The Role of CCL Chemokines in Experimental Staphylococcus aureus Endophthalmitis.

Purpose: To test the hypothesis that (C-C motif) ligand 2 (CCL2) and CCL3 impact retinal function decline and inflammation during Staphylococcus aureus endophthalmitis. Methods: Experimental endophthalmitis was initiated by intravitreal injection of 5000 colony-forming units of S. aureus into the eyes of C57BL/6J, CCL2-/-, or CCL3-/- mice. At 12 and 24 hours post-infection, retinal function, bacterial load, and myeloperoxidase levels were quantified. Results: During S. aureus endophthalmitis, we observed a significant improvement in retinal function in CCL2-/- mice relative to C57BL/6J mice at 12 hours but not at 24 hours. In CCL3-/- mice, retinal function was significantly improved relative to C57BL/6J mice at 12 and 24 hours. The absence of CCL2 did not alter intraocular S. aureus intraocular concentrations. However, CCL3-/- mice had significantly lower intraocular S. aureus at 12 hours but not at 24 hours. No difference in myeloperoxidase levels was observed between C57BL/6J and CCL2-/- mice at 12 hours. CCL3-/- mice had almost no myeloperoxidase at 12 hours. At 24 hours, increased myeloperoxidase was observed in CCL2-/- and CCL3-/- mice relative to C57BL/6J mice. Conclusions: Although the absence of CCL2 resulted in improved retinal function retention at 12 hours, CCL3 deficiency resulted in improved retinal function at 12 and 24 hours. CCL3 deficiency, but not CCL2 deficiency, resulted in almost no inflammation at 12 hours. However, at 24 hours, the absence of CCL2 or CCL3 resulted in significantly increased inflammation. These results suggest that, although both CCL2 and CCL3 impact intraocular infection outcomes, CCL3 may have a more significant impact in S. aureus endophthalmitis.

Complement Cascade 8 - Alpha and Calpain-2 in Extracellular Vesicles of Human Vitreous as Biomarkers of Infectious Endophthalmitis.

Purpose: Extracellular vesicles (EVs) are messenger pigeons of the cells that communicate about cellular microenvironment. In this study, we evaluated the expression of C8α and calpain-2 in EVs from vitreous of patients with bacterial endophthalmitis to assess its utility as a diagnostic marker. Methods: EVs were isolated from vitreous of patients with bacterial endophthalmitis (culture positive and culture negative) and noninfectious control by exosome isolation reagent and characterized, and the levels of C8α and calpain-2 was assessed by enzyme-linked immunosorbent assay in isolated EVs and direct vitreous. The receiver operating characteristic curve was generated to assess the diagnostic performance. Results: Scanning electron microscopy (SEM) and dynamic light scattering (DLS) confirmed the presence of EVs having a diameter (nm) of 275.2 ± 93, 92 ± 22, and 77.28 ± 12 in culture-positive (CP), culture-negative (CN), and control respectively. The expression level (ng/mL) of C8α in the EVs obtained from CP was 144 ± 22 and CN was 31.2 ± 9.8, which was significantly higher (P < 0.01) than control 3.7 ± 2.4. Interestingly, C8α is not expressed directly in the vitreous of CN and controls. Calpain-2 was significantly downregulated (P ≤ 0.0001) in CP (0.94 ± 0.16) and CN (0.70 ± 0.14) than control. The sensitivity and specificity of 1 for C8α and calpain-2 in the EVs implied that its diagnostic accuracy was significant. Conclusions: This study showed that the EV proteins C8α and calpain-2 could be suitable diagnostic markers for endophthalmitis. However, the presence of C8α in the EVs of CN samples but not in direct vitreous promises EVs as the future of diagnostics. Translational Relevance: Expression levels of EV-calpain-2 and EV-C8α could diagnose CN bacterial endophthalmitis.

Aquaporin 5-in Extracellular Vesicles of Human Vitreous as a Potential Marker for Fungal Endophthalmitis.

