Sun Zhaoyang, Liu Wenjian, Zhang Jinghao, Wang Su, Yang Feng, Fang Yi, Jiang Wenrong, Ding Li, Zhao Hu, Zhang Yanmei
Department of Laboratory Medicine, Huadong Hospital, Affiliated With Fudan University, Shanghai, China.
Key Laboratory of Clinical Geriatric Medicine, Shanghai, China.
Front Cell Infect Microbiol. 2021 Apr 12;11:660461. doi: 10.3389/fcimb.2021.660461. eCollection 2021.
Urinary tract infections (UTIs) are one the most common infections. The rapid and accurate identification of uropathogens, and the determination of antimicrobial susceptibility, are essential aspects of the management of UTIs. However, existing detection methods are associated with certain limitations. In this study, a new urinary tract infection high-throughput multiplex genetic detection system (UTI-HMGS) was developed for the semi-quantitative detection of 18 pathogens and the simultaneously screening of nine resistance genes directly from the clinical urine sample within 4 hours.
We designed and optimized a multiplex polymerase chain reaction (PCR) involving fluorescent dye-labeled specific primers to detect 18 pathogens and nine resistance genes. The specificity of the UTI-HMGS was tested using standard strains or plasmids for each gene target. The sensitivity of the UTI-HMGS assay was tested by the detection of serial tenfold dilutions of plasmids or simulated positive urine samples. We also collected clinical urine samples and used these to perform urine culture and antimicrobial susceptibility testing (AST). Finally, all urine samples were detected by UTI-HMGS and the results were compared with both urine culture and Sanger sequencing.
UTI-HMGS showed high levels of sensitivity and specificity for the detection of uropathogens when compared with culture and sequencing. In addition, ten species of bacteria and three species of fungi were detected semi-quantitatively to allow accurate discrimination of significant bacteriuria and candiduria. The sensitivity of the UTI-HMGS for the all the target genes could reach 50 copies per reaction. In total, 531 urine samples were collected and analyzed by UTI-HMGS, which exhibited high levels of sensitivity and specificity for the detection of uropathogens and resistance genes when compared with Sanger sequencing. The results from UTI-HMGS showed that the detection rates of 15 pathogens were significantly higher (P<0.05) than that of the culture method. In addition, there were 41(7.72%, 41/531) urine samples were positive for difficult-to-culture pathogens, which were missed detected by routine culture method.
UTI-HMGS proved to be an efficient method for the direct semi-quantitative detection of 18 uropathogens and the simultaneously screening of nine antibiotic resistance genes in urine samples. The UTI-HMGS could represent an alternative method for the clinical detection and monitoring of antibiotic resistance.
尿路感染（UTIs）是最常见的感染之一。快速准确地鉴定尿路病原体并确定抗菌药物敏感性，是尿路感染管理的重要方面。然而，现有的检测方法存在一定局限性。在本研究中，开发了一种新型尿路感染高通量多重基因检测系统（UTI-HMGS），用于在4小时内直接从临床尿液样本中对18种病原体进行半定量检测，并同时筛查9种耐药基因。
我们设计并优化了一种多重聚合酶链反应（PCR），该反应涉及荧光染料标记的特异性引物，用于检测18种病原体和9种耐药基因。使用针对每个基因靶点的标准菌株或质粒对UTI-HMGS的特异性进行测试。通过检测质粒或模拟阳性尿液样本的系列十倍稀释液来测试UTI-HMGS检测的灵敏度。我们还收集了临床尿液样本，并用于进行尿液培养和抗菌药物敏感性测试（AST）。最后，所有尿液样本均通过UTI-HMGS进行检测，并将结果与尿液培养和桑格测序结果进行比较。
与培养和测序相比，UTI-HMGS在检测尿路病原体方面显示出高灵敏度和特异性。此外，对10种细菌和3种真菌进行了半定量检测，以便准确区分显著菌尿和念珠菌尿。UTI-HMGS对所有靶基因的灵敏度可达每个反应50个拷贝。总共收集了531份尿液样本并通过UTI-HMGS进行分析，与桑格测序相比，该系统在检测尿路病原体和耐药基因方面显示出高灵敏度和特异性。UTI-HMGS的结果表明，15种病原体的检测率显著高于培养方法（P<0.05）。此外，有41份（7.72%，41/531）尿液样本中存在难培养病原体阳性，这些样本被常规培养方法漏检。
UTI-HMGS被证明是一种直接对尿液样本中18种尿路病原体进行半定量检测并同时筛查9种抗生素耐药基因的有效方法。UTI-HMGS可作为临床检测和监测抗生素耐药性的替代方法。
