Development and Clinical Evaluation of a CRISPR/Cas12a-Based Nucleic Acid Detection Platform for the Diagnosis of Keratomycoses.

Objective: The objective of this study was to develop a rapid and accurate clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based molecular diagnostic assay (Rapid Identification of Mycoses using CRISPR, RID-MyC assay) to detect fungal nucleic acids and to compare it with existing conventional mycologic methods for the diagnosis of fungal keratitis (FK). Design: This study was structured as a development and validation study focusing on the creation and assessment of the RID-MyC assay as a novel diagnostic modality for FK. Subjects: Participants comprised 142 individuals presenting with suspected microbial keratitis at 3 tertiary care institutions in South India. Methods: The RID-MyC assay utilized recombinase polymerase amplification targeting the 18S ribosomal RNA gene for isothermal amplification, followed by a CRISPR/Cas12a reaction. This was benchmarked against microscopy, culture, and polymerase chain reaction for the diagnosis of FK. Main Outcome Measures: The primary outcome measures focused on the analytical sensitivity and specificity of the RID-MyC assay in detecting fungal nucleic acids. Secondary outcomes measured the assay's diagnostic sensitivity and specificity for FK, including its concordance with conventional diagnostic methods. Results: The RID-MyC assay exhibited a detection limit ranging from 13.3 to 16.6 genomic copies across 4 common fungal species. In patients with microbial keratitis, the RID-MyC assay showed substantial agreement with microscopy (kappa = 0.714) and fair agreement with culture (kappa = 0.399). The assay demonstrated a sensitivity of 93.27% (95% confidence interval [CI], 86.62%-97.25%) and a specificity of 89.47% (95% CI, 66.86%-98.70%) for FK diagnosis, with a median diagnostic time of 50 minutes (range, 35-124 minutes). Conclusions: The RID-MyC assay, utilizing CRISPR-Cas12a technology, offers high diagnostic accuracy for FK. Its potential for point-of-care use could expedite and enhance the precision of fungal diagnostics, presenting a promising solution to current diagnostic challenges. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Double-masked, sham and placebo-controlled trial of corneal cross-linking and topical difluprednate in the treatment of bacterial keratitis: Steroids and Cross-linking for Ulcer Treatment Trial (SCUT II) study protocol.

INTRODUCTION: Although antibiotics are successful at achieving microbiological cure in infectious keratitis, outcomes are often poor due to corneal scarring. Ideal treatment of corneal ulcers would address both the infection and the inflammation. Adjunctive topical steroid treatment may improve outcomes by reducing inflammation. Corneal cross-linking (CXL) is a novel prospective therapy that may simultaneously reduce both inflammatory cells and bacterial pathogens. The purpose of this study is to determine differences in 6-month visual acuity between standard medical therapy with antibiotics versus antibiotics with adjunctive early topical steroid therapy versus antibiotic treatment plus CXL and early topical steroids. METHODS AND ANALYSIS: This international, randomised, sham and placebo-controlled, three-arm clinical trial randomises patients with smear positive bacterial ulcers in a 1:1:1 fashion to one of three treatment arms: (1) topical 0.5% moxifloxacin plus topical placebo plus sham CXL; (2) topical 0.5% moxifloxacin plus difluprednate 0.05% plus sham CXL; or (3) the CXL group: topical 0.5% moxifloxacin plus difluprednate 0.05% plus CXL. ETHICS AND DISSEMINATION: We anticipate that both adjunctive topical steroids and CXL will improved best spectacle corrected visual acuity and also reduce complications such as corneal perforation and the need for therapeutic penetrating keratoplasty. This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-Funded Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. Our results will be disseminated via ClinicalTrials.gov website, meetings and journal publications. Our data will also be available on reasonable request. TRIAL REGISTRATION NUMBER: NCT04097730.

Descemet Membrane Suturing for Large Descemet Membrane Detachment After Cataract Surgery.

