Unveiling the Innate and Adaptive Immunity Interplay: Global Transcriptomic Profiling of the Host Immune Response in Candida albicans Endophthalmitis in a Murine Model.

Intraocular fungal infection poses a significant clinical challenge characterized by chronic inflammation along with vision impairment. Understanding the host defense pathways involved in fungal endophthalmitis will play a pivotal role in identifying adjuvant immunotherapy. Clinical isolates of Candida albicans (15,000 CFU/µL) were intravitreally injected in C57BL/6 mice followed by enucleation at 24 and 72 h postinfection. Histopathological analysis was performed to evaluate the retinal changes and the disease severity. RNA-seq analysis was conducted on homogenized eyeballs to assess the relevant gene profiles and their differentially expressed genes (DEGs). Pathway enrichment analysis was performed to further annotate the functions of the DEGs. Histopathological analysis demonstrated a higher disease severity with increased inflammatory cells at 72 hpi and transcriptome analysis revealed 27,717 DEGs, of which 1493 were significant (adj p value ≤0.05, FC ≥ 1.5). Among these, 924 were upregulated, and 569 were downregulated. Majority of the upregulated genes were associated with the inflammatory/host immune response and signal transduction and enriched in the T-cell signaling pathway, natural killer cell-mediated cytotoxicity, C-type receptor signaling pathway, and NOD-like receptor signaling pathway. Furthermore, inflammation-associated genes such as T-cell surface glycoprotein CD3, cathelicidin antimicrobial peptide, and lymphocyte cell-specific protein tyrosine kinase were enriched, while pathways such as MAPK, cAMP, and metabolic pathways were downregulated. Regulating the T-cell influx could be a potential strategy to modulate excessive inflammation in the retina and could potentially aid in better vision recovery in fungal endophthalmitis.

Design and Validation of Endophthalmitis Infectivity Measurement Algorithm in Post Cataract Acute Endophthalmitis: EMS Report No. 6.

Purpose: We constructed a clinical clue-based algorithm to identify the microbiology-positive post-cataract surgery endophthalmitis. Methods: The Endophthalmitis Infectivity Measurement Algorithm (EIMA) was constructed using presenting Snellen vision (Letter score [LS]) and Inflammation Score (IS, from the cornea, anterior chamber, iris, and vitreous). Retrospective data (70% for training; 30% for testing) was fitted into CHAID (Chi-squared Automatic Interaction Detection). EIMA was validated with prospective data. EIMA-categorized disease severity was weighed against the symptom duration to detect infecting micro-organisms. Results: EIMA was constructed from 1444 retrospective data. The average LS was 6.03 ± 12.11, median IS was 14 (8-24), and culture positivity was 38%. The accuracy and area under the curve of CHAID were 66.36% and 0.642, respectively. EIMA was validated with 175 prospectively collected data. Microbiology positivity (culture + sequencing) was 58.9%. EIMA sensitivity, specificity, and accuracy against microbiology-positive endophthalmitis were 73.7 (95% confidence interval [CI], 64.19-81.96), 81.9 (95% CI, 71.1-90.02), 77.1 (95% CI, 70.20-83.14), respectively. The positive and negative likelihood ratios were 4.08 (95% CI, 2.46-6.67) and 0.32 (95% CI, 0.22-0.45), respectively. There was higher microbial growth in two days or less than in three- to six-day symptom duration (69.9% vs. 28.2%; P = 0.018) endophthalmitis. Gram-negative infection was higher in two days or less (55.6% vs. 20.2%; P = 0.014), and gram-positive infection was higher in three- to six-day endophthalmitis (62.1% vs. 27.7%; P = 0.027). Conclusions: EIMA identified microbiology-positive endophthalmitis three-quarters of the time. Translational Relevance: EIMA suggested infectivity and the class of microbial infection could help targeted management of endophthalmitis after cataract surgery.

Global Transcriptomic Profiling of Innate and Adaptive Immunity During Aspergillus flavus Endophthalmitis in a Murine Model.

