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.

Mesenchymal stem cell-based adjunctive therapy for Pseudomonas aeruginosa-induced keratitis: A proof-of-concept in-vitro.

PURPOSE: Pseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro. METHODS: The effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay. RESULTS: Compared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2. CONCLUSION: MSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.

Herpes simplex virus keratitis: electronic medical records driven big data analytics report from a tertiary eye institute of South India.

OBJECTIVE: To describe the demographics and clinical profile of Herpes Simplex Virus (HSV) Keratitis in patients presenting to a multi-tier ophthalmology hospital network in South India. METHODS: We have reviewed the medical records of all patients having a clinical diagnosis of any form of HSV keratitis, seen between May 2012 and August 2020 across the L V Prasad Eye Institute network. All the further analyses of the groups were performed using the keywords used for making the diagnosis of HSV keratitis and the data were collected from the electronic medical record system. RESULTS: There were a total of 8308 (N = 8897 eyes) patients. Male: female ratio was 5368 (64.61%):2940 (35.39%). Unilateral involvement was in 7719 (92.91%) patients. The most common age group affected was between the third to fifth decades of life with 1544 (18.58%). 3708 (1.68%) eyes had mild visual impairment (< 20/70) while the rest of them had moderate to severe visual impairment as observed mainly (p ≤ 0.01) in Necrotizing stromal keratitis. 7314 (82.21%) eyes had normal intraocular pressure (10-21 mm Hg) while raised most commonly in keratouveitis (P ≤ 0.01). Epithelial Keratitis, Immune Stromal Keratitis, Endotheliitis, Neurotrophic keratopathy and Keratouveitis were observed in 1875 (17.22%) eyes, 5430 (61.03%) eyes, in 129(1.45%) eyes, 1188 (13.35%) eyes, 148 (1.66%) eyes and 256 (2.88%) eyes respectively. CONCLUSION: Based on our institute-based data, the most common type of HSV keratitis is Immune stromal keratitis followed by epithelial keratitis. Although not representative of the general population, this data provide useful insights related to HSV keratitis from India.

Fusarium Keratitis From a Comprehensive Eye Health Care Facility in South India: Molecular Characterization by MALDI-TOF Versus Polymerase Chain Reaction Sequencing, Species Complex Distribution, and Clinical Correlation.

PURPOSE: Fusarium keratitis possesses significant diagnostic and therapeutic challenges. Medically relevant Fusaria belong to various species complexes and show prominent differences in their antifungal susceptibility profile which may influence the clinical outcome. Rapid diagnostic methods are warranted for precise identification of species complexes for prompt initiation of correct antifungals. The aim of the study was to compare between matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and polymerase chain reaction sequencing for correct species-level identification and to analyze the clinical outcome among different Fusarium species complexes. METHODS: Twenty-nine culture-proven Fusarium keratitis cases were included in this study. A phylogenetic tree was constructed after TEF1α gene sequencing and isolates were subjected to MALDI-TOF MS, followed by database expansion and identification. Clinical outcome and risk association among species complexes were analyzed retrospectively. RESULTS: Maximum likelihood phylogeny categorized 68.9% isolates as Fusarium solani species complex (FSSC), 17.2% as Fusarium dimerum species complex (FDSC), followed by 13.7% as Fusarium fujikuroi species complex (FFSC). With extended database, MALDI-TOF MS could correctly speciate 96.5% (28/29) isolates. Previous antibiotic usage ( P = 0.034) and preoperative antifungal treatment with natamycin, voriconazole, or ketoconazole ( P = 0.025) were significantly higher in the FSSC group. The patients in the FFSC group had a significantly longer duration of symptoms at the time of clinical presentation to the clinic (15 days vs. 5 days, P = 0.030). Among 11 patients with a clinically poor outcome, 9 (31%) had FSSC infection. CONCLUSIONS: Patients infected with the FSSC had more aggressive infection with poor prognosis. MALDI-TOF MS can serve as the best alternative method to conventional molecular identification with reduced turnaround time, which may help the ophthalmologists to consider the appropriate antifungals or early surgical intervention for improved outcome.

Pediatric Pasteurella canis endophthalmitis.

We report an atypical presentation of endophthalmitis in a 2-month-old infant due to a rare infection by Pasteurella canis, small Gram-negative coccobacilli that inhabit the oral cavity and the gastrointestinal tracts of animals, including domesticated cats and dogs. Ocular infections are mainly associated with animal bites and scratches.

Outcomes of therapeutic penetrating keratoplasty in Pythium insidiosum keratitis managed with a combination of antibiotics.

