一种用于鉴定支气管肺泡灌洗液样本中病原体的快速实时定量聚合酶链反应检测方法。

A rapid, real-time quantitative polymerase chain reaction test for the identification of pathogens in bronchoalveolar lavage samples.

机构信息

From the Trauma Research Department (A.O., G.T., C.W.M., D.B.-O.), and Trauma Services Department (C.W.M.), St. Anthony Hospital, Lakewood; Trauma Research Department (A.O., G.T., D.B.-O.), and Trauma Services Department (D.S.S.), Swedish Medical Center, Englewood; and College of Osteopathic Medicine (C.W.M., D.B.-O.), Rocky Vista University, Parker, Colorado.

出版信息

J Trauma Acute Care Surg. 2014 Mar;76(3):651-9; discussion 659-60. doi: 10.1097/TA.0000000000000157.

DOI:10.1097/TA.0000000000000157

PMID:24553531

Abstract

BACKGROUND

Standard bacteriologic culture techniques offer results within 2 days to 3 days, precluding a focused and timely antibiotic therapy in ventilated trauma patients. Our laboratory developed a real-time quantitative polymerase chain reaction (qPCR) test that can detect 25 different bacteria and fungi and methicillin resistance and offers results within 3 hours. The objective of this study was to compare the qPCR method to standard culture techniques.

METHODS

This was a prospective observational cohort study at a Level I trauma center from 2009 to 2012. Adult trauma patients on ventilation, receiving at least one bronchoalveolar lavage (BAL) with culture results were eligible for inclusion. DNA was isolated from the BAL samples and analyzed in 96-well plates using qPCR. Student's t tests were used to examine differences in mean qPCR cycle counts. Sensitivities, specificities, negative predictive values, and positive predictive values were calculated for the qPCR primer sets.

RESULTS

There were 28 BALs in the study. The qPCR method detected a total of 165 organisms, and culture methods found 54. The qPCR test had an overall sensitivity of 85%, specificity of 74%, negative predictive value of 98%, and positive predictive value of 27%. Those organisms that were only identified through qPCR had significantly less DNA than those identified through both qPCR and quantitative culture (28.8 vs. 23.3, p < 0.001). Concurrent antibiotic therapy was found to decrease the qPCR specificity in some primer sets, and methicillin resistance was only found in BAL samples that were concurrent with antibiotics.

CONCLUSION

The qPCR method shows promising initial diagnostic value. Many of the organisms not identified by quantitative culture had late cycle calls, suggesting that they might have been in quantities too low to result in culture identification. Once refined, our qPCR method has the potential to identify pathogens faster and earlier than standard quantitative culture methods, allowing for targeted antibiotic therapy within 3 hours.

LEVEL OF EVIDENCE

Diagnostic test, level II.

摘要

背景

标准细菌培养技术需要 2 到 3 天才能出结果，这使得接受机械通气的创伤患者无法及时得到针对性的抗生素治疗。我们的实验室开发了一种实时定量聚合酶链反应（qPCR）检测方法，可检测 25 种不同的细菌、真菌、耐甲氧西林金黄色葡萄球菌，并在 3 小时内得出结果。本研究旨在比较 qPCR 方法与标准培养技术。

方法

这是 2009 年至 2012 年在一级创伤中心进行的前瞻性观察性队列研究。纳入标准为接受机械通气且至少进行了一次支气管肺泡灌洗（BAL）并有培养结果的成年创伤患者。从 BAL 样本中提取 DNA，在 96 孔板中使用 qPCR 进行分析。采用 Student t 检验比较 qPCR 循环计数的平均值差异。计算 qPCR 引物的灵敏度、特异性、阴性预测值和阳性预测值。

结果

研究中共进行了 28 次 BAL。qPCR 方法共检测到 165 种微生物，而培养方法仅发现 54 种。qPCR 检测的总灵敏度为 85%，特异性为 74%，阴性预测值为 98%，阳性预测值为 27%。仅通过 qPCR 检测到的微生物的 DNA 含量明显低于同时通过 qPCR 和定量培养检测到的微生物（28.8 与 23.3，p<0.001）。同时使用抗生素治疗会降低某些引物组的 qPCR 特异性，而耐甲氧西林金黄色葡萄球菌仅在与抗生素同时进行的 BAL 样本中被发现。