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J Clin Microbiol. 2016 Feb;54(2):368-75. doi: 10.1128/JCM.01965-15. Epub 2015 Dec 4.

Metagenomic Approach for Identification of the Pathogens Associated with Diarrhea in Stool Specimens.

Author information

1
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
2
Department of Pediatrics, University of Mississippi Medical Center, Jackson, Mississippi, USA.
3
McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri, USA.
4
Division of Infectious Disease, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.
5
McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri, USA george.weinstock@jax.org.

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.

PMID:
26637379
PMCID:
PMC4733167
DOI:
10.1128/JCM.01965-15
[Indexed for MEDLINE]
Free PMC Article

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