Study On Mycoplasma Pneumoniae P1Genotyping And The Association With Clinical Severity In Sochow Area

Objective:Mycoplasma pneumoniae(M. pneumoniae) clinical samples were isolated frompediatric patients with respiratory tract infection in Sochow area, the p1gene genotypingwere performed and sequenced.To analyze the genotype and variation of MP in Sochowarea.Collecting the clinical data of different types of MP,to explore the the associationbetween MP genotype and Clinical severity.Methods:Three hundred and twelve patients admitted in Children,s Hospital Affiliated toSuchow University with acute respiratory tract infection between January2012andDecember2013were recruited in the study(including29outpatients with MP infection and264hospitalized patients diagnosed MPP （Mycoplasma penumoniae penumoniae，MPP）)．We selected the respiratory secretions as the object of the study.1.DNA extraction.Collect the throat swab specimen of the out-patient children withMP infection and the respiratory tract secretions of the hospitalized children diagnosedMPP.And extract the DNA of MP for fluorescence PCR.We select the specimens whoseconncentration was2.5*10^3or above(including2.5*10^3),and then drawn the supernatantafter4000rpm/15min centrifugation to1.5ml eppendorf tube,frozen the supernatant in-80°C refrigerator.2.Mycoplasma pneumoniae p1genotype.The type of MP clinical samples wasdetermined by restriction fragment length polymorphism(RFLP) and nested-multiplex PCRtyping of the P1gene．And the application value of the two genotyping ways werecompared.3.MP gene sequencing.Randomly selected the P1type1and P1type2specimens,thereference strain M129and the reference strainMP-FH to sequence.4.Clinical data collection.Collected and compared the clinical data of MPP childrenwith different subtype: sex, age, clinical manifestations, pulmonary complications,labortatory test results,imaging results and treatment conditions,et al.Results:1.Genetyping results showed that:313cases of positive samples in PCR-RFLPshowed that32samples (10.22%) were classified as typeⅠ,1samples (0.32%)wereclassified as type Ⅱ,280samples (89.46%)were classified as neither typeⅠ nor type Ⅱ.Meanwhile, The results of nested-multiplex PCR indicated that304samples(97.12%)wereclassified as typeⅠ,8samples (2.56%)were classified as typeII,1samples (0.32%)wereclassified as V2variant，indicating that during the study period the epidemic genotype ofMP strains is P1-type in Suchow area.By comparing the two method,we conclude that thenested-multiplex PCR is superior to PCR-RFLP.2.Sequence analysis showed that one point mutations was identided in2of3P1type1samples to the registered sequence in GeneBank.The rest of1P1type1samples wascompletely identical to M129strain;3P1type2samples was completely identical toMP-FH strain.3. The clinical data showed that no significant difference in the average duration offever and hospitalization between the P1type1group and the P1type2group（P>0．05）.Also,no significant difference was found in the levels of white bloodcell(wbc),high-sensitivity c-reactive protein(CRP) and CPIS（P>0．05）.But the percentageof neutrophils in the P1type1group is higher than that in the P1type2group(P<0.05).1V2variant showed showed no clinical symptoms of refractoryMycoplasma pneumoniae pneumonia(RMPP).Conculsion:1.Between between January2012and December2013in Sochow area,the epidemicgenotype of MP strains is P1-type.2.Nested-multiplex PCR is a reliable method for Mp genotyping.3.No significant correlation was found between MP subtype and the severity ofpulmonary infection.