Melanin depletion affects Aspergillus flavus conidial surface proteins, architecture, and virulence.

Melanin is an Aspergillus flavus cell wall component that provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile, and virulence of melanized conidia (MC) and non-melanized conidia (NMC) of A. flavus. Kojic acid treatment inhibited melanin synthesis in A. flavus, and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathways were found only in the formic acid extracts of NMC. Scanning electron microscopy (SEM) analysis showed the conidial surface morphology difference between the NMC and MC, indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor white staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining between MC and NMC. Evaluation of the virulence of MC and NMC in the Galleria mellonella model showed NMC was less virulent compared to MC. Our findings showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer, and hence, melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. The G. mellonella virulence assay also confirmed that the NMC were susceptible to host defense as in other Aspergillus pathogens. KEY POINTS: • l-DOPA melanin production was inhibited in A. flavus isolates by kojic acid, and for the first time, scanning electron microscopy (SEM) analysis revealed morphological differences between MC and NMC of A. flavus strains • Proteome profile of non-melanized conidia showed more conidial surface proteins and these proteins were mainly involved in the virulence, oxidative stress, and metabolism pathways • Non-melanized conidia of A. flavus strains were shown to be less virulent than melanised conidia in an in vivo virulence experiment with the G. melonella model.

Comparative proteome analysis identifies species-specific signature proteins in Aspergillus pathogens.

Aspergillus flavus and Aspergillus fumigatus are important human pathogens that can infect the lung and cornea. During infection, Aspergillus dormant conidia are the primary morphotype that comes in contact with the host. As the conidial surface-associated proteins (CSPs) and the extracellular proteins during the early stages of growth play a crucial role in establishing infection, we profiled and compared these proteins between a clinical strain of A. flavus and a clinical strain of A. fumigatus. We identified nearly 100 CSPs in both Aspergillus, and these non-covalently associated surface proteins were able to stimulate the neutrophils to secrete interleukin IL-8. Mass spectrometry analysis identified more than 200 proteins in the extracellular space during the early stages of conidial growth and germination (early exoproteome). The conidial surface proteins and the early exoproteome of A. fumigatus were enriched with immunoreactive proteins and those with pathogenicity-related functions while that of the A. flavus were primarily enzymes involved in cell wall reorganization and binding. Comparative proteome analysis of the CSPs and the early exoproteome between A. flavus and A. fumigatus enabled the identification of a common core proteome and potential species-specific signature proteins. Transcript analysis of selected proteins indicate that the transcript-protein level correlation does not exist for all proteins and might depend on factors such as membrane-anchor signals and protein half-life. The probable signature proteins of A. flavus and A. fumigatus identified in this study can serve as potential candidates for developing species-specific diagnostic tests. KEY POINTS: • CSPs and exoproteins could differentiate A. flavus and A. fumigatus. • A. fumigatus conidial surface harbored more antigenic proteins than A. flavus. • Identified species-specific signature proteins of A. flavus and A. fumigatus.

Expression of immune response genes in human corneal epithelial cells interacting with Aspergillus flavus conidia.

BACKGROUND: Aspergillus flavus, one of the causative agents of human fungal keratitis, can be phagocytosed by human corneal epithelial (HCE) cells and the conidia containing phagosomes mature into phagolysosomes. But the immunological responses of human corneal epithelial cells interacting with A. flavus are not clear. In this study, we report the expression of immune response related genes of HCE cells exposed to A. flavus spores using targeted transcriptomics. METHODS: Human corneal epithelial cell line and primary cultures were grown in a six-well plate and used for coculture experiments. Internalization of the conidia was confirmed by immunofluorescence microscopy of the colocalized endosomal markers CD71 and LAMP1. Total RNA was isolated, and the quantity and quality of the isolated RNA were assessed using Qubit and Bioanalyzer. NanoString nCounter platform was used for the analysis of mRNA abundance using the Human Immunology panel. R-package and nSolver software were used for data analysis. KEGG and FunRich 3.1.3 tools were used to analyze the differentially expressed genes. RESULTS: Different morphotypes of conidia were observed after 6 h of coculture with human corneal epithelial cells and found to be internalized by epithelial cells. NanoString profiling showed more than 20 differentially expressed genes in immortalized human corneal epithelial cell line and more than ten differentially expressed genes in primary corneal epithelial cells. Distinct set of genes were altered in their expression in cell line and primary corneal epithelial cells. KEGG pathway analysis revealed that genes associated with TNF signaling, NF-KB signaling, and Th17 signaling were up-regulated, and genes associated with chemokine signaling and B cell receptor signaling were down regulated. FunRich pathway analysis showed that pathways such as CDC42 signaling, PI3K signaling, and Arf6 trafficking events were activated by the clinical isolates CI1123 and CI1698 in both type of cells. CONCLUSIONS: Combining the transcript analysis data from cell lines and primary cultures, we showed the up regulation of immune defense genes in A. flavus infected cells. At the same time, chemokine signaling and B cell signaling pathways are downregulated. The variability in the expression levels in the immortalized cell line and the primary cultures is likely due to the variable epigenetic reprogramming in the immortalized cells and primary cultures in the absence of any changes in the genome. It highlights the importance of using both cell types in host-pathogen interaction studies.

