Purpose Vernal keratoconjunctivitis (VKC) is a severe form of ocular allergic disease possibly related to an altered ocular surface microbiota. The aim of the study was to determine the bacterial and fungal composition of conjunctival microbiota in VKC compared with healthy controls (HC). Methods Lower fornix conjunctival swabs were obtained from 22 VKC children and 20 age, sex and ethnicity-matched HC. Total DNA was extracted, and used for 16S rRNA and ITS2 gene amplification and sequencing. Results High-throughput sequencing of 16S rRNA and ITS2 amplicon libraries produced a total of 734,157 and 677,115 high-quality reads, respectively. Clustering of similar sequences (>97% of identity) resulted in 1,241 and 933 OTUs, respectively. Alfa and beta diversity metrics highlighted significant differences of conjunctival bacterial and fungal microbiota composition in VKC patients and HC. Proteobacteria, Firmicutes and Actinobacteria phyla were present in all subjects qualifying theme as a putative core microbiome of both HC and VKC groups. In addition, Bacteroidetes and Fusobacteria met the core microbiome’s definition criteria in VKC patients. Of the 132 observed families, Moraxellaceae showed a higher abundance in VKC group than HC. Saccharomycetaceae, Malasseziaceae, and Dipodascaceae were present in all subjects, constituting the fungal core microbiome of both HC and VKC patients. OTUs referred to Malasseziaceae were significantly higher in VKC children compared to HC. Conclusion VKC patients and healthy controls have different conjunctival microbiomes. These results may provide new insights into the complex VKC pathogenesis.

A document by Andrea Leonardi. Click on the document to view its contents.

Purpose Tear fluid N-Glycome from patients affected with vernal (VKC) and atopic keratoconjunctivitis (AKC) was investigated to identify specific changes in tears and to recognize possible glyco-biomarkers. Methods The analysis of N-glycans was performed using matrix-assisted laser desorption ionization mass spectrometry on single tear samples. Tears from control normal subjects (CTRL), VKC and AKC patients were processed and treated with peptide N-glycosidase F (PNGase F) to deglycosylate N-glycoproteins. Released N-glycans were purified, permethylated and analyzed by Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry and tandem Mass Spectrometry (MALDI-TOF MS and MALDI-TOF MS/MS). Results More than 150 complex N-glycans, including highly fucosylated biantennary, triantennary, tetraantennary and bisecting species, were observed in our spectra. Three distinct patterns for CTRL, VKC and AKC patients were identified in terms of relative intensities for some N-glycans structures. Major variations involved bisecting and hyperfucosylated glycoforms. The most intense ions were associated to species at m/z 1907.0 (asialo, agalacto, bisected, biantennary structure – NGA2B) in CTRL MS profiles, at m/z 2605.3 and 2966.5 in VKC, and at m/z 2792.4 in AKC corresponding to a well-known biantennary, disialylated N-glycan. Several peaks were associated to structures bearing one or two Lewis X epitopes. Structures were confirmed by MS/MS analysis. Quantitative differences among the three groups were statistically significant. Conclusions Tear MS profiles are rich in specific glycoforms, particularly those with a high fucosylation degree, indicating both core and peripheral decoration. Tear N-glycome analysis provided important information for a better comprehension of VKC and AKC alterations at the molecular level