肺炎支原体23S rRNA基因测序分析的临床应用价值
|
摘要
目的:探讨儿童呼吸道肺炎支原体(MP) 23S rRNA基因测序分析的临床应用价值。方法:收集MP荧光定量PCR≥1×10~4拷贝数的痰或咽拭子样本,行23S rRNA基因V区测序,并按测序结果将患儿分为突变组和未突变组,比较两组患儿的临床资料;将相同样本行体外培养加药敏实验,与测序进行比较。结果:共收集120例样本,98例23S rRNA基因发生2063位点A-> G突变(突变组),22例未检测到突变(未突变组)。两组患儿在性别、年龄、急性期外周血白细胞等一般资料无显著差异,但住院时间、总发热时间、大环内酯类药物治疗后退热时间、咳嗽时间及疗程方面存在统计学差异,且突变组患儿肺外并发症及大叶性肺炎实变率高于未突变组(P <0. 05)。120例样本中48例MP体外培养阳性(阳性率40%),其中20例检出大环内酯类耐药,耐药率为16. 67%,显著低于23S rRNA基因突变率(P <0. 05)。结论:MP 23S rRNA基因突变率较高。突变组临床症状重、疗程长、并发症多,提示基因突变与MP耐药存在相关性。相对于基因测序,体外培养加药敏实验阳性率及耐药率较低。MP 23S rRNA基因测序可作为耐药检测的手段,具有一定的临床价值。
Objective: To explore the clinical application value of sequencing analysis of 23 S rRNA gene of Mycoplasma Pneumoniae( MP) in children's respiratory tract. Methods: The samples of phlegm or pharynx swab with MP more than 1* 10~4 copies were collected by fluorescence quantitative PCR,and domain V of the 23 S rRNA gene was sequenced. Children were divided into mutation group and non-mutation group according to the sequencing results,and clinical data of the two groups were compared. In vitro culture and drug sensitivity experiments were performed in the same sample to compare the sequencing analysis. Results: A total of 120 samples were collected,98 cases were diagnosed with A2063 G mutation( mutation group),the other 22 samples carried a wild type of 23 S rRNA gene( non-mutation group). There were no differences in the general information such as gender,age,acute peripheral blood leukocytes and so on,but there were statistic differences in the hospitalization duration,total fever period,fever duration after macrolide therapy,cough period and the course of treatment. Compared to mutation group,the rates of extra-pulmonary complications and lobar pneumonia were higher in non-mutation group( P < 0. 05). Among the 120 samples,48 of the samples( 40%) were positive in MP vitro culture. 20 samples were resistant to macrolides, and the drug resistance rate was 16. 67%, which significantly lower than the mutation rate of 23 S rRNA( P < 0. 05). Conclusion: The mutation rate of MP 23 S rRNA was high. The mutation group had more serious clinical symptoms,longer course of treatment and more complications,suggesting that gene mutation was related to MP resistant. Compared with gene sequencing,the positive rate and drug resistant rate were lower in vitro culture and drug susceptibility tests. MP 23 S rRNA gene sequencing could be used as a means of drug resistant detection and had clinical value.
Objective: To explore the clinical application value of sequencing analysis of 23 S rRNA gene of Mycoplasma Pneumoniae( MP) in children's respiratory tract. Methods: The samples of phlegm or pharynx swab with MP more than 1* 10~4 copies were collected by fluorescence quantitative PCR,and domain V of the 23 S rRNA gene was sequenced. Children were divided into mutation group and non-mutation group according to the sequencing results,and clinical data of the two groups were compared. In vitro culture and drug sensitivity experiments were performed in the same sample to compare the sequencing analysis. Results: A total of 120 samples were collected,98 cases were diagnosed with A2063 G mutation( mutation group),the other 22 samples carried a wild type of 23 S rRNA gene( non-mutation group). There were no differences in the general information such as gender,age,acute peripheral blood leukocytes and so on,but there were statistic differences in the hospitalization duration,total fever period,fever duration after macrolide therapy,cough period and the course of treatment. Compared to mutation group,the rates of extra-pulmonary complications and lobar pneumonia were higher in non-mutation group( P < 0. 05). Among the 120 samples,48 of the samples( 40%) were positive in MP vitro culture. 20 samples were resistant to macrolides, and the drug resistance rate was 16. 67%, which significantly lower than the mutation rate of 23 S rRNA( P < 0. 05). Conclusion: The mutation rate of MP 23 S rRNA was high. The mutation group had more serious clinical symptoms,longer course of treatment and more complications,suggesting that gene mutation was related to MP resistant. Compared with gene sequencing,the positive rate and drug resistant rate were lower in vitro culture and drug susceptibility tests. MP 23 S rRNA gene sequencing could be used as a means of drug resistant detection and had clinical value.
引文
[1]YANG H J,SONG D J,SHIM J Y.Mechanism of resistance acquisition and treatment of macrolide-resistant Mycoplasma pneumoniae pneumonia in children[J].Korean J Pediatr,2017,60(6):167-174.
[2]付晓燕,辛徳莉,秦选光.儿童肺炎支原体感染流行病学、临床特点、发病机制及治疗研究进展[J].山东医药,2015,55(4):96-99.
[3]赵汉青,闫超,孙红妹,等.肺炎支原体快速液体培养法的评价和存在的问题[J].中华微生物学和免疫学杂志,2016,36(7):517-518.