Purpose: To describe the clinical outcomes of therapeutic penetrating keratoplasty (TPK) in patients with Pythium insidiosum keratitis following treatment with anti-pythium therapy (APT) consisting of linezolid and azithromycin. Methods: A retrospective review of medical records from May 2016 to December 2019 of patients with P. insidiosum keratitis was carried out. Patients who were treated with APT for a minimum of 2 weeks and then subsequently underwent TPK were included in the study. Data on demographic characteristics, clinical features, microbiology characteristics, and intraoperative details, postoperative outcomes were documented. Results: A total of 238 cases of Pythium keratitis were seen during the study period and 50 cases that satisfied the inclusion criteria were included. The median of the geometric mean of the infiltrate was 5.6 mm (IQR 4.0-7.2 mm). The patients received topical APT for a median of 35 days (IQR 25-56) prior to surgery. The most common indication of TPK was worsening keratitis (41/50, 82%). No recurrence of infection was observed. An anatomically stable globe was noted in 49/50 eyes (98%). The median graft survival rate was 2.4 months. A clear graft was present in 10 eyes (20%) with a final median visual acuity of 20/125 after a median follow-up period of 18.4 months (IQR 11-26 months). Graft size of less than 10 mm [OR: 5.824 (CI:1.292-41.6), P = 0.02] was found to be significantly associated with a clear graft. Conclusion: Performing TPK following the administration of APT has good anatomical outcomes. A smaller graft of <10 mm was associated with a higher chance of graft survival.

Clinical Profile and Demographic Distribution of Acanthamoeba Keratitis: An Electronic Medical Record-Driven Data Analytics from an Eye Care Network in India.

OBJECTIVE: To describe the clinical profile of patients presented with Acanthamoeba keratitis (AK) to a multi-tier ophthalmology hospital network in India. METHODS: This cross-sectional hospital-based study included 1945339 new patients registering between September 2016 and May 2022. Patients with clinically confirmed diagnosis of AK in one or both eyes were included in the study. All the relevant data were documented using an electronic medical record (EMR) system. RESULTS: A total of 245 (0.013%) patients were diagnosed with AK and majority were male (62.86%) with unilateral (99.59%) affliction. The most common age group was during the fourth decade of life, 65 (26.53%) patients and predominantly were adults (95.51%). The prevalence of the infection was higher in patients from a lower socioeconomic status (43.27%) from rural geography (52.24%) and in agriculture-related work (28.16%). The most common inciting factor was injury with vegetative matter (8.98%), dust (7.76%) and contact lens wear (4.49%). The majority of the eyes had blindness (20/400 to 20/1200) in 116 (47.15%) eyes with a presenting visual acuity (logMAR) of 2.14 ± 1.04. Among the surgical interventions, therapeutic keratoplasty was performed in 41 (16.67%) eyes, penetrating keratoplasty in 22 (8.94%) eyes, and evisceration in 2 (0.81%) eyes. CONCLUSION: AK more commonly affects males presenting during the fourth decade of life from lower socio-economic status and is predominantly unilateral. A fourth of the affected eyes underwent keratoplasty and the majority had significant visual impairment at presentation.

Randomized Double-Masked Placebo-Controlled Trial for the Management of Pythium Keratitis: Combination of Antibiotics Versus Monotherapy.

PURPOSE: The aim of this study was to compare the efficacy of monotherapy (topical linezolid 0.2%) versus a combination of antibiotics (topical linezolid 0.2% and topical azithromycin 1%) for the treatment of Pythium insidiosum keratitis. METHODS: Cases of P. insidiosum keratitis were prospectively randomized into group A on topical 0.2% linezolid along with topical placebo (sodium carboxymethyl cellulose [CMC] 0.5%) and group B on a combination of topical 0.2% linezolid and topical 1% azithromycin. Both groups were compared by proportion of both clinical resolution and worsening of keratitis along with the number of therapeutic penetrating keratoplasty (TPK) performed at 3 months. RESULTS: We initially planned N = 66 patients but later limited to 20 (N = 10 in each group) patients owing to one interim analysis. The average size of the infiltrate in group A and B was 5.6 ± 1.5 mm and 4.8 ± 2.0 mm, respectively, with a mean Logarithm of the Minimum Angle of Resolution (logMAR) visual acuity of 2.74 ± 0.55 and 1.79 ± 1.19. At 3 months, from group A, 7 (70%) patients needed TPK and 2 patients had signs of resolution, whereas from group B, 6 (60%) patients achieved complete resolution ( P = 0.0003) and 2 were improving while only 1 needed TPK ( P = 0.02). The median duration of treatment in group A and B, with the study drugs, was 31 days (17.8-47.8) and 101.5 days (80-123.3), P value = 0.003, respectively. Final visual acuity at 3 months was 2.50 ± 0.81 and 0.75 ± 0.87, P = 0.02, respectively. CONCLUSIONS: A combination of topical linezolid and topical azithromycin was found to have superior efficacy than the monotherapy with topical linezolid for the management of Pythium keratitis.

