Whole-Genome Analysis of Exserohilum rostratum from an Outbreak of Fungal Meningitis and Other Infections

Abstract

Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ϳ136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum. Beginning in September, 2012, the United States experienced one of the largest outbreaks of health care-associated infections ever reported. More than 13,000 people were exposed to three lots of contaminated methylprednisolone acetate (MPA) produced by a single compounding pharmacy, and Ͼ750 developed fungal infections following their MPA injections (1-3). Infections included meningitis, as well as localized epidural, paraspinal, and peripheral joint infections, complicated by arachnoiditis and abscess formation (4, 5). Although several fungal species were implicated in the outbreak, the vast majority of infections were caused by Exserohilum rostratum, a saprobic mold found in soil and plant debris worldwide (2, 6-10). Most species of Exserohilum are plant pathogens. Exserohilum rostratum and the species formerly known as Exserohilum longirostratum and Exserohilum mcginnisii are morphologically similar and are the only species known to cause human infections; however, their taxonomic status remains under investigation (11-13).DNA fingerprinting has been used as an epidemiological tool to answer questions pertaining to strain relatedness (14, 15). However, no methodologies for DNA fingerprinting of E. rostratum existed at the time of the outbreak. While whole-genome sequencing (WGS) is a relatively new tool of molecular epidemiology, it has quickly been embraced as a mechanism for inferring phylogenetic relationships among organisms. This has translated to the recent use of whole-genome single nucleotide polymorphism (SNP) typing (whole-genome sequence typing [WGST]) as a mechanism for inferring relatedness of both bacteria (16)(17)(18)(19) and fungi (20, 21) during outbreak investigations. One of the benefits of WGST is that prior knowledge of the genome i...

Local population structure and patterns of Western Hemisphere dispersal forCoccidioides spp., the fungal cause of Valley Fever

“…A later comparative genomics study, based on the analysis of 10 genomes of each species, determined that the separation of the two species has not remained fully complete and documented possible presence of introgression from hybridization events following species separation (11). As rapid DNA sequencing analysis has become more accessible to research and public health laboratories, approaches such as whole-genome SNP typing (WGST) have been shown to be useful for identifying clonal fungal outbreaks (12)(13)(14)(15). However, genomic epidemiology is also needed to help establish the location of exposure of a nonclonal outbreak and robust phylogenomic analysis is needed for in-depth investigation of the local population structure to better understand pathogen emergence, dispersal, and expansion (15).…”

In Vitro Activities of Eight Antifungal Drugs against a Global Collection of Genotyped Exserohilum Isolates

“…The different susceptibilities to voriconazole in the present study could be attributed to strains tested from various geographical locations. Recently, using whole-genome analysis, significant genomic variability has been reported among E. rostratum strains unrelated to the outbreak strains (21).…”

Emerging issues, challenges, and changing epidemiology of fungal disease outbreaks

“…68 Close collaboration between local, state, and federal health agencies and clinicians resulted in rapid public health actions, including recall of the contaminated methylprednisolone, notification of nearly 14 000 people who were potentially exposed to the product, and development and dissemination of diagnostic and treatment guidance for this unfamiliar clinical entity. 69,70 These public health actions were estimated to save more than 100 lives. 69 Although no molecular typing methods for E rostratum or knowledge of its population structure existed at the time of the outbreak, WGST analysis of 28 isolates from patients and the contaminated methylprednisolone indicated nearly identical genomes.…”

“…69 Although no molecular typing methods for E rostratum or knowledge of its population structure existed at the time of the outbreak, WGST analysis of 28 isolates from patients and the contaminated methylprednisolone indicated nearly identical genomes. 70 No more than two single nucleotide polymorphisms (SNPs) separated any of the isolates, whereas substantial genetic diversity (more than 20 000 SNPs) was observed among unrelated control isolates. 70 …”