Purpose: Fungal endophthalmitis is characterized by chronic inflammation leading to the partial or complete vision loss. Herein, we analyzed the transcriptomic landscape of Aspergillus flavus (A. flavus) endophthalmitis in C57BL/6 mice to understand the host-pathogen interactions. Methods: Endophthalmitis was induced by intravitreal injection of A. flavus spores in C57BL/6 mice and monitored for disease progression up to 72 hours. The enucleated eyeballs were subjected to histopathological analysis and mRNA sequencing using the Illumina Nextseq 2000. Pathway enrichment analysis was performed to further annotate the functions of differentially expressed genes (DEGs) and validation of cytokines was performed in vitreous of patients with fungal endophthalmitis using multiplex ELISA. Results: Transcriptomic landscape of A. flavus endophthalmitis revealed upregulated T-cell receptor signaling, PI3K-AKT, MAPK, NF-κB, JAK-STAT, and NOD like receptor signaling pathways. We observed significant increase in the T-cells during infection especially at 72 hours infection along with elevated expression levels of IL-6, IL-10, IL-12, IL-18, IL-19, IL-23, CCR3, and CCR7. Furthermore, host-immune response associated genes, such as T-cell interacting activating receptor, TNF receptor-associated factor 1, TLR1, TLR9, and bradykinin receptor beta 1, were enriched. Histopathological assessment validated the significant increase in inflammatory cells, especially T-cells at 72 hours post-infection along with increased disruption in the retinal architecture. Additionally, IL-6, IL-8, IL-17, TNF-α, and IL-1ß were also significantly elevated, whereas IL-10 was downregulated in vitreous of patients with Aspergillus endophthalmitis. Conclusions: Regulating T-cell influx could be a potential strategy to modulate the excessive inflammation in the retina and potentially aid in better vision recovery in fungal endophthalmitis.

Antibiotic susceptibility in Endophthalmitis Management Study and intravitreal antibiotic practice trend in India-EMS Report #5.

AIM: Analyze antibiotic susceptibility in the Endophthalmitis Management Study (EMS) and compare it with the current intravitreal antibiotic practice trend of members of the Vitreoretinal Society of India (VRSI) practicing in India. METHODS: The microbiology work-up of undiluted vitreous included microscopy, culture-susceptibility, polymerase chain reaction (PCR), and next-generation sequencing (NGS). VRSI members were invited to the survey. The EMS conventional culture-susceptibility (PCR and NGS excluded) results were compared vis-a-vis gram-positive cocci (GPC), gram-negative bacilli (GNB), and less commonly used antibiotics with the current recommended intravitreal antibiotics. p < 0.05 was considered significant. RESULTS: Culture and positivity (culture + PCR/NGS) positivity was 28.8% and 56.1%, respectively. GPC was most susceptible to cefazolin, linezolid, and vancomycin; GNB was most susceptible to amikacin, ceftazidime, colistin, and imipenem. There was no susceptibility difference between cefazolin and vancomycin (p = 0.999) and between ceftazidime and imipenem (p = 1.0). Colistin was superior to ceftazidime (p = 0.047) against GNB. The GNB resistant to amikacin (n = 14) were equally susceptible to ceftazidime and colistin; resistant to ceftazidime (n = 16) were susceptible to colistin; and resistant to colistin (n = 7) were susceptible to ceftazidime. The preference of VRSI members (n = 231) practicing in India was a vancomycin-ceftazidime combination (82%), vancomycin for GPC (94%), ceftazidime for GNB (61%), and voriconazole for fungi (74%). CONCLUSION: In EMS, GPC had good susceptibility to vancomycin; GNB had good susceptibility to ceftazidime and colistin. Given the lower resistance of colistin, a vancomycin-colistin combination could be an alternative empiric treatment in post-cataract endophthalmitis in India.

Evaluating the utility of inflammation score in post-cataract surgery endophthalmitis. Results from a prospective study in India. EMS Report #4.