Nocardia keratitis presenting as an anterior chamber ball of exudates and its management.

A man in late 40s presented with corneal ulcer of the right eye of 1 month duration. He had a central corneal epithelial defect measuring 4.6×4.2 mm with an underlying 3.6×3.5 mm anterior to mid stromal patchy infiltrate and 1.4 mm hypopyon. Gram stain of the colonies on chocolate agar showed presence of confluent thin branching, gram-positive beaded filaments, which were positive after 1% acid fast stain. This confirmed our organism to be Nocardia sp. Topical amikacin was started but continued worsening of the infiltrate and presence of a ball of exudates in the anterior chamber, prompted the use of systemic trimethoprim-sulfamethoxazole. There was a dramatic improvement in the signs and symptoms, with complete resolution of infection over a period of 1 month.

Clinico-microbiological Features and Treatment Outcomes of Serratia Keratitis and Comparison with Pseudomonas aeruginosa Keratitis.

PURPOSE: To describe clinico-microbiological features and outcomes of Serratia keratitis and to compare them with Pseudomonas aeruginosa keratitis. METHODS: Cases of microbiologically proven Serratia keratitis and P. aeruginosa keratitis were reviewed. Data regarding demographic and clinical characteristics, and outcomes were recorded. RESULTS: 39 patients with pure Serratia keratitis were included. Median presenting vision was 1.8 logMAR (IQR, 0.8-2.4) and median infiltrate size was 5 mm (IQR 3-7.8 mm). An ocular risk factor was present in 35 (89.7%) cases. S. marcescens was the most common species (31/39, 79.5%). Medical resolution was observed in 36/39 (92.3%) cases, while three (7.7%) eyes needed penetrating keratoplasty. On comparing with P. aeruginosa keratitis (58 eyes), no difference in outcomes (p = .14) was noted. CONCLUSION: Serratia keratitis usually occurs in eyes with a compromised surface and has good resolution with medical therapy. Both Serratia and P. aeruginosa keratitis have similar outcomes.

Challenges in management of microbial keratitis during COVID-19 pandemic related lockdown: a comparative analysis with pre pandemic data.

PURPOSE: To study the challenges of managing microbial keratitis(MK) during the COVID19 pandemic related lockdown and assess the outcomes of treatment at a tertiary cornea service. METHODS: Retrospective, non comparative study of electronic medical records of MK presenting to a network of four tertiary care cornea services. The medical history, presenting clinical features, microbiology work up and treatment outcomes were analyzed. The primary outcome measure was final outcome at last follow up. Secondary outcomes measures were non-compliance to treatment due to travel restrictions, therapeutic PKP not done due non availability of corneal tissues. Results- MK was noted in 330 eyes of 330 patients between April and May 2020. Of these 237(71.8%) were males. Median age was 45 years(IQR, 33-56). Low socioeconomic status noted in 102(30.9%). Patients travelling beyond the district from where the hospital was located comprised of 64.9%(n=214). At a median follow up of 32 days(IQR, 9-54), 118(35.8%) patients had resolved, with medical management, 73(22.1%) patients were under active treatment, 139(42.1%) were lost to follow up. Sixty-six patients(20%) were non-compliant to treatment of which 59 could not follow appointment schedule due to travel restrictions. Therapeutic PKP (TPK) was planned in 48/128 (37.5%) patients, but was performed in only 34/48 (70.8%) due to non-availability of donor corneas. CONCLUSIONS: Abnormal social circumstances due to the COVID pandemic and the ensuing impediments to travel for access to health care affected compliance to treatment of ocular emergencies such as microbial keratitis.

Trends in the Microbiological Spectrum of Nonviral Keratitis at a Single Tertiary Care Ophthalmic Hospital in India: A Review of 30 years.

