Rapid Molecular Characterization of Clostridium difficile and Assessment of Populations of C-difficile in Stool Specimens

Wadsworth Center, New York State Department of Health, Albany, NY 12201-2002, USA.
Journal of clinical microbiology (Impact Factor: 3.99). 05/2009; 47(7):2142-8. DOI: 10.1128/JCM.02498-08
Source: PubMed


Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n = 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n = 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n = 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates.

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    • "CDI, however, is not an infection exclusive to hospitalized patients. It has been reported that nearly half of all healthcare-onset cases of CDI occur in long-term care facilities (LTCFs) [9] [10]. In 1993, Simor, et al. found an 8% point prevalence of CDI among LTCF residents receiving antibiotics [11]. "
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    ABSTRACT: Introduction The incidence and severity of Clostridium difficile infection (CDI) has been increasing and long-term care facility (LTCF) residents are at high risk given their age, co-morbidities, and high antibiotic exposure. Infection control policies are crucial for controlling CDI, but there are currently no regulatory guidelines in the United States. Therefore, we evaluated infection control policies in local LTCFs to define the CDI-specific policies and the administrative and staff understanding of CDI, so as to identify perceived barriers for compliance. Methods IRB approval was sought and exemption granted, all 8 local LTCFs were asked to participate. Each facility was visited by study personnel who interviewed the administrative Infection Control Practitioner (ICP) and 3 - 4 Licensed Practical Nurses (LPNs) with distinct survey format. Infection control policies were then compared to the SHEA recommendations for CDI in LTCFs. Results Of the eligible facilities, 75% (n = 6) participated. ICP (n = 6) and LPNs (n = 21) were interviewed. All facilities accept residents with active CDI and 2 had written CDI-specific infection control policies. All facilities had hand hygiene or glove use policies and 2 had policies for the use of sporicidal environmental cleaning. No facility restricted antibiotic use. Each facility has a policy to instruct their staff through in-services, either annually or upon new hire, but 33% (n = 7) LPNs reported no facility-based CDI training. While 80% (n = 17) of LPNs felt comfortable with the facility CDI policies, only 11 accurately restated it. ICPs felt the most relevant barrier to staff compliance was time constraints (n = 4, 67%), however, LPNs felt it was limited knowledge (n = 10, 48%) and poor communication (n = 2, 10%). Discussion and Conclusions With the increasing incidence and severity of CDI in LCTF, few of the facilities surveyed had CDI-specific policies. Despite CDI-specific training, there is a perceived knowledge and communication gap for staff caring for residents with CDI.
    International Journal of Clinical Medicine 04/2014; 5(7):414-419. DOI:10.4236/ijcm.2014.57056
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    • "Briefly, amplification reactions were performed in 50 μl reaction volumes, composed of 5 μl of buffer 2 (PE), 2.5 mM MgCl 2 , 16 μM dNTPs, 0.5 μM of both forward and reverse primers, Table 1 Primer and probe sequences used in real-time multiplex PCR assay. Letters in red indicate changes from Wroblewski et al. (2009) "
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    ABSTRACT: The increased prevalence of hypervirulent ribotype 027 Clostridium difficile requires rapid identification of isolates in order to implement timely infection control strategies. High resolution melt (HRM) analysis of PCR products can identify strain variation amongst genera of bacteria. The intergenic (16S-23S rDNA) spacer region contains sequence regions conserved within genera and other sequence region variables between species within genera. We wished to investigate whether HRM analysis of PCR ribotyping products could identify ribotype 027 C. difficile. Ribotyping was performed on 93 clinical isolates and five control strains and band patterns were analysed using GelCompar II (Applied Maths, USA). Real-time PCR using ribotyping primers was performed and normalised melt curves were generated. The HRM data was then imported into ScreenClust software (QIAGEN) to generate principal component analysis graphs depicting clustered relationships of strains. Ribotyping produced clear PCR bands for 88/98 isolates tested. Dendrograms generated by GelCompar showed a diversity of ribotype patterns amongst these 88 isolates with 18 groups identified with 70% homology. One clinical isolate showed 100% homology with the control 027 strains. ScreenClust analysis of the same 88 HRM results showed clustering of isolates, with 027 strains identifiable as a unique cluster. HRM analysis correctly identified the control 027 stains and the clinical isolate shown to be 027. HRM combined with ScreenClust analysis of real-time PCR products of the 16S-23S rDNA spacer region successfully identified ribotype 027 strains. For infection control purposes this was achieved within 2-3 h of colony isolation.
    Journal of microbiological methods 03/2012; 89(2):87-94. DOI:10.1016/j.mimet.2012.02.011 · 2.03 Impact Factor
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    • "Therefore several groups have reported in-house real-time PCR assays. All had very good sensitivity, specificity and short turnaround times but special procedures for elimination of PCR inhibitors during DNA extraction were needed (Alonso et al., 1999; Bélanger et al., 2003; Van den Berg et al., 2005; Eastwood et al., 2009; Wroblewski et al., 2009). Currently several realtime PCR assays that detect toxigenic C. difficile directly from stool are commercially available. "
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    ABSTRACT: Two commercial real-time PCR assays for the detection of Clostridium difficile, BD GeneOhm Cdiff assay (BD Diagnostics) and Xpert C. difficile assay (Cepheid), were compared to each other and to toxigenic culture, which was used as a gold standard, on a set of 194 clinical stools submitted for routine diagnostic analysis. Of 28 (14.4 %) toxigenic culture positive samples 23 were positive with both assays, the BD and the Cepheid real-time PCR assays, 4 were positive only by Cepheid Xpert C. difficile assay and 1 sample was negative by both PCR assays, resulting in sensitivity, specificity, positive predictive value and negative predictive value of 82.1, 98.2, 88.5 and 97.0 %, respectively, for the BD GeneOhm Cdiff assay, and 96.4, 97.3, 87.1 and 99.3 %, respectively, for the Cepheid Xpert C. difficile assay. Altogether 26 out of 194 (13.4 %) samples were reported invalid by Cepheid. Toxigenic C. difficile positive samples contained 15 different PCR ribotypes distributed into toxinotype 0 and 2 different variant toxinotypes (III, IV). Clinical data were available for 24 out of 28 (85.7  %) toxigenic C. difficile positive patients and 18 (75.0 %) of them were diagnosed with diarrhoea, while others had other symptoms or risk factors related to possible C. difficile infection (antibiotics, bloody stool, peritonitis, Crohn's disease).
    Journal of Medical Microbiology 03/2011; 60(Pt 8):1131-6. DOI:10.1099/jmm.0.030163-0 · 2.25 Impact Factor
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