The Clinical Significance Of Multilocus Variable-Number Tandem-Repeat Analysis In Genotyping Of Mycoplasma Pneumoniae From Clinical Samples

With the development of Mycoplasma pneumoniae (MP) genomics technique, the relationship between the MP genotypes and MP variation、disease progression、 macrolides resistance or treatment have received more and more attention in recent years, The genotyping of MP isolates, at the molecular level, benefit the epidemiological surveillance and clinical study of infection. At present, application of traditional genotyping methods were limited because of their low discrimination, which is caused by genetical homogeneity of MP, few open reading frames which contain point mutations and little polymorphism in the housekeeping genes or structural genes. Multiple-locus variable-number tandem-repeat analysis (MLVA) is a new molecular typing method by polymerase chain reaction, based on the variable number of tandemly repeated sequences, called VNTRs. MLVA has a higher discrimination potential of MP, thus benefits clinical diagnosis, treatment and epidemiological surveillance.In order to establish a complete set of MLVA genotyping system and apply it to clinical research, we collected and isolated MP strains from oropharyngeal swabs(OP) of outpatient, inpatient and children from two primary schools where MP epidemic took place. These strains were genotyped by real-time PCR, PCR-RFLP on PI adhension protein and MLVA, aiming to understand distribution of MP genotypes, the relationship between MP genotypes and clinical characteristics.Methods:(1) MP isolates and MLVA genotyping:We set a series of steps for OP collection, confirmed culture conditions of MP standard and clinical strains and explored best procedures of PCR and MLVA.(2) MLVA application:We collected 1131 OPs from outpatients or inpatient children from children’s hospital of Zhejiang university school of medicine, and 82 OPs from two primary schools where there was a MP outbreak. We isolated MP strains, confirmed MP infection by double probe real-time PCR, and genotyped MP by MLVA and Pl-RFLP, respectively. Finally, we compared the clinical data of MP children with different MLVA genotypes like respiratory symptoms, fever duration, maximum temperature, application of glucocorticoids and severity.Results:(1) Double probe real-time PCR and the MP commercial detection kits tested 217 clinical MP specimens. Taking the result of the MP commercial detection kits as reference, we found the sensitivity and specificity of double probe real-time PCR is 92.3%and 91.7%, respectively;(2) We isolated 164 MP strains. Taking double probe real-time PCR as reference, the MP isolating rate was 66.67%(164/246).146 strains were obtained from outpatients and inpatients.18 strains were collected from two primary schools with MP outbreaks;(3) The MLVA type of 146 MP strains showed:MLVA type A (13 cases), MLVA type B (1 case), MLVA type E (36 cases), MLVA type H (1 case), MLVA type J (27 cases), MLVA type S (1 case), MLVA type P (39 cases), MLVA type U (20 cases), MLVA type X (6 cases), MLVA 1-4-5-7-3(1 cases), heterozygote(l cases).18 MP strains from two primary schools revealed strains from the same school were the same type(5 cases from school A were type J,13 cases from school B were type U);(4) Type U infection tended to cause more severe MP infection.Conclusion:(1) The double probe real-time PCR of our study can be used for clinical detection;(2) The MP isolating rate was 66.67%, similar to the result reported by foreign laboratory; the establishment of MP culture was successful;(3) Type U infection tended to cause more severe MP infection.