DEVELOPMENT AND APPLICATION OF MOLECULAR PHYLOGENETICS OF FUNGI TO ENHANCE FOOD SAFETY AND FOOD SECURITY
Location: Bacterial Foodborne Pathogens & Mycology Research Unit
Title: 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
O Donnell, Kerry
| Sarver, Brice |
| Brandt, Mary - CDC ATLANTA, GA |
| Chang, Douglas - CDC ATLANTA, GA |
| Nobel-Wang, Judith - CDC ATLANTA, GA |
| Park, Benjamin - CDC ATLANTA, GA |
| Sutton, Deanna - UNIV OF TX HLTH SCI CNTR |
| Benjamin, Lynette - CDC ATLANTA, GA |
| Lindsley, Mark - CDC ATLANTA, GA |
| Padhye, Arvind - CDC ATLANTA, GA |
| Geiser, David - PENN STATE UNIV, UNIV PRK |
Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 3, 2007
Publication Date: July 1, 2007
Citation: O Donnell, K., Sarver, B., Brandt, M., Chang, D.C., Nobel-Wang, J., Park, B.J., Sutton, D., Benjamin, L., Lindsley, M., Padhye, A., Geiser, D.M., Ward, T.J. 2007. 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. Journal of Clinical Microbiology. 45(7):2235-2248.
Interpretive Summary: A study conducted by the Centers for Disease Control and Prevention demonstrated the outbreak of Fusarium corneal infections that peaked in April 2006 in the United States were associated with the use of Bausch & Lomb’s ReNu with MoistureLoc (Rochester, NY) brand of contact lens solution. This outbreak was newsworthy in that corneal infections caused by Fusarium are rare in the developed world where they are typically associated with ocular trauma. Accurate identification of the pathogens causing fungal infections is critical to advancing our understanding of the environmental reservoir of each Fusarium species, and they may provide guidance to the most effective regimen of antifungal therapy. Therefore, the objectives of this study were threefold: (i) investigate the full spectrum of fusaria associated with the outbreak of corneal infections within the U.S. and Puerto Rico, by assessing the genetic diversity of 87 corneal and 104 environmental isolates associated the outbreaks, using DNA sequence data from 3 genes; (ii) assess how the outbreak isolates fell within the full spectrum of Fusarium, using nucleotide sequence data from the RNA polymerase II second largest subunit gene; and (iii) develop and validate a high through-put, molecular diagnostic assay for the simultaneous detection and identification of human pathogenic fusaria in a single well using flow cytometry. These results will be of interest to and benefit medical personnel concerned with public health in that they provide strong support for the hypothesis that fungal contamination occurred at the contact lens/lens solution point of use, rather than at the contact lens solution production facility, and they provide the first robust molecular diagnostic assay for medically important fusaria, thereby allowing medical microbiologists to rapidly and accurately identify these pathogens for the first time.
In 2005-06, 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 3 loci (RPB2, EF-1', and nuclear ribosomal rRNA) totaling 3.48 kb were obtained from 87 corneal isolates and 104 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 species in a single-well assay using flow cytometry and fluorescent microsphere technology. The multilocus data revealed that one haplotype from each of the 3 most common species comprised over 50% of CDC’s corneal and environmental isolates, and that the corneal isolates comprised 33 haplotypes distributed among 17 species. The high degree of phylogenetic diversity represented among the corneal isolates is consistent with multiple sources of contamination.