PURPOSE: To investigate if inflammation score (IS), calculated from the cornea, anterior chamber, iris, and vitreous, indicates endophthalmitis severity. METHODS: In a prospective study, consecutive adults with a clinical diagnosis of post-cataract endophthalmitis within 6 weeks of surgery were recruited. Patients were allocated to IS-based primary treatment (IS < 10: intravitreal injection and IS ≥ 10: vitrectomy) and randomized to two intravitreal antibiotics combinations (vancomycin + ceftazidime and vancomycin + imipenem). Undiluted vitreous microbiology work-up included culture susceptibility, polymerase chain reaction, Sanger sequencing, and targeted next-generation sequencing. RESULTS: The average age of 175 people was 63.4 ± 10.7 years and included 52.6% small incision cataract surgery and 47.4% phacoemulsification surgery. Severe endophthalmitis (IS ≥ 20), diagnosed in 27.4% of people, had a shorter time to symptoms (average 5.4 vs 8.7 days; P = 0.018), poorer presenting vision (all ≤ hand motion), higher culture positivity (50% vs 30.7%; P = 0.032), and higher Gram-negative bacterial infection (70.8% vs 46.2%; P = 0.042). For IS ≥ 20 discriminant and Gram-negative infection, Spearman's coefficient was 0.7 [P < 0.0001, 95% confidence interval (CI) 0.59-0.82], with an area under the receiver operating characteristic curve of 0.9 (95% CI 0.85-0.94, P < 0.0001), a Youden index J of 0.74, a sensitivity of 87.2%, and a specificity of 87.5%. The final vision of >20/400 and >20/100 was regained in 50.2% and 29.1% of people, respectively. The susceptibility of common Gram-positive cocci and Gram-negative bacilli was the highest for vancomycin (95.0%) and colistin (88.6%), respectively. NGS detected polymicrobial infection in 88.5% of culture-negative endophthalmitis. CONCLUSIONS: Higher inflammation scores indicated severe disease and Gram-negative infection in post-cataract endophthalmitis.

Endophthalmitis Management Study-A Prospective Randomized Clinical Trial on Postoperative Endophthalmitis Management in India: An Interim Analysis. Endophthalmitis Management Study Report #3.

PURPOSE: An interim analysis of the Endophthalmitis Management Study to examine the outcome of inflammation score (IS)-based treatment and antibiotic susceptibility. DESIGN: A prospective randomized study. PATIENTS AND METHODS: IS was measured on a 0-4 scale from presenting signs in 4 cardinal ocular tissues. The eyes with IS <10 received vitreous tap and intravitreal antibiotics, whereas eyes with IS ≥10 received vitrectomy and intravitreal antibiotics. These eyes were randomized to 2 intravitreal antibiotic combinations: (1) vancomycin and ceftazidime and (2) vancomycin and imipenem. Microbiology workup of undiluted vitreous included microscopy, culture-susceptibility, Sanger, and targeted next-generation sequencing. The clinical and microbiology outcomes were analyzed for advanced (IS = ≥20) and less advanced (IS = <10) endophthalmitis. RESULTS: Interim analysis was performed after the Endophthalmitis Management Study recruited 56.85% (248/436) of patients and completed 54.6% (238/436) of microbiology workup. A 90-day follow-up was completed in 90.8% (168/185) of eligible people. In eyes with IS ≥20, the time to symptoms was shorter (5.8 ± 6.7 vs 8.5 ± 9.1 d; P = 0.015), and the need for additional treatment was higher (95.8% vs 53.1%; P = 0.0267). Good final vision was associated with good presenting vision (r = 0.30) and IS-based treatment decisions (r = 0.170). Microbiology positivity was 55.9%. Eyes with IS <10 had a higher Gram-positive cocci (33.9% vs 4.8%; P = 0.013) infection. Gram-positive cocci were most susceptible to vancomycin (95.7%), and Gram-negative bacilli to colistin (95.7%). CONCLUSIONS: Considering both IS and presenting vision, rather than only one of them, helps in making appropriate management decisions for acute postoperative endophthalmitis.

Trachipleistophora hominis: molecular characterization of a rare pathogen causing microsporidial stromal keratitis.

The authors report the clinical and microbiological findings of a unique case of stromal keratitis caused by a rare microsporidium, Trachipleistophora hominis. This case of stromal keratitis was in a 49-year-old male with a history of COVID-19 infection and diabetes mellitus. Corneal scraping specimens revealed numerous microsporidia spores upon microscopic examination. PCR of the corneal button revealed the presence of T. hominis infection, which could be controlled by penetrating keratoplasty surgery. The graft was clear with no recurrence of infection until the last follow-up 6 weeks postsurgery. This is the first case of human stromal keratitis caused by this organism in a post-COVID infection, confirmed by molecular diagnosis.

Clinical and Microbiological Spectra and Therapeutic Outcomes of Polymicrobial Keratitis.

