Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae, with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis. While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (tox). Nontoxigenic tox-bearing (NTTB) Corynebacterium 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 tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis. Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for tox, Diph_rpoB, and CUP_rpoB targets, respectively. Twenty-nine NTTB Corynebacterium 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 tox or the promoter region. This new Corynebacterium 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.

中文翻译：

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通过新型实时荧光定量PCR检测和表征携带​​白喉毒素基因的棒状杆菌。

呼吸道白喉病的特征是牢牢附着的假膜，是由产毒素的白喉杆菌菌株引起的，偶尔会产生类似的疾病，由产毒性溃疡杆菌或假结核杆菌引起。尽管白喉实验室确认需要使用培养方法来确定毒素原性，但实时PCR（RT-PCR）提供了一种检测毒素基因（tox）的更快方法。毒的TOX荷瘤（NTTB）棒状杆菌分离株进行了说明，但在分子诊断的准确性这些分离物的影响没有得到很好的表征。在这里，我们描述了一种新的三重RT-PCR检测方法从密切相关的物种C.溃疡和C.假结核中分离和区分白喉C .。与使用690个先前鉴定的微生物分离株进行的培养相比，评估了分析的敏感性和特异性。新的三重测定法可以准确地鉴定棒状杆菌菌株，对所有靶标的分析灵敏度均为100％。分离物对tox，Diph_ rpoB和CUP_ rpoB靶标的分析特异性分别为94.1％，100％和99.5％。二十九个NTTB棒状杆菌通过RT-PCR检测到分离株，占测试的494种非毒素分离株的5.9％。NTTB分离株的全基因组测序揭示变化的突变推测底层缺乏毒素的生产，以及8个分离株中没有突变TOX或启动子区域。这种新的棒状杆菌RT-PCR方法提供了一种快速的工具，可以直接从标本中筛选分离株并鉴定可能的白喉病例。但是，NTTB分离株的零星出现加强了白喉培养诊断继续提供最准确的病例确认的观点。