PURPOSE: Extracellular vesicles (EVs) are lipid-bilayered nanoparticles that play an important role in cellular cross-talk, and as received attention for their role as diseases biomarker. Aquaporin-5 (AQP5) is a small integral membrane protein that help in the migration of cells, proliferation, and invasion. However, the association of AQP5 with fungal diseases is still unknown. The aim of this study was to evaluate the expression of AQP5 in EVs (EV-AQP5) extracted from the vitreous of patients with Fungal Endophthalmitis (FE). METHODS: Vitreous fluid was collected from 20 patients clinically suspected as FE, 10 patients from non-infectious conditions, and 10 patients with bacterial endophthalmitis as controls. EVs were isolated from human vitreous and characterized by dynamic light scattering, and scanning electron microscopy. Human Aquaporin-5 levels were evaluated using a commercial ELISA Kit. The Receiver Operating Characteristic (ROC) curves and its significance were correlated with microbiology data. RESULTS: Isolated EVs size were approx.250-380 nm in diameter. The measured levels of EV-AQP5 resulted significantly higher in FE patients (mean=216±15pg/ml; 95% confidence interval (CI): 182-250) in comparison to controls (mean=130±12pg/ml; 95%CI: 111-166)(p = .001). However, AQP5 levels in EVs derived from culture-proven bacteria patients were insignificant compared to controls (mean=169±4 pg/ml; 95%CI: 161-177). ROC curve was used to define the optimal cut-off level of the test at 180 pg/ml with an AUC of 98% (95%CI: 95-100) (p = .03), with a sensitivity of 100% and specificity of 90%. Additionally, the AQP5 level in EVs derived from culture-negative vitreous was above the threshold value (200 ± 10 pg/ml (95%CI: 180-230) in comparison to the control group (p < .001) However, no significant association was found between age or visual acuity and the level of AQP5 in FE. CONCLUSION: Our results reveal that the vitreous EV-AQP5 levels can aid in differentiating FE from non-infectious retinal conditions, mainly when the cultures are negative.

Proteomic landscape of extracellular vesicles in human retinal cells infected with Staphylococcus aureus and Pseudomonas aeruginosa: Role in endophthalmitis.

Extracellular Vesicles (EVs) have evolved as a promising entity for developing diagnostic and therapeutic biomarkers. We profiled global EV proteome of EVs from Human retinal cells (ARPE-19) infected with S. aureus and P. aeruginosa. EVs were isolated by ultracentrifugation and subjected to LC-MS/MS for proteome analysis. In S. aureus infection, sequest identified 864 proteins, of which 81 were differentially expressed in comparison to control. Similarly, in P. aeruginosa infection, of 516 proteins identified, 86 were differentially expressed. Additionally, 38 proteins were exclusive to infected sets. KEGG and Gene Ontology revealed crucial dysregulated pathways involving proteins such as complement cascades, annexins and calpain-2, all playing major role in the pathogenesis of the disease. This study provides insight into the global EV proteome of S. aureus and P. aeruginosa endophthalmitis with their functional correlation and distinctive pattern of expression. Calpain-2 and C8a are attractive biomarkers for bacterial endophthalmitis.

Proteomic profiling of extracellular vesicles derived from ARPE-19 cells challenged with Aspergillus flavus and Candida albicans: application in fungal endophthalmitis.

Extracellular vesicles (EVs) are nano-sized-particles that play an important role in cellular cross-talk. The aim of this study was to understand the proteomic cargo of EVs, released by Retinal Pigment Epithelial (RPE) cells challenged with Candida albicans (C-CA) and Aspergillus flavus (C-AF). EVs were isolated from culture supernatant of retinal cells infected with fungal pathogens and characterized by dynamic light scattering, SEM, and western blot. EV proteome was then evaluated by mass spectrometry (LC-MS/MS). Isolated EVs were approximately 120-150 nm and higher in number in infected group compared to control. Proteomic profiling of EVs from infected cells, showed a total of 419 and 254 differentially expressed proteins, of which 218 were upregulated in C-CA group and 81 proteins were upregulated in C-AF group. Gene ontology revealed majority of proteins associated with transport, cell migration, and in activation of innate immune response. Proteins identified were annexins, calpain, and Sorcin proteins. Additionally, KEGG analysis unveiled involvement of MAPK, HIF-1, and PI3K-AKT signalling pathways. Proteomic results indicate that EVs cargo derived from fungal-infected retinal cells can activate immune signalling pathways and might contribute to the pathogenesis of endophthalmitis, indicating the potential use of EVs as theranostic marker for management of fungal infections.

Proteomic profiling of aspergillus flavus endophthalmitis derived extracellular vesicles in an in-vivo murine model.

Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell communication, and are involved in potential pathological and physiological cellular processes. The aim of this study was to understand the proteomic cargo of these vesicles, in a murine model of Aspergillus flavus (AF) endophthalmitis. EVs were isolated from A. flavus infected C57BL/6 mice eyes by differential ultracentrifugation at 24 h post infection (p.i) and isolated EVs were characterized by Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), Exocet assay, and western blot. Proteomic profiling of EVs was then evaluated by mass spectrometry (LC-MS/MS) and compared it with control uninfected mice. The average size of the EVs were 180-280 nm by DLS and the number of EVs increased to 1.55 × 1010 in infected mice in comparison to EVs from uninfected eye (1.24 × 109). Western blot was positive for CD9, CD63, and CD81 confirming the presence of EVs. LC-MS/MS analysis, identified 81 differentially expressed proteins, of these 22 were up-regulated and 59 were down-regulated. Gene Ontology (GO) analysis revealed enrichment of lipid metabolism, protein complex binding, and transferase activity, and the proteins associated were Aquaporin-5, CD177 antigen, Solute carrier family-25, and Calcium/calmodulin-dependent protein kinase. Additionally, KEGG pathway analysis indicated that glucagon signalling, metabolic, and PPAR signalling pathway were significantly associated with EVs from A. flavus infected mice eyes. The protein cargo in EVs from A. flavus endophthalmitis provides new insights into the pathogenesis of fungal endophthalmitis and validation of these proteins can serve as diagnostic and/or prognostic biomarkers for patients with a clinical suspicion of fungal endophthalmitis. LAY SUMMARY: EVs play an important role in cell communication. In our study proteomic profiling of EVs isolated from A. flavus infected mice provided new insights into the understanding of the pathobiology of A. flavus endophthalmitis and validation of these proteins can serve as biomarkers.

Characterization and proteome profiling of extracellular vesicles in a murine model of Staphylococcus aureus endophthalmitis.

Endophthalmitis is a vision-threatening complication of intraocular surgery or penetrating injury of which Staphylococcus aureus is an important etiological agent. Extracellular vesicles (EVs) hold a tremendous possibility for developing diagnostic and therapeutic biomarkers due to their role in the pathogenesis of various infections. The aim of this study was to characterise the protein cargo of EVs, isolated from a murine (C57BL/6) model of S. aureus endophthalmitis by LC-MS/MS. Contralateral eye injected with sterile media served as control and both eyes were enucleated after 24 h, followed by extraction of EVs by ultracentrifugation. EVs were characterized by DLS and western blotting with tetraspanin markers, CD9 and CD81 and quantified by ExoCet quantification kit. Proteomic analysis identified 1964 proteins (FDR ≤ 0.01) in EVs from infected mice eyes, of which 40 proteins varied significantly in their amounts in comparison to EVs obtained from control eyeballs (P-value ≤ 0.05). The results of this study provide insight into the global EV proteome of S. aureus endophthalmitis with their functional correlation and differential protein amounts between infected and control set. Annexin A5, cathepsin D and C5a play a pivotal role in disease pathogenesis and could possibly play a role as a prognostic marker in endophthalmitis.

Proteomic profiling of exosomes in a mouse model of Candida albicans endophthalmitis.

Exosomes play pivotal roles in intercellular communication, and pathophysiological functions. In this study, we aimed to understand the role of exosomal proteome derived from C. albicans infected mice (C57BL/6) eyeball. Exosomes were characterized by Dynamic Light Scattering and Western blot, quantified and subjected to LC-MS/MS and cytokine quantification by ELISA. The average size of exosomes was 170-200 nm with number of exosomes amounted to 1.42 × 1010 in infected set compared to control (1.24 × 109). Western blot was positive for CD9, CD63 and CD81 confirming the presence of exosomes. IL-6, IL1ß, TNF-α, and IFN-γ levels were significantly elevated in infected eye at 72 h.p.i. Proteomic analysis identified 42 differentially expressed proteins, of these 37 were upregulated and 5 were downregulated. Gene Ontology (GO) revealed enrichment of cell adhesion, cytoskeleton organisation, and cellular response proteins such as aquaporin-5, gasdermin-A, CD5 antigen-like, Catenin, V-ATPase, and vesicle associated protein. Additionally, KEGG pathway analysis indicated the association of metabolic and carbon signalling pathways with exosomes from C. albicans infected eye. The protein cargo in exosomes released during endophthalmitis with C. albicans seems to play a unique role in the pathogenesis of the disease and further validations with larger cohort of patients is required to confirm them as biomarkers.