PURPOSE: The aims of this study were to assess the trends in microorganisms from patients with infectious keratitis and to assess their antibiogram patterns at a tertiary eye care center in India. METHODS: In this retrospective observational case series, microbiological records of all corneal ulcers were reviewed from 1991 to 2020 and assessed for trends in keratitis and antibiotic susceptibility using the χ 2 test. RESULTS: Of the total of 51,747 patients, 51.13% were culture positive. A decrease in bacteria was noted from 56% to 38%, with a parallel increase in fungal isolates from 24% to 51%. Gram-positive bacteria accounted for 70.8% of the total bacteria, a trend in rise of Streptococcus pneumoniae (31%) and a decreasing trend in prevalence of Staphylococcus epidermidis was observed over 30 years . Pseudomonas aeruginosa (55.5%) was the most prevalent gram-negative pathogen, whereas Fusarium spp . (33.1%) and Aspergillus spp. (32.4%) were the most common fungal isolates. The susceptibility of gram-positive organisms to cefazolin decreased from 95.5% to 66% ( P = 0.0001), amikacin from 88% to 55% ( P = 0.0001), and vancomycin from 98.9% to 90.7% ( P < 0.05). A similar decrease in susceptibility was also significant for gram-negative organisms with piperacillin/tazobactam and chloramphenicol ( P < 0.05). A significant trend toward increasing resistance against fluoroquinolones was also observed for ciprofloxacin (gram-positive organisms: 16% to 50%; gram-negative organisms: 11.5% to 18.7%), gatifloxacin (38% to 47%), and moxifloxacin (9.4% to 29%). CONCLUSIONS: The spectrum of keratitis has changed, and fungus is now the predominant etiology. An increasing trend in resistance to all antibiotics studied would affect the empiric treatment, also suggesting regular surveillance.

Use of Decellularized SMILE (Small-Incision Lenticule Extraction) Lenticules for Engineering the Corneal Endothelial Layer: A Proof-of-Concept.

PURPOSE: To demonstrate the suitability of using decellularized SMILE (Small-incision Lenticule Extraction) lenticules for culturing and transplanting the corneal endothelium (CE). METHODS: The SMILE lenticules, obtained during refractive surgery, were decellularized by incubating in CE culture medium and fetal bovine serum. Decellularization was confirmed by hematoxylin and eosin staining, DAPI staining, and gel electrophoresis. The amount of DNA per milligram of dry tissue weight was calculated to quantify the residual nuclear content. The transparency of the decellularized lenticules was determined by calculating the modulation transfer function. Immunostaining for stromal collagens and glycosaminoglycan was performed using specific antibodies. Engineered tissue was constructed by culturing the CE cells on lenticules and staining for ZO-1, Na/K ATPase, and N-cadherin. The functionality of the engineered tissues was assessed by transplanting them onto edematous human donor corneas and perfusing for 10 days ex-vivo. RESULTS: The residual DNA per milligram of dry tissue weight was found to be significantly reduced (p < 0.0001) in serum (0.255 µg/mg) and Opti-MEM (0.140 µg/mg) when compared to fresh lenticules (3.9 µg/mg). Decellularization did not alter the arrangement of the collagen fibers or the transparency of the lenticules. CE cells attached and matured to express ZO-1, Na/K ATPase, and N-cadherin at two weeks after seeding. The engineered tissue upon transplantation significantly reduced the corneal edema (p < 0.05) and the transplanted cells remained intact on the SMILE lenticule post-transplantation. CONCLUSION: This study demonstrates the suitability of using SMILE lenticules decellularized using a simple, chemical-free method for engineering the corneal endothelium for transplantation.

Case Report: Poor Outcome Despite Aggressive Management in Pythium insidiosum Endophthalmitis.

Pythium insidiosum is a parasitic oomycete that can cause keratitis and closely resembles fungus, both clinically and morphologically. It requires a trained microbiologist for its differentiation from fungal filaments and has poor response to antifungal therapy. We present a case of primary isolation of the organism from the vitreous cavity in a case of endophthalmitis. The infection spread quickly and involved all the ocular tissues. The eye had poor visual outcome despite a sequence of rapidly completed retinal and corneal surgeries along with initiation of anti-Pythium therapy.

Pythium insidiosum keratitis: Review of literature of 5 years' clinical experience at a tertiary eye care center.

BACKGROUND: Pythium insidiosum is an important cause of infectious keratitis from tropical and sub-tropical countries. Due to its closely mimicking clinical and microbiological features with fungus, it remained unidentified and managed as fungal keratitis for a long time. Previously all patients had poor outcomes with antifungal therapy and needed surgical treatment with higher rates of recurrences of infection leading to loss of an eye. Thus, a novel approach was required to treat it and, in this article, we would like to elaborate on the drastic change which these 5 years have brought in the management of this condition. METHODS: In view of making a consolidated article comprising all the required information and also our clinical experience in the management of Pythium keratitis, we extensively reviewed several articles available on it over PubMed and Google scholars. Relevant literature describing details about Pythium, its clinical correlation, and recent advances from 52 articles including 12 articles from our group were finally included. RESULTS: Our group identified and highlighted the unique clinical and microbiological features of Pythium insidiosum, performed several in-vitro, in-vivo studies along with clinical trials, and proposed the strategic way of its diagnosis and treatment. The use of antifungals was replaced with antibacterial medications and this resulted in better medical and surgical outcomes. CONCLUSION: The diagnosis and management of Pythium insidiosum is constantly evolving with several recent works pointing out the possible changes in the practice patterns for the management of this challenging form of keratitis.

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.