Badran Samir, Chen Ming, Coia John E
Research Unit in Clinical Microbiology, Department of Clinical Diagnostics, Hospital South West Jutland, University Hospital of Southern Denmark, Esbjerg, Denmark.
Department of Regional Health Research, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark.
JMIR Res Protoc. 2021 Dec 13;10(12):e33746. doi: 10.2196/33746.
Blood cultures are the cornerstone of diagnosis for detecting the presence of bacteria or fungi in the blood, with an average detection time of 48 hours and failure to detect a pathogen occurring in approximately 50% of patients with sepsis. Rapid diagnosis would facilitate earlier treatment and/or an earlier switch to narrow-spectrum antibiotics.
The aim of this study is to develop and implement a multiplex droplet digital polymerase chain reaction (ddPCR) assay as a routine diagnostic tool in the detection and identification of pathogens from whole blood and/or blood culture after 3 hours of incubation.
The study consists of three phases: (1) design of primer-probe pairs for accurate and reliable quantification of the most common sepsis-causing microorganisms using a multiplex reaction, (2) determination of the analytical sensitivity and specificity of the multiplex ddPCR assay, and (3) a clinical study in patients with sepsis using the assay. The QX200 Droplet Digital PCR System will be used for the detection of the following species-specific genes in blood from patients with sepsis: coa (staphylocoagulase) in Staphylococcus aureus, cpsA (capsular polysaccharide) in Streptococcus pneumoniae, uidA (beta-D-glucuronidase) in Escherichia coli, oprL (peptidoglycan-associated lipoprotein) in Pseudomonas aeruginosa, and the highly conserved regions of the 16S rRNA gene for Gram-positive and Gram-negative bacteria. All data will be analyzed using QuantaSoft Analysis Pro Software.
In phase 1, to determine the optimal annealing temperature for the designed primer-probe pairs, results from a gradient temperature experiment will be collected and the limit of detection (LOD) of the assay will be determined. In phase 2, results for the analytical sensitivity and specificity of the assay will be obtained after an optimization of the extraction and purification method in spiked blood. In phase 3, clinical sensitivity and specificity as compared to the standard blood culture technique will be determined using 301 clinical samples.
Successful design of primer-probe pairs in the first phase and subsequent optimization and determination of the LOD will allow progression to phase 3 to compare the novel method with existing blood culture methods.
INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/33746.
血培养是诊断血液中细菌或真菌存在的基石，平均检测时间为48小时，约50%的脓毒症患者无法检测到病原体。快速诊断将有助于更早开始治疗和/或更早改用窄谱抗生素。
本研究的目的是开发并实施一种多重液滴数字聚合酶链反应（ddPCR）检测方法，作为一种常规诊断工具，用于在孵育3小时后从全血和/或血培养物中检测和鉴定病原体。
该研究包括三个阶段：（1）设计引物-探针组，通过多重反应准确可靠地定量最常见的引起脓毒症的微生物；（2）确定多重ddPCR检测方法的分析灵敏度和特异性；（3）使用该检测方法对脓毒症患者进行临床研究。QX200液滴数字PCR系统将用于检测脓毒症患者血液中以下物种特异性基因：金黄色葡萄球菌中的coa（葡萄球菌凝固酶）、肺炎链球菌中的cpsA（荚膜多糖）、大肠杆菌中的uidA（β-D-葡萄糖醛酸酶）、铜绿假单胞菌中的oprL（肽聚糖相关脂蛋白），以及革兰氏阳性和革兰氏阴性细菌16S rRNA基因的高度保守区域。所有数据将使用QuantaSoft Analysis Pro软件进行分析。
在第一阶段，为确定设计的引物-探针组的最佳退火温度，将收集梯度温度实验的结果并确定检测限（LOD）。在第二阶段，在优化加标血液中的提取和纯化方法后，将获得该检测方法的分析灵敏度和特异性结果。在第三阶段，将使用301份临床样本确定与标准血培养技术相比的临床灵敏度和特异性。
在第一阶段成功设计引物-探针组，随后进行优化并确定检测限，将使研究进展到第三阶段，以将新方法与现有的血培养方法进行比较。
国际注册报告识别码（IRRID）：PRR1-10.2196/33746。