Human Corneal Epithelial Cells Internalize Aspergillus flavus Spores by Actin-Mediated Endocytosis.

Human corneal epithelial (HCE) cells play a significant role in the innate immune response by secreting cytokines and antimicrobial peptides when they encounter fungal pathogens. But the detailed mechanism of attachment and engulfment of the fungal conidia by HCE cells is not well understood. Here, we show the phagocytosis of Aspergillus flavus conidia by RCB2280 cells and primary HCE cultures using confocal microscopy and proteomic analysis of conidium-containing phagosomes. Phalloidin staining showed actin polymerization, leading to an actin ring around engulfed conidia. Cytochalasin D inhibited the actin-mediated endocytosis of the conidia. Immunolabeling of the early endosomal markers CD71 and early endosomal antigen (EEA1) and the late endosomal markers lysosome-associated membrane protein 1 (LAMP1), Rab7, and cathepsin G showed that endosomal proteins were recruited to the site of conidia and showed maturation of the conidium-containing phagosomes. Lysotracker red DND 99 labeling showed the acidification of the phagosomes containing conidia. Phagosome-specific proteome analysis confirmed the recruitment of various phagosomal and endosomal proteins to the conidium-containing phagosomes. These results show that the ocular surface epithelium contributes actively to antifungal defense by the phagocytosis of invading fungal conidia.

Genotyping and phylogenetic analysis of Pythium insidiosum causing human corneal ulcer.

Pythium insidiosum belongs to the Oomycetes, which are known to cause serious life-threatening infectious condition in humans and animals. Corneal infections caused by P. insidiosum are rare and difficult to treat. The molecular-based diagnosis of Pythium is employed for the species identification and to study molecular phylogenetic relationship. Based on Cytochrome oxidase II (cox II) gene, P. insidiosum is categorized into three clades or groups: Clade-I or ATH (American strains), Clade-II or BTH (American, Asian, and Australian strains), and Clade-III or CTH (mostly Thailand strains). This study focused on the molecular identification of Pythium insidiosum from patients with corneal ulcer using ITS regions and clade identification by cox II gene sequencing and correlated with the clinical outcome. The isolates were collected from Aravind Eye Hospital, Madurai, India, from April to December 2018. Through the microbiological laboratory reports, 15 isolates of Pythium sp. from keratitis patient were selected, followed by DNA extraction, ITS, and cox II gene sequencing and phylogenetic analysis using the reference sequences from NCBI database. All 15 P. insidiosum isolates were phylogenetically clustered together as a single group and where also placed distantly from other Pythium species (outgroup). Most ocular isolates fell into either clade BTH or clade CTH, and none of our ocular isolates were in clade ATH. Two of the strains were very distinct and did not match any of the clusters indicating different lineages. There was no significant difference between clinical outcome and genotype of P. insidiosum.

Autophagy induced by type III secretion system toxins enhances clearance of Pseudomonas aeruginosa from human corneal epithelial cells.