Susceptibility of bacterial endophthalmitis isolates to vancomycin, ceftazidime, and amikacin.

Bacterial endophthalmitis is a rare intraocular infection, and prompt administration of intravitreal antibiotics is crucial for preventing severe vision loss. The retrospective study is to investigate the in vitro susceptibility to the antibiotics vancomycin, amikacin, and ceftazidime of bacterial endophthalmitis isolates in specimens at a tertiary referral center from January 1996 to April 2019 in Taiwan. Overall, 450 (49.9%) isolates were Gram positive, 447 (49.6%) were Gram negative, and 4 (0.4%) were Gram variable. In Gram-positive isolates, coagulase-negative staphylococci were the most commonly cultured bacteria (158, 35.1%), followed by Streptococci (100, 22.2%), Enterococci (75, 16.7%), and Staphylococcus aureus (70, 15.6%). In Gram-negative isolates, they were Klebsiella pneumoniae (166, 37.1%) and Pseudomonas aeruginosa (131, 29.3%). All Gram-positive organisms were susceptible to vancomycin, with the exception of one Enterococcus faecium isolate (1/450, 0.2%). Of the Gram-negative isolates, 96.9% and 93.7% were susceptible to ceftazidime and amikacin, respectively. Nine isolates (9/447, 2.0%) were multidrug-resistant Gram-negative bacteria, comprising K. pneumoniae (4/164, 2.4%), Acinetobacter baumannii (2/3, 67%), and Stenotrophomonas maltophilia (3/18, 17%). In conclusion, in vitro susceptibility testing revealed that vancomycin remains the suitable antibiotic treatment for Gram-positive endophthalmitis. Ceftazidime and amikacin provide approximately the same degree of Gram-negative coverage. Multidrug-resistant bacterial endophthalmitis was uncommon.

Genomic and phenotypic diversity of Enterococcus faecalis isolated from endophthalmitis.

Enterococcus faecalis are hospital-associated opportunistic pathogens and also causative agents of post-operative endophthalmitis. Patients with enterococcal endophthalmitis often have poor visual outcomes, despite appropriate antibiotic therapy. Here we investigated the genomic and phenotypic characteristics of E. faecalis isolates collected from 13 patients treated at the University of Pittsburgh Medical Center Eye Center over 19 years. Comparative genomic analysis indicated that patients were infected with E. faecalis belonging to diverse multi-locus sequence types (STs) and resembled E. faecalis sampled from clinical, commensal, and environmental sources. We identified known E. faecalis virulence factors and antibiotic resistance genes in each genome, including genes conferring resistance to aminoglycosides, erythromycin, and tetracyclines. We assessed all isolates for their cytolysin production, biofilm formation, and antibiotic susceptibility, and observed phenotypic differences between isolates. Fluoroquinolone and cephalosporin susceptibilities were particularly variable between isolates, as were biofilm formation and cytolysin production. In addition, we found evidence of E. faecalis adaptation during recurrent endophthalmitis by identifying genetic variants that arose in sequential isolates sampled over eight months from the same patient. We identified a mutation in the DNA mismatch repair gene mutS that was associated with an increased rate of spontaneous mutation in the final isolate from the patient. Overall this study documents the genomic and phenotypic variability among E. faecalis causing endophthalmitis, as well as possible adaptive mechanisms underlying bacterial persistence during recurrent ocular infection.

Machine Learning Approach for Intraocular Disease Prediction Based on Aqueous Humor Immune Mediator Profiles.

