评估VITEK 2-Compact GN13测定肺炎克雷伯菌体外亚胺培南药敏及高级专家系统修正的可靠性

摘要

目的评估VITEK 2-Compact GN13测定肺炎克雷伯菌体外亚胺培南药敏及高级专家系统(AES)修正的可靠性.方法回顾性研究2014-2015年南昌大学第一附属医院分离的157株肺炎克雷伯菌,分别采用纸片扩散法、VITEK 2-Compact GN13法及以微量肉汤稀释法作为参考方法测定该菌对亚胺培南的敏感性.分别评估纸片扩散法、VITEK 2-Compact GN13与参考方法的分类一致率(CA%).对经AES修正药敏的肺炎克雷伯菌采用改良Hodge试验等表型确证试验及PCR、测序法等分子生物学方法,对AES中提示的耐药机制进行验证,评估AES修正GN13测定肠杆菌科细菌体外亚胺培南药敏试验结果的可靠性.结果157株菌中64株经微量肉汤稀释法检测为耐药株,8株为中介;纸片扩散法检测52株为耐药株,10株为中介;VITEK 2-Compact GN13检测54株为耐药株,13株为中介,而经AES修正后70株为耐药株,3株为中介.纸片扩散法和VITEK 2-Compact GN13与参考方法的一致率均>90%;但在使用VITEK 2-Compact GN13与纸片扩散法测定亚胺培南时会出现一定比例的大错误(ME,3.8%)和极大错误(VME,0.6%).AES对16株菌株进行了亚胺培南药敏修正,虽然消除了0.6%的VME,但却增加1.3%的ME和1.9%的小错误(MIE)出现.表型验证显示75.0%(12/16)表现为产ESBL联合产碳青霉烯酶,与AES推测相符,PCR及测序显示62.5%(10/16)为IMP-4/KPC-2/NDM-1联合产ESBL.结论无论采用VITEK 2-Compact GN13还是纸片扩散法,对肺炎克雷伯菌亚胺培南体外药敏的测定均较可靠,但都应注意可能出现ME及VME,使用AES修正虽可避免VME的出现,但会增加ME和MIE的出现,这或与碳青霉烯酶表达降低有关.%Objective To evaluate the performance of VITEK 2-Compact GN13 methods for testing imipenem susceptibility of Klebsiella pneumoniae and assess the reliability of its Advanced Expert System (AES).Methods A retrospective study was conducted with a total of 157K. pneumoniae strains, which were isolated from blood and intra-abdominal infections in the First Affiliated Hospital of Nanchang University from 2014 to 2015. Thein vitro minimum inhibitory concentration (MIC) values of imipenem were determined by disc diffusion, VITEK 2-Compact GN13 and broth microdilution methods, respectively. Categorical agreement (CA) rates of disc diffusion and VITEK 2-Compact GN13 methods were determined using broth microdilution as reference method. The genes encoding ESBLs and carbapenemase were screened by PCR and sequencing analysis. The phenotypic confirmatory tests such as modified Hodge test, PCR and DNA sequencing were used to confirm the resistance mechanism and evaluate the reliability of AES in interpreting the imipenem susceptibility of K. pneumoniae.Results Among the 157 isolates, 64 and 8 were identified as resistant and intermediate strains by broth microdilution method, respectively; 52 and 10 were tested as resistant and intermediate strains by disc diffusion method, respectively; 54 and 13 were determined as resistant and intermediate strains by VITEK 2-Compact GN13 method, respectively, while 70 and 3 were judged as resistant and intermediate strains by VITEK 2-Compact GN13 method plus AES validation. The CA of disc diffusion and VITEK 2-Compact GN13 methods compared with broth microdilution method were all higher than 90 %. However, the major error (ME) rate was 3.8 % and very major error (VME) rates were all 0.6 % in imipenem susceptibility testing by VITEK 2-Compact GN13 and disc diffusion. The imipenem susceptibility of 16 strains were modified by the AES, which eliminated 0.6 % VME, but increased major error by 1.3 % and minor error by 1.9 %. Phenotypic confirmatory tests showed that 75 % (12/16) of these strains were validated as producers of both ESBLs and carbapenemase, which was consistent with the result of AES validation. PCR and DNA sequencing analysis proved that 62.5 % (10/16) of these strains produce IMP-4/KPC-2 /NDM-1 and ESBLs.Conclusions Both disc diffusion and VITEK 2-Compact GN13 methods can be used for testing the imipenem susceptibility of K. pneumoniae isolates with reliable and accurate results. Attention should be paid to the possibility of ME and VME when testing imipenem susceptibility. The VME can be avoided by the AES mechanism. However, AES intervention will increase ME and minor error, which may be associated with decreased expression of carbapenemase.