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|Katherine Bowden, Ashley Simon, Margaret Williams, Virgina Stringer, Pamela Cassiday, Tejpratap Tiwari, Jessica Waller, Maureen Diaz, Jonas Winchell and M Lucia Tondella|
|Centers for Disease Control and Prevention, USA|
|ScientificTracks Abstracts: J Bacteriol Parasitol|
|Statement of the Problem: Toxigenic strains of Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis are capable of eliciting diphtheria toxin that causes symptoms of diphtheria, a vaccine-preventable disease. Historically, diagnosis of toxbearing Corynebacterium species and verification of toxin production required an isolate. The purpose of this study was to validate a new triplex real-time PCR assay to both detect the tox gene and differentiate C. diphtheriae from the 2 other toxbearing Corynebacterium species directly from clinical specimens. Methodology & Theoretical Orientation: The triplex assays detect the Corynebacterium tox gene, C. diphtheriae rpoB, and the rpoB ortholog in C. ulcerans and C. pseudotuberculosis. A total of 101 archived clinical specimens (throat and nasal swabs) from suspected diphtheria cases, 20 Corynebacterium spp. and 15 other respiratory pathogen isolates were used to examine sensitivity and specificity of the triplex assay. Comprehensive in silico analysis of the oligo sequences was performed to confirm specificity. When available, results were compared to previous culture, CDC singleplex tox real-time PCR, and toxin production results. Positivity was determined with a Ct less than 40. Findings: The triplex assay demonstrated an LOD of 10 genomic copies, 10X more sensitive than the current singleplex CDC assay. No cross-reactivity was found with 15 respiratory pathogens, including other Corynebacterium spp. Three specimens that tested negative with the current CDC assay were found to harbor the tox gene using the triplex assay, 2 of which were confirmed as C. diphtheriae. Conclusion & Significance: The new triplex assay successfully differentiates C. diphtheriae from other toxigenic Corynebacterium species directly from clinical specimens and is more sensitive than the current CDC assay. Although a bacterial isolate is needed to confirm toxin production, when an isolate is not available, this assay can be used to identify a potentially toxigenic strain of Corynebacterium in clinical specimens. Further analysis will be conducted to determine if this assay is a good surrogate for identifying truly toxigenic strains.|
Katherine Bowden received her BS in Microbiology and PhD in Genetics from the University of Georgia. Over the past 4 years, she has led numerous projects under the scope of molecular diagnostics in the Pertussis and Diphtheria Lab at the Centers for Disease Control and Prevention. These projects include real-time PCR method validation, analysis of molecular epidemiology of pertussis epidemics, and development of a whole genome Multi Locus Sequence Typing (wgMLST) for pertussis. Additionally, she has coordinated numerous international trainings to build in-country capacity for both pertussis and diphtheria diagnostics in Latin American and Caribbean nations.
Email: [email protected]
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