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Understanding fungal keratitis pathogenesis through a reverse-translational approach.

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Posterboard#: B0343

Abstract Number: 865 - B0343

AuthorBlock: Kevin K. Fuller1,2, Marina Evette Brown8, David Giacalone7, Jay Dunlap4, Tom Lietman5, Robert Cramer7, K Dharmalingam6, Michael Zegans3,7
1Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States; 2Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States; 3Surgery, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire, United States; 4Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States; 5Ophthalmology, University of California at San Francisco, San Francisco, California, United States; 6Proteomics, Aravind Medical Research Foundation, Madurai, India; 7Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States; 8Ophthalmology, Dean McGee Eye Institute, Oklahoma City, Oklahoma, United States;

DisclosureBlock: Kevin K. Fuller, None; Marina Evette Brown, None; David Giacalone, None; Jay Dunlap, None; Tom Lietman, None; Robert Cramer, None; K Dharmalingam, None; Michael Zegans, None;

Purpose
Fungal keratitis is an important cause of ocular morbidity worldwide. As a non-candidate (unbiased) means to understand fungal keratitis pathogenesis, we initiated a clinical association study that exploits (1) variability in the clinical outcomes across keratitis patients and (2) variability in phenotypes across the fungal isolates obtained from those patients (i.e. strain heterogeneity). By correlating the two, we seek to identify fungal properties (phneotypes) that are predicitve of patient outcome and could therefore serve as a target for novel antifungal therapy.

Methods
The NEI-funded Mycotic Ulcer Treatment Trial (MUTT) enrolled 323 patients across South India to compare topical voriconazole and natamycin monotherapies. 128 isolates (40%) were identified as Fusarium in the clinic and were analyzed further in this work. Species-level identification of the isolates was performed at Dartmouth and OUHSC by sequencing the internal- transcribed spacer (ITS) and translation elongation factor-1 (tef-1) loci. Phenotypic analyses were also performed at Dartmouth and OUHSC by spot inoculating microconidia onto various growth media and analyzing colony diameters following incubation at variable times and temperatures. Statistical comparisons of the phenotypic and patient data sets were performed at UCSF.

Results
Molecular genotyping revealed that the majority of Fusarium isolates (80%) belong to the Fusarium solani species complex (FSSC). Not only were the FSSC organisms most prevalent, they were statistically correlated with worst baseline scar size compared to the non-FSSC organisms (p=.031). The FSSC isolates displayed marked variability in growth rate at 30oC and 37oC, but only growth at the latter temperature was correlated with patient outcome; specifically, increased growth rate was predictive of longer re-epithelization time.

Conclusions
Our data implicate FSSC species as the most common and most severe agents in Fusarium keratitis. The phenotypic analyses so-far suggest that this fungus experiences thermal stress at the ocular surface, and that metabolic pathways that cope with this stress are important for keratitis pathogenesis. This further serves as a proof-of-principle for our study design, and continued analyses should inform our understanding of (1) microbial factors that influence patient outcome, and (2) fungal pathways that can serve as targets for novel therapeutics.

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