Epidemiology, risk factors, and clinical outcomes in severe microbial keratitis in South India.

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.

Persistence of Innate Immune Pathways in Late Stage Human Bacterial and Fungal Keratitis: Results from a Comparative Transcriptome Analysis.

Microbial keratitis (MK) is a major cause of blindness worldwide. Despite adequate antimicrobial treatment, tissue damage can ensue. We compared the human corneal transcriptional profile in late stage MK to normal corneal tissue to identify pathways involved in pathogenesis. Total RNA from MK tissue and normal cadaver corneas was used to determine transcriptome profiles with Illumina HumanHT-12 v4 beadchips. We performed differential expression and network analysis of genes in bacterial keratitis (BK) and fungal keratitis (FK) compared with control (C) samples. Results were validated by RTqPCR for 45 genes in an independent series of 183 MK patients. For the microarray transcriptome analysis, 27 samples were used: 12 controls, 7 BK culture positive for Streptococcus pneumoniae (n = 6), Pseudomonas aeruginosa (n = 1), and 8 FK, culture positive for Fusarium sp. (n = 5), Aspergillus sp. (n = 2), or Lasiodiplodia sp. (n = 1). There were 185 unique differentially expressed genes in BK, 50 in FK, and 339 common to both [i.e., genes with fold-change (FC) < -4 or ≥4 and false discovery rate (FDR) adjusted P < 0.05]. MMP9 had the highest FC in BK (91 FC, adj p = 3.64 E-12) and FK (FC 64, adj. p = 6.10 E-11), along with other MMPs (MMP1, MMP7, MMP10, MMP12), pro-inflammatory cytokines (IL1B, TNF), and PRRs (TLR2, TLR4). HIF1A and its induced genes were upregulated uniquely in BK. Immune/defense response and extracellular matrix terms were the most enriched Gene Ontology terms in both BK and FK. In the network analysis, chemokines were prominent for FK, and actin filament reorganization for BK. Microarray and RTqPCR results were highly correlated for the same samples tested with both assays, and with the larger RTqPCR series. In conclusion, we found a great deal of overlap in the gene expression profile of late stage BK and FK, however genes unique to fungal infection highlighted a corneal epithelial wound healing response and for bacterial infection the prominence of HIF1A-induced genes. These sets of genes may provide new targets for future research into therapeutic agents.

In vivo confocal microscopy appearance of Fusarium and Aspergillus species in fungal keratitis.

BACKGROUND: Clinical outcomes in fungal keratitis vary between Fusarium and Aspergillus spp, therefore distinguishing between species using morphological features such as filament branching angles, sporulation along filaments (adventitious sporulation) or dichotomous branching may be useful. In this study, we assessed these three features within Heidelberg Retina Tomograph 3 in vivo confocal microscopy (IVCM) images from culture-positive Fusarium and Aspergillus spp keratitis participants. METHODS: Prospective observational cohort study in Aravind Eye Hospital (February 2011-February 2012). Eligibility criteria: age ≥18 years, stromal infiltrate ≥3 mm diameter, Fusarium or Aspergillus spp culture-positive. EXCLUSION CRITERIA: previous/current herpetic keratitis, visual acuity <6/60 in fellow eye, >80% corneal thinning. IVCM was performed and images analysed for branch angle, presence/absence of adventitious sporulation or dichotomous branching by a grader masked to the microbiological diagnosis. RESULTS: 98 participants were included (106 eligible, 8 excluded as no measurable branch angles); 68 were positive for Fusarium spp, 30 for Aspergillus spp. Mean branch angle for Fusarium spp was 59.7° (95% CI 57.7° to 61.8°), and for Aspergillus spp was 63.3° (95% CI 60.8° to 65.8°), p=0.07. No adventitious sporulation was detected in Fusarium spp ulcers. Dichotomous branching was detected in 11 ulcers (7 Aspergillus spp, 4 Fusarium spp). CONCLUSIONS: There was very little difference in the branching angle of Fusarium and Aspergillus spp. Adventitious sporulation was not detected and dichotomous branching was infrequently seen. Although IVCM remains a valuable tool to detect fungal filaments in fungal keratitis, it cannot be used to distinguish Fusarium from Aspergillus spp and culture remains essential to determine fungal species.