Rapid NETosis Is an Effector Mechanism to Combat Ocular Herpes Infection.

Purpose: Neutrophils are known mediators of innate immunity, yet their effector function in herpesvirus infections remains poorly understood. Here, we elucidate the mechanistic action and pivotal role of neutrophil extracellular traps (NETs) during herpes simplex virus type 1 (HSV-1) ocular infection. Methods: Neutrophils were collected from mice for HSV-1 infection, fluorescence imaging, and immunoblotting assay. Tear samples from healthy subjects and patients with HSV-1 and mice were collected at L. V. Prasad Eye Institute, India, and at the University of Illinois, USA, respectively. For the in vivo study, C57BL/6 mice as well as diversity outbred mice were infected with HSV-1 (McKrae strain) followed by tear fluid collection at various time points (0-10 days). Samples were used for Flow cytometry, ELISA, and immunofluorescence assay. Human transcriptomic profile of keratitis dataset was used evaluate NETosis signaling pathways. We also performed neutrophil depletion studies. Results: Our data revealed a discernible temporal NET formation (NETosis) predominantly in the infected eye, across normal and diversity outbred murine models and human cases of HSV-1 infection. HSV-1 instigates swift NETosis governed by caspase-1 activation and myeloperoxidase secretion. Distinct accumulations of neutrophils, remaining unengaged in NET release in the contralateral eye post-infection, hinting at a proactive defensive posture in the uninfected eye. Moreover, neutrophil depletion accentuated ocular pathology, augmented viral load, and escalated disease scores, substantiating the protective effects of NETs in curtailing viral replication. Conclusions: Our report uncovers a previously unexplored mechanism of NETosis through pro-inflammatory cell death in response to ocular HSV-1 infection, and HPSE up-regulation, identifying new avenues for future studies.

Exploring Heparanase Levels in Tears: Insights From Herpes Simplex Virus-1 Keratitis Patients and Animal Studies.

Purpose: Heparanase (HPSE) cleaves heparan sulfate proteoglycans during herpes simplex virus-1 (HSV-1) infection, aiding in viral egress and disease progression. Its action has been well established in in vitro and in vivo models, but its relevance in human patients remains unclear. This study aimed to specifically evaluate tear HPSE levels of patients with herpes simplex keratitis (HSK) and to correlate these findings with a commonly used murine model. Methods: Tear samples from patient and mice samples were collected at LV Prasad Eye Institute, Hyderabad, India, and at the University of Illinois, Chicago, IL, respectively. Tears were collected from HSV-1 patients, bacterial/fungal keratitis cases, and healthy individuals. For in vivo study, C57BL/6 mice were infected with HSV-1 (McKrae strain) followed by tear fluid collection at various time points (0-10 days). Results: The HSV-1, bacterial keratitis, fungal keratitis, and healthy control groups each had 30 patients. There was a significant difference in HPSE expression in the HSV-1 infected eyes (1.55 ± 0.19 units/mL) compared to HSV-1 contralateral eyes (1.23 ± 0.13 units/mL; P = 0.82), bacterial keratitis eyes (0.87 ± 0.15 units/mL; P = 0.0078), fungal keratitis eyes (0.64 ± 0.09 units/mL; P < 0.00001), and normal controls (0.53 ± 0.06 units/mL; P < 0.00001). C57BL/6 mice tear HPSE expression in infected eyes was 0.66 to 5.57 ng heparan sulfate (HS) removed per minute when compared to non-infected eye (range, 0.70-3.67 ng HS removed per minute). Conclusions: To the best of our knowledge, this study is the first to report elevated HPSE levels in the tears of patients with different forms of HSV-1 keratitis, and it confirms similar findings in a murine model, providing a valuable basis for future in vivo and clinical research on HSV-1 ocular infection.

Evaluation of Vitreous Galactomannan and (1, 3) ß-D-Glucan Levels in the Diagnosis of Fungal Endophthalmitis in Southern India.

