Department of Emergency Medicine, Johns Hopkins University, Baltimore, MD, USA.
Acad Emerg Med. 2010 Jul;17(7):741-7. doi: 10.1111/j.1553-2712.2010.00790.x.
Conventional laboratory diagnosis of bacterial meningitis based on microscopy followed by culture is time-consuming and has only moderate sensitivity.
The objective was to define the limit of detection (LOD), analytic specificity, and performance characteristics of a broad-based quantitative multiprobe polymerase chain reaction (PCR) assay for rapid bacterial detection and simultaneous pathogen-specific identification in patients with suspected meningitis.
A PCR algorithm consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by pathogen identification using either pathogen-specific probes or Gram-typing probes, was employed to detect pathogens. The 16S rRNA gene, which contains both conserved and variable regions, was chosen as the target. Pathogen-specific probes were designed for Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes. Gram-positive and -negative typing probes were designed based on conserved regions across all eubacteria. The LOD and time to detection were assessed by dilutional mocked-up samples. A total of 108 convenience cerebrospinal fluid (CSF) clinical samples obtained from the Johns Hopkins Hospital (JHH) microbiology laboratory were tested, and results were compared with hospital microbiologic culture reports.
The LOD of the assay ranged from 10(1) to 10(2) colony-forming units (CFU)/mL. Pathogen-specific probes showed no cross-reactivity with other organisms. Time to detection was 3 hours. In clinical specimens, the universal probe correctly detected 16 of 22 culture-positive clinical specimens (sensitivity = 72.7%; 95% confidence interval [CI] = 49.8% to 89.3%), which were all correctly characterized by either pathogen-specific or Gram-typing probes. Adjusted sensitivity after removing probable microbiologic laboratory contaminants was 88.9% (95% CI = 65.3% to 98.6%). The universal probe was negative for 86 of 86 culture-negative specimens.
A broad-based multiprobe PCR assay demonstrated strong analytic performance characteristics. Findings from a pilot clinical study showed promise in translation to human subjects, supporting potential utility of the assay as an adjunct to traditional diagnostics for early identification of bacterial meningitis.
基于显微镜检查和培养的传统实验室诊断细菌性脑膜炎耗时且灵敏度仅为中等。
本研究旨在定义一种广谱定量多重探针聚合酶链反应（PCR）检测方法的检测限（LOD）、分析特异性和性能特征，以快速检测疑似脑膜炎患者的细菌，并进行病原体特异性鉴定。
采用广谱检测的方法，即通过通用探针检测真细菌纲，然后根据病原体特异性探针或革兰氏染色探针进行病原体鉴定。选择 16S rRNA 基因作为靶标，该基因包含保守区和可变区。针对肺炎链球菌、脑膜炎奈瑟菌、流感嗜血杆菌、表皮葡萄球菌、金黄色葡萄球菌、大肠杆菌和单核细胞增生李斯特菌设计了病原体特异性探针，基于所有真细菌的保守区设计了革兰氏阳性和阴性分型探针。通过模拟稀释样本评估 LOD 和检测时间。共检测了来自约翰霍普金斯医院（JHH）微生物实验室的 108 份方便的脑脊液（CSF）临床样本，并将结果与医院微生物培养报告进行比较。
该检测方法的 LOD 范围为 10(1) 至 10(2) 菌落形成单位（CFU）/mL。病原体特异性探针与其他生物体无交叉反应。检测时间为 3 小时。在临床标本中，通用探针正确检测了 22 份培养阳性临床标本中的 16 份（敏感性=72.7%；95%置信区间[CI]为 49.8%至 89.3%），这些标本均通过病原体特异性或革兰氏染色探针正确鉴定。去除可能的微生物学实验室污染后，调整后的敏感性为 88.9%（95%CI 为 65.3%至 98.6%）。通用探针对 86 份培养阴性标本均为阴性。
广谱多重探针 PCR 检测方法具有很强的分析性能特征。一项初步临床研究的结果显示出转化为人体研究的前景，支持该检测方法作为传统诊断方法的辅助手段，用于早期识别细菌性脑膜炎的潜在应用价值。
