Improving polymerase chain reaction diagnostic rates for herpes simplex keratitis: results of a pilot study.

Background: Laboratory confirmation is crucial for diagnosis and management of herpes simplex virus (HSV) keratitis. However, the sensitivity of polymerase chain reaction (PCR) in keratitis is low (25%) compared with that of mucocutaneous disease (75%). We developed an educational intervention aimed at improving the diagnostic yield of PCR. Methods: The medical records of keratitis cases seen at the emergency department of a London tertiary ophthalmic referral hospital over two distinct periods, before and after an educational program on swab technique, were reviewed retrospectively. Results: A total of 252 HSV cases were included. Increases in the laboratory-confirmed diagnosis of HSV-1 were observed, in both first presentations (11.1%-57.7%) and recurrent cases (20%-57.6%). The rate of positive HSV-1 PCR in eyes with an epithelial defect increased from 19% pre-intervention to 62% post intervention. Notably, 3% were positive for varicella zoster virus DNA, and there was a single case of Acanthamoeba keratitis. Conclusion: Our results suggest that, with proper swabbing technique, PCR may be more sensitive than previously reported.

PCR testing for herpesviruses in aqueous humor samples from patients with and without clinical corneal endothelial graft rejection.

To compare prevalence of positive PCR tests for herpesviruses between patients with and without a history of clinical corneal endothelial allograft rejection (AGR). Retrospective cross-sectional study with two-group comparison. A total of 307 aqueous humor (AH) samples from 235 Patients and 244 eyes who underwent penetrating keratoplasty or Descemet membrane endothelial keratoplasty or had a diagnostic AH aspiration due to clinical AGR between 2019 and 2023 were tested for DNA of herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV). PCR test results were compared between the two groups (with/without AGR). Another sub-analysis examined the results of patients without a history of herpetic keratitis. A total of 8% of eyes with clinical AGR (9/108) had a positive PCR result for one of the herpesviruses (HSV:3, CMV:3, EBV:2, VZV:1). All patients in the group without AGR had negative PCR results for all previous viruses (0/136). The difference was statistically significant (p < 0.001). The sub-analysis of eyes without a history of herpetic keratitis also revealed significantly more positive herpes PCR results (7/87) in eyes with AGR than in eyes without AGR (0/42, p = 0.005). Clinical AGR after keratoplasty shows a significant correlation to viral replication. Herpetic infection and AGR could occur simultaneously and act synergistically. Timely differentiation between active herpetic infection and/or AGR is pivotal for proper treatment and graft preservation.

UVB induced reactivation leads to HSV1 in the corneas of virtually all latently infected mice and requires STING to develop corneal disease.

Reactivation of latent herpes simplex type 1 results in virus returning to the cornea leading to recurrent herpetic stromal keratitis (rHSK). We compare two competing models to reactivate viruses from latency, UV-B irradiation and cyclophosphamide (CP). Results revealed that while both result in corneal recrudescence, only UV-B irradiation results in rHSK. To better understand the dynamics of reactivation, we analyzed corneas for both the presence of infectious viruses and the dynamics of exposure to multiple reactivations using UV-B. We noted that multiple reactivations result in progressively worse corneal disease. We also noted that expression of IFNα and STING, surragate markers for the presence of virus, are induced by the presence of reactivated virus. Studies to determine the importance of STING to the development of HSK revealed that in the absence of STING, mice do not develop significant HSK and the magnitude of the infiltrate of CD45+ cells in these corneas is significantly reduced. The resulting paucity of CD45+CD11b+GR-1+F4/80-neutrophils, and to a lesser extent CD45+CD11b+GR-1-F4/80+ macrophages in B6-STING KO mice following reactivation is likely the underlying cause for lack of rHSK as has been noted by ourselves and others. These results underscore the critical importance of STING's role in developing rHSK.

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.

Herpes Simplex Keratitis as a Complication of Pterygium Surgery.

