.