| Background and objectiveMycoplasma pneumoniae is one of the common causative organisms of community-acquired respiratory tract infection, which can cause bronchitis and atypical pneumonia, and lead to many extra-pulmonary complications. The infection and drug resistance rates of this pathogen are rising in recent years, which causes serious harm to the adolescent’s health. Previous studies indicate that macrolides resistance of M. pneumoniae is a serious issue, while no clinical isolates resistant to tetracyclines or quinolones has been reported. However, clinical isolates resistant to tetracyclines or quinolones may appear with increasing application of these drugs.This study focuses on the surveillance of M. pneumoniae infection and drug resistance in China during the year of 2014. Besides, we aim to develop a new Real-time quantitative PCR assay, which is rapid, specific, sensitive, and accurate for M. pneumoniae detection, and evaluate its clinical application value.Methods1. Surveillance of M. pneumoniae Infection and Drug Resistance in ChildrenAll throat swab specimens were collected from patients attending hospitals in Beijing from January 2014 to December 2014. Every swab was cultured and isolates’ minimal inhibitory concentrations(MICs) were detected. Apart from that, samples were extracted DNA samples and detected drug resistance genes.2. Development and Clinical Application of Real-time Quantitative PCR AssayThe sequence of the 23 S r RNA gene in MP type strain FH was selected as amplified regions, and specific primers were designed. Then the related plasmids were extracted as standards, and the absolute quantitative standard curve wasestablished. The sensitivity and specificity of the fluorescence quantitative PCR assay was compared with the nest-PCR and kit; To calculate correlation coefficient, coincidence rate and kappa coefficient, clinical samples were detected using above-mentioned methods and cultivation, respectively.Results1. Surveillance of M. pneumoniae Infection and Drug Resistance in Children(1) In totle 827 samples were collected from children infected with respiratory tract infection in Beijing during 2014, and 822 were enrolled. The positive rate was41.48%, 341 samples showing positive to V domain of 23 S r RNA in PCR testing.Among them, 105 strains were sensitive(30.79%), and of 236 drug-resistant strains(69.21%), 199 were A2063G(58.36%), 25 were A2064G(7.33%), and 12 harbored both(3.52%). Eventually a total of 99(29.03%) positive culture were obtained.(2) The incidence of M. pneumoniae infection showed a downtrend in 2014, with high level of drug-resistant rate in spring and winter. The infection rate increased with age, with highest level in school aged children. But there was no significance in gender.(3) Children with pneumonia showed higher incidence of M. pneumoniae infection and drug-resistant rate than upper respiratory tract infection and bronchitis;The culture positive rates in children with bronchitis and pneumonia were significantly higher than upper respiratory tract infection.(4) Eight M. pneumoniae isolations showed sensitive to macrolides, tetracyclines and quinolones antibiotics, 57 M. pneumoniae isolations showed resistant to macrolides, while sensitive to tetracyclines and quinolones antibiotics.2. Development and Clinical Application of Real-time Quantitative PCR Assay(1) The standard curve was established: y=-3.5692x+40.786,R2=0.998.(2) The detection sensitivity of Real-time Quantitative PCR and nest-PCR was 10 copies of FH DNA, while the kit 100 copies. The M. pneumoniae sample was detected positive, while mycoplasma hominis and other four bacteria were all negative, showing a high specificity.(3) Comparing Real-time Quantitative PCR with nest-PCR, kit and culture, the total consistency rates were all >80%, and Kappa coefficients were all >0.65. The correlation coefficient of Real-time Quantitative PCR and the kit was 0.923,P<0.001.Conclusions1. Surveillance of M. pneumoniae Infection and Drug Resistance in Children(1) The incidence of M. pneumoniae infection showed a downtrend in 2014; the infection rate increased with age, with highest level in school aged children; but there was no significance in gender, which is in agreement with available data reported abroad and at home.(2) M. pneumoniae showed high resistance to macrolide antibiotics, and the key mechanism was still point mutation at domain V of 23 S r RNA, including A2063 G,A2064G or both. While they were all sensitive to tetracyclines and quinolones.2. Development and Clinical Application of Real-time Quantitative PCR AssayCompared with other methods, the Real-time Quantitative PCR assay could be used to detect mycoplasma pneumoniae quickly and economically, with high sensitivity and specificity, little contamination, and beyond the restriction of age,disease course and immune function of patients, revealing great utility value on scientific and clinical values. |
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