PURPOSE: To study the outcomes of Descemet membrane (DM) suturing for moderate to severe Descemet membrane detachment (DMD) after cataract surgery. METHODS: This is a retrospective case review of all patients who underwent DM suturing for moderate to severe DMD after cataract surgery in a tertiary training eye care hospital from January 2017 to December 2017. Demographic details, type of cataract, type of cataract surgery, intraoperative complications, severity of DMD, time to repair, and post-DM suturing visual and clinical outcomes were documented. RESULTS: Forty-nine patients were included during the study period with a mean age of 67.7 ± 9.7 years. In total, 79.5% had immature cataracts. Median precataract LogMAR visual acuity was 0.78 (IQR 0.69-1.13). In total, 61.2% underwent manual small-incision cataract surgery, 32.6% had phacoemulsification, 2% had extracapsular cataract extraction (ECCE), and 6% underwent small-incision cataract surgery with trabeculectomy. In total, 44.9% were operated on by trainees and 55.1% were operated on by consultants. In total, 22.4% had primary air descemetopexy during the cataract surgery and failed treatment. All patients had attached DM in the immediate postoperative period. None of them had secondary angle closure glaucoma. At the final follow-up, 42 (85.7%) had clear corneas and 7 (14.3%) had corneal decompensation needing endothelial keratoplasty. Of these 7 patients, 2 (28.5%) had Fuchs dystrophy and 3 (42.8%) had associated DM loss. The median LogMAR visual acuity at the final follow-up was 0.39 (IQR 0.3-0.78). CONCLUSIONS: DM suturing can reattach DM, especially in moderate to severe, recurrent DMD with fewer postoperative complications and thereby reduce the need for corneal transplantation.

Toxic Keratitis After Application of Custard Apple Seed for Head Lice Infestation.

PURPOSE: To report the clinical features and outcomes of toxic keratitis after application of powdered custard apple seeds for hair washing for head lice infestation. METHODS: Retrospective review of all patients with toxic keratitis after application of powdered custard apple seed for head lice infestation during the time period from January 2015 to December 2017. Demographic details, clinical features, and visual outcomes were documented. RESULTS: Thirty-one eyes of 19 patients with toxic keratitis after application of crushed custard apple seeds for head lice infestation were included in the study. Eighteen females and 1 male with a median age of 14 years [interquartile range (IQR) 12-34 years] presented with severe epiphora, congestion, photophobia, and defective vision (median logMar visual acuity 0.4, IQR 0.2-0.8) after application of custard apple seed powder for hair washing. Ten eyes (32.2%) had an epithelial defect (median size 9 mm, IQR 5-12 mm), and 21 (67.7%) eyes had punctate epithelial erosions. All the patients were treated with topical antibiotics, and at 3 days follow-up, all of them had resolution of symptoms and signs with a median logarithm of the minimum angle of resolution (logMAR) visual acuity of 0 (IQR 0-0.2). CONCLUSIONS: Health education about the harmful effect of this traditional practice for head lice infestation will prevent further similar events.

The Asia Cornea Society Infectious Keratitis Study: A Prospective Multicenter Study of Infectious Keratitis in Asia.

