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

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.

Infectious crystalline keratopathy-clinical-microbiological profile and outcome of management.

PURPOSE: To describe the clinical features, management, and long-term outcome of Infectious crystalline keratopathy (ICK). METHODS: The medical records of clinically diagnosed and microbiologically proven cases of ICK were reviewed from January 2011 to December 2022. Clinical characteristics include the presence of whitish needle-like projections with branching, limited to anterior-mid stroma. Keratoplasty being the most common risk factor, graft-related microbial keratitis during the same period was also studied. The demography, clinical profile, microbiology, treatment, and outcome were analyzed, and compared with secondary graft infiltrate(GI). RESULTS: Medical records of 24 cases with ICK were reviewed. The mean age was 49.3 ± 20.1 years, with 15(62.5%) males. Prior keratoplasty was done in 18 (75%) cases, with a mean graft size of 10.1 ± 1.5 mm, and mean interval between the last graft and presentation was 9.7 ± 6.2 (3-90) months. In comparison to GI (n = 24), ICK patients (n = 18,75%) were less symptomatic, presented late (7.3 ± 6.5 days vs 16.3 ± 19.4, p = 0.003), using frequent topical steroids (> 3 times/day, p = 0.006), smaller infiltrate size < 4 mm (p = 0.008), central (p = 0.02), less associated with epithelial defect (p = 0.0001), hypopyon (p = of 0.0002), corneal perforation (p = 0.0006), and surgical management (p = 0.03). On microbiology, 22 (91.6%) ICK cases were culture positive, 14 (63.6%) gram-positive, 3 (13.6%) gram-negative, 2 (9%) mixed bacteria, and 3 (13.6%) fungus, comparable with GI. CONCLUSION: ICK affects poor ocular surfaces usually following keratoplasty with larger graft size, the use of steroids being the most common association, and it responds to medical management as compared to GI.

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.

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.

Elucidating the clinical, microbiological and molecular diagnostic aspects of Macrophomina phaseolina keratitis.

This study reports the clinico-microbiological features of Macrophomina phaseolina keratitis. Clinically diagnosed as microbial keratitis, six patients underwent microbiological evaluation. Fungal culture isolates from cornea were subjected to DNA sequencing of the ITS region, phylogenetic analysis and reconfirmation by polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) of six antifungal drugs were determined by microbroth dilution method against the six isolates. All patients were treated with antifungals. Failed medical therapy necessitated therapeutic penetrating keratoplasty (TPK). Corneal buttons were processed for histopathology. In all patients, the corneal scraping showed septate hyaline fungal filaments. The BLAST analysis for ITS sequences of all six fungal isolates suggested M. phaseolina, however, when limited to sequences from type material, they matched M. pseudophaseolina. Phylogenetic analysis could not differentiate between these two species and clustered in a single clade. PCR assay of specific gene sequence [MpCal (calmodulin)] reconfirmed all isolates as M. phaseolina. The MICs of voriconazole and posaconazole were lowest (0.03 to 2 and 0.1 to 2µg/mL respectively) and all isolates were susceptible to natamycin. Except for case 1, which healed with a scar on treatment, all other cases worsened, despite medical treatment, necessitating TPK. Histopathology of 3 out of 4 buttons showed the presence of fungal filaments. While direct microscopic examination of corneal scrapings is helpful in diagnosis, identification of M. phaseolina in culture is challenging. Although MICs of commonly used antifungals are low response to medical therapy is not encouraging; patients may require TPK for resolution of infection in M. phaseolina keratitis.

DNA sequencing, phylogenetic analysis and specific PCR confirmed Macrophomina phaseolina keratitis in six patients. Although antifungal susceptibility showed the organisms to be susceptible to natamycin five patients did not respond to treatment and needed keratoplasty.

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.

Linezolid shows high safety and efficacy in the treatment of Pythium insidiosum keratitis in a rabbit model.

Efficacy and safety of three antibiotics (Linezolid-LZ, 0.2%; Azithromycin-AZ, 1%; Tigecycline-TG, 1%) were determined in the treatment of Pythium insidiosum keratitis in rabbits. Infection of right eye of 38 rabbits was induced by standard intracorneal injection of P. insidiosum zoospores (left eye, intracorneal saline). Corneal infection developed in all right eyes. One hourly eye drops of one of the three antibiotics was instilled in both eyes (3 groups of 12 rabbits each) except in controls. Half of the rabbits in each group received intracorneal injection of the respective antibiotic after 4 days of starting eye drops. Clinical scoring of eyes was done over next 3 weeks. The reduction in scores post-treatment was significant for each drug (LZ: p < 0.025, AZ: p < 0.025, TG: p < 0.01). Scores with LZ (median change of 3) was significantly (p = 0.013) higher than TG (median change of 2) and comparable (p = 0.06) to AZ (median change of 3). Reduction in clinical scores in eyes receiving intracorneal antibiotics was not significantly different from the eyes that did not receive intracorneal antibiotics (p = 0.73). While no adverse effect of LZ was seen in the control corneas, 66-100% of rabbits showed reaction to AZ and TG. Histopathology showed severe inflammation in all infected corneas and intraocular extension in some of the rabbits with poor response. The success rate was noted to be 16.7%, 25% and 50% in AZ, TG and LZ respectively (p = 0.45). LZ demonstrated superior efficacy and safety and can be considered for trial in human disease.