[4]CHAN K H,TO K K,CHAN BW,et al.Comparison of pyrosequencing,Sanger sequencing,and melting curve analysis for detection of low-frequency macrolide resistant mycoplasma pneumoniae quasispecies in respiratory specimens[J].J Clin Microbiol 2013,51(8):2592-2598.
[5]中华医学会儿科学分会呼吸学组,《中华实用儿科临床杂志》编辑委员会.儿童肺炎支原体肺炎诊治专家共识(2015年版)[J].中华实用儿科临床杂志,2015,30(17):1304-1308.
[6]潘芬,张泓.肺炎支原体耐药性及分子流行病学研究进展[J].上海交通大学学报:医学版,2014,34(8):1248-1253.
[7]MOROZUMI M,IWATA S,HASEGAWA K,et al.Increased macrolide resistance of mycoplasma pneumoniae in pediatric patients with community-acquired pneumonia[J].Antimicrob Agents Chemother,2008,52(1):348-350.
[8]LIU Y,YE X,ZHANG H,et al.Antimicrobial susceptibility of mycoplasma pneumoniae isolates and molecular analysis of macrolide-resistant strains from Shanghai,China[J].Antimicrob Agents Chemother,2009,53(5):2160-2162.
[9]LEE E,CHO H J,HONG S J,et al.Prevalence and clinical manifestations of macrolide resistant mycoplasma pneumoniae pneumonia in Korean children[J].Korean J Pediatr,2017,60(5):151-157.
[10]姚慧生,张睿,刘立云,等.肺炎支原体耐药基因检测与难治性肺炎支原体肺炎的相关性分析[J].国际儿科学杂志,2016,43(6):492-496.
[11]施李芬,陈俐丽,余坚,等.24例23SrRNAA2063G基因突变肺炎支原体肺炎临床分析[J].中国小儿急救医学,2017,24(3):205-209.
[12]ZHOU Y,ZHANG Y,SHENG Y,et al.More complications occur in macrolide-Resistant than in macrolide-Sensitive Mycoplasma pneumoniae pneumonia[J].Antimicrob Agents Chemother,2014,58(2):1034-1038.
[13]NARITA M.Classification of extrapulmonary manifestations due to mycoplasma pneumoniae infection on the basis of possible pathogenesis[J].Front Microbiol,2016,7:23.
[14]曹波,郁飞,张宪伟,等.3种检测肺炎支原体感染方法的比较分析[J].东南大学学报:医学版,2016,35(5):770-772.
[2]付晓燕,辛徳莉,秦选光.儿童肺炎支原体感染流行病学、临床特点、发病机制及治疗研究进展[J].山东医药,2015,55(4):96-99.
[3]赵汉青,闫超,孙红妹,等.肺炎支原体快速液体培养法的评价和存在的问题[J].中华微生物学和免疫学杂志,2016,36(7):517-518.
[4]CHAN K H,TO K K,CHAN BW,et al.Comparison of pyrosequencing,Sanger sequencing,and melting curve analysis for detection of low-frequency macrolide resistant mycoplasma pneumoniae quasispecies in respiratory specimens[J].J Clin Microbiol 2013,51(8):2592-2598.
[5]中华医学会儿科学分会呼吸学组,《中华实用儿科临床杂志》编辑委员会.儿童肺炎支原体肺炎诊治专家共识(2015年版)[J].中华实用儿科临床杂志,2015,30(17):1304-1308.
[6]潘芬,张泓.肺炎支原体耐药性及分子流行病学研究进展[J].上海交通大学学报:医学版,2014,34(8):1248-1253.
[7]MOROZUMI M,IWATA S,HASEGAWA K,et al.Increased macrolide resistance of mycoplasma pneumoniae in pediatric patients with community-acquired pneumonia[J].Antimicrob Agents Chemother,2008,52(1):348-350.
[8]LIU Y,YE X,ZHANG H,et al.Antimicrobial susceptibility of mycoplasma pneumoniae isolates and molecular analysis of macrolide-resistant strains from Shanghai,China[J].Antimicrob Agents Chemother,2009,53(5):2160-2162.
[9]LEE E,CHO H J,HONG S J,et al.Prevalence and clinical manifestations of macrolide resistant mycoplasma pneumoniae pneumonia in Korean children[J].Korean J Pediatr,2017,60(5):151-157.
[10]姚慧生,张睿,刘立云,等.肺炎支原体耐药基因检测与难治性肺炎支原体肺炎的相关性分析[J].国际儿科学杂志,2016,43(6):492-496.
[11]施李芬,陈俐丽,余坚,等.24例23SrRNAA2063G基因突变肺炎支原体肺炎临床分析[J].中国小儿急救医学,2017,24(3):205-209.
[12]ZHOU Y,ZHANG Y,SHENG Y,et al.More complications occur in macrolide-Resistant than in macrolide-Sensitive Mycoplasma pneumoniae pneumonia[J].Antimicrob Agents Chemother,2014,58(2):1034-1038.
[13]NARITA M.Classification of extrapulmonary manifestations due to mycoplasma pneumoniae infection on the basis of possible pathogenesis[J].Front Microbiol,2016,7:23.
[14]曹波,郁飞,张宪伟,等.3种检测肺炎支原体感染方法的比较分析[J].东南大学学报:医学版,2016,35(5):770-772.