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Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples.
Ann Lab Med. 2015 May; 35(3):306-13.AL

Abstract

BACKGROUND

The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile.

METHODS

The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR.

RESULTS

A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 10(3) colony forming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%.

CONCLUSIONS

The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.

Authors+Show Affiliations

Department of Microbiology, Gulhane Military Medical Academy, Etlik, Ankara, Turkey. ; Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Houston, TX, USA. ; St Luke's Episcopal Hospital, Houston, TX, USA.Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Houston, TX, USA.St Luke's Episcopal Hospital, Houston, TX, USA.Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Houston, TX, USA.Department of Microbiology, Gulhane Military Medical Academy, Etlik, Ankara, Turkey.St Luke's Episcopal Hospital, Houston, TX, USA.Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Houston, TX, USA. ; St Luke's Episcopal Hospital, Houston, TX, USA.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25932438

Citation

Kilic, Abdullah, et al. "Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium Difficile From Stool Samples." Annals of Laboratory Medicine, vol. 35, no. 3, 2015, pp. 306-13.
Kilic A, Alam MJ, Tisdel NL, et al. Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples. Ann Lab Med. 2015;35(3):306-13.
Kilic, A., Alam, M. J., Tisdel, N. L., Shah, D. N., Yapar, M., Lasco, T. M., & Garey, K. W. (2015). Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples. Annals of Laboratory Medicine, 35(3), 306-13. https://doi.org/10.3343/alm.2015.35.3.306
Kilic A, et al. Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium Difficile From Stool Samples. Ann Lab Med. 2015;35(3):306-13. PubMed PMID: 25932438.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples. AU - Kilic,Abdullah, AU - Alam,Mohammad J, AU - Tisdel,Naradah L, AU - Shah,Dhara N, AU - Yapar,Mehmet, AU - Lasco,Todd M, AU - Garey,Kevin W, Y1 - 2015/04/01/ PY - 2014/09/02/received PY - 2014/10/23/revised PY - 2015/01/28/accepted PY - 2015/5/2/entrez PY - 2015/5/2/pubmed PY - 2016/12/15/medline KW - Binary toxin KW - Clostridium difficile KW - Multiplex real-time PCR KW - Toxin variant strains SP - 306 EP - 13 JF - Annals of laboratory medicine JO - Ann Lab Med VL - 35 IS - 3 N2 - BACKGROUND: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. METHODS: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. RESULTS: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 10(3) colony forming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. CONCLUSIONS: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run. SN - 2234-3814 UR - https://www.unboundmedicine.com/medline/citation/25932438/Multiplex_Real_Time_PCR_Method_for_Simultaneous_Identification_and_Toxigenic_Type_Characterization_of_Clostridium_difficile_From_Stool_Samples_ L2 - http://www.annlabmed.org/journal/viewJournal.html?year=2015&vol=35&page=306 DB - PRIME DP - Unbound Medicine ER -