分离胶法与十二烷基硫酸钠法在血培养阳性瓶基质辅助激光解析电离飞行时间质谱病原菌直接鉴定中的应用

Application of direct determination of pathogens in positive blood culture bottles with matrix-assisted laser desorption ionization-time of flight mass spectrometry by Separation Gel Coagulation tube method and Sodium Dodecyl Sulfonate method

目的评估分离胶促凝管法和十二烷基硫酸钠(SDS)法两种阳性血培养瓶前处理方法联合基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)快速鉴定病原菌的临床应用。方法收集2016年11月至2017年5月首都医科大学宣武医院微生物室阳性血培养需氧瓶120例和厌氧瓶80例(经革兰染色镜检确认为单一菌),利用分离胶促凝管和0.5%SDS前处理后与转种纯培养菌落MALDITOFMS鉴定比较,评价两种方法的鉴定准确率。结果分离胶法和SDS法质谱鉴定与培养鉴定的符合率为86.0%和87.0%;分离胶法鉴定需氧瓶和厌氧瓶中病原菌的准确率为86.7%和85.0%,SDS法为85.0%和90.0%,两者鉴定需氧瓶中病原菌的鉴定率差异无统计学意义,SDS法鉴定厌氧瓶中细菌的准确率显著高于分离胶法(χ~2=11.13、P<0.05);分离胶法和SDS法对革兰阴性杆菌的鉴定准确率(94.3%和94.3%)显著高于革兰阳性球菌(80.6%和83.3%)和真菌(50.0%和63.6%),差异均有统计学意义(χ~2=24.7、40.3、15.1,P均<0.01);分离胶法和SDS法鉴定革兰阴性菌和阳性菌的准确率差异无统计学意义,而SDS法鉴定真菌和厌氧菌的准确率显著高于分离胶法(χ~2=11.05、P <0.01,χ~2=14.05、P <0.05);分离胶法和SDS法鉴定分值> 2.0分者分别占37.5%和45.0%(χ~2=20.48、P <0.05),1.6~2.0分者分别占33.0%和25.0%(χ~2=11.14、P <0.05),差异均具有统计学意义;而<1.6分者分别占15.5%和17.0%,差异无统计学意义(χ~2=5.9、P> 0.05)。结论分离胶法和SDS法均可快速直接鉴定阳性血培养瓶中常见病原菌,显著缩短鉴定时间,在真菌和厌氧菌鉴定时以SDS法前处理MS鉴定效果较好。

Objective To evaluate the clinical application of fast identification of pathogens by two pretreatments of separation gel coagulation tube and sodium dodecyl sulfonate（SDS） on positive blood culture bottles with matrix-assisted laser desorption ionization-time of flight mass spectrometry（MALDITOF MS）. Methods Positive blood samples in 120 aerobic bottles and 80 anaerobic bottles（confirmed single bacteria by Gram＇s staining technique） were collected from November 2016 to May 2017 in microbiology laboratory of Xuanwu Hospital of Capital Medical University. The identification accuracy of the two methods was evaluated by comparing the pre-treatment of the separated gelatinizing tube and 0.5% SDS with the identification of MALDI-TOF MS in pure culture colonies. Results The coincidence rates of identification with MALDI-TOF MS by the pretreatment of the separation gel coagulation tube method and the SDS method were 86.0% and 87.0%, respectively. In the Separation Gel Coagulation tube method, the coincidences by aerobic bottle and anaerobic bottle were 86.7% and 85.0%, respectively, which were 85.0% and 90.0% for SDS method. No significant difference was identified in terms of coincidence with aerobic blood bottles for the two methods. However, the accuracy by SDS method was clearly superior to that of Separation Gel Coagulation tube method with anaerobic bottles（χ^2 = 11.14, P 〈0.05）. The accuracy rates for Gram-negative bacteria（94.3%, 94.3%） were significantly higher than those of Gram-positive bacteria（80.6%, 83.3%） and fungi（50.0%, 63.6%）, with significant differences（χ^2 = 24.7, 40.3, 15.1; all P〈 0.01）. The two methods had no significant difference in accuracy of determining Gram-negative and Gram-positive bacteria. However, the identification accuracy of fungi and anaerobic bacteria by SDS method was significantly higher than that of the separation gel coagulation tube method（χ^2 = 11.05, P 0.01; χ^2 = 14.05, P 〈0.05）. The scores 2.0 identified by pretreatment of separation gel and SDS method were 37.5% and 45.0%（χ^2 = 20.48, P 〈0.05）, which were 33.0% and 25.0% for scores 1.6-2.0（χ^2 = 11.14, P 〈0.05）, and 15.5% and 17.0% for score 1.6（χ^2 = 5.9, P 〉0.05）. Conclusions The common pathogens in positive blood culture bottles can be quickly and directly identified by pretreatment of separation gel and SDS method, which significantly reduce the identification period. SDS method is better for identification of fungi and anaerobes.