PURPOSE: To evaluate vitreous Galactomannan(GM) and 1,3 ß-D-Glucan (BDG) levels in the diagnosis of fungal endophthalmitis, with emphasis on culture-negative cases. METHODS: Vitreous from 31 clinically suspected fungal endophthalmitis patients and 11 controls were evaluated for GM and BDG using ELISA Kits. The Receiver Operating Characteristic (ROC) curves and diagnostic significance was calculated. RESULTS: The median vitreous GM in culture-positive (60.83pg/ml) and culture-negative (59.9pg/ml) samples were higher than the (51.2pg/ml) control group. The median vitreous BDG in culture-positive (1.47pg/ml) and culture-negative (1.52pg/ml) samples were also similar, and higher than the control group (1.18pg/ml). ROC analysis showed that at a cut-off of 51.35pg/ml, the sensitivity and specificity for GM were 0.88 and 0.73.Similarly, for BDG at a cut-off of 1.18pg/ml, the sensitivity and specificity were 0.94 and 0.82 respectively. CONCLUSION: Vitreous GM and BDG above the indicated threshold level could suggest a fungal infection, even when cultures are negative.

Retrobulbar Hyaluronidase Injection: Does It Reach the Eye?

PURPOSE: To determine if retrobulbar injection of hyaluronidase reaches the vitreous cavity, and to determine its concentration in the vitreous. METHODS: Prospective case-control study. Patients undergoing evisceration with implant for noninfective blind eyes were enrolled in the study. Before the evisceration, a retrobulbar injection of 3,000 IU of hyaluronidase (2 ml) was injected. Time from injection to in vivo sampling of posterior vitreous was noted. Vitreous samples from controls were obtained from patients undergoing vitrectomy for retinal detachment or diabetic retinopathy. Concentration of hyaluronidase was assessed in all 30 samples. An ELISA-based microtiter-technique was used to evaluate the activity of hyaluronidase by an avidin-peroxidase-based procedure using an ELISA reader. Incubations were carried out at room temperature and at 37°C. All the samples were analyzed in duplicates, and the mean of each sample was plotted on a scatter plot. RESULTS: Total of 30 vitreous samples were analyzed, of which 15 were controls and 15 were test samples. Of the 15 test samples, injection-to-sampling time was 0 to 20 minutes in 4 samples, 20 to 40 minutes in 6 samples, and 40 to 60 minutes in 5 samples. The highest concentration of hyaluronidase detected in control and test samples were 2.9 and 3.0 µg/ml, and the lowest concentration was 1.7 and 1.5 µg/ml (SD 0.3), respectively. There was no significant difference between control and test groups. CONCLUSION: Retrobulbar injection did not result in higher concentration of hyaluronidase in the posterior vitreous compared with controls when measured up to 60 minutes following injection.

Virulence factors in multidrug (MDR) and Pan-drug resistant (XDR) Pseudomonas aeruginosa: a cross-sectional study of isolates recovered from ocular infections in a high-incidence setting in southern India.

BACKGROUND: Global concerns have been raised due to upward trend of Multi-drug Resistant (MDR) Pseudomonas aeruginosa reports in ocular infections. Our aim was to characterize the virulence determinants of MDR P. aeruginosa causing ocular infections. METHODS: P. aeruginosa strains were isolated from 46 patients with conjunctivitis (2), endophthalmitis (11) and active keratitis (25) seen at our Institute, between 2016 and 2020. The isolates were identified by Vitek-2 and characterized based on growth kinetics, biofilm formation, motility, pyoverdine and pyocyanin production, phospholipase and catalase activity, urease production along with expression of exotoxins (exo-A, exo-U and exo-S) and correlated to its antibiotic profiles. RESULTS: Of the 46 P. aeruginosa isolates, 23 were MDR and were significantly (p = 0.03) associated with older (> 65) patients, along with higher production of pyoverdine (58.3%), pyocyanin (30.4%), phospholipase (91.6%) and protease (62.5%) activity, formed strong biofilms and exo-A (30.4%). No significant relation between motility, urease and catalase production with antibiotic susceptibility was observed. Heatmap and PCoA analysis confirmed this unique virulence profile associated with MDR-PA strains. CONCLUSION: Phenotypic characteristics of P.aeruginosa might be responsible for increased colonization and antibiotic resistance observed in vivo and understanding these differences may lead to development of clinical guidelines for the management of MDR infections.