PURPOSE: The aim of this study was to evaluate the risk factors, microbiology, and treatment outcomes of polymicrobial keratitis (PMK). METHODS: We retrospectively analyzed 65 patients diagnosed with PMK between 2016 and 2019. The clinical and microbiological characteristics and outcomes were evaluated. The primary outcome measure was clinical resolution of keratitis. Absolute success (group A) was defined as resolution of infection with medical treatment, qualified success (group B) was resolution with medical therapy and additional minor procedures, and failure (group C) was defined as worsening of infection and presence of corneal melting or perforation requiring therapeutic penetrating keratoplasty or evisceration. RESULTS: Of 4764 cases of microbial keratitis, PMK was identified in 65 eyes of 65 patients (1.36%). Infiltrate was central in 60% (39 eyes). Predisposing factors were higher in group C and included uncontrolled diabetes mellitus in 25% (7/28), history of trauma/foreign body in 57.1% (16/28), and poor presenting visual acuity (0.9 for group A vs. 1.79 for group B vs. 3.00 logarithm of the minimum angle of resolution for group C; P = 0.02). Microbiological profiling revealed that a majority (95%: 62/65) had bacterial with fungal keratitis. High fluoroquinolone resistance was noted (57%; 34/59). Absolute success was achieved in 28.3% (17/60), qualified success in 16.6% (10/60), and treatment failure was noted in 55% (33/60). The final best corrected visual acuity in group A was significantly better than that in group B and C (0.44 vs. 3.00 vs. 2.78; P < 0.001). CONCLUSIONS: PMK is extremely rare but responds poorly to medical therapy. Bacterial with fungal etiology is predominant. Early surgical intervention to improve overall outcome is advocated.

Virulence factors in multidrug (MDR) and Pan-drug resistant (XDR) Pseudomonas aeruginosa: a cross-sectional study of isolates recovered from ocular infections in a high-incidence setting in southern India.

BACKGROUND: Global concerns have been raised due to upward trend of Multi-drug Resistant (MDR) Pseudomonas aeruginosa reports in ocular infections. Our aim was to characterize the virulence determinants of MDR P. aeruginosa causing ocular infections. METHODS: P. aeruginosa strains were isolated from 46 patients with conjunctivitis (2), endophthalmitis (11) and active keratitis (25) seen at our Institute, between 2016 and 2020. The isolates were identified by Vitek-2 and characterized based on growth kinetics, biofilm formation, motility, pyoverdine and pyocyanin production, phospholipase and catalase activity, urease production along with expression of exotoxins (exo-A, exo-U and exo-S) and correlated to its antibiotic profiles. RESULTS: Of the 46 P. aeruginosa isolates, 23 were MDR and were significantly (p = 0.03) associated with older (> 65) patients, along with higher production of pyoverdine (58.3%), pyocyanin (30.4%), phospholipase (91.6%) and protease (62.5%) activity, formed strong biofilms and exo-A (30.4%). No significant relation between motility, urease and catalase production with antibiotic susceptibility was observed. Heatmap and PCoA analysis confirmed this unique virulence profile associated with MDR-PA strains. CONCLUSION: Phenotypic characteristics of P.aeruginosa might be responsible for increased colonization and antibiotic resistance observed in vivo and understanding these differences may lead to development of clinical guidelines for the management of MDR infections.

Transcriptomic and Histological Analysis of Exacerbated Immune Response in Multidrug-Resistant Pseudomonas aeruginosa in a Murine Model of Endophthalmitis.

Multidrug-resistant (MDR) endophthalmitis is a serious threat to the whole spectrum of therapeutic procedures associated with the risk of managing and preventing vision loss. We have earlier shown the interplay of immune mediators in patients with MDR Pseudomonas aeruginosa (PA) endophthalmitis leading to worse outcome. Expanding on these findings, a murine model of endophthalmitis was developed to explore the effects of drug resistance on the pathogenesis by analyzing the temporal changes in retinal morphology along with its transcriptomic signatures. Clinical isolates of susceptible (S-PA) and multidrug-resistant PA (MDR-PA) were injected intravitreally in C57BL/6 mice followed by enucleation at 6 and 24 h time points postinfection. Disease progression and retinal changes were monitored by clinical and histological assessment and transcriptome analysis in a pair-wise manner. Histological assessment of MDR-PA eyeball revealed higher disease severity (p < 0.05), CD45+ cells (p = 0.007), MPO+ cells (p = 0.01), GFAP+ (p = 0.02), along with higher retinal cell death in mice infected with MDR-PA (p = 0.008). Temporal transcriptome analysis revealed differential expression of nearly 923 genes at 6 h p.i. and 2,220 genes at 24 h p.i. (FC ≥2, adjusted p-value <0.05). Pathway enrichment analysis identified differential regulation of chemokine- and cytokine-mediated, MAPK, and NF-Ðºß signaling pathways. In conclusion, rapid deterioration of retinal architecture and immune exacerbation was significantly associated with the MDR endophthalmitis, suggesting the need for immunomodulatory agents to strengthen host cell functions and support antibiotics to save the retinal structure from inevitable deterioration and restoration of the vision.

