PDF(Pubmed)
Abstract：
Traditional drug susceptibility testing cannot be performed in clinical laboratories due to the slow-growing characteristics of Mycoplasma genitalium when cultured in vitro. Sanger sequencing is the standard method for detecting drug resistance-associated mutations. It has been used in some laboratories to guide the choice of macrolide antibiotics for Mycoplasma genitalium infected patients. Furthermore, resistance to fluoroquinolone has become another emerging clinical challenge.
Sequencing analysis can detect unknown mutations, but it is time-consuming, requires professional analytical skills and the appropriate testing equipment. The main objective of this study was to establish a nested real-time PCR method for the simultaneous detection of 23S rRNA and parC genotypes in relation to the macrolide and fluoroquinolone resistance.
105 MG-positive samples and 27 samples containing other pathogens were used for validation. The limit of the nested real-time PCR detection was 500 copies/reaction and there was no cross-reaction with Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis, Neisseria gonorrhoeae, Human papillomavirus, Herpes simplex virus, Candida albicans and Ureaplasma parvum, but the 23S rRNA assay cross-reacted with Mycoplasma pneumoniae. Compared with sequencing results, the sensitivity of 23S rRNA was 100% (95% CI; 93.3 -100), the specificity was 94.3% (95% CI; 79.4 - 99.0), the overall consistency was 98% (95% CI; 92.5 - 99.7) and kappa value was 0.96 (P < 0.001); the sensitivity of parC was 100% (95% CI; 93.4 - 100), the specificity was 89.7% (95% CI; 71.5 - 97.3) and the overall consistency was 96.9% (95% CI; 90.7 - 99.2) with a kappa value of 0.92 (P < 0.001).
The results of this sensitive and rapid alternative for identifying resistant genotypes of Mycoplasma genitalium are intuitive and easy to interpret, especially for mixed MG populations. Although the relevant 23S rRNA primers need further adjustment, this reliable method would provide an effective diagnostic tool for the selection of antibiotics in clinical practice.
Sequencing analysis can detect unknown mutations, but it is time-consuming, requires professional analytical skills and the appropriate testing equipment. The main objective of this study was to establish a nested real-time PCR method for the simultaneous detection of 23S rRNA and parC genotypes in relation to the macrolide and fluoroquinolone resistance.
105 MG-positive samples and 27 samples containing other pathogens were used for validation. The limit of the nested real-time PCR detection was 500 copies/reaction and there was no cross-reaction with Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis, Neisseria gonorrhoeae, Human papillomavirus, Herpes simplex virus, Candida albicans and Ureaplasma parvum, but the 23S rRNA assay cross-reacted with Mycoplasma pneumoniae. Compared with sequencing results, the sensitivity of 23S rRNA was 100% (95% CI; 93.3 -100), the specificity was 94.3% (95% CI; 79.4 - 99.0), the overall consistency was 98% (95% CI; 92.5 - 99.7) and kappa value was 0.96 (P < 0.001); the sensitivity of parC was 100% (95% CI; 93.4 - 100), the specificity was 89.7% (95% CI; 71.5 - 97.3) and the overall consistency was 96.9% (95% CI; 90.7 - 99.2) with a kappa value of 0.92 (P < 0.001).
The results of this sensitive and rapid alternative for identifying resistant genotypes of Mycoplasma genitalium are intuitive and easy to interpret, especially for mixed MG populations. Although the relevant 23S rRNA primers need further adjustment, this reliable method would provide an effective diagnostic tool for the selection of antibiotics in clinical practice.
摘要：
■由于生殖支原体在体外培养时具有缓慢生长的特征，因此无法在临床实验室中进行传统的药物敏感性测试。Sanger测序是检测耐药性相关突变的标准方法。它已在一些实验室中用于指导生殖器支原体感染患者的大环内酯类抗生素的选择。此外，氟喹诺酮耐药已成为另一个新兴的临床挑战.
■测序分析可以检测未知突变，但这很耗时，需要专业的分析技能和适当的测试设备。本研究的主要目的是建立巢式实时PCR方法，用于同时检测与大环内酯和氟喹诺酮抗性有关的23SrRNA和parC基因型。
■105个MG阳性样本和27个含有其他病原体的样本用于验证。巢式实时PCR检测的极限为500拷贝/反应，并且与解脲支原体没有交叉反应，人型支原体,沙眼衣原体,淋病奈瑟菌,人乳头瘤病毒,单纯疱疹病毒,白色念珠菌和细小脲原体，但23SrRNA检测与肺炎支原体发生交叉反应.与测序结果相比，23SrRNA的敏感性为100%(95%CI;93.3-100)，特异性为94.3%(95%CI；79.4-99.0)，总体一致性为98％（95％CI;92.5-99.7），κ值为0.96（P<0.001）；parC的敏感性为100％（95％CI;93.4-100），特异性为89.7%(95%CI;71.5~97.3),总体一致性为96.9%(95%CI;90.7~99.2),kappa值为0.92(P<0.001).
■这种用于鉴定生殖支原体抗性基因型的敏感和快速替代方法的结果直观且易于解释，特别是对于混合MG人群。尽管相关的23SrRNA引物需要进一步调整，这种可靠的方法将为临床实践中抗生素的选择提供有效的诊断工具。
■测序分析可以检测未知突变，但这很耗时，需要专业的分析技能和适当的测试设备。本研究的主要目的是建立巢式实时PCR方法，用于同时检测与大环内酯和氟喹诺酮抗性有关的23SrRNA和parC基因型。
■105个MG阳性样本和27个含有其他病原体的样本用于验证。巢式实时PCR检测的极限为500拷贝/反应，并且与解脲支原体没有交叉反应，人型支原体,沙眼衣原体,淋病奈瑟菌,人乳头瘤病毒,单纯疱疹病毒,白色念珠菌和细小脲原体，但23SrRNA检测与肺炎支原体发生交叉反应.与测序结果相比，23SrRNA的敏感性为100%(95%CI;93.3-100)，特异性为94.3%(95%CI；79.4-99.0)，总体一致性为98％（95％CI;92.5-99.7），κ值为0.96（P<0.001）；parC的敏感性为100％（95％CI;93.4-100），特异性为89.7%(95%CI;71.5~97.3),总体一致性为96.9%(95%CI;90.7~99.2),kappa值为0.92(P<0.001).
■这种用于鉴定生殖支原体抗性基因型的敏感和快速替代方法的结果直观且易于解释，特别是对于混合MG人群。尽管相关的23SrRNA引物需要进一步调整，这种可靠的方法将为临床实践中抗生素的选择提供有效的诊断工具。
