Comparison of repetitive extragenic palindromic sequence-based PCR with PCR ribotyping and pulsed-field gel electrophoresis in studying the clonality of Clostridium difficile  T. Pasanen, S.M. Kotila, J. Horsma, A. Virolainen, J. Jalava, S. Ibrahem, J. Antikainen, S. Mero, E. Tarkka, M. Vaara, P. Tissari  Clinical Microbiology and Infection  Volume 17, Issue 2, Pages 166-175 (February 2011) DOI: 10.1111/j.1469-0691.2010.03221.x Copyright © 2011 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 1 Repetitive extragenic palindromic sequence-based PCR (rep-PCR) results for the 181 clinical isolates analyzed. Of PCR ribotype 027 belonging to (DL 1) and 001 (DL 2), a representative set of five isolates is included (between red dashed lines). The six largest rep-PCR groups (DL 1–6) with >90% similarity are numbered. The typical electropherograms of PCR ribotypes 001 and 027 are shown. (Unknown, intralaboratory ribotype name; ribotype, PCR ribotype according to Cardiff-ECDC; PFGE, pulsed-field electrophoresis; DL, DiversiLab). Clinical Microbiology and Infection 2011 17, 166-175DOI: (10.1111/j.1469-0691.2010.03221.x) Copyright © 2011 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 2 Repetitive extragenic palindromic sequence-based (rep-PCR) profiles of the PCR ribotype 001 isolates. Within the PCR ribotype 001 isolates, repetitive extragenic palindromic sequence-based PCR allows the identification of four subgroups (Sub. 1–4) and one outlier (isolate ID 60603), when 90% similarity is used as threshold. Clinical Microbiology and Infection 2011 17, 166-175DOI: (10.1111/j.1469-0691.2010.03221.x) Copyright © 2011 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 3 Example of contradictory results. Discrepant results were obtained for some isolates when using the three typing methods [PCR ribotyping, repetitive extragenic palindromic sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis]. An example of this phenomenon is the grouping of PCR ribotypes 002, 014 and 020 as presented. Isolates 12–25 cluster in the third largest rep-PCR group DL 3 in Fig. 1. DL, DiversiLab. Clinical Microbiology and Infection 2011 17, 166-175DOI: (10.1111/j.1469-0691.2010.03221.x) Copyright © 2011 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 4 Analysis of the large tcdC deletions. Repetitive extragenic palindromic sequence-based PCR (rep-PCR) types of the isolates with large tcdC deletions clustered in two rep-PCR groups according to the size of the deletion. Most of the isolates with tcdC-Avar cluster in one major rep-PCR group, whereas the isolates with tcdC-A appear more heterogeneous in rep-PCR. Clinical Microbiology and Infection 2011 17, 166-175DOI: (10.1111/j.1469-0691.2010.03221.x) Copyright © 2011 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 5 Reproducibility of the repetitive extragenic palindromic sequence-based PCR profiles for a set of isolates (PCR ribotypes 027, 001, 078 and ‘unknown 55'). As a conclusion of analysing the effect of technician skills and the freezing/thawing step (1–3 amplifications), we chose 90% similarity for reliable grouping of isolates. Clinical Microbiology and Infection 2011 17, 166-175DOI: (10.1111/j.1469-0691.2010.03221.x) Copyright © 2011 European Society of Clinical Infectious Diseases Terms and Conditions