title: Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study.
abstract: The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targets mapA and ceuE for the detection of Campylobacter jejuni and Campylobacter coli, leading global causes of bacterial gastroenteritis. In silico analyses of mapA and ceuE primer and probe sequences from 1,713 genetically diverse C. jejuni and C. coli genomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of the mapA-ceuE assay was the result of interspecies diversity and intraspecies conservation of the target genes in C. jejuni and C. coli Rare instances of a lack of specificity among C. coli isolates were due to introgression in mapA or sequence diversity in ceuE The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software.
authors.affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom.|NIHR Health Protection Research Unit in Gastrointestinal Infections, University of Oxford, Oxford, United Kingdom.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Jansen van Rensburg
authors.affiliation: NIHR Health Protection Research Unit in Gastrointestinal Infections, University of Oxford, Oxford, United Kingdom.|Gastrointestinal Bacteria Reference Unit, Public Health England, London, United Kingdom.
authors.forename: Craig
authors.initials: C
authors.lastname: Swift
authors.affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom.|NIHR Health Protection Research Unit in Gastrointestinal Infections, University of Oxford, Oxford, United Kingdom.
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Cody
authors.affiliation: NIHR Health Protection Research Unit in Gastrointestinal Infections, University of Oxford, Oxford, United Kingdom.|Gastrointestinal Bacteria Reference Unit, Public Health England, London, United Kingdom.
authors.forename: Claire
authors.initials: C
authors.lastname: Jenkins
authors.affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom martin.maiden@zoo.ox.ac.uk.|NIHR Health Protection Research Unit in Gastrointestinal Infections, University of Oxford, Oxford, United Kingdom.
authors.forename: Martin C J
authors.identifier: 0000-0001-6321-5138
authors.initials: MC
authors.lastname: Maiden
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002169:Campylobacter Infections / Q000175:diagnosis* / Q000188:drug therapy / Q000382:microbiology; D017000:Campylobacter coli / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D016123:Campylobacter jejuni / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D002352:Carrier Proteins / Q000235:genetics*; D005759:Gastroenteritis / Q000175:diagnosis* / Q000382:microbiology; D016680:Genome, Bacterial / Q000235:genetics*; D006801:Humans; D033862:Iron-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D008565:Membrane Proteins; C499680:ceuE protein, Campylobacter coli; C490272:mapA protein, Campylobacter jejuni