Corneal ulcers caused by Pseudomonas aeruginosa may lead to severe visual disability due to impaired bacterial clearance from corneal tissues. Our purpose was to study the role of autophagy in the intracellular clearance of P. aeruginosa from human corneal epithelial cells (HCET) and its regulation by the bacterial type III secretion system (T3SS) toxins. Nine different corneal ulcer isolates of P. aeruginosa, PAO1 and T3SS mutants of PAO1 were used to infect HCET cells. Induction of autophagy (Immunofluorescence and Western blot) and pro-inflammatory gene expression (real time PCR) by P. aeruginosa and the role of autophagy in intracellular bacterial clearance were studied in the context of T3SS genotypes. The clinical isolates and PAO1 induced autophagy irrespective of the T3SS genotype, whereas the T3SS mutants were relatively defective in inducing autophagy and becn1 gene expression. External induction of autophagy significantly reduced the intracellular load of P. aeruginosa strains that were associated with worst clinical outcomes. The T3SS negative isolate and PAO1ΔexoST were less sensitive to pharmacological modulation of autophagy and had relatively higher replication potential, suggesting a possible mechanism of bacterial survival in the absence of T3SS toxins. Overall, our results highlight a selective role for autophagy in bacterial clearance from corneal epithelial cells and emphasize the pro-autophagic role of bacterial toxins in the context of corneal ulcers.

Genotypic and phenotypic characterization of Pseudomonas aeruginosa isolates from post-cataract endophthalmitis patients.

Endophthalmitis caused by Pseudomonas aeruginosa is associated with rapid disease progression and poor visual outcome due to high virulence of the organism. Our aim was to characterize the virulence determinants of P. aeruginosa causing post-operative endophthalmitis. Repetitive sequence analysis (ERIC PCR) was done to study the clonal relatedness of the 17 P. aeruginosa isolates. Type 3 secretion system (T3SS) genotypes were determined and the isolates were further classified as invasive or cytotoxic based on gentamicin survival, trypan blue dye exclusion and MTT assays. Phenotypically, the strains were characterized based on bacterial motility patterns, biofilm formation, phospholipase production and antibiotic susceptibility patterns. Most of our ocular isolates were invasive in nature and nearly half of them were multi-drug resistant. 47% of the isolates formed a strong biofilm, whereas the rest formed moderate to weak biofilms, which may account for an increased colonization and antibiotic resistance. Although the T3SS genotypes correlated well with the invasive/cytotoxic nature of the strains, none of the genotypes were associated with any particular phenotypic trait. To the best of our knowledge, this is the first report on the phenotypic characteristics of P. aeruginosa strains causing post-operative endophthalmitis. Our findings demonstrate that these strains have higher invasive potential and an ability to form biofilm which possibly contributes to an increased ocular virulence.

Biomarker Detection and Validation for Corneal Involvement in Patients With Acute Infectious Conjunctivitis.

Importance: Infectious conjunctivitis can lead to corneal involvement and result in ocular morbidity. The identification of biomarkers associated with corneal involvement has the potential to improve patient care. Objective: To identify biomarkers in patients with acute infectious conjunctivitis. Design, Setting, and Participants: This cross-sectional study took place from December 2016 to March 2024. Analyses were performed in 3 phases. First, logistic regression and machine learning algorithms were used to predict the probability of demonstrating corneal involvement in patients with presumed infectious conjunctivitis. Second, quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to confirm the most important biomarker gene identified by the algorithm. Third, the biomarker gene was validated in prospectively collected conjunctival samples of adult patients from 3 outpatient centers in Thailand and 1 in India. Patients with signs and symptoms of infectious conjunctivitis and onset within less than 14 days were eligible. Exclusion criteria were the inability to consent, presumed toxicity, or allergic conjunctivitis. Exposures: Acute infectious conjunctivitis. Main Outcomes and Measures: The identification and validation of ocular surface gene expression associated with corneal findings on slitlamp examination. Results: Thirteen genes exhibited a 1.5-log2 fold change in expression in patients with corneal involvement compared to patients without corneal involvement. Using the 13 genes to train and cross validate, logistic regression produced the highest mean area under the receiver operating characteristic curve (AUROC; 0.85; 95% CI, 0.84-0.86) for corneal involvement. The removal of apolipoprotein E (APOE) from the gene ensemble led to a decline in predictive performance of the logistic regression classifier (from mean AUROC 0.85 [95% CI, 0.84-0.86] to 0.74 [95% CI, 0.73-0.75]; adjusted P = .001 [Tukey test]). Orthogonal testing of APOE expression level with RT-qPCR showed that APOE expression was higher in patients with corneal involvement compared to patients without (median [IQR], 0.23 [0.04-0.47] vs 0.04 [0.02-0.06]; P = .004 [Mann-Whitney U test]). Using a Youden index of 0.23 Δ threshold cycle, APOE had a sensitivity of 56% (95% CI, 33-77) and a specificity of 88% (95% CI, 79-93) in 106 samples with conjunctivitis at Aravind, India (P < .001 [Fisher exact test]). When applied to a different patient population in Thailand, the same criteria could discriminate between disease states (58 samples; sensitivity, 47%; 95% CI, 30-64 and specificity, 93%; 95% CI, 77-99; P = .001 [Fisher exact test]). Conclusions and Relevance: The results from this study suggest that the host conjunctival immune response can be meaningfully interrogated to identify biomarkers for ocular surface diseases.

