Estimating risk of c. difficile transmission from pcr positive but cytotoxin negative cases

Abstract Background: The use of molecular methods to diagnose Clostridium difficile infection (CDI) has improved diagnostic yield compared to conventional methods. However, PCR testing can detect colonization and has introduced several practical challenges pertaining to need for treatment and isolation of cases

PCR threshold cycle value (Ct) for forty-two samples tested by Cytotoxin assay (CYT) and PCR.

<p>PCR threshold cycle value (Ct) for forty-two samples tested by Cytotoxin assay (CYT) and PCR.</p

Minimum spanning tree of MLVA data from study isolates (n = 47).

<p>Letter symbol in the center of the circle represents the results for testing by cytotoxin assay for 42 samples included in the analysis (C + and C −). Five samples were not tested by CYT (NT). Each circle represents a distinct MLVA type and numbers between the circles represent the STRD [Summed tandem repeat difference]. Isolates with a STRD<10 are highlighted in colored clouds representing clusters (genetically related clonal complexes). <i>tcdC</i> sequencing is depicted by color coding within circles with <i>tcdC 1</i> (corresponding to NAP1) strain represented in red (reference in right corner).</p

Plasmid pKp28.

<p>An 83.4 kb mosaic plasmid with 99% identity to portions of 4 unique plasmids, (blue) pKPC-727; (yellow) pKpQIL; (red) pHg; (green), pKPN-262. (A) Linear depiction of open reading frames (B) circular depiction of pKp28 sequence with >99% nucleotide identity to indicated plasmids. Black arrows indicate PCR primers specific to pKp28.</p

Timeline of 51 Kp isolates collected from January 2011 –July 2013.

<p>Dates indicated in days relative to collection of first isolate (0–930); isolate source indicated by shape–circle, patient isolate; diamond, endoscope; genotype indicated by color; underlined IDs depict isolates with WGS; superscripts ^{a, b, c, d, e} represent multiple isolates from the same patient as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144310#pone.0144310.s002" target="_blank">S1 Table</a>.</p

Genomic Epidemiology of an Endoscope-Associated Outbreak of <i>Klebsiella pneumoniae</i> Carbapenemase (KPC)-Producing <i>K</i>. <i>pneumoniae</i>

<div><p>Increased incidence of infections due to <i>Klebsiella pneumoniae</i> carbapenemase (KPC)-producing <i>Klebsiella pneumoniae</i> (KPC-Kp) was noted among patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) at a single hospital. An epidemiologic investigation identified KPC-Kp and non-KPC-producing, extended-spectrum β-lactamase (ESBL)-producing Kp in cultures from 2 endoscopes. Genotyping was performed on patient and endoscope isolates to characterize the microbial genomics of the outbreak. Genetic similarity of 51 Kp isolates from 37 patients and 3 endoscopes was assessed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Five patient and 2 endoscope isolates underwent whole genome sequencing (WGS). Two KPC-encoding plasmids were characterized by single molecule, real-time sequencing. Plasmid diversity was assessed by endonuclease digestion. Genomic and epidemiologic data were used in conjunction to investigate the outbreak source. Two clusters of Kp patient isolates were genetically related to endoscope isolates by PFGE. A subset of patient isolates were collected post-ERCP, suggesting ERCP endoscopes as a possible source. A phylogeny of 7 Kp genomes from patient and endoscope isolates supported ERCP as a potential source of transmission. Differences in gene content defined 5 ST258 subclades and identified 2 of the subclades as outbreak-associated. A novel KPC-encoding plasmid, pKp28 helped define and track one endoscope-associated ST258 subclade. WGS demonstrated high genetic relatedness of patient and ERCP endoscope isolates suggesting ERCP-associated transmission of ST258 KPC-Kp. Gene and plasmid content discriminated the outbreak from endemic ST258 populations and assisted with the molecular epidemiologic investigation of an extended KPC-Kp outbreak.</p></div

PCR of pKp28 and gene content of 35 ST258 outbreak isolates.

<p>PCR of pKp28 and gene content of 35 ST258 outbreak isolates.</p

Gene differences among 7 <i>K</i>. <i>pneumoniae</i>.

<p>Gene differences among 7 <i>K</i>. <i>pneumoniae</i>.</p

PFGE of 35 Cluster B ST258 isolates with S1 nuclease digests depicting plasmid profiles (PP).

<p>Superscripts (b-e) indicate isolates from the same patient. Boxed, bold IDs have WGS data; endoscope IDs are bold and underlined (K-28, also boxed).</p