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J Clin Microbiol. 2015 Apr;53(4):1192-7. doi: 10.1128/JCM.03591-14. Epub 2015 Jan 28.

Evaluation of portability and cost of a fluorescent PCR ribotyping protocol for Clostridium difficile epidemiology.

Author information

1
Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, USA.
2
College of Pharmacy, University of Houston, Houston, Texas, USA.
3
Department of Medicine, Division of Infectious Diseases, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
4
Department of Internal Medicine, Division of Infectious Diseases, University of Michigan, Ann Arbor, Michigan, USA Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA Division of Infectious Diseases, Veterans Affairs Ann Arbor Healthcare System, Ann Arbor, Michigan, USA.
5
Department of Internal Medicine, Division of Infectious Diseases, University of Michigan, Ann Arbor, Michigan, USA.
6
Department of Internal Medicine, Division of Infectious Diseases, University of Michigan, Ann Arbor, Michigan, USA Division of Infectious Diseases, Veterans Affairs Ann Arbor Healthcare System, Ann Arbor, Michigan, USA.
7
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
8
Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, USA seth.walk@montana.edu.

Abstract

Clostridium difficile is the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e., portable data) to help characterize isolates during epidemiologic investigations of C. difficile outbreaks and sporadic cases of disease. The most popular C. difficile-typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands of C. difficile isolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database (http://walklab.rcg.montana.edu) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing of C. difficile pathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified.

PMID:
25631804
PMCID:
PMC4365229
DOI:
10.1128/JCM.03591-14
[Indexed for MEDLINE]
Free PMC Article

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