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.

A double-masked, sham-controlled trial of rose bengal photodynamic therapy for the treatment of fungal and acanthameoba keratitis: Rose Bengal Electromagnetic Activation with Green Light for Infection Reduction (REAGIR) Study.

Background: Infectious keratitis secondary to fungus or acanthamoeba often has a poor outcome despite receiving the best available medical therapy. In vitro Rose Bengal Photodynamic therapy (RB-PDT) appears to be effective against fungal and acanthamoeba isolates.22,23 In one published series RB-PDT reduced the need for therapeutic penetrating keratoplasty in severe bacterial, fungal, and acanthameoba keratitis not responsive to medical therapy. Methods: This international, randomized, sham and placebo controlled 2-arm clinical trial, randomizes patients with smear positive fungal and acanthameoba and smear negative corneal ulcers in a 1:1 fashion to one of two treatment arms: 1) Topical antimicrobial plus sham RB-PDT or 2) Topical antimicrobial plus RB-PDT. Discussion: We anticipate that RB-PDT will improve 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 upon reasonable request. Trial Registration: NCT, NCT05110001, Registered November 5, 2021. https://www.clinicaltrials.gov/study/NCT05110001.

Molecular diagnosis and ocular imaging of West Nile virus retinitis and neuroretinitis.

PURPOSE: To describe the ocular features of West Nile virus (WNV) infection proven by serology and molecular diagnostic techniques. DESIGN: Prospective case series. PARTICIPANTS: Fifty-two patients who presented to the uveitis clinic with ocular inflammatory signs and history of fever preceding ocular symptoms between January 2010 and January 2012 were enrolled for laboratory diagnosis. Serum samples were collected from 30 healthy controls from the same geographic area. METHODS: Patients were tested for all endemic infectious diseases that can cause ocular inflammation by serology or molecular diagnostics. When patients had positive antibodies for WNV, serum/plasma samples were tested by real-time reverse transcription (RT) polymerase chain reaction (PCR) and RT loop-mediated isothermal gene amplification assays. The PCR product was subjected to nucleotide sequencing. Fundus fluorescence angiography (FFA), optical coherence tomography (OCT), and indocyanine green angiography were performed. Visual prognosis was analyzed. MAIN OUTCOME MEASURES: Clinical signs (retinitis, neuroretinitis, and choroiditis) and ocular complications (decrease in vision). RESULTS: A total of 37 of 52 patients (71%) showed positive results for at least 2 laboratory tests for WNV. Fundus examination revealed discrete, superficial, white retinitis; arteritis; phlebitis; and retinal hemorrhages with or without macular star. The FFA revealed areas of retinal inflammation with indistinct borders, vascular and optic disc leakage, vessel wall staining, or capillary nonperfusion. Indocyanine green angiography confirmed choroidal inflammation in 1 of the patients who was diabetic. The OCT scan of the macula revealed inner retinal layer edema in active inflammation and retinal atrophy in late stage. At the final visit, 43% of patients had visual acuity better than 6/12. CONCLUSIONS: In addition to previously reported clinical signs, retinitis, neuroretinitis, and retinal vasculitis were seen in this population. Atrophy of the inner retinal layer was seen on OCT after resolution of inflammation. Visual prognosis was good in patients with focal retinitis and poor in patients with occlusive vasculitis. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Incidence, Clinical Profile, and Management of Keratitis Caused by Uncommon Species of Pseudomonas at a Tertiary Eye Care Center.

