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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Available from: Ghinwa Dumyati, Oct 02, 2015
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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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    • "Author's personal copy assays showed to have a high analytical sensitivity; the LOD calculated from the spiking experiments were in agreement with positive results that were obtained for the lowest sample concentration (100 CFU/ml) in the QCMD 2008 proficiency panel. The analytical sensitivity of both assays are favorably comparable with most other published real-time PCR assays targeting C. difficile in stool specimens (Belanger et al., 2003; Stamper et al., 2009; van den Berg et al., 2004; Wroblewski et al., 2009 "
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    ABSTRACT: We have developed and validated a rapid molecular screening protocol for toxigenic Clostridium difficile, that also enables the identification of the hypervirulent epidemic 027/NAP1 strain. We describe a multiplex real-time PCR assay, which detects the presence of the tcdA and tcdB genes directly in stool samples. In case of positive PCR results, a separate multiplex real-time PCR typing assay was performed targeting the tcdC gene frame shift mutation at position 117. We prospectively compared the results of the screening PCR with those of a cytotoxicity assay (CTA), and a rapid immuno-enzyme assay for 161 stool samples with a specific request for diagnosis of C. difficile infection (CDI). A total of 16 stool samples were positive by CTA. The screening PCR assay confirmed all 16 samples, and gave a PCR positive signal in eight additional samples. The typing PCR assay detected the tcdC Δ117 mutation in 2/24 samples suggesting the presence of the epidemic strain in these samples. This was confirmed by PCR ribotyping and sequencing of the tcdC gene. Using CTA as the "gold standard", the sensitivity, specificity, positive predictive value, and negative predictive value, for the screening PCR were 100%, 94.4%, 66.7%, and 100%, respectively. In conclusion, PCR may serve as a rapid negative screening assay for patients suspected of having CDI, although the low PPV hamper the use of PCR as a standalone test. However, PCR results may provide valuable information for patient management and minimising the spread of the epidemic 027/NAP1 strain.
    Journal of microbiological methods 10/2010; 83(1):59-65. DOI:10.1016/j.mimet.2010.07.017 · 2.03 Impact Factor
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