Surface chlorophyll anomalies associated with Indian Ocean Dipole and El Niño Southern Oscillation in North Indian Ocean: a case study of 2006-2007 event.

North Indian Ocean witnesses varied dynamical response due to independent climate modes such as Indian Ocean Dipole (IOD)/El Niño Southern Oscillations (ENSO) and their co-occurrences. These modes have a significant impact on ocean productivity, which in turn shows feedback for the strengthening of these patterns. Keeping this in view, the present work attempts to analyze the biological activity during the combined influence of positive IOD with El Niño during 2006-2007 event. To divulge the biological variability along with the dynamical response, the study includes intra-annual variability surface chlorophyll anomaly with D20 anomaly using satellite observations. Here, the individual role of IOD and ENSO on both surface chlorophyll and D20 is segregated through partial regression analysis for a period of 25 years (1993-2017). By the regression method, it can be seen varied chlorophyll response for the 2006-2007 event with the IOD forcing leads to the major spatial and temporal variability with positive anomalies in Eastern Equatorial Indian Ocean (EEIO) (generally oligotrophic), Northwestern Bay of Bengal (NWBoB), and Northwestern Arabian Sea (NAS2) where production begins in fall intermonsoon and peaks up during November. On the other hand, negative anomalies are observed around the southern tip of India (SBoB) and the Northern Arabian Sea (NAS1). While ENSO depicts the high surface chlorophyll variability in the Western Indian Ocean (WIO1, WIO2) with negative anomalies of surface chlorophyll. This study observed an asymmetric response of chlorophyll variability over the North Indian Ocean during the 1997-1998 and 2006-2007 events with a major influence of IOD mode compared with the El Niño. Therefore, understanding the chlorophyll anomalies during different climate modes will help us to better understand the interannual variability and improve the predictability of chlorophyll productivity regions.

Magneto-thermally activated spin-state transition in La0.95Ca0.05CoO3: magnetically-tunable dipolar glass and giant magneto-electricity.

The magneto-dielectric spectroscopy of La0.95Ca0.05CoO3 covering the crossover of spin states reveals the strong coupling of its spin and dipolar degrees of freedom. The signature of the spin-state transition at 30 K clearly manifests in the magnetization data at a 1 Tesla optimal field. Our Co L3,2-edge X-ray absorption spectrum on the doped specimen is consistent with its suppressed low-to-intermediate spin-state transition temperature at ∼30 K compared to ∼150 K, documented for pure LaCoO3. The dispersive activation step in the dielectric constant with the associated relaxation peak in imaginary permittivity characterize the allied influence of coexistent spin-states on the dielectric character. Dipolar relaxation in the low-spin regime below the transition temperature is partly segmental (Vogel-Fulcher-Tamman (VFT) kinetics) and features magnetic-field tunability, whereas in the low/intermediate-spin disordered state above ∼30 K, it is uncorrelated (Arrhenic kinetics) and almost impervious to the magnetic field H. Kinetics-switchover defines the dipolar-glass transition temperature Tg(H) (=27 K|0T), below which their magneto-thermally-activated cooperative relaxations freeze out by the VFT temperature T0(H) (=15 K|0T). An applied magnetic field facilitates thermal activation in toggling the low spins up into the intermediate states. Consequently, the downsized dipolar-glass segments in the low-spin state and the independent dipoles in the intermediate state exhibit accelerated dynamics. A critical 5 Tesla field collapses the entire relaxation kinetics into a single Arrhenic behaviour, signaling that the dipolar glass is completely devitrified under all higher fields. The magneto-electricity (ME) spanning sizeable thermo-spectral range registers diverse signatures here in kinetic, spectral, and field behaviors, in contrast to the static/perturbative ME observed close to the spin-ordering in typical multiferroics. Intrinsic magneto-dielectricity (50%) along with vanishing magneto-loss is obtained at (27 K/50 kHz)9T. The sub-linear deviant and field-hysteretic split seen in above 4 Tesla suggests the emergence of robust dipoles organized into nano-clusters, induced by the internally-generated high magneto-electric field. An elaborate ω-T multi-dispersions diagram maps the rich variety of phase/response patterns, revealing highly-interacting magnetic and electric moments in the system.