PURPOSE: The purpose of this study was to study the incidence, demographic features, clinical course, profiling, and management of uncommon species of Pseudomonas keratitis (other than Pseudomonas aeruginosa ) at a tertiary eye care center. METHODS: Thirty cases of culture-proven uncommon species of Pseudomonas keratitis between January 2017 and December 2021 were retrospectively studied. The incidence, demographic and clinical profile, predisposing factors, microbial results, treatment, and visual outcomes were analyzed. We evaluated the risk factors for poor treatment outcomes. RESULTS: Among bacterial keratitis cases, uncommon species of Pseudomonas keratitis occurred at a rate of 2.2%. The mean age at presentation was 51.37 years, and the most common predisposing factor was corneal trauma (36.7%). The mean best corrected visual acuity (BCVA) [in log of minimum angle of resolution (logMAR)] at presentation was 1.99, and the mean ulcer size was 5.75 mm. On culture, 56.7% of the cases were identified as Pseudomonas putida , 26.7% as Pseudomonas stutzeri , 10% as Pseudomonas mendocina, and 3.3% each of Pseudomonas oryzihabitans and Pseudomonas alcaligenes . We recorded good treatment responses in 66.7% of cases with the medical therapy of a combination of broad-spectrum antibiotics, whereas 33.3% of cases required surgical intervention. The risk factors for poor clinical outcome were older age, ocular trauma, previous ocular surgeries, poor BCVA at presentation, large ulcer size, delayed treatment, hypopyon, and early complications such as perforation, limbal involvement, and total ulcer. CONCLUSIONS: Uncommon species of pseudomonas keratitis was more closely related to predisposing factors such as corneal trauma and other factors such as previous ocular surgeries, older age, large ulcers, longer duration of treatment, early surgical intervention in complicated cases, and poor visual outcome.

Apollo Rising: Acute Conjunctivitis Outbreak in India, 2022.

Purpose: To identify pathogens associated with the 2022 conjunctivitis outbreak in Tamil Nadu, India. Methods: This prospective study was conducted in November of 2022. Patients with presumed acute infectious conjunctivitis presenting to the Aravind Eye Clinic in Madurai, India were eligible. Anterior nares and conjunctival samples from participants were obtained and processed for metagenomic RNA deep sequencing (RNA-seq). Results: Samples from 29 patients were sequenced. A pathogen was identified in 28/29 (97%) patients. Coxsackievirus A24v, a highly infectious RNA virus, was the predominant pathogen and detected in 23/29 patients. Human adenovirus D (HAdV-D), a DNA virus commonly associated with conjunctivitis outbreaks, was detected in the remaining patients (5/29). Hemorrhagic conjunctiva was documented in both HAdV-D and coxsackievirus A24v affected patients but was not the predominant clinical presentation. Phylogenetic analysis of coxsackievirus A24v revealed a recent divergence from the 2015 outbreak. Conclusions: Coxsackievirus A24v and HAdV-D were co-circulating during the 2022 conjunctivitis outbreak in Tamil Nadu, India. Clinical findings were similar between patients with HAD-V and coxsackievirus A24v associated conjunctivitis. As high-throughput technologies become more readily accessible and cost-effective, unbiased pathogen surveillance may prove useful for outbreak surveillance and control.

Antiamoebic Susceptibility in Acanthamoeba Keratitis: Comparison of Isolates From South India and Northern California.

PURPOSE: Outcomes of Acanthamoeba keratitis are often worse in India than in the United States. The goal of this study was to determine whether antiamoebic susceptibility patterns were different when comparing Acanthamoeba isolates from India with those of the United States. METHODS: Acanthamoeba isolates were obtained from corneal scrapings of 43 patients with infectious keratitis seen at the Francis I. Proctor Foundation (N = 23) and Aravind Eye Hospital (N = 20) from 2008 through 2012 and plated on growth media. A previously described minimum cysticidal concentration (MCC) assay was performed by a single laboratory technician to assess susceptibility to 5 antiamoebic agents for all isolates. Testing was conducted in triplicate, with the median MCC chosen for analyses. RESULTS: The MCC (µg/mL) of polyhexamethylene biguanide was 6.25 [IQR 5.47-12.5] for Aravind isolates and 6.25 [IQR 6.25-9.375] for Proctor isolates ( P = 0.75), corresponding values were 6.25 [IQR 3.125-6.25] and 3.125 [IQR 3.125-9.375] for chlorhexidine ( P = 0.81), 2500 [IQR 2500-5000] and 5000 [IQR 1250-20,000] for voriconazole ( P = 0.25), 15.6 [IQR 15.6-39.0625] and 15.6 [IQR 15.6-31.25] for hexamidine ( P = 0.92), and 15.6 [IQR 7.81-15.6] and 15.6 [IQR 7.81-31.25] for propamidine ( P = 0.42). CONCLUSIONS: This study found no statistically significant differences in antiamoebic susceptibility of Indian versus US samples from Acanthamoeba keratitis clinical isolates. These findings suggest that differences in antiamoebic susceptibility are likely not responsible for differential outcomes in Acanthamoeba keratitis between the 2 locations.

