应用通用引物PCR检测诊断细菌和真菌性中枢神经系统感染的价值

Value of universal primer PCR for diagnosing bacterial and fungal infection of central nervous system

摘要:

目的了解细菌和真菌性中枢神经系统（CNS）感染病原谱，并探讨通用引物聚合酶链反应（PCR)检测技术的病原学诊断价值。方法收集某院2009年1月—2015年3月疑似或确诊CNS细菌或真菌性感染患者资料，分析其脑脊液培养病原菌种类，采用细菌16S rRNA和真菌28S rRNA通用引物对患者脑脊液DNA进行PCR扩增和序列测定，并与同期脑脊液培养及鉴定结果进行比较。结果共收集确诊或疑似CNS细菌或真菌感染者400例，其中脑脊液培养阳性132例。共分离病原菌150株，其中革兰阳性菌48株，革兰阴性菌90株，真菌12株；居前3位的细菌为鲍曼不动杆菌（32株）、凝固酶阴性葡萄球菌（16株）和肺炎克雷伯菌（13株）；最常见真菌为新生隐球菌（8株）。选取88份感染患者脑脊液标本和20例非感染患者脑脊液进行PCR扩增，PCR扩增法灵敏度（35.23％，31/88）高于培养法（28.41%，25/88）（χ2＝4.17，P＜0.05）。PCR扩增和培养法阴性预测值分别为25.97%、24.10%，两种方法的特异度、阳性预测值均为100.00%。PCR扩增测序与培养结果符合率为84.00%（21/25）；PCR检测平均报告时间（48 h）较培养结果报告时间（细菌平均约72 h，真菌平均约96 h）更快速。结论CNS 感染病原分布广泛，以革兰阴性菌为主；通用引物PCR检测具有快速、灵敏、准确等特点，具有良好的临床推广和应用价值。

Abstract:

ObjectiveTo understand pathogen spectrum of bacterial and fungal infection of central nervous system (CNS), and evaluate the etiological diagnostic value of universal primer polymerase chain reaction (PCR).MethodsData about patients with suspected or confirmed bacterial and fungal infection of CNS from January 2009 to March 2015 were collected, species of pathogens from cerebrospinal fluid (CSF) were analyzed, DNA from patients’ CSF were performed PCR amplification and sequencing with universal primers of bacterial 16S rRNA and fungal 28S rRNA, PCR detection results were compared with CSF culture during the same period.ResultsA total of 400 patients were with confirmed or suspected bacterial or fungal infection of CNS, 132 of whom were with positive CSF culture.150 pathogenic isolates were detected, including 48 isolates of grampositive bacteria, 90 gramnegative bacteria, and 12 fungi; the top three isolated bacteria were Acinetobacter baumannii (n=32), coagulase negative staphylococcus (n=16) and Klebsiella pneumoniae (n=13); the most common fungus was Cryptococcus neoformans (n=8). CSF from 88 infected patients and 20 noninfected patients were selected for PCR amplification, the sensitive of PCR amplification assay was higher than the culture method (35.23% ［31/88］ vs 28.41%［25/88］, χ2＝4.17，P＜0.05）. Negative predictive value of PCR amplification assay and culture method were 25.97% and 24.10% respectively, the specificity and positive predictive value of two methods were both 100.00%. The coincidence rate of PCR amplification sequencing and culture result was 84.00%(21/25); the average reporting time of PCR (48 h) was more rapid than that of culture (72 h for bacteria and 96h for fungi).ConclusionPathogens of CNS infections are widely distributed and the main are gramnegative bacteria; universal primer PCR has the characteristics of rapid, high sensitivity, and accuracy, which has a good clinical popularization and application value.