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BackgroundSeasonal outbreaks of infectious conjunctivitis remain a public health issue. Determination of outbreak etiologies in the context of a worldwide pandemic may provide useful information to guide public health strategies. The aim of this study was to identify pathogens associated with outpatient infectious conjunctivitis during the COVID-19 Delta surge.MethodsThis prospective study was conducted from April 2021 to September 2021. All outpatients presenting to the Aravind Eye Center (Madurai, India) with signs and symptoms consistent with acute infectious conjunctivitis were eligible. Three swabs were obtained from each participant: one from each conjunctiva and one from the anterior nares. Samples were processed for metagenomic RNA deep sequencing (RNA-seq).ResultsSamples from 106 study participants were sequenced. The most common presenting symptoms were tearing (86%) and itching (71%). Preauricular lymphadenopathy was present in 38% of participants. 20% of participants had close contacts with similar symptoms. Systemic symptoms such as coughing, runny nose, vomiting or diarrhea were uncommonly reported. 60% of all participants used some medicated eye drops upon enrollment. 75% of study participants demonstrated infection with human adenovirus D (HAdV-D). 11% of conjunctivitis was associated with SARS-CoV-2. 15% had no definitive pathogen detected. 8% of all participants had codetection of more than one pathogen on RNA-seq.ConclusionsDuring the COVID-19 Delta surge in India, HAdV-D was the most common pathogen associated with infectious conjunctivitis. SARS-CoV-2 was the second most common associated pathogen. Seasonal surveillance may be necessary for the determination of emerging and reemerging pathogens responsible for infectious conjunctivitis.

PURPOSE: To determine if smartphone photography could be a useful adjunct to blindness prevalence surveys by providing an accurate diagnosis of corneal opacity. METHODS: A total of 174 patients with infectious keratitis who had undergone corneal culturing over the past 5 years were enrolled in a diagnostic accuracy study at an eye hospital in South India. Both eyes had an ophthalmologist-performed slit lamp examination, followed by anterior segment photography with a handheld digital single lens reflex (SLR) camera and a smartphone camera coupled to an external attachment that provided magnification and illumination. The diagnostic accuracy of photography was assessed relative to slit lamp examination. RESULTS: In total, 90 of 174 enrolled participants had a corneal opacity in the cultured eye and no opacity in the contralateral eye, and did not have a penetrating keratoplasty or missing photographs. Relative to slit lamp examination, the sensitivity of corneal opacity diagnosis was 68% (95%CI 58-77%) using the smartphones default settings and 59% (95%CI 49-69%) using the SLR, and the specificity was 97% (95%CI 93-100%) for the smartphone and 97% (95%CI 92-100%) for the SLR. The sensitivity of smartphone-based corneal opacity diagnosis was higher for larger scars (81% for opacities 2 mm in diameter or larger), more visually significant scars (100% for eyes with visual acuity worse than 20/400), and more recent scars (85% for eyes cultured in the past 12 months). CONCLUSION: The diagnostic performance of a smartphone coupled to an external attachment, while somewhat variable, demonstrated high specificity and high sensitivity for all but the smallest opacities.

Abstract Our previous study demonstrated hsa-miR-143-3p as one of the highly expressed miRNAs in enriched corneal epithelial stem cells (CESCs). Hence this study aims to elucidate the regulatory role of hsa-miR-143-3p in the maintenance of stemness in CESCs. The target genes of hsa-miR-143-3p were predicted and subjected to pathway analysis to select the targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-143-3p mimic, inhibitor or scrambled sequence using Lipofectamine 3000. The transfected cells were analysed for (i) colony forming potential, (ii) expression of stem cell (SC) markers/ transcription factors (ABCG2, NANOG, OCT4, KLF4, ΔNp63), (iii) differentiation marker (Cx43), (iv) predicted five targets of hsa-miR-143-3p (DVL3, MAPK1, MAPK14, KRAS and KAT6A), (v) MAPK signaling regulators and (vi) Wnt-β-catenin signaling regulators by qPCR, immunofluorescence staining and/or Western blotting. High expression of hsa-miR-143-3p increased the colony forming potential (10.04 ± 1.35%, p

Abstract 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.

