MAST D73C组合纸片法和PCR基因检测儿童碳青霉烯类耐药肠杆菌科细菌菌株中碳青霉烯酶的价值

Value of MAST D73C combined paper disk method and PCR gene testing indetermining carbapenemase in CRE strains in children

目的探讨儿童碳青霉烯类耐药肠杆菌科细菌(CRE)菌株中碳青霉烯酶与其分型的检测方法和价值,本研究所用检测方法包括MAST D73C组合纸片法、聚合酶链式反应(PCR)基因检测法。方法MAST D73C组合纸片法与PCR基因检测结果显示,分离出CRE菌株碳青霉烯酶基因菌株数量为240株,所选菌株为2018年6月至2019年10月采集,针对MAST D73C在碳青霉烯酶中的检测灵敏度与特异度进行分析。结果PCR基因检测中,240株CRE菌株中检测到碳青霉烯酶基因菌株数量为233株,MAST D73C组合纸片MβL酶检出率达47.16%、OXA-48酶检出率达27.07%、KPC酶检出率达8.73%,无法解释其他17.03%细菌的抑菌圈结果。以PCR结果为金标准,以MAST D73C分别计算MβL酶及OXA-48酶的灵敏度和特异度,灵敏度>80%,特异度>95%。通过Youden指数与Kappa值可知,区分产MβL酶及OXA-48酶菌株均具有较好的一致性和真实性。KPC酶特异度达98.85%,灵敏度为32.14%,一致性及真实性均较差。通过MAST D73C组合纸片法总计检出CRE 39株,无法对所得结果进行准确解释,PCR检测结果确认CRE产KPC酶总计31株、产OXA-48酶CRE数量为8株。若B-A、C-A及D-A不足5 mm,E超过10 mm,对法罗培南耐药时可判定为产KPC酶,可使KPC酶检测灵敏度得到显著提高。结论儿童分离CRE菌株所检出的碳青霉烯酶类型包括NDM-1酶及OXA-232酶,MAST D73C组合纸片法可以简便、快速、准确地对碳青霉烯酶进行检测,还能够迅速分型,而且操作简单,值得应用。

Objective To determine carbapenemase in carbapenem-resistant Enterobacteriaceae(CRE)strains in children and its typing and clinical application value using MAST D73C combined paper disk method and gene testing with polymerase chain reaction(PCR)method.Methods The MAST D73C combined paper disk method and PCR gene testing were performed to determine the carbapenemase gene in 240 clinically isolated CRE strains that were collected from children from June 2018 to October 2019.The sensitivity and specificity of MAST D73C in detecting carbapenemase were analyzed by statistical methods.Results The results of PCR gene testing revealed that carbapenemase gene was detected in 233 out of 240 CRE strains.The detection rate of MβL enzyme,OXA-48 enzyme and KPC enzyme was 47.16%,27.07% and 8.73% respectively by MAST D73C combined paper disk method,and 17.03% bacteriostatic ring result could not be interpreted.The sensitivity was calculated>80%and specificity>95% for MβL enzyme and OXA-48 enzyme by MAST D73C using PCR results as the gold standard.Youden index and Kappa value showed good consistency and authenticity in distinguishing MβL enzyme and OXA-48 enzyme producing strains.The specificity of KPC enzyme was 98.85% and sensitivity was 32.14%,with poor consistency and authenticity.The detection results of 39 CRE strains by the MAST D73C combined paper disk method could not be interpreted,and PCR determined the number of KPC enzyme producing CRE as 31 strains and the number of OXA-48 enzyme producing CRE as 8 strains.If B-A,C-A and D-A would be less than 5 mm and E more than 10 mm,it could be judged as KPC enzyme-producing when it is resistant to faropenem,which could significantly improve the sensitivity of KPC enzyme detection.Conclusion NDM-1 enzyme and OXA-232 enzyme are mainly detected in CRE strains isolated from children,and MAST D73C combined paper disk method can detect carbapenemase easily,rapidly and accurately and type them,which is worthy of application.