PURPOSE: To survey the demographics, risk factors, microbiology, and outcomes for infectious keratitis in Asia. DESIGN: Prospective, nonrandomized clinical study. METHODS: Thirteen study centers and 30 sub-centers recruited consecutive subjects over 12-18 months, and performed standardized data collection. A microbiological protocol standardized the processing and reporting of all isolates. Treatment of the infectious keratitis was decided by the managing ophthalmologist. Subjects were observed for up to 6 months. Main outcome measures were final visual acuity and the need for surgery during infection. RESULTS: A total of 6626 eyes of 6563 subjects were studied. The majority of subjects were male (n = 3992). Trauma (n = 2279, 34.7%) and contact lens wear (n = 704, 10.7%) were the commonest risk factors. Overall, bacterial keratitis was diagnosed in 2521 eyes (38.0%) and fungal keratitis in 2166 eyes (32.7%). Of the 2831 microorganisms isolated, the most common were Fusarium species (n = 518, 18.3%), Pseudomonas aeruginosa (n = 302, 10.7%), and Aspergillus flavus (n = 236, 8.3%). Cornea transplantation was performed in 628 eyes to manage ongoing infection, but 289 grafts (46%) had failed by the end of the study. Moderate visual impairment (Snellen vision less than 20/60) was documented in 3478 eyes (53.6%). CONCLUSION: Demographic and risk factors for infection vary by country, but infections occur predominantly in male subjects and are frequently related to trauma. Overall, a similar percentage of bacterial and fungal infections were diagnosed in this study. Visual recovery after infectious keratitis is guarded, and corneal transplantation for active infection is associated with a high failure rate.

Fungal keratitis: The Aravind experience.

Research becomes very significant and meaningful when it addresses a significant public health problem of a region. Fungal keratitis is a serious problem affecting the agrarian poor and hence requires attention from public health specialists. The approach to a public health issue should focus not only on treatment but also prevention or at least show a significant thrust to reduce the morbidity of the problem. At our institution, we have developed a special interest in fungal keratitis and tried to study it in a multitude of aspects. As we put the pieces of the puzzle together, we believe that interest will be rekindled among policymakers, clinicians, microbiologists, pharmaceutical industry, and basic scientists to work together to join forces and take up an integrative approach to managing this problem. It is also believed that the article underscores the need and importance of having a focused approach to ensuring a successful career in clinical research.

Clinical and demographic study of microsporidial keratoconjunctivitis in South India: a 3-year study (2013-2015).

AIM: To study the demographic profile, clinical features, treatment outcome and ocular morbidity of smear-positive microsporidial keratoconjunctivitis. METHODS: Retrospective case series of all patients with clinical features of microsporidial keratoconjunctivitis and who were smear positive for the same on Gram stain from January 2013 to December 2015. Demographic data, predisposing factors, microbiological investigations, clinical course and visual outcome were analysed. RESULTS: Of the 10 655 patients with conjunctivitis, 550 (5.2%) patients were positive for microsporidia on Gram stain during this time period. The disease was prevalent throughout the year with an increased incidence from July to December. Bilateral involvement was seen in 27 (4.9%) patients. There was no predisposing risk factor in 428 (77.8%) patients. 384 (69.1%) patients had received prior treatment before presentation to us with the most common drug being antibiotic in 285 (49.4%) patients. All the patients underwent diagnostic corneal debridement and received topical 0.3% fluconazole eye drops four times a day. Of the 296 patients who followed-up, 187 (63.1%) patients had complete resolution without sequelae. 68 (22.9%) had persistent superficial punctate keratopathy, 30 (10.1%) developed subepithelial nummular keratitis. No significant change in visual acuity was seen in 255 (80.7%) eyes, two or more line improvement was seen in 48 (15.2%) eyes, while two or more line worsening was seen in 13 (4.1%) eyes. CONCLUSIONS: Microsporidial keratoconjunctivitis is prevalent in South India throughout the year. The characteristic clinical signs and simple microbiological investigation help us to differentiate it from adenoviral keratoconjunctivitis. The visual prognosis is good.

Clinical and microbiological study of paediatric infectious keratitis in South India: a 3-year study (2011-2013).

