Direct Screening of Blood by PCR and Pyrosequencing for a 16S rRNA Gene Target from Emergency Department and Intensive Care Unit Patients Being Evaluated for Bloodstream Infection.
Direct Screening of Blood by PCR and Pyrosequencing for a 16S rRNA Gene Target from Emergency Department and Intensive Care Unit Patients Being Evaluated for Bloodstream Infection.
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通过 PCR 和焦磷酸测序直接筛查来自急诊科和重症监护病房接受血流感染评估的患者的血液中的 16S rRNA 基因靶标。
DOI：
10.1128/jcm.02394-15
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发表时间：
2016
影响因子：
9.4
通讯作者：
Jordan,JA
中科院分区：
文献类型：
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作者：
Moore,MS;McCarroll,MG;McCann,CD;May,L;Younes,N;Jordan,JA
在这里，我们将 PCR/焦磷酸测序的结果与直接从血液中检测细菌的培养结果进行了比较。从 913 名疑似菌血症患者（入选标准为医生要求的血培养和全血细胞计数 [CBC]）和 102 名对照者（健康献血者）的 1,130 份血液样本中提取了 DNA。对所有 1,232 份提取物进行了 β-珠蛋白和通用 16S rRNA 基因靶标的实时 PCR 测定。通过通用 16S rRNA 基因 PCR/焦磷酸测序鉴定为含有葡萄球菌、链球菌或肠革兰氏阴性杆菌的样本进行了目标特异性 PCR/焦磷酸测序。 99.1% (1,120/1,130) 的患者提取物和 100% (102/102) 的对照组出现可扩增的 β-珠蛋白（熔解温度 [Tm]，87.2°C ± 0.2°C）。对于通用 16S rRNA 基因靶标，PCR/焦磷酸测序与培养之间的一致性为 96.9% (1,085/1,120)，阳性率分别为 9.4% (105/1,120) 和 11.3% (126/1,120)。培养的细菌包括葡萄球菌（59/126，46.8%）、革兰氏阴性杆菌（34/126，27%）、链球菌（32/126，25.4%）和革兰氏阳性杆菌（1/126，0.8%）。所有对照均通过 PCR/焦磷酸测序筛选为阴性。通用 16S rRNA 基因 PCR/焦磷酸测序的临床表现特征（95% 置信区间 [CI]）包括敏感性 77.8%（69.5 至 84.7）、特异性 99.3%（98.6 至 99.7）、阳性预测值 (PPV) 93.3%（86.8 至 97.3）和阴性预测值 (NPV) 97.2% （96.0 至 98.2）。使用通用 16S PCR/焦磷酸测序技术可准确鉴定出 77.8% (98/126) 培养确认的脓毒症样本中的细菌，而使用后续目标特异性 PCR/焦磷酸测序技术可准确识别出 76.4% (96/126) 的细菌。初始 PCR/焦磷酸测序需要 ∼5.5 小时才能完成，如果包括目标特异性 PCR/焦磷酸测序则需要 ∼7.5 小时，而革兰氏染色需要 27.9 ± 13.6 小时，表型鉴定需要 81.6 ± 24.0 小时。总之，这种分子方法在超过四分之三的所有培养确诊的菌血症病例中直接从血液中检测出致病细菌，所需时间明显短于标准培养，但不能用于排除感染。
Here we compared the results of PCR/pyrosequencing to those of culture for detecting bacteria directly from blood. DNA was extracted from 1,130 blood samples from 913 patients suspected of bacteremia (enrollment criteria were physician-ordered blood culture and complete blood count [CBC]), and 102 controls (healthy blood donors). Real-time PCR assays for beta-globin and Universal 16S rRNA gene targets were performed on all 1,232 extracts. Specimens identified by Universal 16S rRNA gene PCR/pyrosequencing as containing staphylococci, streptococci, or enteric Gram-negative rods had target-specific PCR/pyrosequencing performed. Amplifiable beta-globin (melting temperature [Tm], 87.2°C ± 0.2°C) occurred in 99.1% (1,120/1,130) of patient extracts and 100% (102/102) of controls. Concordance between PCR/pyrosequencing and culture was 96.9% (1,085/1,120) for Universal 16S rRNA gene targets, with positivity rates of 9.4% (105/1,120) and 11.3% (126/1,120), respectively. Bacteria cultured included staphylococci (59/126, 46.8%), Gram-negative rods (34/126, 27%), streptococci (32/126, 25.4%), and a Gram-positive rod (1/126, 0.8%). All controls screened negative by PCR/pyrosequencing. Clinical performance characteristics (95% confidence interval [CI]) for Universal 16S rRNA gene PCR/pyrosequencing included sensitivity of 77.8% (69.5 to 84.7), specificity of 99.3% (98.6 to 99.7), positive predictive value (PPV) of 93.3% (86.8 to 97.3), and negative predictive value (NPV) of 97.2% (96.0 to 98.2). Bacteria were accurately identified in 77.8% (98/126) of culture-confirmed sepsis samples with Universal 16S PCR/pyrosequencing and in 76.4% (96/126) with follow-up target-specific PCR/pyrosequencing. The initial PCR/pyrosequencing took ∼5.5 h to complete or ∼7.5 h when including target-specific PCR/pyrosequencing compared to 27.9 ± 13.6 h for Gram stain or 81.6 ± 24.0 h for phenotypic identification. In summary, this molecular approach detected the causative bacteria in over three-quarters of all culture-confirmed cases of bacteremia directly from blood in significantly less time than standard culture but cannot be used to rule out infection.