title: Metagenomic Approach for Identification of the Pathogens Associated with Diarrhea in Stool Specimens.
abstract: The potential to rapidly capture the entire microbial community structure and/or gene content makes metagenomic sequencing an attractive tool for pathogen identification and the detection of resistance/virulence genes in clinical settings. Here, we assessed the consistency between PCR from a diagnostic laboratory, quantitative PCR (qPCR) from a research laboratory, 16S rRNA gene sequencing, and metagenomic shotgun sequencing (MSS) for Clostridium difficile identification in diarrhea stool samples. Twenty-two C. difficile-positive diarrhea samples identified by PCR and qPCR and five C. difficile-negative diarrhea controls were studied. C. difficile was detected in 90.9% of C. difficile-positive samples using 16S rRNA gene sequencing, and C. difficile was detected in 86.3% of C. difficile-positive samples using MSS. CFU inferred from qPCR analysis were positively correlated with the relative abundance of C. difficile from 16S rRNA gene sequencing (r(2) = -0.60) and MSS (r(2) = -0.55). C. difficile was codetected with Clostridium perfringens, norovirus, sapovirus, parechovirus, and anellovirus in 3.7% to 27.3% of the samples. A high load of Candida spp. was found in a symptomatic control sample in which no causative agents for diarrhea were identified in routine clinical testing. Beta-lactamase and tetracycline resistance genes were the most prevalent (25.9%) antibiotic resistance genes in these samples. In summary, the proof-of-concept study demonstrated that next-generation sequencing (NGS) in pathogen detection is moderately correlated with laboratory testing and is advantageous in detecting pathogens without a priori knowledge.
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Yanjiao
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Kristine M
authors.initials: KM
authors.lastname: Wylie
authors.affiliation: Department of Pediatrics, University of Mississippi Medical Center, Jackson, Mississippi, USA.
authors.forename: Rana E
authors.initials: RE
authors.lastname: El Feghaly
authors.affiliation: McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Kathie A
authors.initials: KA
authors.lastname: Mihindukulasuriya
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Alexis
authors.initials: A
authors.lastname: Elward
authors.affiliation: Division of Infectious Disease, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.
authors.forename: David B
authors.initials: DB
authors.lastname: Haslam
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Storch
authors.affiliation: McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri, USA george.weinstock@jax.org.
authors.forename: George M
authors.initials: GM
authors.lastname: Weinstock
mesh_terms: D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D016000:Cluster Analysis; D003967:Diarrhea / Q000175:diagnosis* / Q000382:microbiology*; D004352:Drug Resistance, Microbial; D005243:Feces / Q000382:microbiology* / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D054892:Metagenome; D056186:Metagenomics / Q000379:methods; D064307:Microbiota; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D012336:RNA, Ribosomal, 16S