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      Molecular Epidemiologic Investigation of an Anthrax Outbreak among Heroin Users, Europe

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          Abstract

          Heroin may have been accidentally contaminated by an animal-derived source along a major drug trafficking route.

          Abstract

          In December 2009, two unusual cases of anthrax were diagnosed in heroin users in Scotland. A subsequent anthrax outbreak in heroin users emerged throughout Scotland and expanded into England and Germany, sparking concern of nefarious introduction of anthrax spores into the heroin supply. To better understand the outbreak origin, we used established genetic signatures that provided insights about strain origin. Next, we sequenced the whole genome of a representative Bacillus anthracis strain from a heroin user (Ba4599), developed Ba4599-specific single-nucleotide polymorphism assays, and genotyped all available material from other heroin users with anthrax. Of 34 case-patients with B. anthracis–positive PCR results, all shared the Ba4599 single-nucleotide polymorphism genotype. Phylogeographic analysis demonstrated that Ba4599 was closely related to strains from Turkey and not to previously identified isolates from Scotland or Afghanistan, the presumed origin of the heroin. Our results suggest accidental contamination along the drug trafficking route through a cutting agent or animal hides used to smuggle heroin into Europe.

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          Aggressive assembly of pyrosequencing reads with mates

          Motivation: DNA sequence reads from Sanger and pyrosequencing platforms differ in cost, accuracy, typical coverage, average read length and the variety of available paired-end protocols. Both read types can complement one another in a ‘hybrid’ approach to whole-genome shotgun sequencing projects, but assembly software must be modified to accommodate their different characteristics. This is true even of pyrosequencing mated and unmated read combinations. Without special modifications, assemblers tuned for homogeneous sequence data may perform poorly on hybrid data. Results: Celera Assembler was modified for combinations of ABI 3730 and 454 FLX reads. The revised pipeline called CABOG (Celera Assembler with the Best Overlap Graph) is robust to homopolymer run length uncertainty, high read coverage and heterogeneous read lengths. In tests on four genomes, it generated the longest contigs among all assemblers tested. It exploited the mate constraints provided by paired-end reads from either platform to build larger contigs and scaffolds, which were validated by comparison to a finished reference sequence. A low rate of contig mis-assembly was detected in some CABOG assemblies, but this was reduced in the presence of sufficient mate pair data. Availability: The software is freely available as open-source from http://wgs-assembler.sf.net under the GNU Public License. Contact: jmiller@jcvi.org Supplementary information: Supplementary data are available at Bioinformatics online.
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            Global Genetic Population Structure of Bacillus anthracis

            Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064–6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated.
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              Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing.

              Phylogenetic reconstruction using molecular data is often subject to homoplasy, leading to inaccurate conclusions about phylogenetic relationships among operational taxonomic units. Compared with other molecular markers, single-nucleotide polymorphisms (SNPs) exhibit extremely low mutation rates, making them rare in recently emerged pathogens, but they are less prone to homoplasy and thus extremely valuable for phylogenetic analyses. Despite their phylogenetic potential, ascertainment bias occurs when SNP characters are discovered through biased taxonomic sampling; by using whole-genome comparisons of five diverse strains of Bacillus anthracis to facilitate SNP discovery, we show that only polymorphisms lying along the evolutionary pathway between reference strains will be observed. We illustrate this in theoretical and simulated data sets in which complex phylogenetic topologies are reduced to linear evolutionary models. Using a set of 990 SNP markers, we also show how divergent branches in our topologies collapse to single points but provide accurate information on internodal distances and points of origin for ancestral clades. These data allowed us to determine the ancestral root of B. anthracis, showing that it lies closer to a newly described "C" branch than to either of two previously described "A" or "B" branches. In addition, subclade rooting of the C branch revealed unequal evolutionary rates that seem to be correlated with ecological parameters and strain attributes. Our use of nonhomoplastic whole-genome SNP characters allows branch points and clade membership to be estimated with great precision, providing greater insight into epidemiological, ecological, and forensic questions.
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                Author and article information

                Journal
                Emerg Infect Dis
                Emerging Infect. Dis
                EID
                Emerging Infectious Diseases
                Centers for Disease Control and Prevention
                1080-6040
                1080-6059
                August 2012
                : 18
                : 8
                : 1307-1313
                Affiliations
                [1]Northern Arizona University, Flagstaff, Arizona, USA (E.P. Price, M.L. Seymour, D.S. Sarovich, S.R. Wolken, K.P. Drees, S.M. Beckstrom-Sternberg, R.T. Okinaka, W.-K. Chung, J.M. Schupp, D.M. Wagner, J.T. Foster, T. Pearson, P. Keim);
                [2]Health Protection Agency, London, UK (J. Latham, J. Mason, G. Vincent, R. Vipond, T. Brooks);
                [3]National Biodefense Analysis and Countermeasures Center, Frederick, Maryland, USA (A.M. Phillippy, S. Koren, N. H. Bergman, J. Burans);
                [4]and Translational Genomics Research Institute, Phoenix, Arizona, USA (S.M. Beckstrom-Sternberg, J.M. Schupp, P. Keim)
                Author notes
                Address for correspondence: Paul Keim, Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-4073, USA; email: paul.keim@ 123456nau.edu
                Article
                11-1343
                10.3201/eid1808.111343
                3414016
                22840345
                3be72e4c-9e83-4dfc-8ca5-c991c8ab9fa0
                History
                Categories
                Research
                Research

                Infectious disease & Microbiology
                trans-eurasian,epidemic,bacteria,outbreak,epidemiology,anthrax,cansnp,heroin,phylogenetic,bacillus anthracis,scotland

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