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Journal of Clinical Microbiology, July 2007, p. 2235-2248, Vol. 45, No. 7
0095-1137/07/$08.00+0     doi:10.1128/JCM.00533-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Phylogenetic Diversity and Microsphere Array-Based Genotyping of Human Pathogenic Fusaria, Including Isolates from the Multistate Contact Lens-Associated U.S. Keratitis Outbreaks of 2005 and 2006{triangledown} ,{ddagger}

Kerry O'Donnell,1*,{dagger} Brice A. J. Sarver,1,{dagger},§ Mary Brandt,2 Douglas C. Chang,2 Judith Noble-Wang,2 Benjamin J. Park,2 Deanna A. Sutton,3 Lynette Benjamin,2 Mark Lindsley,2 Arvind Padhye,2 David M. Geiser,4 and Todd J. Ward1

Microbial Genomics and Bioprocessing Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois,1 Centers for Disease Control and Prevention, Atlanta, Georgia,2 Department of Pathology, University of Texas Health Science Center, San Antonio, Texas,3 Department of Plant Pathology, The Pennsylvania State University, University Park, Pennsylvania4

Received 9 March 2007/ Returned for modification 25 April 2007/ Accepted 2 May 2007

In 2005 and 2006, outbreaks of Fusarium keratitis associated with soft contact lens use occurred in multiple U.S. states and Puerto Rico. A case-control study conducted by the Centers for Disease Control and Prevention (CDC) showed a significant association between infections and the use of one particular brand of lens solution. To characterize the full spectrum of the causal agents involved and their potential sources, partial DNA sequences from three loci (RPB2, EF-1{alpha}, and nuclear ribosomal rRNA) totaling 3.48 kb were obtained from 91 corneal and 100 isolates from the patient's environment (e.g., contact lens and lens cases). We also sequenced a 1.8-kb region encoding the RNA polymerase II second largest subunit (RPB2) from 126 additional pathogenic isolates to better understand how the keratitis outbreak isolates fit within the full phylogenetic spectrum of clinically important fusaria. These analyses resulted in the most robust phylogenetic framework for Fusarium to date. In addition, RPB2 nucleotide variation within a 72-isolate panel was used to design 34 allele-specific probes to identify representatives of all medically important species complexes and 10 of the most important human pathogenic Fusarium in a single-well diagnostic assay, using flow cytometry and fluorescent microsphere technology. The multilocus data revealed that one haplotype from each of the three most common species comprised 55% of CDC's corneal and environmental isolates and that the corneal isolates comprised 29 haplotypes distributed among 16 species. The high degree of phylogenetic diversity represented among the corneal isolates is consistent with multiple sources of contamination.

* Corresponding author. Mailing address: Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604-3999. Phone: (309) 681-6383. Fax: (309) 681-6672. E-mail: Kerry.odonnell{at}ars.usda.gov

{triangledown} Published ahead of print on 16 May 2007.

{ddagger} Supplemental material for this article may be found at http://jcm.asm.org/.

{dagger} K.O. and B.A.J.S. contributed equally to this study.

§ Present address: Washington University in St. Louis, St. Louis, MO 63117.

Journal of Clinical Microbiology, July 2007, p. 2235-2248, Vol. 45, No. 7
0095-1137/07/$08.00+0     doi:10.1128/JCM.00533-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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