淋病奈瑟菌药敏试验刃天青微量稀释法的建立与评价

李梦欢; 杨桂琴; 王有为; 雍刚; 王红仁; 别明江; 王国庆

doi:10.12182/20240160209

淋病奈瑟菌药敏试验刃天青微量稀释法的建立与评价

Establishment and Evaluation of a Resazurin-Based Microdilution Assay for Microbial Sensitivity Test of Neisseria gonorrhoeae

摘要

摘要:
目的建立一种基于刃天青显色的淋病奈瑟菌药敏试验方法并对其进行评价。
方法在肉汤微量稀释法的基础上，添加刃天青作为活细菌指示剂，采用参考株WHO G 进行试验以优化加入刃天青后细菌的培养时间，肉眼观察颜色变化以读取结果。用琼脂稀释法（金标准）和刃天青微量稀释法对3株淋球菌参考株和32株分离株分别进行阿奇霉素、头孢曲松和大观霉素最低抑菌浓度（minimum inhibitory concentration, MIC）的测定，从基本一致性（essential agreement, EA）（反映对MIC值的一致性）、分类一致性（category agreement, CA）（反映对耐药、中介和敏感判定的一致性）、极重大误差（very major error, VME）（反映假敏感性）和重大误差（major error, ME）（反映假耐药性）等指标进行分析，评价刃天青微量稀释法用于淋球菌药敏试验的准确性。CA率和EA率≥90%，VME率和ME率≤3%被认为是可接受的标准。
结果加入刃天青后反应6 h读取的结果与琼脂稀释法一致，以此参数建立刃天青微量稀释法。刃天青微量稀释法测量阿奇霉素、头孢曲松和大观霉素MIC结果的EA率分别为97.1%、91.5%和94.3%，CA率分别为88.6%、94.3%和94.3%，VME率均为0%，ME率分别为11.4%、5.7%和5.7%。
结论本研究建立的刃天青微量稀释法用于测量抗生素对淋病奈瑟菌MIC时，与琼脂稀释法具有良好的一致性，敏感结果可信度极高，仅通过肉眼观察即可方便读取结果。但应慎重对待耐药结果，需要继续进行参数的优化。

Abstract:
Objective To establish and evaluate a microbial sensitivity test method for Neisseria gonorrhoeae based on resazurin coloration.
Methods Based on the broth microdilution method, resazurin was added as a live bacteria indicator. WHO G, a WHO gonococcal reference strain, was used to optimize the incubation time for resazurin-stained bacteria and the color change was visually observed to obtain the results. Agar dilution method (the gold standard) and resazurin-based microdilution assay were used to determine the minimum inhibitory concentration (MIC) of azithromycin, ceftriaxone, and spectinomycin for 3 reference strains and 32 isolates of Neisseria gonorrhoeae. The results were analyzed based on essential agreement (EA), which reflected the consistency of the MIC values, category agreement (CA), which reflected the consistency in the determination of drug resistance, intermediary, and sensitivity, very major error (VME), which reflected false sensitivity, and major error (ME), which reflected pseudo drug resistance, to evaluate the accuracy of resazurin-based microdilution assay as a microbial sensitivity test of of Neisseria gonorrhoeae. CA and EA rates≥90% and VME and ME rates≤3% were found to be the acceptable performance rates.
Results The results obtained 6 hours after resazurin was added were consistent with those of the agar dilution method and the resazurin-based microdilution assay was established accordingly based on this parameter. The EA of resazurin-based microdilution assay for measuring the MIC results of azithromycin, ceftriaxone, and spectinomycin was 97.1%, 91.5%, and 94.3%, respectively, and the CA was 88.6%, 94.3%, and 94.3%, respectively. The VME was 0% for all three antibiotics, while the ME was 11.4%, 5.7%, and 5.7%, respectively.
Conclusion The resazurin-based microdilution assay established in this study showed good agreement with agar dilution method for measuring the MIC of antibiotics against Neisseria gonorrhoeae. Moreover, the sensitivity results of this method were highly reliable and could be easily obtained through naked eye observation. Nonetheless, the results of drug resistance should be treated with caution and the optimization of parameters should be continued.

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