Aspergillus flavus and Fusarium solani are the predominant causative agents of mycotic keratitis in the tropical part of the world. Tear proteins play a major role in the innate immune response against these fungal infections as has been shown by the presence of complement proteins and neutrophil extracellular trap proteins in keratitis patients tear. In this study, we established the presence of the components of the alternate pathway of complement system and their functional state in the tear film of mycotic keratitis patients. The complement proteins namely, C3 and CFH were found only in the open-eye tear of patients but not in control individuals. In vitro analysis showed binding of purified C3b and CFH to fungal spores, which confirmed that the spores can provide a foreign surface for forming the complement complex. Analysis of spore bound tear proteins by mass spectrometry exhibited the presence of known proteins of the alternate pathway complement cascade in keratitis patient tear. Hemolytic assay using rabbit RBC confirmed the presence of a functional alternate pathway of complement cascade in the tear proteome of the patients. The presence of negative regulators, CFH and CFI, in the patient tear indicate that the complement activity is tightly regulated during fungal infection. Mass spectrometry data show vitronectin and clusterin, two known inhibitors of the membrane attack complex only in the patient tear. These data demonstrate the activation of the alternate pathway of complement cascade during the early stages of infection. Interestingly, the production of multiple negative regulators of complement cascade implies the pathogen can effectively evade the host complement system during infection.

Aim: To analyse the outcome of high volume cataract surgery in a developing country, community based, high volume eye hospital. Methods: In a non-comparative interventional case series, the authors reviewed the surgical outcomes of 593 patients with cataract operated upon by three high volume surgeons on six randomly selected days. There were 318 female (54%) and 275 male (46%) patients. Their mean age was 59.57 (SD 10.13) years. The majority of the patients underwent manual small incision cataract surgery (manual SICS). Extracapsular cataract extraction with posterior chamber intraocular lens (ECCE-PCIOL) and intracapsular cataract extraction (ICCE) were also done on a few patients as clinically indicated. Results: Best corrected visual acuity of ⩾6/18 was achieved in 94% of the 520 patients who could be followed up on the 40th postoperative day (88% follow up rate). Intraoperative and immediate postoperative complications as defined by OCTET occurred in 11 (1.9%) and 75 (12.6%) patients, respectively. Average surgical time of 3.75 minutes per case (16-18 cases per hour) was achieved. Statistically significant risk factors for outcomes were found to be age >60, sex, and surgeon. Conclusion: High volume surgery using appropriate techniques and standardised protocols does not compromise quality of outcomes. [ABSTRACT FROM AUTHOR]

Fungal keratitis is a major sight-threatening corneal infection: and mycotic keratitis is more common in tropical parts of the world including India. Aspergillus flavus and Fusarium are the predominant causative agents of corneal infection. We extracted conidial surface proteins of A. flavus from saprophyte and clinical isolates and analyzed the proteins using high resolution mass spectrometry. The data revealed ecotype specific alteration in surface proteome since the proteome profile of the clinical isolates and saprophyte showed significant differences. Detailed examination of the mass spec data of RodA proteins extracted from polyacrylamide gels revealed the presence of two proteoforms of this protein. We also identified the mechanism of formation of these two isoforms. Detailed analysis of this data and the conclusions derived are described in the article, “Identification of the proteoforms of surface localized Rod A of A. flavus and determination of the mechanism of proteoform generation” [1].

