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

Abstract

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-1alpha, 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.

FIG. 1.

One of 186 MPTs inferred from analysis of the design panel RPB2 data set, rooted with the outgroup sequence of Lecanicillium lecanii (consistency index = 0.52, retention index = 0.93). Numbers above nodes represent the frequency (%) with which they were recovered from 1,000 bootstrap pseudoreplicates of the data. The six medically important species complexes are indicated by bold internodes. Six unnamed species within the FSSC are indicated by numbers. Isolate numbers that are shaded represent the three most common haplotypes: FOSC 3-a, FSSC 1-a, and FSSC 2-d. Arrowheads identify each of the 18 allele-specific probes used in the identification of species complexes and species in the design (Table 2) and experimental panels (Table 3).