PURPOSE: To study the risk factors, microbiological profile and clinical outcomes of infectious keratitis affecting paediatric patients. STUDY DESIGN: Retrospective case series. METHODS: Review of case records of paediatric patients (0-16 years) diagnosed with infectious keratitis who presented to Aravind Eye Hospital, Madurai, India during January 2011 to December 2013. Demographic details, predisposing factors, microbiological investigations, clinical course and visual outcome were analysed. RESULTS: In this time period, 240 eyes of 234 children had a diagnosis of infectious keratitis. One hundred and twenty-five (53.4%) children had a history of trauma. Smears were obtained in 220 eyes, while culture was performed in 191 eyes. The culture results were positive in 142 (74.3%) eyes. Fungi was the most common infectious agent isolated in culture (54.2%) followed by bacteria (40.8%) and acanthamoeba (2.1%). Successful healing of the keratitis with appropriate medical therapy occurred in 223 (92.9%) eyes, while 17 (7.1%) eyes required therapeutic keratoplasty. Of the 151 patients with preliminary and final visual acuity, vision improved by 2 lines in 68 eyes (45%), stayed the same in 75 eyes (49.6%) and worsened in 8 eyes (5.3%). CONCLUSIONS: Contrary to previous reports, fungi are the most common aetiological organism in the causation of infectious keratitis in children in our study population. Fusarium was the most common fungal species isolated. These data are similar to the data obtained from adult patients with infectious keratitis in this region. While microbiological investigations are important to initiate appropriate antimicrobial therapy, the findings from our study need to be kept in mind, especially while initiating empirical therapy in this population.

Corneal Cross-linking as an Adjuvant Therapy in the Management of Recalcitrant Deep Stromal Fungal Keratitis: A Randomized Trial.

PURPOSE: To assess the efficacy of corneal cross-linking (CXL) as an adjuvant to appropriate antifungal therapy in nonresolving deep stromal fungal keratitis. DESIGN: Randomized clinical trial. METHODS: Eyes with culture-positive deep stromal fungal keratitis not responding to appropriate medical therapy for a period of 2 weeks were randomized to receive either adjuvant CXL or no additional treatment. Antifungal medical therapy was continued in both groups. The prespecified primary outcome was treatment failure at 6 weeks after enrollment, defined as perforation and/or increase in ulcer size by ≥2 mm. RESULTS: The trial was stopped before full enrollment because of a marked difference in the rate of perforation between the 2 groups. Of the 13 cases enrolled in the study, 6 were randomized to the CXL group and 7 to the non-CXL group. Five eyes in the CXL group and 3 eyes in the non-CXL group experienced treatment failure by 6 weeks (P = .56). In a secondary analysis, the CXL group experienced more perforations than the non-CXL group (4 vs 0, respectively; P = .02). CONCLUSION: CXL used as adjuvant therapy for recalcitrant deep stromal fungal keratitis did not improve outcomes.

Economic analysis of corneal ulcers in South India.

PURPOSE: To estimate the costs of treating corneal ulcers at a tertiary eye care center in south India from a patient perspective. METHODS: A prospective cohort study design at a tertiary eye care center in south India. Each subject presenting with corneal ulcers to the center had a detailed clinical and laboratory workup including slit-lamp biomicroscopy, direct smear examination with appropriate stains, and cultures. Data on direct costs including costs of consultation, studies to confirm diagnosis, and treatment were collected at baseline and follow-up visits. We also collected indirect costs including travel, accommodation, food, and loss of wages of both the subject and accompanying persons. RESULTS: We recruited 498 consecutive cases for the study. The mean duration of onset of symptoms before presentation at the cornea service was 13.1 +/- 19.9 days. The mean follow-up duration was 34.8 +/- 28.2 days. The total overall cost for accessing and receiving care was 85.8 US dollars +/- 4.6 (95% confidence interval: 76.4 US dollars, 94.6). The mean total cost to diagnose and appropriately treat 1 case of keratitis such that the patient had vision better than 6/18 at final follow-up was 56.2 US dollars +/- 3.6 (95% confidence interval: 49.0 US dollars, 63.3). CONCLUSIONS: : The costs to the patient to receive appropriate care for corneal ulcers in this population are much higher than the average monthly wage for this population. There is an urgent need to devise strategies that may help reduce costs of care for corneal ulcers in this population.