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Jaya D Chidambaram, Namperumalsamy Venkatesh Prajna, Palepu Srikanthi, Manisha Shah, Lalitha Prajna, Elakkiya Shanmugam, Julien Bauer, Martin Holland, Matthew John Burton; Gene expression analysis of severe bacterial, fungal and acanthamoeba keratitis: role of immune and inflammatory biomarkers of disease. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4065.
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© ARVO (1962-2015); The Authors (2016-present)
Microbial keratitis (MK) is a leading cause of blindness worldwide. Excessive host inflammatory responses are thought to be the cause of tissue damage in MK. In this study, we investigated the expression of 45 genes involved in immune and inflammatory pathways, and extracellular matrix (ECM) modulation in corneal cells taken from late stage human bacterial (BK), fungal (FK) and acanthamoeba keratitis (AK), as compared to normal cadaver corneal tissue.
Corneal swab samples from 239 patients presenting to Aravind Eye Hospital Cornea Clinic in India from Feb 2012 to Feb 2013 with MK (>=3mm diameter stromal infiltrate). Final outcome was recorded at 21 days post-enrolment (i.e. “non-healing”=perforation or descemetocoele or intervention, e.g. corneal glue, transplant or intrastromal antifungal). Cadaver corneal tissue (n=13) was collected from Aravind Eye Bank. Total RNA was extracted from swabs/tissue (Qiagen, Netherlands), and RTqPCR performed with custom Taqman Low Density Arrays (Life Technologies, USA). Genes were normalized to HPRT1. Pairwise comparisons between BK, FK or AK versus Controls, BK versus FK and FK healed versus FK non-healed were performed and statistical significance of differential expression assessed with Wilcoxon rank sum test in Stata 12.1 (Bonferroni adjusted p-values).
218 patients were microbiologically positive for fungus (n=185, mainly Fusarium sp. or Aspergillus flavus), bacteria (n=20, mainly Streptococcus pneumoniae) and Acanthamoeba sp. (n=13). Differential expression (FC >2 or <2, p<0.0005) was found in 15, 9 and 3 genes in FK, BK and AK versus Controls, with no significant difference between BK and FK expression. Upregulated genes included ECM modifiers MMP9 (BK), MMP10 (FK, BK) and COL5A1 (BK, FK, AK); cytokines IL8 (BK, FK), IL18 (FK), and neutrophil antimicrobial effectors S100A9 and PI3 (all 3). In FK, PI3 was upregulated in non-healing ulcers (FC 3.6, p=0.002).
Several MMPs and immune effectors are significantly upregulated in BK, FK and AK. PI3, expressed in healing epithelia and known to be an immune modulator, correlates with poor prognosis in other diseases (e.g. cutaneous graft-vs-host disease). Further research is needed into PI3 as a possible prognostic biomarker for FK.
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