Corynebacterium Keratitis: Pure Versus Mixed Infection and Antibiotic Susceptibility Patterns From Different Tertiary Eye Care Centers.

PURPOSE: The objective of this study was to compare the clinical and microbiological profiles of culture-proven pure Corynebacterium keratitis with mixed infection and their antibiotic susceptibility patterns over a 2-year period. METHODS: A retrospective analysis of culture-proven cases of Corynebacterium keratitis over a 2-year period was performed in 3 different tertiary eye care centers. All isolates were tested for antibiotic susceptibility in vitro using the disc-diffusion method for 7 antibiotics. RESULTS: Altogether 108 cases were identified as culture-positive Corynebacterium keratitis in 3 tertiary eye care centers. Of these, 60.2% (n = 65) and 39.8% (n = 43) of cases were due to pure Corynebacterium and mixed infection, respectively. The mean duration of symptoms was 23.2 ± 29.6 days. In the mixed-infection group, fungus was identified as the coexistent pathogen in 22 cases (51.1%). Ocular surface disorder was the most common risk factor (33.9%) in Corynebacterium keratitis. The most frequently isolated species was Corynebacterium amycolatum (22.2%) in both groups. Therapeutic keratoplasty was performed in 8.3% of cases. There was no significant difference in the outcome between the 2 groups. Cefazolin resistance was seen in 13.9% of patients, and all isolates were susceptible to vancomycin. The resistance pattern showed emerging resistance toward fluoroquinolone because the isolates were resistant to gatifloxacin (58.3%), moxifloxacin (47.2%), ciprofloxacin (54.6%), and ofloxacin (45.4%). CONCLUSIONS: Ocular surface disorder is the most common risk factor in Corynebacterium keratitis. Although fluoroquinolones are commonly used as first-line therapy in microbial keratitis, the in vitro resistance pattern indicates that these are less likely to be effective in infection with Corynebacterium species.

Pythium insidiosum keratitis - A review.

Pythium insidiosum is an oomycete and is also called "parafungus" as it closely mimics fungal keratitis. The last decade saw an unprecedented surge in Pythium keratitis cases, especially from Asia and India, probably due to growing research on the microorganism and improved diagnostic and treatment modalities. The clinical features such as subepithelial infiltrate, cotton wool-like fluffy stromal infiltrate, satellite lesions, corneal perforation, endoexudates, and anterior chamber hypopyon closely resemble fungus. The classical clinical features of Pythium that distinguish it from other microorganisms are reticular dots, tentacular projections, peripheral furrowing, and early limbal spread, which require a high index of clinical suspicion. Pythium also exhibits morphological and microbiological resemblance to fungus on routine smearing, revealing perpendicular or obtuse septate or aseptate branching hyphae. Culture on blood agar or any other nutritional agar is the gold standard for diagnosis. It grows as cream-colored white colonies with zoospores formation, further confirmed using the leaf incarnation method. Due to limited laboratory diagnostic modalities and delayed growth on culture, there was a recent shift toward various molecular diagnostic modalities such as polymerase chain reaction, confocal microscopy, ELISA, and immunodiffusion. As corneal scraping (10% KOH, Gram) reveals fungal hyphae, antifungals are started before the culture results are available. Recent in vitro molecular studies have suggested antibacterials as the first-line drugs in the form of 0.2% linezolid and 1% azithromycin. Early therapeutic keratoplasty is warranted in nonresolving cases. This review aims to describe the epidemiology, clinical features, laboratory and molecular diagnosis, and treatment of Pythium insidiosum keratitis.

A comparative study on endothelial cell loss in nanophthalmic eyes undergoing cataract surgery by phacoemulsification.