Risk factors, clinical features and outcomes of Neisseria keratitis.

OBJECTIVE: To describe the clinical features, risk factors and outcomes of Neisseria keratitis. METHODS: This is a retrospective observational study wherein medical records of cases with microbiologically proven Neisseria keratitis were reviewed. Data pertaining to the underlying predisposing factors, clinical characteristics of the corneal ulcer, antibiotic susceptibility of the Neisseria species isolate from the corneal scraping, the treatment given, and outcomes were collected and analyzed. RESULTS: Medical records of 60 patients (60 eyes) with Neisseria keratitis were reviewed. Among the causes of poor ocular surface as predisposing factor, vernal keratoconjunctivitis (n = 6 eyes), along with use of topical corticosteroids (n = 18 eyes) was the most common. The ulcer was characterized by a central infiltrate (31/60, 51.7%) involving up to the mid-stroma (43/60, 71.7%). Of the forty-four (73.3%) eyes with pure Neisseria keratitis, 31 eyes (72.1%) resolved with medical therapy alone while five eyes (11.6%) underwent therapeutic penetrating keratoplasty and in two (4.6%) eyes evisceration was performed. The other 5/44 (11.6%) patients were lost to follow-up. Resolution with medical therapy was found to be similar in cases with pure infection and mixed infection (p = 0.58). CONCLUSIONS: Neisseria keratitis most commonly causes a mild form of keratitis and is often associated with the poor ocular surface or prior steroid use. In most cases medical therapy is sufficient for complete resolution of the keratitis.

One-stage femtolaser-assisted lamellar keratoplasty and foreign body removal.

PURPOSE: To report the use of femtosecond laser platform to remove a glass foreign body (FB) and remove scar by performing sutureless lamellar keratoplasty. CASE REPORT: A 34-year-old man presented with a posttraumatic anterior stromal scar and retained corneal glass FB after injury to his left eye, sustained in a prior vehicular accident. Right eye examination was essentially normal. Best-corrected visual acuity of the left eye was 20/320. He underwent femtosecond laser-assisted sutureless anterior lamellar keratoplasty using the 500-kHz femtosecond laser machine. The host bed and donor lenticule diameters were 7.9 and 7.8 mm and the thickness of the lenticule was 125 and 150 µm, respectively. The recipient lenticule was obtained by placing the lamellar incision that encompassed the FB. When the recipient lenticule was lifted, partially embedded FB present in the stroma was removed after dislodging it with a 26-gauge needle. The femtosecond-dissected donor button was placed onto the stromal bed with a snug fit. No sutures were taken. Postoperatively, best-corrected visual acuity improved to 20/50 at 1 month and 20/30 at 7 months of follow-up. CONCLUSIONS: Femtosecond laser-assisted sutureless anterior lamellar keratoplasty is a promising modality for lamellar keratoplasty, and as the depth of the incision can be obtained with precision, impacted stromal FBs can be removed in a single procedure along with scars.

Long-term outcome of minor salivary gland transplantation for pediatric congenital alacrimia.

A 3-year-old boy presented with repeated episodes of corneal erosions and ulceration and absence of tear secretion since birth. Examination under anesthesia revealed a dry ocular surface, with corneal scarring and vascularization. Computed tomography of the orbit showed complete absence of lacrimal glands bilaterally. Minor salivary gland transplantation was performed in the right eye, which was more severely affected. This procedure has been used to manage severe dry eyes but not previously for congenital alacrimia. On long-term follow-up, 5 years after surgery, we observed considerable improvement in the photophobia, corneal transparency, and neovascularization.

Nocardia keratitis after small incision lenticule extraction (SMILE).

We report the case of a female patient in her late 20s who visited the clinic with concerns about poor vision, redness, watering and a burning sensation in her left eye 2 weeks after undergoing a small incision lenticule extraction. She had no history of systemic illness or immunosuppressed status. On slit lamp examination, she was found to have corneal stromal infiltrates in the interface at multiple locations. Given the clinical diagnosis of microbial keratitis, corneal scraping of the interface infiltrate was performed and sent for microbiological examination revealing gram-positive, thin, beaded filaments that were acid-fast positive and later identified by growth in culture media as Nocardia species. This case was managed successfully with the use of topical amikacin and systemic trimethoprim-sulfamethoxazole with complete resolution of infection.

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.

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.

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.