BACKGROUND Infectious keratitis after pterygium surgery is a rare but potentially devastating complication. The present study presents 5 cases of herpes simplex keratitis (HSK) after pterygium surgery. CASE REPORT This study was conducted in our clinic in a 5-year period from February 2017 to September 2021. The 5 patients were men, aged between 42 and 73 years, with no prior history of herpes simplex virus (HSV) infections. Symptoms appeared near 1 month (median 30 days, range 10 to 70 days) after primary pterygium surgery. Diagnosis was based on clinical symptoms and laboratory test results, such as tear HSV-sIgA, corneal tissue polymerase chain reaction, and next-generation sequencing of metagenomics. The epithelial (1/5) and stromal (4/5) subtypes of HSK were identified. The patients received topical ganciclovir gel, immunosuppressive eyedrops, and oral acyclovir tablets, along with additional surgical interventions if necessary. Three were healed with conservative therapy, 1 eye required amniotic membrane transplantation due to corneal melt, and 1 was perforated and followed by corneal grafting. Finally, a literature review of previous publications on HSK after ocular surgeries was conducted. CONCLUSIONS HSK is a rare but serious complication that can arise after uneventful pterygium surgery. It is worthy of attention that both epithelial and stromal forms can occur. Timely diagnosis and treatment are crucial to prevent unfavorable outcomes. Consequently, routine corneal fluorescein staining, tear sIgA examination, and corneal scraping for polymerase chain reaction or next-generation sequencing of metagenomics should be performed in any suspected cases.

Integrative Analysis of miRNA and circRNA Expression Profiles and Interaction Network in HSV-1-Infected Primary Corneal Epithelial Cells.

PURPOSE: Circular RNAs (circRNAs) are products of alternative splicing with roles as competitive endogenous RNAs or microRNA sponges, regulating gene expression and biological processes. However, the involvement of circRNAs in herpes simplex keratitis remains largely unexplored. METHODS: This study examines circRNA and miRNA expression profiles in primary human corneal epithelial cells infected with HSV-1, compared to uninfected controls, using microarray analysis. Bioinformatic analysis predicted the potential function of the dysregulated circRNAs and microRNA response elements (MREs) in these circRNAs, forming an interaction network between dysregulated circRNAs and miRNAs. RESULTS: A total of 332 circRNAs and 16 miRNAs were upregulated, while 80 circRNAs and six miRNAs were downregulated (fold change ≥2.0 and p < 0.05). Gene ontology (GO) and KEGG pathway analyses were performed on parental genes of dysregulated circRNAs to uncover potential functions in HSV-1 infection. Notably, miR-181b-5p, miR-338-3p, miR-635, and miR-222-3p emerged as pivotal miRNAs interacting with multiple dysregulated circRNAs. CONCLUSIONS: This comprehensive study offers insights into differentially expressed circRNAs and miRNAs during HSV-1 infection in corneal epithelial cells, shedding light on circRNA-miRNA interactions' potential role in herpes simplex keratitis pathogenesis.

Targeted delivery of herpes simplex virus glycoprotein D to CD169+ macrophages using ganglioside liposomes alleviates herpes simplex keratitis in mice.

Herpes simplex keratitis (HSK) is a common blinding corneal disease caused by herpes simplex virus type 1 (HSV-1) infection. Antiviral drugs and corticosteroids haven't shown adequate therapeutic efficacy. During the early stage of HSV-1 infection, macrophages serve as the first line of defense. In particular, CD169+ macrophages play an important role in phagocytosis and antigen presentation. Therefore, we constructed GM-gD-lip, a ganglioside GM1 liposome vaccine encapsulating HSV-1 glycoprotein D and targeting CD169+ macrophages. After subconjunctival injection of the vaccine, we evaluated the survival rate and ocular surface lesions of the HSK mice, as well as the virus levels in the tear fluid, corneas, and trigeminal ganglia. We discovered that GM-gD-lip reduced HSV-1 viral load and alleviated the clinical severity of HSK. The GM-gD-lip also increased the number of corneal infiltrating macrophages, especially CD169+ macrophages, and polarized them toward M1. Furthermore, the number of dendritic cells (DCs) and CD8+ T cells in the ocular draining lymph nodes was significantly increased. These findings demonstrated that GM-gD-lip polarized CD169+ macrophages toward M1 to eliminate the virus while cross-presenting antigens to CD8+ T cells via DCs to activate adaptive immunity, ultimately attenuating the severity of HSK. The use of GM-gD-lip as an immunotherapeutic method for the treatment of HSK has significant implications.

Metabolic signatures of tear extracellular vesicles caused by herpes simplex keratitis.

