Simultaneous detection and characterization of toxigenic Clostridium difficile directly from clinical stool specimens

Abstract

We employed a multiplex polymerase chain reaction (PCR) coupled with capillary electrophoresis (mPCR-CE) targeting six Clostridium difficile genes, including tpi, tcdA, tcdB, cdtA, cdtB, and a deletion in tcdC for simultaneous detection and characterization of toxigenic C. difficile directly from fecal specimens. The mPCR-CE had a limit of detection of 10 colony-forming units per reaction with no cross-reactions with other related bacterial genes. Clinical validation was performed on 354 consecutively collected stool specimens from patients with suspected C. difficile infection and 45 isolates. The results were compared with a reference standard combined with BD MAX Cdiff, real-time cell analysis assay (RTCA), and mPCR-CE. The toxigenic C. difficile species were detected in 36 isolates and 45 stool specimens by the mPCR-CE, which provided a positive rate of 20.3% (81/399). The mPCR-CE had a specificity of 97.2% and a sensitivity of 96.0%, which was higher than RTCA (x² = 5.67, P = 0.017) but lower than BD MAX Cdiff (P = 0.245). Among the 45 strains, 44 (97.8%) were determined as non-ribotype 027 by the mPCR-CE, which was fully agreed with PCR ribotyping. Even though ribotypes 017 (n = 8, 17.8%), 001 (n = 6, 13.3%), and 012 (n = 7, 15.6%) were predominant in this region, ribotype 027 was an important genotype monitored routinely. The mPCR-CE provided an alternative diagnosis tool for the simultaneous detection of toxigenic C. difficile in stool and potentially differentiated between RT027 and non-RT027.

Keywords

Clostridium difficile / multiplex PCR / capillary electrophoresis / detection / characterization

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Acknowledgements

This work was supported by the Programs for Major Science and Technology Medicine and Healthcare in Zhejiang (No. WKJ-ZJ-1507), the Key Research and Development Program of Zhejiang (Nos. 2015G03048 and 2017C03028), the National Natural Science Foundation of China (No. 81471998), the Project of Medical Health and Technology in Zhejiang (No. 2016DTB005), and the National Cancer Institute at the National Institutes of Health Support Grant (No. P30 CA008748).

Compliance with ethics guidelines

Hanjiang Lai, Chen Huang, Jian Cai, Julian Ye, Jun She, Yi Zheng, Liqian Wang, Yelin Wei, Weijia Fang, Xianjun Wang, Yi-Wei Tang, Yun Luo, and Dazhi Jin declare that they have no conflict of interest. This study as a part of the Programs for Major Science and Technology Medicine and Healthcare in Zhejiang (No. WKJ-ZJ-1507) has been approved by the Institutional Review Boards of ZJCDC. This study was presented in part at the 114th American Society for Microbiology Annual Meeting, Boston, MA, 17 to 20 May 2014.

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