Purpose: The purpose of this study is to compare the endothelial cell loss (ECL) in nanophthalmic eyes and age-matched controls undergoing cataract surgery by phacoemulsification and also to identify the risk factors influencing the endothelial cell density (ECD). This was a prospective comparative interventional case series. Methods: We enrolled 19 nanophthalmic eyes (study group) and 42 age-matched cataract controls (control group) undergoing phacoemulsification after meeting the inclusion criteria. Ocular parameters like best-corrected visual acuity, intraocular pressure, pachymetry, specular microscopy, and slit lamp findings were noted preoperatively and at month 1 and 3 postsurgery. All nanophthalmic eyes underwent cataract surgery with concomitant prophylactic posterior sclerostomy. Results: The median percentage endothelial loss in nanophthalmic eyes was 4.0 (IQR 0-23.5), 7.4 (IQR 1.0--22.4) at 1 and 3 months postoperatively compared to 6.3 (IQR 1.7-14.1) and 6.4 (IQR 2.6--12.1) in age controlled normal eyes (P = 0.94, P = 0.46, respectively). Linear regression analysis showed increasing age as the only variable influencing the percentage decrease in corneal ECD in the study group (P = 0.001). Nanophthalmic eyes with ACD <2.5 mm had a significantly greater reduction in ECD at 3 months postcataract surgery compared to baseline (P = 0.039). Visual outcomes and IOP reduction in the study group with ACD >2.5 mm were significantly better postcataract surgery (P = 0.02 and P = 0.002, respectively). Conclusion: The percentage of ECL in nanophthalmic eyes undergoing phacoemulsification is equivalent to normal eyes. However, in the nanophthamic eyes with AC depth <2.5 mm, the percentage cell loss was significantly higher warranting the need for extensive intraoperative care. Increasing age was found to be the only significant risk factor influencing the ECD in short eyes.

Periconia species - An unusual fungal pathogen causing mycotic keratitis.

PURPOSE: The purpose of this study is to determine the epidemiology, risk factors, clinical features, and treatment outcome of molecularly diagnosed Periconia keratitis. METHODS: Clinical records of all culture proven fungal ulcers with molecular identification suggestive of Periconia species who presented to a single tertiary referral center from January 2012 to December 2013 were retrospectively analysed. RESULTS: Among 1356 cases of keratomycosis, 8 (0.6%) patients were affected due to Periconia species. The mean age of presentation was 59 years with males (n = 6; 75%) were more commonly affected than females (n = 2; 25%). Significant history of trauma was present only in one patient. The infiltrate size was less than 5 mm in majority of patients 75% (n = 6). 50% (n = 4) responded to antifungal, 12.5% (n = 1) responded to antibacterial, 12.5% (n = 1) required therapeutic penetrating keratoplasty, 25% (n = 2) lost to follow up after first visit. The mean duration of treatment in healed cases was 20 days. CONCLUSION: This is the first report on Periconia sp causing human corneal ulcer. This study signifies the importance of molecular identification in the diagnosis of rare fungi which will improve our understanding on disease pathology and outcome. Visual prognosis appears good if the infection is diagnosed and topical antifungal interventions started early.

Clinical and demographic study of non-tuberculous mycobacterial ocular infections in South India.

PURPOSE: To describe demographics, risk factors, antibiotic susceptibility, management and outcomes of ocular infections caused by non-tuberculous mycobacteria (NTM). METHODS: A retrospective review of medical case records and microbiology records of patients with ocular infections that were culture positive for non-tuberculous Mycobacteria from January 2014 to December 2018 was done. Antibiotic susceptibility profile was done based on the CLSI guidelines. Laboratory diagnosis for the NTM Species was done by conventional microbiological methods. The species identification was done for stored isolated utilizing polymerase chain reaction targeting 16S rDNA and rpoB gene, followed by DNA sequencing and phylogenetic analysis. RESULTS: Twenty patients with NTM ocular infections were identified during the study period. A majority of cases presented as 12 infectious keratitis (60%) and three suture-related corneal infiltrates (15%). Common risk factors were history of trauma in 9 (45%) patients and history of ocular surgery in 5 (25%) patients. Patients were treated with combination of amikacin and flouroquinolones/chloramphenicol (70%) and surgical interventions were performed in 25% cases. Only twelve isolates were stored and ten isolates were identified as the M. abscessus subsp. abscessus and two isolates as M. abscessus subsp. massiliense by sequencing and phylogenetic analysis. Majority of the NTM were sensitive to amikacin (75%) followed by moxifloxacin, ciprofloxacin, cephotaxime and tobramycin (35%). CONCLUSION: High degree of clinical suspicion, multidrug antibiotic therapy and timely surgical intervention in patients with NTM infections, are advised for better clinical outcomes. Prior ocular trauma, prior ocular surgery and presence of biomaterials were the major predisposing factors. Earlier surgical intervention in cases where abscesses or biomaterials are involved, is necessary for rapid recovery.