PURPOSE: Herpes simplex keratitis (HSK), caused by type 1 herpes simplex virus (HSV) reactivation, is a severe infectious disease that leads to vision loss. HSV can trigger metabolic reprogramming in the host cell and change the extracellular vesicles (EV) cargos; however, little is known about the EV metabolic signatures during ocular HSV infection. Here, we aimed to depict the EV-associated metabolic landscape in HSK patients' tears. METHODS: We collected 82 samples from 41 participants with unilateral HSK (contralateral unaffected tears were set as negative control), including subtype cohorts of 13 epithelial, 20 stromal, and 8 endothelial HSK. We isolated tear EVs via our previously established platform and conducted metabolic analysis using LC-MS/MS. The metabolic signatures for recognizing HSK and subtypes were assessed through differential analysis and machine learning algorithms. RESULTS: Hypopsia and increased extracellular CD63 levels were observed in affected eyes. We identified 339 metabolites based on sEVs isolated from tears. Differential analysis revealed alterations in energy and amino acid metabolism, as well as the infectious microenvironment. Furthermore, we observed dysregulated metabolite such as methyldopa, which is associated with inappropriate neovascularization and corneal sensation loss, contributing to the HSK severity particularly in the stromal subtype. Moreover, machine learning classification also suggested a set of EV metabolic signatures that have potential for pan-keratitis detection. CONCLUSIONS: Our findings demonstrate that tear EV metabolites can serve as valuable indicators for comprehending the underlying pathological mechanisms. This knowledge is expected to facilitate the development of liquid biopsy means and therapeutic target discovery.

[Chinese expert consensus on the diagnosis and treatment of herpes simplex keratitis (2023)].

As one of the common infectious corneal diseases, herpes simplex keratitis (HSK) has diverse clinical manifestations, is prone to recurrence, and can lead to blindness. In recent years, as new virus detection technologies, treatment drugs and surgical methods have emerged, there are more options for the diagnosis and treatment of HSK, but many problems still exist. In order to further standardize the clinical diagnosis and treatment of HSK and provide guidance and reference for clinical work, the Ocular Infection Group of Chinese Ophthalmologist Association has gathered relevant domestic experts, and reached this consensus on the diagnosis, treatment, and prevention of HSK through full discussion, on the basis of previous opinions, and in consideration of the latest research progress, relevant guidelines abroad and expert recommendations regarding the clinical care of patients with HSK.

Diagnostic performance of real-time quantitative PCR in tear samples in various subtypes of herpes simplex keratitis.

Diagnosis of herpes simplex keratitis (HSK) is mostly based on clinical findings, yet biological confirmation supports management of challenging cases. This study evaluated the place of real-time quantitative PCR (RT-qPCR) on tear samplings in the management of HSK. Clinical records of patients who underwent tear sampling tested by RT-qPCR for herpes simplex virus type 1 for an acute episode of corneal inflammation or defect between January 2013 and December 2021 were retrospectively reviewed, and results were compared to clinical diagnosis (i.e., HSK or not) based on biomicroscopic findings and medical history. Of 465 tested tear samples from 364 patients, a clinical diagnosis of active (ongoing) HSK was recorded in 240 cases, among which 76 were RT-qPCR positive (global sensitivity of 31.6%, specificity of 99.5%). Sensitivity of RT-qPCR was higher in epithelial (97.4%) and stromal keratitis with ulceration (48.7%), compared to other types of HSK (23.5% in keratouveitis, 13.6% in endotheliitis, 11.1% in postherpetic neurotrophic keratopathy, and 8.1% in stromal keratitis without ulceration). The highest viral loads were detected from epithelial and stromal keratitis with ulceration, while in HSK with no epithelial involvement, the viral load detected was 196-fold lower, on average. The proportion of clinically characterized HSK patients with negative tear samples was higher in patients receiving antiviral treatment (P < 0.0001). RT-qPCR, performed on tear samples, can help in confirming diagnosis in case of presumed HSK, including clinical forms with no obvious epithelial involvement. The sensitivity of tear sampling is much higher whenever epithelial keratitis is present.

Loss of TRPM8 Exacerbate Herpes Simplex Keratitis Infection in Mice by Promoting the Infiltration of CD11b+ Ly6G+ Cells and Increasing the Viral Load in the Cornea.

