RT-PCR快速检测产KPC型碳青霉烯酶肺炎克雷伯菌临床应用评价

Clinical application of RT-PCR for Klebsiella pneumoniae producing KPC carbapenemase

目的评估荧光定量聚合酶链反应(RT-PCR)和聚合酶链反应(PCR)扩增测序法检测产KPC型碳青霉烯酶肺炎克雷伯菌的一致性,分析RT-PCR直接检测临床样本中肺炎克雷伯菌和blaKPC基因的性能。方法收集碳青霉烯类耐药肺炎克雷伯菌(CRKP)非重复临床分离株89株,采用RT-PCR检测和常规blaKPC基因PCR扩增一代测序检测,比较2种方法的检出率和一致性。收集临床送检的痰液样本226份,采用RT-PCR检测肺炎克雷伯菌外膜磷脂蛋白blaphoE基因和blaKPC基因,同时进行传统细菌培养和体外药物敏感性试验,比较二者的符合率。结果RT-PCR和PCR扩增测序均从89株CRKP中检出87株携带blaKPC基因,2株未检出blaKPC基因,2种分子检测方法符合率为100%,blaKPC检出率为97.8%,blaphoE检出率为100%。226份痰液样本中,以培养法为标准,RT-PCR检测肺炎克雷伯菌blaphoE的敏感性为100%,特异性为99.38%,2种方法符合率为99.56%。以药物敏感性试验结果为标准,RT-PCR直接检测痰样本blaKPC基因的符合率为98.67%,敏感性为95.56%,特异性为99.48%。结论RT-PCR可快速筛查产KPC型碳青霉烯酶肺炎克雷伯菌,可快速为重症感染患者提供抗感染治疗的依据。

Objective To evaluate the consistency of fluorescent quantitative real-time polymerase chain reaction(RT-PCR)and polymerase chain reaction(PCR)amplification for carbapenem-resistant Klebsiella pneumoniae(CRKP)producing KPC carbapenemase,and to evaluate the performance of RT-PCR for the direct determination of Klebsiella pneumoniae and blaKPC gene in clinical specimens.Methods Totally,89 clinical isolates of CRKP were collected.The determination rates and consistency of the 2 methods were compared.A total of 226 sputum specimens were collected.The blaKPC gene and blaphoE gene of outer membrane phospholipid protein of Klebsiella pneumoniae in clinical specimens were determined by RT-PCR.Traditional culture and drug susceptibility test were carried out to evaluate consistency.Results Among the 89 CRKP isolates,the determination rate of blaphoE gene was 100%,and 2 isolates did not determine blaKPC gene by RT-PCR and PCR amplification.The determination rate of blaKPC was 97.8%,and the consistency of the 2 methods was 100%.In the 226 sputum specimens,for the determination of blaphoE gene of Klebsiella pneumoniae by RT-PCR,using the results of culture as standard,the sensitivity,specificity and consistency of the 2 methods were 100%,99.38%and 99.56%,respectively.Using the results of drug susceptibility test as standard,for the determination of blaKPC gene of Klebsiella pneumoniae by RTPCR,the sensitivity,specificity and consistency of the 2 methods were 95.56%,99.48%and 98.67%,respectively.Conclusions Klebsiella pneumoniae producing KPC carbapenemase can be rapidly screened by RT-PCR,which can provide a reference for rapid anti-infection treatment in patients with severe infection.