Fungal keratitis is one of the leading causes of blindness in the tropical countries affecting individuals in their most productive age. The host immune response during this infection is poorly understood. We carried out comparative tear proteome analysis of Aspergillus flavus keratitis patients and uninfected controls. Proteome was separated into glycosylated and non-glycosylated fractions using lectin column chromatography before mass spectrometry. The data revealed the major processes activated in the human host in response to fungal infection and reflected in the tear. Extended analysis of this dataset presented here complements the research article entitled “Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection [1]” (Jeyalakhsmi Kandhavelu, Naveen Luke Demonte, Venkatesh Prajna Namperumalsamy, Lalitha Prajna, Chitra Thangavel, Jeya Maheshwari Jayapal, Dharmalingam Kuppamuthu, 2016). The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE:PXD003825.

Virulence-factor encoding genes (VFGs) and antimicrobial resistance genes (ARGs) of ocular Methicillin-Resistant Staphylococcus aureus (MRSA), are the reason behind the common cause of severe and untreatable ocular infection and are largely unknown. The unavailability of the complete genome sequence of ocular MRSA strains hinders the unambiguous determination of ARGs and VRGs role in disease pathogenesis and their genomic location. To fulfill this critical need, we achieved the high-quality complete genome of four ocular MRSA strains (AMRF3 - AMRF6) by combining MinION nanopore sequencing technology, followed by polishing with Illumina sequence reads. We obtained a single chromosome and a plasmid in each strain. Sequence typing revealed that AMRF3 and AMRF5 strains harbored ST772, whereas AMRF4 and AMRF6 harbored ST 2066. All plasmids carried heavy metal cadmium resistance genes cadC and cadD, while cadA was detected only in the plasmid pSaa6159 of AMRF4 and AMRF6 strains. Further, pSaa6159 contains a complete Tn552 transposon with beta-lactamase genes, blaI, blaR1, and blaZ. Interestingly, pSaa6159 in AMRF6 carried five copies of Tn552 transposon. Several exotoxins and enterotoxins were identified across ocular MRSA strains and ST2066 strains found to be not carried any enterotoxins; this finding suggests that these two strains are exotoxigenic. Besides, ST2066 strains carried serine proteases (splA, splB, splD, splE and spIF) and exotoxin (seb and set 21) for their virulence, while ST772 carried antimicrobial resistance genes (blaZ, dfrG, msrA, mphC and fosB) and enterotoxin sec for virulence, suggesting sequence type-specific resistance and virulence. Also, we identified many VFGs and ARGs, that provided multi-drug resistance, enterotoxigenic, exotoxigenic, biofilm-forming, host tissue adhesion and immune response evasion in ocular MRSA strains. Thus, our study provides a better insight into the genomes of ocular MRSA strains that would provide more effective treatment strategies for ocular MRSA infection., (Copyright © 2021. Published by Elsevier Ltd.)

Purpose: Here, we report risk factors associated with outcome in severe bacterial keratitis (BK), fungal keratitis (FK), and Acanthamoeba keratitis (AK) in India., Methods: Prospective observational cohort study conducted in Aravind Eye Hospital, India. Adults presenting with severe microbial keratitis (MK) were enrolled (size ≥3 mm) and followed to 21 days post-enrolment. Ulcer clinical features were recorded at presentation. Outcomes by final visit were classified as good (completely healed or reduced infiltrate size) or poor (enlarged infiltrate size, perforated, or surgery performed)., Results: Of 252 participants with severe MK, 191 had FK, 18 had AK, 19 had BK, 4 had mixed BK/FK, and 20 were microbiologically negative. Median age was 50 years (interquartile range [IQR]: 37-60 years), 64% were male, 63% were agriculturalists, and 45% had no formal education. Corneal trauma occurred in 72%, and median symptom duration before presentation was 7 days (IQR: 5-15 days). Clinical features associated with FK were feathery margins (p < 0.001), raised profile (p = 0.039), or dry surface (p = 0.007). Hypopyon was more likely in BK (p = 0.001) and ring infiltrate in AK (p < 0.001). Ulcers with poor outcome (n = 106/214) were more likely to be larger (odds ratio [OR]: 1.63, 95% confidence interval [CI]: 1.30-2.05, p < 0.001), involve the posterior cornea at presentation (OR: 2.31, 95% CI: 1.16-4.59, p = 0.017), involve Aspergillus sp. (OR: 3.23, 95% CI: 1.26-8.25, p = 0.014), or occur in females (OR: 2.04, 95% CI: 1.03-4.04, p = 0.04). Even after treatment, 34% (n = 76/221) had severe visual impairment by the final visit., Conclusions: Severe MK occurred predominantly in agriculturalists post-corneal trauma and often had poor outcomes. Provision of community-based eyecare may allow earlier treatment and improve outcomes.