Purpose: To reveal the role of transient receptor potential cation subfamily M member 8 (TRPM8) channels in herpes simplex keratitis (HSK). Methods: HSK models were established using TRPM8 knockout (TRPM8-/-) mice and their wild-type (WT) littermates. The infected corneas were graded and harvested to evaluate the mRNA levels of inflammatory factors through quantitative real-time polymerase chain reaction (RT-PCR), as well as the infiltration of inflammatory cells through immunofluorescence staining and flow cytometry. Viral titers were determined by plaque assay and absolute quantitative method. RNA-sequencing was conducted to elucidate the transcriptome of corneal epithelium in response to TRPM8 knockout after infection. The anti-inflammatory effect of TRPM8 agonist menthol was documented via subconjunctival administration. Results: Compared to their wild-type counterparts, TRPM8-deficient mice exhibited exacerbated infection symptoms and thicker corneas in HSK models. Infection in TRPM8-deficient mice resulted in significant lymphocyte infiltration, primarily consisting of Ly6G+ CD11b+ cells. Additionally, TRPM8-deficient mice displayed increased levels of corneal viral titers after infection, along with decreased expression of interferon-stimulated genes (ISGs). Subconjunctival administration of menthol effectively alleviated infection-induced symptoms and Ly6G+ CD11b+ cell infiltration in herpes simplex virus type 1 (HSV-1)-treated mice. Conclusions: TRPM8 promoted host resistance to HSV-1 infection by suppressing the accumulation of Ly6G+ CD11b+ cells and virus replication. These findings suggest that targeting TRPM8 could be valuable for therapeutic interventions against HSV-1 infections.

Steroids in the Management of Infectious Keratitis.

PURPOSE: To summarize the evidence base on the use of topical corticosteroids for infectious keratitis. METHODS: Narrative review. RESULTS: Infectious keratitis is a painful condition that often results in visually significant corneal stromal scarring, even when antimicrobial therapy is successful. Corticosteroids may reduce inflammation and subsequent scar formation and while relieving the acute ocular pain associated with a corneal ulcer. However, corticosteroids also reduce the host immune response, which could hinder the ability to clear infection. The safety and effectiveness of corticosteroids depends to a large part on the efficacy of the antimicrobials being used to treat the underlying infection. Randomized trials have found that corticosteroids are safe and effective for herpetic keratitis when used with appropriate antiviral therapy, and are safe for bacterial keratitis when used with broad spectrum topical antibiotics. The effectiveness of corticosteroids for bacterial keratitis has not been shown conclusively, although more advanced bacterial corneal ulcers may do better with corticosteroids. No randomized trials have assessed the safety and effectiveness of steroids for fungal or acanthamoeba keratitis. Animal studies suggest corticosteroids may be harmful in fungal keratitis, and observational human studies have found that steroids are harmful for fungal and acanthamoeba keratitis when started prior to anti-amoebics. CONCLUSIONS: Topical corticosteroids, when used as an adjunct to antimicrobial therapy, may be beneficial if the antimicrobial being used can effectively clear or suppress the infection, such as in bacterial and herpetic keratitis. Randomized trials would be helpful to further delineate the role of corticosteroids for infectious keratitis.

New-Onset of Herpes Simplex Keratitis After Blepharoplasty, Case Series and Review of the Literature.

PURPOSE: To report 3 cases of new-onset herpes simplex keratitis (HSK) after uncomplicated extraocular plastic surgery and discuss potential risk factors. METHODS: This case series includes 3 patients who underwent uncomplicated blepharoplastic surgery. Within 2 weeks postoperatively, all patients reported ocular discomfort, and their ophthalmic examinations revealed corneal lesions suspicious of HSK. One case was confirmed as an active herpes infection, and the other 2 cases were clinically diagnosed with HSK. The patients were treated with oral acyclovir and followed up for up to 6 weeks. RESULTS: All patients demonstrated improvement without sequelae at follow-up visits from 5 days to 4 weeks after initiating acyclovir treatment. CONCLUSIONS: Risk factors for new-onset HSK after uncomplicated extraocular surgeries may be related to an immunocompromised state, postoperative administration of topical or periocular corticosteroids, or environmental factors such as psychological stress. Ophthalmologists, particularly plastic surgeons, should be vigilant for ocular discomfort following eyelid surgeries and consider the possibility of herpes infection. This report highlights the importance of recognizing and managing HSK in the context of extraocular plastic surgery.