PURPOSE: Various immune mediators have crucial roles in the pathogenesis of intraocular diseases. Machine learning can be used to automatically select and weigh various predictors to develop models maximizing predictive power. However, these techniques have not yet been applied extensively in studies focused on intraocular diseases. We evaluated whether 5 machine learning algorithms applied to the data of immune-mediator levels in aqueous humor can predict the actual diagnoses of 17 selected intraocular diseases and identified which immune mediators drive the predictive power of a machine learning model. DESIGN: Cross-sectional study. PARTICIPANTS: Five hundred twelve eyes with diagnoses from among 17 intraocular diseases. METHODS: Aqueous humor samples were collected, and the concentrations of 28 immune mediators were determined using a cytometric bead array. Each immune mediator was ranked according to its importance using 5 machine learning algorithms. Stratified k-fold cross-validation was used in evaluation of algorithms with the dataset divided into training and test datasets. MAIN OUTCOME MEASURES: The algorithms were evaluated in terms of precision, recall, accuracy, F-score, area under the receiver operating characteristic curve, area under the precision-recall curve, and mean decrease in Gini index. RESULTS: Among the 5 machine learning models, random forest (RF) yielded the highest classification accuracy in multiclass differentiation of 17 intraocular diseases. The RF prediction models for vitreoretinal lymphoma, acute retinal necrosis, endophthalmitis, rhegmatogenous retinal detachment, and primary open-angle glaucoma achieved the highest classification accuracy, precision, and recall. Random forest recognized vitreoretinal lymphoma, acute retinal necrosis, endophthalmitis, rhegmatogenous retinal detachment, and primary open-angle glaucoma with the top 5 F-scores. The 3 highest-ranking relevant immune mediators were interleukin (IL)-10, interferon-γ-inducible protein (IP)-10, and angiogenin for prediction of vitreoretinal lymphoma; monokine induced by interferon γ, interferon γ, and IP-10 for acute retinal necrosis; and IL-6, granulocyte colony-stimulating factor, and IL-8 for endophthalmitis. CONCLUSIONS: Random forest algorithms based on 28 immune mediators in aqueous humor successfully predicted the diagnosis of vitreoretinal lymphoma, acute retinal necrosis, and endophthalmitis. Overall, the findings of the present study contribute to increased knowledge on new biomarkers that potentially can facilitate diagnosis of intraocular diseases in the future.

Venom Ophthalmia and Ocular Complications Caused by Snake Venom.

Little is known about the detailed clinical description, pathophysiology, and efficacy of treatments for ocular envenoming (venom ophthalmia) caused by venom of the spitting elapid and other snakes, as well as ocular complications caused by snake venom injection. In this paper, we review clinical information of case reports regarding venom ophthalmia and snake venom injection with associated ocular injuries in Asia, Africa, and the United States. We also review the literature of snake venom such as their compositions, properties, and toxic effects. Based on the available clinical information and animal studies, we further discuss possible mechanisms of venom ophthalmia derived from two different routes (Duvernoy's gland in the mouth and nuchal gland in the dorsal neck) and the pathophysiology of snake venom injection induced ocular complications, including corneal edema, corneal erosion, cataract, ocular inflammation, retinal hemorrhage, acute angle closure glaucoma, as well as ptosis, diplopia, and photophobia. Finally, we discuss the appropriate first aid and novel strategies for treating venom ophthalmia and snake envenoming.

Association of the 1q25 Diabetes-Specific Coronary Heart Disease Locus With Alterations of the γ-Glutamyl Cycle and Increased Methylglyoxal Levels in Endothelial Cells.

A chromosome 1q25 variant (rs10911021) has been associated with coronary heart disease (CHD) in type 2 diabetes. In human umbilical vein endothelial cells (HUVECs), the risk allele "C" is associated with lower expression of the adjacent gene GLUL encoding glutamine synthase, converting glutamic acid to glutamine. To further investigate the mechanisms through which this locus affects CHD risk, we measured 35 intracellular metabolites involved in glutamic acid metabolism and the γ-glutamyl cycle in 62 HUVEC strains carrying different rs10911021 genotypes. Eight metabolites were positively associated with the risk allele (17-58% increase/allele copy, P = 0.046-0.002), including five γ-glutamyl amino acids, ß-citryl-glutamate, N-acetyl-aspartyl-glutamate, and ophthalmate-a marker of γ-glutamyl cycle malfunction. Consistent with these findings, the risk allele was also associated with decreased glutathione-to-glutamate ratio (-9%, P = 0.012), decreased S-lactoylglutathione (-41%, P = 0.019), and reduced detoxification of the atherogenic compound methylglyoxal (+54%, P = 0.008). GLUL downregulation by shRNA caused a 40% increase in the methylglyoxal level, which was completely prevented by glutamine supplementation. In summary, we have identified intracellular metabolic traits associated with the 1q25 risk allele in HUVECs, including impairments of the γ-glutamyl cycle and methylglyoxal detoxification. Glutamine supplementation abolishes the latter abnormality, suggesting that such treatment may prevent CHD in 1q25 risk allele carriers.

Aspergillus endophthalmitis: Potential role for vitreous galactomannan testing?

