Detection and Characterization of Diphtheria Toxin Gene-Bearing <i>Corynebacterium</i> Species through a New Real-Time PCR Assay

Abstract

Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of <i>Corynebacterium diphtheriae</i>, with similar illness produced occasionally by toxigenic <i>Corynebacterium ulcerans</i> or, rarely, <i>Corynebacterium pseudotuberculosis</i> While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (<i>tox</i>). Nontoxigenic <i>tox</i>-bearing (NTTB) <i>Corynebacterium</i> isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect <i>tox</i> and distinguish <i>C. diphtheriae</i> from the closely related species <i>C. ulcerans</i> and <i>C. pseudotuberculosis</i> Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized <i>Corynebacterium</i> isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for <i>tox</i>, Diph_<i>rpoB</i>, and CUP_<i>rpoB</i> targets, respectively. Twenty-nine NTTB <i>Corynebacterium</i> isolates, representing 5.9% of 494 nontoxigenic isolates tested, were detected by RT-PCR. Whole-genome sequencing of NTTB isolates revealed varied mutations putatively underlying their lack of toxin production, as well as eight isolates with no mutation in <i>tox</i> or the promoter region. This new <i>Corynebacterium</i> RT-PCR method provides a rapid tool to screen isolates and identify probable diphtheria cases directly from specimens. However, the sporadic occurrence of NTTB isolates reinforces the viewpoint that diphtheria culture diagnostics continue to provide the most accurate case confirmation.