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Objective:To investigate drug resistance gene in Mycoplasma pneumoniae(MP) and the distribution of 13 respiratory pathogens in bronchoalveolar lavage fluid(BALF) of children with Mycoplasma pneumoniae pneumonia(MPP).Methods:A total of 100 BALF of children with MPP in Peking University Third Hospital and Peking University First Hospital from January 2018 to January 2019 were collected.Fluorogenic quantitative PCR was used to detect nucleic acid and it′s drug resistance gene of MP and multiple PCR method was adopted to detect influenza A virus, influenza A virus-H 1N 1, influenza A virus-H 3N 2, influenza B, human parainfluenza virus, adenovirus, human bocavirus, human rhinovirus, Chlamydia pneumoniae, human metapneumovirus, MP, human coronavirus, and respi-ratory syncytial virus gene, and the results were compared by using Chi square test. Results:In 100 BALF samples, MP and drug resistance gene were detected by fluorogenic quantitative PCR.Totally, 83 cases (83.00%) were MP positive and 78 cases (93.98%) were drug resistant.All of them had the point mutations A2063G in V region of 23S rRNA domain.A total of 13 kinds of respiratory pathogens were detected by multiplex PCR method, and 89 cases (89.00%) were positive.Totally, 79 cases (79.00%) were MP positive, of which 74 cases (74.00%) detected only MP, and 5 cases (5.00%) detected MP combined with other pathogens.Other pathogens were detected in 10 cases (10.00%). The virus detection rate of 0-4 years old group was higher than that of >4-6 years old group ( P=0.042) and >6 years old group ( P=0.002), and the differences were statistically significant. Conclusions:MP can be detected in most BALF samples of MPP children, the drug resistance phenomenon is serious, and the main point mutation is A2063G.There were other respiratory pathogens and 2 or 3 pathogens were detected in a small number of BALF samples.
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Objective To investigate the antibacterial effect of Fusidic acid on Mycoplasma pneumoniae and antibiotic resistant Mycoplasma pneumoniae in vitro.Methods Twenty-eight clinical strains of Mycoplasma pneumoniae isolated from patients with respiratory tract infection at Beijing Friendship Hospital Affiliated to the Capital University of Medical Sciences from January to December 2016 and 2 Mycoplasma pneumoniae reference strains were enrolled.The minimum inhibitory concentration (MIC) of Fusidic acid and Azithromycin were determined by using micro-dilution ration method.The chessboard method was used to check the antibacterial effect of combination between Fusidic acid and Azithromycin.The antibacterial activity of the Fusidic acid was evaluated by measuring the antibacterial rate of different concentrations.Results One isolate showed no mutation in 23SrRNA,26 isolates had one point mutation in loci 2063 and 1 isolate had one point mutation in loci 2064 among the 28 clinical isolates.The findings by micro-dilution method results showed that the MIC values of all the clinical isolates with mutations associated with macrolide resistance to Azithromycin were ＞ 1.000 0 mg/L,and the MIC values of all the clinical isolates with no mutations to azithromycin were ＜ 0.500 0 mg/L.The findings by micro-dilution method results showed that the MIC value of Fusidic acid for Mycoplasma pneumonia and drug resistance Mycoplasma pneumoniae was 1.000 0 mg/L.The Fractional Inhibitory Concentration index of Fusidic acid and Azithromycin combination was ≤0.500 0 mg/L.When the concentration of the Fusidic acid was lower than or equal to 32 MIC,the antibacterial effect of Fusidic acid against Mycoplasma pneumoniae increased with its higher concentration.When the concentration of the Fusidic acid was lower than or equal to 8 MIC,the longer the strain was exposed to the drug,the stronger antibacterial effect was against Mycoplasma pneumoniae.Conclusion If the treatment of Mycoplasma pneumoniae infection is not effective or the infection of patient is combined with bacteria,the application or combination of Fusidic acid may inhibit pathogenic bacteria effectively.Of course,how to use Fusidic acid in clinical treatment needs further study and discussion.
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Objective To developed A laboratory diagnosis of Moraxella catarrhalis by an laboratories diagnostic method real-time fluorescence quantitative PCR assay. Methods The specific primers and probes were designed based on the sequence of outer membrane protein CopB(copB)gene in Moraxella catarrhalis,and the Taqman probe RT-PCR method was developed to detect the Moraxella catarrhalis.The standard plasmids ex-tracted from the Moraxella catarrhalis standard strains were used to constitute the standard samples,and compared with these standard samples,the sensitivity of the fluorescence quantitative PCR assay was tested by the estab-lished standard curves.The specificity of the fluorescence quantitative PCR assay was tested by the DNA samples of other bacterias in the laboratory.Meanwhile,321 throat swab samples from inpatient and outpatient child pa-tients,with asthma infection were collected as clinical samples to validate the fluorescence quantitative PCR as-say.Results The standard curve was drawn in the real-time PCR by the Taqman fluorescence reporter.During the sensitivity tests,the newly-developed real-time fluorescence PCR could detect at least 10 copies of Moraxella catarrhalis,and could successfully distinguish several DNAs of the pathogens.On the basis of the validation result of the 321 throat swab samples,there are 25 Moraxella catarrhalis with 7.79 % positive rate.Conclusion The fluorescence quantitative PCR assay is of great sensitivity and specificity,and it can be widely used for the detec-tion of Moraxella catarrhalis.
ABSTRACT
Mycoplasma pneumoniae(MP) is a common pathogen found in respiratory tract infections of children and is susceptible to macrolides, tetracycline, aminogly-cosides, and quinoloines . Of all these antibiotics, macrolides is the frist choice for children. However,in recent years, strains which are resistant to common drugs have been selected in vitro and isolated from patients Point mutation at antibitics target site of these 8trains is one of the molecular mechanisms.The study of MP resistance can provide theoretical guidance for rational choice of antibiotics and application.