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

Abstract

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, and χ(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.