Purpose: To evaluate the in vitro, extended drug reservoir function of human amniotic membrane (HAM) of different thicknesses impregnated with moxifloxacin., Methods: HAM buttons (12 mm) were soaked with freshly prepared 0.5% wt/vol topical moxifloxacin at different soaking time intervals: 3 hours (group I), 6 hours (group II), 12 hours (group III), 24 hours (group IV), and 48 hours (group V). They were then transferred into 1 mL of fresh simulated tear fluid (pH-7.4) and incubated at 37°C. The release kinetics of moxifloxacin was studied by analyzing the amount of drug in simulated tear fluid collected at different time intervals from each pretreated HAM for 3 weeks. In another experiment, thin and thick HAMs were selected based on weight and soaked with moxifloxacin for 24 hours, and the release kinetics was studied for 7 weeks. All samples were stored at -80°C until analysis by high-performance liquid chromatography., Results: No significant difference was observed between different soaking times and the release of moxifloxacin. The cumulative amount of moxifloxacin released from thick HAM was found to be statistically significant compared with thin HAM (P < 0.05)., Conclusions: Our in vitro data showed that the sustained release of moxifloxacin from HAM was achieved up to 7 weeks. The entrapment efficiency of moxifloxacin was significantly higher in thicker HAM than in thin HAM. Moxifloxacin-impregnated HAM application can be considered in bacterial keratitis to provide sustained drug delivery through a biological bandage system for up to a period of 7 weeks.

Aspergillus flavus is one of the predominant causative organisms of mycotic keratitis in tropical parts of the world. Extracellular proteins are the earliest proteins that come in contact with the host and have a role in the infection process. Exoproteins of A. flavus isolated from infected cornea, sputum and a saprophyte were pooled and identified using high resolution mass spectrometry in order to get the total exoproteome from cultures isolated from different sources. A total of 637 proteins was identified from the pooled A. flavus exoproteome. Analysis based on GO annotations of the 637 identified proteins revealed that hydrolases form the predominant class of proteins in the exoproteome. Interestingly, a greater proportion of the exoproteins seem to be secreted through the non-classical pathways. This data represent the first in-depth analysis of the representative A. flavus exoproteome of a large set of isolates from distinct sources. This data have been deposited to the ProteomeXchange with identifier PXD001296.

Fusarium is the major causative agent of fungal infections leading to corneal ulcer (keratitis) in Southern India and other tropical countries. Keratitis caused by Fusarium is a difficult disease to treat unless antifungal therapy is initiated during the early stages of infection. In this study tear proteins were prepared from keratitis patients classified based on the duration of infection. Among the patients recruited, early infection (n = 35), intermediate (n = 20), late (n = 11), samples from five patients in each group were pooled for analysis. Control samples were a pool of samples from 20 patients. Proteins were separated on difference gel electrophoresis (DIGE) and the differentially expressed proteins were quantified using DeCyder software analysis. The following differentially expressed proteins namely alpha-1-antitrypsin, haptoglobin α2 chain, zinc-alpha-2-glycoprotein, apolipoprotein, albumin, haptoglobin precursor - β chain, lactoferrin, lacrimal lipocalin precursor, cystatin SA III precursor, lacritin precursor were identified using mass spectrometry. Variation in the expression level of some of the proteins was confirmed using western blot analysis. This is the first report to show stage specific tear protein profile in fungal keratitis patients. Validation of this data using a much larger sample set could lead to clinical application of these findings.