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2007 OMIG, Abstract 22

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Genotypic Identification of Fusarium sp From Ocular Sources in South Florida
RA Oechsler, M Feilmeier, D Ledee, D Miller, ME Fini, J Fell, E Alfonso.
Bascom Palmer Eye Institute – Miller School of Medicine – University of Miami

Introduction: Keratomycosis is a prevalent cause of ocular morbidity throughout the world.  Current diagnostic standards are limited in sensitivity and rapidity.  Failure to rapidly and accurately identify the etiologic agent may delay the initiation of appropriate therapy, thereby increasing ocular morbidity.  Ultimately, a more rapid and accurate diagnostic test is necessary to improve patient outcomes. Classification of  Fusarium at the subgenus level has been problematic, but increasing reports of Fusarium as a human pathogen in ocular infections has generated an interest in finding a more consistent basis for classification.  The purpose of this study is: 1) to determine whether or not genotypic data support the morphological classification of strains of Fusarium sp, and 2) to determine whether sequence analysis of Internal Transcribed Spacer regions (ITS1 and 2) and  5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections. 
Methods: Thirty one strains of Fusarium sp were retrieved from the Bascom Palmer’s ocular microbiology laboratory and grown in pure culture.  DNA was then extracted and purified, a targeted region was amplified using selected primers, and sequence analysis was performed. Lasergene program was used to create consensus sequences, alignments, bootstrapping and phylogenetic trees.  Distinct sequence types were identified based on parsimony and distance analyses. 
Results:  All samples were amplified successfully using the selected primers, indicating successful molecular identification in all cases. Sequence analysis demonstrated that the majority of sequence variation localized to the ITS regions. The grouping showed that 27 of 31 strains (87%) were distributed among 4 main clades, supported by bootstrap values of ≥ 73%. Interstrain sequence differences were within 0 and 11.2% among the 31 strains.
Conclusions: Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others, we propose that the ITS sequences provide a consistent quantitative basis for a new classification scheme for the Fusarium genus. 



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