儿童肺炎支原体感染咽拭子核酸检测方法的比较

Comparison of nucleic acid detection methods in pharyngeal swabs of Mycoplasma pneumoniae infection in children

目的:探讨咽拭子核酸检测方法在儿童肺炎支原体(MP)感染病原学诊断中的应用价值。方法:选取2018年7至10月在浙江大学医学院附属儿童医院呼吸科住院的454例(男210例、女244例)肺炎患儿进行前瞻性研究,入院当天或第2天采集患儿的咽拭子和静脉血,分别采用DNA荧光定量扩增、RNA恒温扩增、MP培养和MP-IgM进行MP病原检测,以MP培养阳性或其余3项检测中2项阳性为MP感染诊断标准并进行二次检测,筛选出MP感染患儿于出院前再次采集咽拭子,采用DNA荧光定量扩增、RNA恒温扩增、MP培养3种方法再次进行MP病原学检测。比较3种检测方法随病情变化的检出率及MP菌量变化,组间比较采用χ^2检验。 结果:454例住院肺炎患儿采用DNA荧光定量扩增、RNA恒温扩增、MP培养和MP-IgM的检出率分别为43.6%(198/454)、43.2%(196/454)、40.0%(180/454)、30.6%(139/454),差异有统计学意义(χ^2=20.8, P<0.05)。DNA荧光定量扩增和RNA恒温扩增检出率差异无统计学意义(χ^2=0.018, P=0.900),较MP-IgM及MP培养高。以MP培养阳性或其余3项检测中2项阳性为MP感染诊断标准,筛选出MP感染患儿209例。209例MP感染患儿在出院前的第2次MP检验中,MP培养阳性率67.5%(141/209),39.4%(13/33)由阴性转为阳性,27.3%(48/176)由阳性转为阴性。RNA恒温扩增阳性率82.3%(172/209),16.2%(31/191)由阳性转为阴性,66.7%(12/18)由阴性转为阳性。DNA荧光定量扩增阳性率67.0%(140/209),52.9%(18/34)由阴性转为阳性,30.3%(53/175)由阳性转为阴性。DNA荧光定量扩增阳性检测标本中,141例(67.5%)二次检测菌量减少,68例(32.5%)菌量增加。4种方法在非重症组和重症组患儿中的检出率相近,各组间差异均无统计学意义( P均>0.05),在二次检测中,重症组转阳比例仅有RNA恒温扩增高于非重症组,差异有统计学意义(7比5例, P=0.038)。 结论:咽拭子核酸检测方法在儿童MP感染急性期病原学诊断中具有较高的敏感性和应用价值,DNA荧光定量扩增、RNA恒温扩增的检测结果具有高度一致性,较MP-IgM检测更具优势。急性期重复检测有助于检出第一次检测结果为阴性的MP感染患儿。急性期部分患儿经抗感染治疗后MP菌量未下降。

Objective To explore the value of nucleic acid detection methods in pharyngeal swabs in the etiological diagnosis of Mycoplasma pneumoniae(MP)infection in children.Methods Four hundred and fifty-four(male 210,female 244)children with pneumonia in Department of Pulmonology,Children′s Hospital of Zhejiang University School of Medicine were enrolled from July,2018 to October,2018.Pharyngeal swabs and venous blood were obtained on the first or the second day after hospitalization.Fluorescence detection quantitative amplification of DNA,thermostatic amplification of RNA,MP culture and MP-IgM were used to detect MP simultaneously.MP infection is diagnosed if MP culture is positive or the two of the other three methods are positive.Pharyngeal swabs were acquired and detected using fluorescence quantitative amplification of DNA,thermostatic amplification of RNA and MP culture again for children with confirmed MP infection before discharge.The detection rates and quantitative changes of the three methods were compared,andχ^2 test was used for comparison among groups.Results A total of 454 hospitalized children with pneumonia were included in this study.The detection rates of fluorescence quantitative amplification of DNA,thermostatic amplification of RNA,MP culture and MP-IgM IgM were 43.6%(198/454),43.2%(196/454),40.0%(180/454)and 30.6%(139/454)respectively.The difference of detection rates of the four methods was statistically significant(χ^2=20.8,P<0.05),but no significant difference between the detection rates of fluorescence quantitative amplification of DNA and thermostatic amplification of RNA was found(χ^2=0.018,P=0.900).They both had higher detection rates than MP-IgM or MP culture.MP infection is diagnosed if MP culture is positive or the two of the other three methods are positive,and two hundred and nine children were diagnosed as MP infection.In the second test of MP infection in 209 children before discharge,the positive rate of MP culture was 67.5%(141/209),with 39.4%(13/33)changed from negative to positive,and 27.3%(48/176)changed from positive to negative.The positive rate of thermostatic amplification of RNA was 82.3%(172/209),with 16.2%(31/191)turned from positive to negative,and 66.7%(12/18)turned from negative to positive.The positive rate of fluorescence quantitative amplification of DNA was 67.0%(140/209),with 52.9%(18/34)cases changed from negative to positive,and 30.3%(53/175)cases changed from positive to negative.MP-DNA load decreased in 141 cases(67.5%)and increased in 68 cases(32.5%)in the second test among the positive samples tested by fluorescence quantitative amplification of DNA.The detection rates of the four methods in the non-severe group and the severe group were similar,and the differences among the groups were not statistically significant(all P>0.05).In the second test,the proportion of changing from negative to positive in the severe group was higher than that in the non-severe group,but only the difference in the thermostatic amplification of RNA was statistically significant(P=0.038)and the cases of changing from negative to positive of thermostatic amplification of RNA in the severe group and non-severe group are 7 and 5 respectively.Conclusions The methods of pharyngeal swab nucleic acid detection have high sensitivity and application value in the etiological diagnosis of acute MP infection in children.The results of fluorescence quantitative amplification of DNA and thermostatic amplification of RNA are highly consistent,and they are both more advantageous than MP-IgM.Repeated testing in the acute phase is helpful to find MP infection children whose first test is negative.The load of MP-DNA did not decrease in some children in the acute stage after antibiotic treatment.