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: 4.23). 05/2009; 47(7):2142-8. DOI: 10.1128/JCM.02498-08
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Clostridium difficile (CD)-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing CD had been difficult as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAAT) are quickly becoming the standard of care. We compared the performance of two automated Investigational/Research-Use-Only (IUO/RUO) NAAT for the detection of CD toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD MAX™ Cdiff Assay (MAX). A prospective analysis of 111 stool specimens received in the laboratory for CD testing by the laboratory's test of record (TOR), BD GeneOhm™ Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for CD, were included in this study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods; 9 were positive by TOR and MAX, and 3 were positive by TOR only. One negative specimen was initially inhibitory by MAX. The remaining 95 specimens were negative by all methods. Toxigenic CD culture was performed on the 12 discrepant samples. True CD positive was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for MAX and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance and each have individual benefits, which will ensure that they will both have a niche in clinical laboratories.
    Journal of clinical microbiology 02/2014; · 4.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    International Journal of Clinical Medicine 04/2014; 5(7):414-419.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The presence of gene 16S rRNA and genes encoding toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA/cdtB) of Clostridium difficile in stool samples from children with (110) and without (150) diarrhea was determined by using a TaqMan system. Fifty-seven (21.9%) out of 260 stool samples harbored the 16S rRNA gene. The genetic profile of tcdA+/tcdB- and cdtA+/cdtB+ was verified in one C. difficile-positive diarrhea sample and of tcdA+/tcdB+ in three C. difficile-positive nondiarrhea samples. The presence of tcdA+/tcdB+ in stools obtained from children without diarrhea, suggests that they were asymptomatic carriers of toxigenic strains.
    Scientifica. 01/2014; 2014:594014.

Full-text (2 Sources)

Available from
May 20, 2014