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.

In vivo CRISPR gene editing in patients with herpetic stromal keratitis.

In vivo CRISPR gene therapy holds large clinical potential, but the safety and efficacy remain largely unknown. Here, we injected a single dose of herpes simplex virus 1 (HSV-1)-targeting CRISPR formulation in the cornea of three patients with severe refractory herpetic stromal keratitis (HSK) during corneal transplantation. Our study is an investigator-initiated, open-label, single-arm, non-randomized interventional trial at a single center (NCT04560790). We found neither detectable CRISPR-induced off-target cleavages by GUIDE-seq nor systemic adverse events for 18 months on average in all three patients. The HSV-1 remained undetectable during the study. Our preliminary clinical results suggest that in vivo gene editing targeting the HSV-1 genome holds acceptable safety as a potential therapy for HSK.

Application of topical 2% cyclosporine A in inflammatory ocular surface diseases.

PURPOSE: To report our experience with the 2% cyclosporin A (CsA) in a series of challenging inflammatory ocular surface diseases due to different etiologies. METHODS: The records of patients who received topical 2% CsA for different indications were reviewed retrospectively. Demographic characteristics, indications for treatment, patient symptoms and clinical findings were recorded. RESULTS: Fifty-two eyes of 52 patients were included. Mean age was 43.2 ± 14.3 (11-66) years with a F/M ratio of 34/18. Indications included pediatric acne rosacea (n = 4), adenoviral corneal subepithelial infiltrates (n = 12), filamentary keratitis (n = 14), pterygium recurrence (n = 15), herpetic marginal keratitis (n = 2) and graft versus host disease (n = 5 patients). Mean duration of treatment was 7.3 ± 2.8 (3-10) months. Forty-three (83%) patients reported favorable outcome with improvement in symptoms after a mean time of 4.4 ± 2.7 (2-6) months. CONCLUSIONS: Topical 2% CsA may address the needs of different cases with ocular surface inflammation, as a safe option for long-term therapy.

Double Trouble: An Unusual Keratitis Case of Herpes Simplex Virus and Varicella-Zoster Virus Co-infection.

PURPOSE: The aim of this study was to describe a case of herpes simplex virus (HSV) and varicella-zoster virus (VZV) corneal co-infection in a patient with systemic immunosuppression. METHODS: A 77-year-old White man who was recently administered pembrolizumab present with reduction in visual acuity in his left eye from 20/25 to 20/50. There was a known history of ocular HSV keratitis. Slit-lamp examination showed superficial dendritic lesions suggestive of VZV. RESULTS: Viral polymerase chain reaction testing was positive for both HSV and VZV, confirming clinical diagnosis of VZV keratitis in the setting of recurrent HSV keratitis. The infection responded to treatment with topical trifluridine. Two months later, he had another episode of keratitis based on his symptoms reported through telephone encounter which resolved with trifluridine. Unfortunately, the patient committed suicide 4 months after onset. CONCLUSIONS: This is the first case of keratitis with HSV and VZV co-infection likely related to systemic immunosuppression. Clinicians should have a high suspicion for viral co-infections in the setting of systemic immunosuppression.

Decision-making in Herpes Simplex viral keratitis grafts.

Background: Keratoplasty after healed herpes simplex viral keratitis is a challenge due to problems arising preoperatively, intraoperatively, and postoperatively. Purpose: In this video, we describe the necessary challenges and the steps, which can be taken to prevent and manage those cases of healed herpes simplex virus (HSV) keratitis that would require a keratoplasty. Synopsis: The video talks about both the typical and atypical features of HSV keratitis, clinical examination, the scenarios which would require a keratoplasty, problems arising intraoperatively and how to manage them, and finally how to approach these high-risk grafts postoperatively. Highlights: Our video highlights the diagnosis of HSV keratitis, which cases are ready for surgery, and preoperative, intraoperative, and postoperative considerations before corneal transplantation in healed HSV keratitis. Decision-making before corneal transplant in HSV grafts can become more structured if these points are followed. Video Link: https://youtu.be/xMT7Ki8vuc4.