Rapid typing of Coxiella burnetii

Coxiella burnetii has the potential to cause serious disease and is highly prevalent in the environment. Despite this, epidemiological data are sparse and isolate collections are typically small, rare, and difficult to share among laboratories as this pathogen is governed by select agent rules and fastidious to culture. With the advent of whole genome sequencing, some of this knowledge gap has been overcome by the development of genotyping schemes, however many of these methods are cumbersome and not readily transferable between institutions. As comparisons of the few existing collections can dramatically increase our knowledge of the evolution and phylogeography of the species, we aimed to facilitate such comparisons by extracting SNP signatures from past genotyping efforts and then incorporated these signatures into assays that quickly and easily define genotypes and phylogenetic groups. We found 91 polymorphisms (SNPs and indels) among multispacer sequence typing (MST) loci and designed 14 SNP-based assays that could be used to type samples based on previously established phylogenetic groups. These assays are rapid, inexpensive, real-time PCR assays whose results are unambiguous. Data from these assays allowed us to assign 43 previously untyped isolates to established genotypes and genomic groups. Furthermore, genotyping results based on assays from the signatures provided here are easily transferred between institutions, readily interpreted phylogenetically and simple to adapt to new genotyping technologies

Whole-genome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections

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 ∼136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum

High prevalence and two dominant host-specific genotypes of Coxiella burnetii in U.S. milk

BackgroundCoxiella burnetii causes Q fever in humans and Coxiellosis in animals; symptoms range from general malaise to fever, pneumonia, endocarditis and death. Livestock are a significant source of human infection as they shed C. burnetii cells in birth tissues, milk, urine and feces. Although prevalence of C. burnetii is high, few Q fever cases are reported in the U.S. and we have a limited understanding of their connectedness due to difficulties in genotyping. Here, we develop canonical SNP genotyping assays to evaluate spatial and temporal relationships among C. burnetii environmental samples and compare them across studies. Given the genotypic diversity of historical collections, we hypothesized that the current enzootic of Coxiellosis is caused by multiple circulating genotypes. We collected A) 23 milk samples from a single bovine herd, B) 134 commercial bovine and caprine milk samples from across the U.S., and C) 400 bovine and caprine samples from six milk processing plants over three years.ResultsWe detected C. burnetii DNA in 96% of samples with no variance over time. We genotyped 88.5% of positive samples; bovine milk contained only a single genotype (ST20) and caprine milk was dominated by a second type (mostly ST8).ConclusionsThe high prevalence and lack of genotypic diversity is consistent with a model of rapid spread and persistence. The segregation of genotypes between host species is indicative of species-specific adaptations or dissemination barriers and may offer insights into the relative lack of human cases and characterizing genotypes

Whole Genome Sequence Typing to Investigate the <em>Apophysomyces</em> Outbreak following a Tornado in Joplin, Missouri, 2011

<div><p>Case reports of <em>Apophysomyces</em> spp. in immunocompetent hosts have been a result of traumatic deep implantation of <em>Apophysomyces</em> spp. spore-contaminated soil or debris. On May 22, 2011 a tornado occurred in Joplin, MO, leaving 13 tornado victims with <em>Apophysomyces trapeziformis</em> infections as a result of lacerations from airborne material. We used whole genome sequence typing (WGST) for high-resolution phylogenetic SNP analysis of 17 outbreak <em>Apophysomyces</em> isolates and five additional temporally and spatially diverse <em>Apophysomyces</em> control isolates (three <em>A. trapeziformis</em> and two <em>A. variabilis</em> isolates). Whole genome SNP phylogenetic analysis revealed three clusters of genotypically related or identical <em>A. trapeziformis</em> isolates and multiple distinct isolates among the Joplin group; this indicated multiple genotypes from a single or multiple sources. Though no linkage between genotype and location of exposure was observed, WGST analysis determined that the Joplin isolates were more closely related to each other than to the control isolates, suggesting local population structure. Additionally, species delineation based on WGST demonstrated the need to reassess currently accepted taxonomic classifications of phylogenetic species within the genus <em>Apophysomyces</em>.</p> </div

List of strains used in study, by isolation site, species and geographic location, along with sequencing coverage results.

<p>NA = Not Applicable; N/A = Not Available; C = Cluster;</p>*<p>indicates multiple isolates from the same patient.</p

WGST phylogeny of outbreak and background Apophysomyces isolates.

<p>A single maximum parsimony tree was reconstructed using ∼28K SNPs from 22 whole genome sequences, resulting in a CI of 0.62. The tree was rooted with Apo-7449 (A. variabilis). The root was derived from an expanded phylogenetic analyses that included a distant outgroup, which enabled the identification of the most basal member of the samples in the dataset described in this figure (data not shown). Genomes are labeled with a Strain ID # (APO-XXXX) and a Cluster ID #-Letter (CX-A). Clusters of identical and nearly identical genome SNP profiles are also labeled. Branch lengths represent genetic distance based on the number of SNP differences; bar represents 1000 SNPs. Tree constructed using MEGA5.</p

<i>De novo</i> assembled reference statistics for Strain Apo-097.

<p><i>De novo</i> assembled reference statistics for Strain Apo-097.</p

Assessment of cognition in early dementia

Better tools for assessing cognitive impairment in the early stages of Alzheimer’s disease (AD) are required to enable diagnosis of the disease before substantial neurodegeneration has taken place and to allow detection of subtle changes in the early stages of progression of the disease. The National Institute on Aging and the Alzheimer’s Association convened a meeting to discuss state of the art methods for cognitive assessment, including computerized batteries, as well as new approaches in the pipeline. Speakers described research using novel tests of object recognition, spatial navigation, attentional control, semantic memory, semantic interference, prospective memory, false memory and executive function as among the tools that could provide earlier identification of individuals with AD. In addition to early detection, there is a need for assessments that reflect real-world situations in order to better assess functional disability. It is especially important to develop assessment tools that are useful in ethnically, culturally and linguistically diverse populations as well as in individuals with neurodegenerative disease other than AD