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Rapid and Direct Real-Time Detection of blaKPC and blaNDM from Surveillance Samples

机译:从监视样本中快速直接地实时检测blaKPC和blaNDM

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摘要

We assessed the performance of a duplex real-time PCR assay for blaKPC and blaNDM performed directly (D-PCR) on perianal and perirectal swabs and stool. Spiked specimens and 126 clinical surveillance swabs (comprising a sensitivity panel of 46 perirectal double swabs previously determined to be culture positive for blaKPC-PCR-positive Enterobacteriaceae and a specificity panel of 80 perianal swabs from patients at risk of carbapenemase-producing Enterobacteriaceae [CPE] colonization) were studied. For the surveillance swabs, D-PCR was compared to PCR after broth enrichment (BE-PCR) and two culture-based methods: the HardyCHROM ESBL agar (HC-A) and the CDC screening (CDC-A) methods. PCR was performed on morphologically distinct colonies that were isolated by culture. All of the initial PCR testing was done without extraction using a simple lysis procedure. The analytical sensitivities of D-PCR for blaKPC were 9 CFU/μl (for swabs) and 90 CFU/μl (for stool), and for blaNDM, it was 1.9 CFU/μl (for both swabs and stool). In the clinical sensitivity panel, D-PCR and BE-PCR were initially positive for blaKPC in 41/46 (89.1%) and 43/46 (93.5%) swabs, respectively. The swabs that were initially negative by D-PCR (n = 5) and BE-PCR (n = 3) were visibly stool soiled; all swabs were blaKPC positive upon repeat testing after lysate extraction. The CDC-A and HC-A yielded blaKPC-positive Enterobacteriaceae from 36/46 (78.3%) and 35/46 (76.1%) swabs, respectively (sensitivities of D-PCR/BE-PCR postextraction of soiled specimens versus HC-A, P = 0.0009, and versus CDC-A, P = 0.0016). All swabs in the specificity panel were negative for CPE by all four methods. D-PCR allows for the timely detection of blaKPC and blaNDM carriage with excellent sensitivity when specimens visibly soiled with stool undergo preparatory extraction.
机译:我们评估了对肛周和直肠周拭子和粪便直接进行blaKPC和blaNDM(D-PCR)的双重实时PCR分析的性能。尖刺标本和126个临床监测拭子(包括46个直肠直肠双拭子的敏感性面板,先前确定其对blaKPC-PCR阳性肠杆菌科细菌培养呈阳性,而特异性小组则由80个肛周拭子特异性面板组成,这些患者来自有发生碳青霉烯酶的肠杆菌科[CPE]的患者定居)。对于监视拭子,将D-PCR与富集肉汤后的PCR(BE-PCR)和两种基于培养的方法进行比较:HardyCHROM ESBL琼脂(HC-A)和CDC筛选(CDC-A)方法。在通过培养分离的形态上不同的菌落上进行PCR。使用简单的裂解程序,无需提取即可完成所有初始PCR测试。 D-PCR对blaKPC的分析灵敏度为9 CFU /μl(拭子)和90 CFU /μl(粪便),而blaNDM的分析灵敏度为1.9 CFU /μl(拭子和粪便)。在临床敏感性小组中,D-PCR和BE-PCR最初分别在41/46(89.1%)和43/46(93.5%)拭子中对blaKPC呈阳性。最初通过D-PCR(n = 5)和BE-PCR(n = 3)阴性的拭子明显被粪便弄脏;裂解物提取后重复测试,所有拭子均为blaKPC阳性。 CDC-A和HC-A分别从36/46(78.3%)和35/46(76.1%)拭子中产生blaKPC阳性肠杆菌科(D-PCR / BE-PCR提取后的污染标本相对于HC-A的敏感性) ,P = 0.0009,与CDC-A相比,P = 0.0016)。通过所有四种方法,特异性面板中的所有拭子对CPE均为阴性。当可见地被粪便弄脏的标本经过预备提取时,D-PCR可以以优异的灵敏度及时检测blaKPC和 bla NDM载体。

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