Eye damage during invasive aspergillosis is rarely described and biological diagnosis remains challenging. Here we report the case of a heart transplant recipient with ocular aspergillosis complicating disseminated aspergillosis. Although voriconazole was rapidly given, a decrease in visual acuity of the right eye was consistent with endophthalmitis, resulting in an emergency vitrectomy. The diagnosis was rapidly confirmed: laboratory results showed the presence of Aspergillus fumigatus in a vitreous sample. A series of systemic antifungal medications (liposomal amphotericin B, caspofungin, and voriconazole), several liposomal amphotericin B ocular injections, and pars plana vitrectomy resulted in a limited positive clinical outcome. Interestingly although standard mycological follow-up procedures were negative, Aspergillus antigen testing gave an index of 5.92 on vitreous humour, thus a new intraocular injection of liposomal amphotericin B was performed and voriconazole reinitiated. Ten other vitreous samples from patients without fungal infections were also tested, all showing indexes below 0.25. Although larger studies are needed, this case illustrates that galactomannan testing of vitreous humour could be useful for the diagnosis of fungal endophthalmitis if these data are confirmed in other patients, in particular, if standard mycology is negative and PCR is not available.

Anti-microbial Effect and in Vivo Ocular Delivery of Ciprofloxacin-loaded Liposome through Rabbit's Eye.

Purpose: The main purpose of this study was to increase the concentration and bioavailability of Ciprofloxacin (CPX) in the rabbit eye by liposomal formulation. Methods: CPX- loaded liposomes with and without Carbomer 934 (carbomer) were prepared by a thin-layer hydration method. Liposomal formulations after evaluation for characters such as particle size and entrapment efficiency were used in in-vivo experimental for installation into the rabbit's eyes. This experimental study consisted of 10 rabbits divided into two groups. Group 1 (liposomes without coating) and group 2 (carbomer coated liposomes) received one drop per h of liposomes consists of 0.3% CPX in the right eye and commercial CPX eye drop in the left eye until 6 h. Aqueous humor and vitreous samples were collected from all rabbits at the baseline, 1, 3 and 6 h and the drug concentration determined by high pressure liquid chromatography (HPLC). On the other hand, minimum inhibitor concentration (MIC) and minimum bactericidal concentration (MBC) of CPX-loaded in liposomes were determined. Results: liposomal formulations increased ocular bioavailability of CPX around four-folds compared with a commercial CPX eye drop. The increase in the ocular bioavailability may be effective and help to treat bacterial endophthalmitis as well as can be used in prophylaxis of post-operative endophthalmitis. Conclusion: The concentrations of CPX on the aqueous humor and vitreous after liposomes application were more than MIC of CPX against pseudomonas auroginosa and staphylococcus aurous but for commercial eye drop was less than MIC. Therefore liposomes modified the pharmacokinetics of CPX and improved pharmacodynamics property.

Povidone-iodine pharmacokinetics and study design.

Dr. Gnanasekaran et al. reported the bactericidal activity of various concentrations of povidone iodine (PI) solution in an agar plate experiment of respiratory flora. The study design and the pharmacokinetic properties of PI solution ensured that dilute PI would not be effective in this study. These results may not replicate the typical clinical situation and are significantly different than a previously reported agar plate experiment, again owing to subtle but very significant differences in methodology.

Vitreous D-Lactate Levels as a Biomarker in the Diagnosis of Presumed Infectious Culture Negative Endophthalmitis.

Purpose: Microbiological investigations of vitreous fluid have often failed to detect the causative agent in infectious endophthalmitis resulting in a clinical dilemma. D-Lactate is a byproduct of bacterial metabolism, and its accumulation in sterile body fluids indicates bacterial infection. The aim of the study was to evaluate the measurement of vitreous fluid D-lactate for the diagnosis of infectious endophthalmitis and to define an optimal D-lactate concentration for the differentiation from non-infectious samples.Methods: Vitreous samples of 41 patients clinically diagnosed as endophthalmitis and 20 patients with non-infectious disorders, as controls, between October 2018 and February 2019 were included in the study. D-lactate levels were determined by a D-lactate colorimetric assay kit (MAK058 Sigma-Aldrich) and the receiver operating characteristic curves (ROC) of D-lactate were calculated. The clinical finding of D-lactate production in bacterial endophthalmitis was also verified in a mouse model of bacterial endophthalmitis.Results: Of the 41 patients included in the infectious group, 25 had culture-positive infections of which 13/25 were gram-positive organisms and 12/25 grew gram-negative bacilli. Based on the ROC curve, the sensitivity of D-lactate was found to be 80% and specificity 100% and a cut-off value of above 47.06 ng/µl for D-lactate was defined as positive or true infectious in vitreous samples for diagnosis of endophthalmitis. In-vivo, a mouse model of bacterial endophthalmitis showed the significant production of D-lactate levels in retina and vitreous. Interestingly the levels were elevated in Gram-negative infections compared to Gram-positive bacterial endophthalmitis.Conclusion: Our clinical and in-vivo mouse model data showed that vitreous fluid D-lactate could be used as a bacterial-specific biomarker in the diagnosis of most infectious endophthalmitis and could be implemented for the evaluation of treatment success.

