Rapid Diagnosis Of Respiratory Viruses And Mycoplasma Pneumoniae In Children And Detection Of Drug Resistance Gene Of Mycoplasma Pneumoniae

Part1 Clinical application of FilmArray Respiratory PanelObjective:To evaluate the clinical diagnostic value of FilmArray Respiratory Panel(FilmArray RP)in children and analyze the prevalence of respiratory viruses.Methods:Nasopharyngeal swabs or sputum samples were collected from hospitalized children with acute respiratory tract infection(ARTI)in Shanghai Children's Medical Center from December 1,2016 to November 30,2017.FilmArray RP were used to detect adenovirus(ADV),human rhinovirus/enterovirus(HR/EV),influenza virus A(IFVA),influenza virus B(IFVB),parainfluenza virus 1-4(PIV1-4),human metapneumovirus(HMPV),Coronavirus HUKI/OC43/229E/NL63(CoV-HUKI/OC43/229E/NL63),Respiratory syncytial virus(RSV),Mycoplasma pneumoniae(MP),Chlamydophila pneumoniae(CP)and B.pertussis(B.P)in the respiratory specimens.Distribution of respiratory pathogens was analyzed using statistical methods.Results:Among the 775 specimens,428(55.2%,428/775)were positive for single pathogen,198(25.5%,198/775)were positive for multiple pathogens,and 149specimens were negative(19.2%,149/775).The overall positive rate was 80.8%.HR/EV was the most common pathogen(25.5%,198/775),followed by RSV(19.5%,151/775)and PIV3(14.8%,115/775).There was no significant difference in the overall positive rate among children in different age groups(c~2=1.314,p=0.726).MP was detected during the entire year,and the detection rate of B.P was higher in March and May.The detection of the respiratory virus was most significant in the winter and spring.Mixed infection accounted for 31.6%(198/626)of the positive specimens,of which HR/EV and PIV3 were the most common mixed infection(10.6%,21/198),followed by HR/EV and ADV mixed infection(6.1%,12/198).The mixed infection of HR/EV and RSV was also one of the most common types(5.6%,11/198).Conclusions:FilmArray RP could quickly detect a variety of respiratory pathogens and provide the region's respiratory spectrum of infectious diseases and epidemiological characteristics.Part 2 Examination of the drug resistance genes and analysis of the clinical features of Mycoplasma pneumoniae in childrenObjective:To establish a method for detecting Mycoplasma pneumoniae(MP)resistance genes and analyze the clinical features of the mutant and non-mutant groups.To understand the resistance of MP in this area and provide a reference for the selection of antibiotics.Methods:Respiratory specimens of children diagnosed as MP infection by FilmArray RP were selected and QIAamp DNA Mini Kit was used to extract the DNA from these specimens.Primers were designed based on the 2063 and 2064 sites of the MP 23S rRNA and PCR was performed.Sequencing results of PCR-positive specimens were compared with MP129 standard strain registered in NCBI.The clinical data of these children were collected and the clinical features of the mutant and non-mutant groups were statistically analyzed.Results:75 specimens were positive as detected by MP 23S rRNA PCR,and 17 of them were with no 23S rRNA mutation while 58 specimens with an A2063G mutation,with the mutation rate 77.3%.There was no significant difference regarding the age,sex,total fever time,hospitalization time,early inflammation index or complication incidence between the mutant group and the non-mutant group.The fever time before the use of macrolides in the mutation group was shorter than that in the non-mutation group(t=2.067,p=0.042).After macrolide treatment,the time required for fever withdrawal in the non-mutated group was shorter(t=6.399,p=0.000).Conclusions:The drug resistance of MP was serious.It was difficult to distinguish the resistance mutation group from the non-mutation group in terms of age,sex,routine laboratory examination,etc.It took longer time for the patients in the mutation group to reduce the fever after treatment with macrolide.Part 3 Establishment of loop-mediated isothermal amplification assay for Mycoplasma pneumoniae detectionObjective:To preliminarily establish a simple,rapid and loop-mediated isothermal amplification(LAMP)-based method for Mycoplasma pneumoniae detection.Methods:4 specific primers for the MP P1 protein gene were designed and the LAMP reaction using the Bst 2.0 DNA polymerase was established to detect MP.The reaction temperature and reaction time were optimized by using real-time LAMP reaction,and the reaction end point was determined by gel electrophoresis.Results:The reaction temperature and reaction time were optimized by real-time LAMP.The optimal reaction condition was determined at 64°C for 15 min,which was consistent with the electrophoresis results.We established a preliminary MP LAMP reaction system.Conclusions:In this study,the detection method of MP LAMP was established.The optimum condition was reaction at 64°C for 15 min.The result could be judged by gel electrophoresis.The operation was very simple and it could be used for the rapid detection of MP.