MALDI-TOF MS直接鉴定血流感染病原菌的前处理方法比较与优化

Comparison and optimization of pretreatment methods for direct identification of pathogenic bacteria in bloodstream infections by MALDI-TOF MS

摘要

目的 对比分离胶分离法与裂解法处理后的阳性血培养标本质谱鉴定的准确率,寻找最优前处理方法,助力临床血流感染快速精准诊疗。方法 收集2024年5月-2025年1月沂南县人民医院检验科血培养阳性标本348份,分别采用分离胶分离法(直涂法、甲酸法)和裂解法(配套试剂法、红细胞裂解液法)对阳性血培养进行前处理后,直接用质谱检测。以培养菌落自动化鉴定药敏仪鉴定结果为标准评估直接质谱鉴定结果的准确性,无法鉴定或鉴定结果与镜下形态不符者进行16S rDNA基因测序为最终对照标准。并用来自临床标本和质控菌株的112例模拟念珠菌阳性样本验证优势方法的可靠性。结果 分离胶分离法中符合率高的甲酸法(89.37%)鉴定准确率低于裂解法中符合率低的红细胞裂解液法(95.69%),差异有统计学意义(χ²=10.059,P<0.05)。采用直接鉴定难度较高的模拟念珠菌样本对裂解法进行验证,结果显示配套试剂法和红细胞裂解液法符合率分别为88.39%和86.61%,差异无统计学意义(χ²=0.163,P>0.05)。结论 红细胞裂解液法是更具优势的前处理方法,准确率高,对革兰阴性菌、革兰阳性菌、念珠菌的鉴定准确率分别为96.84%、90.48%、86.61%,与配套试剂效果相当;与配套试剂(两千元/盒)相比,红细胞裂解液无需额外高价购买,操作简便,经济实用性强,可较好助力血流感染早期精准诊疗。

Abstract

Objective Comparing the accuracy of mass spectrometry identification for positive blood culture samples processed by separator gel method versus lysis method,to identify the optimal pretreatment approach,thereby facilitating rapid and precise clinical diagnosis and treatment of bloodstream infections. Methods From May 2024 to January 2025,348 positive blood culture specimens were collected from the Clinical Laboratory of Yinan County People′s Hospital.After pretreatment of the positive culture broths using the separation gel method(direct smear method and formic acid method)and the lysis method(commercial kit method and red blood cell lysis method),they were directly analyzed by mass spectrometry.The accuracy of the direct mass spectrometry identification results was evaluated against the automated colony identification and drug susceptibility instrument results as the standard.For samples that could not be identified or whose identification results were inconsistent with microscopic morphology,16S rDNA gene sequencing was performed as the final reference standard.Additionally,the reliability of the superior method was validated using 112 simulated Candida-positive samples derived from clinical specimens and quality control strains. Results In the separator gel method,the formic acid approach,which demonstrated a high concordance rate(89.37%),showed a lower identification accuracy compared to the red blood cell lysis method(95.69%)in the lysis method,despite the latter having a lower concordance rate.The difference was statistically significant(χ²=10.059,P<0.05).Validation was performed using simulated Candida samples with high difficulty in direct identification to assess the lysis method.The results showed that the concordance rates of the supporting reagent method and the red blood cell lysis method were 88.39% and 86.61%,respectively,with no statistically significant difference(χ²=0.163,P>0.05). Conclusion The red blood cell lysis method is a superior pretreatment method,offering high accuracy with identification accuracy rates of 96.84% for Gram-negative bacteria,90.48% for Gram-positive bacteria,and 86.61% for Candida,demonstrating comparable performance to the supporting reagent.Compared with the supporting reagent(which costs 2 000 yuan per kit),the red blood cell lysis method eliminates the need for additional high-cost purchases,is easy to operate,and is highly cost-effective,and can effectively facilitate early and accurate diagnosis and treatment of bloodstream infections.

关键词

基质辅助激光解吸电离飞行时间质谱 / 血流感染 / 直接鉴定 / 前处理方法

Key words

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry / Bloodstream infections / Direct identification / Pretreatment method

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基金资助

济宁医学院贺林院士新医学临床转化工作站科研基金(JYHL2019MS04)