Multicenter evaluation of the Verigene Clostridium difficile nucleic acid assay.

The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as "hypervirulent"; 53 were confirmed as ribotype 027, and all 59 possessed Δ 117 in tcdC. Time to results was approximately 2.5 h per specimen. The Verigene CDF test is a novel nucleic acid microarray that reliably detects both C. difficile toxins A and B in unformed stool specimens and appears to adequately identify ribotype 027 isolates.

Optimal media for use in air sampling to detect cultivable bacteria and fungi in the pharmacy.

Current guidelines for air sampling for bacteria and fungi in compounding pharmacies require the use of a medium for each type of organism. U.S. Pharmacopeia (USP) chapter <797> (http://www.pbm.va.gov/linksotherresources/docs/USP797PharmaceuticalCompoundingSterileCompounding.pdf) calls for tryptic soy agar with polysorbate and lecithin (TSApl) for bacteria and malt extract agar (MEA) for fungi. In contrast, the Controlled Environment Testing Association (CETA), the professional organization for individuals who certify hoods and clean rooms, states in its 2012 certification application guide (http://www.cetainternational.org/reference/CAG-009v3.pdf?sid=1267) that a single-plate method is acceptable, implying that it is not always necessary to use an additional medium specifically for fungi. In this study, we reviewed 5.5 years of data from our laboratory to determine the utility of TSApl versus yeast malt extract agar (YMEA) for the isolation of fungi. Our findings, from 2,073 air samples obtained from compounding pharmacies, demonstrated that the YMEA yielded >2.5 times more fungal isolates than TSApl.

Photorhabdus asymbiotica, a pathogen emerging on two continents that proves that there is no substitute for a well-trained clinical microbiologist.

A 54-year-old ranch hand presented to the emergency room with an alleged spider bite and multiple abscesses. Both wound and blood cultures grew Photorhabdus asymbiotica, an enteric gram-negative rod that was initially misidentified by the hospital's rapid identification system. Clinical laboratories should be aware of the limitations of their rapid identification systems and always use them as an adjunct to analysis of morphological and phenotypic traits.