Aqueous level abatement profiles of intracameral antibiotics: A comparative mathematical model of moxifloxacin, cefuroxime, and vancomycin with determination of relative efficacies.

PURPOSE: To create a model of the abatement profiles of the three most commonly employed endophthalmitis prophylaxis intracameral (IC) antibiotics-cefuroxime, vancomycin, and moxifloxacin-to enable comparison of their durations of efficacy against common endophthalmitis pathogens. SETTINGS: Humber River Hospital and The Eye Foundation of Canada, Toronto, Ontario, the University of Toronto, Ontario, and McGill University, Montreal, Quebec, Canada. DESIGN: Literature review, as well as review of our clinical experience with 4797 consecutive cases with IC vancomycin, followed by 9185 consecutive cases with IC moxifloxacin. METHODS: A detailed review of the prophylactic antibiotic literature was performed. Exponential decay models of the selected IC antibiotics were updated from previous work by the study authors with decay constants adjusted to agree with the available published objective data. RESULTS: The graphs generated by the study data demonstrate the relative duration of IC bactericidal activity of moxifloxacin, cefuroxime, and vancomycin. They suggest that at present, IC moxifloxacin, when administered in appropriate doses, is the most effective agent in preventing postoperative endophthalmitis. Unlike vancomycin and cefuroxime, bacterial resistance to moxifloxacin is dose-dependent, and it is overcome in the vast majority of cases with doses that can safely be achieved intracamerally. The graphs can serve as a useful tool to assess the expected efficacy of each antibiotic in reference to local pathogen resistances. CONCLUSION: The model shows IC moxifloxacin, cefuroxime, and vancomycin durations of bactericidal efficacy post-cataract surgery, which correlate well with the published objective data.

S-layer Impacts the Virulence of Bacillus in Endophthalmitis.

Purpose: Bacillus causes a sight-threating infection of the posterior segment of the eye. The robust intraocular inflammatory response in this disease is likely activated via host innate receptor interactions with components of the Bacillus cell envelope. S-layer proteins (SLPs) of some Gram-positive pathogens contribute to the pathogenesis of certain infections. The potential contributions of SLPs in eye infection pathogenesis have not been considered. Here, we explored the role of a Bacillus SLP (SlpA) in endophthalmitis pathogenesis. Methods: The phenotypes and infectivity of wild-type (WT) and S-layer deficient (ΔslpA) Bacillus thuringiensis were compared. Experimental endophthalmitis was induced in C57BL/6J mice by intravitreally injecting 100-CFU WT or ΔslpA B. thuringiensis. Infected eyes were analyzed by bacterial counts, retinal function analysis, histology, and inflammatory cell influx. SLP-induced inflammation was also analyzed in vitro. Muller cells (MIO-M1) were treated with purified SLP. Nuclear factor-κB (NF-κB) DNA binding was measured by ELISA and expression of proinflammatory mediators from Muller cells was measured by RT-qPCR. Results: Tested phenotypes of WT and ΔslpA B. thuringiensis were similar, with the exception of absence of the S-layer in the ΔslpA mutant. Intraocular growth of WT and ΔslpA B. thuringiensis was also similar. However, eyes infected with the ΔslpA mutant had significantly reduced inflammatory cell influx, less inflammatory damage to the eyes, and significant retention of retinal function compared with WT-infected eyes. SLP was also a potent stimulator of the NF-κB pathway and induced the expression of proinflammatory mediators (IL6, TNFα, CCL2, and CXCL-1) in human retinal Muller cells. Conclusions: Taken together, our results suggest that SlpA contributes to the pathogenesis of Bacillus endophthalmitis, potentially by triggering innate inflammatory pathways in the retina.