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目的比较青霉素结合蛋白2b(pbp2b)与自溶素(ply)、溶血素(lyt A)基因和脑脊液细菌培养在诊断国内儿童化脓性脑膜炎中肺炎链球菌感染及青霉素耐药性方面的情况,并了解肺炎链球菌青霉素结合蛋白基因型特征。方法 2006年1月至2009年9月在北京儿童医院感染内科临床严格诊断为细菌性脑膜炎患儿161例,用pbp2b及其他2个肺炎链球菌种特异的基因(ply、lyt A)巢式PCR法来检测细菌性脑膜炎患儿脑脊液中的肺炎链球菌,对不同方法结果进行比较。对其中pbp2b阳性的标本进行青霉素敏感性PCR(分别用耐药、敏感的引物进行PCR检测)、测序,并与青霉素耐药表型进行比较和评估。结果 161例中不同方法确诊的肺炎链球菌共有25例,3种基因检测均阳性的共有11例。pbp2b检测阳性16例,lyt A检测阳性16例,ply检测阳性14例。脑脊液培养阳性9例。pbp2b阳性的16例标本中,青霉素敏感和青霉素耐药基因型各占一半。16例中4例有培养结果,其中3例基因型和耐药型相符合。敏感性测定PCR和测序结果相一致。测序结果与NCBI基因库(http://blast.ncbi.nlm.nih.gov/Blast.cgi)相比,有2例为新的氨基酸序列型,但是无新的氨基酸点突变发生。由培养结果检测的肺炎链球菌脑膜炎的青霉素耐药率为66.67%(6/9),敏感性PCR检测的肺炎链球菌脑膜炎的青霉素耐药率为50%(8/16),包括脑脊液培养阴性的病例。结论 3种基因检测的敏感度均高于传统的脑脊液培养,其中pbp2b可以作为一个推荐的基因位点筛查肺炎链球菌感染,并在一定程度上预测青霉素耐药性情况。
Objective To investigate the relationship between pbp2b, ply, lyt A gene and cerebrospinal fluid bacterial culture in the diagnosis of S. pneumoniae infection and penicillin resistance in children with purulent meningitis in China. And to understand the characteristics of the pneumococcal penicillin-binding protein genotype. Methods From January 2006 to September 2009, 161 children with bacterial meningitis were clinically diagnosed clinically in Beijing Children’s Hospital for Infectious Diseases. The pbp2b and other 2 Streptococcus pneumoniae-specific genes (ply, lyt A) PCR method to detect S. pneumoniae in cerebrospinal fluid of children with bacterial meningitis and compare the results of different methods. The pbp2b-positive samples were subjected to penicillin-sensitive PCR (PCR detection with resistant and sensitive primers respectively), sequenced and compared with the penicillin resistant phenotype. Results There were 25 cases of Streptococcus pneumoniae diagnosed by different methods in 161 cases, and 11 cases were positive for all three genes. Positive pbp2b test in 16 cases, lyt A positive 16 cases, ply positive in 14 cases. Cerebrospinal fluid culture positive in 9 cases. Of the 16 specimens positive for pbp2b, penicillin-sensitive and penicillin-resistant genotypes each accounted for half. Among 16 cases, 4 cases had culture results, of which 3 cases were consistent with drug resistance. Sensitivity assays PCR and sequencing results are consistent. Sequencing results Compared with the NCBI gene bank (http://blast.ncbi.nlm.nih.gov/Blast.cgi), 2 cases were new amino acid sequence type, but no new amino acid point mutations occurred. The penicillin-resistant rates of S. pneumoniae meningitis detected by culture results were 66.67% (6/9), penicillin-resistant rates of S. pneumoniae meningitis detected by sensitive PCR were 50% (8/16), including cerebrospinal fluid Cultivate negative cases. Conclusion The sensitivity of three kinds of gene detection is higher than that of traditional cerebrospinal fluid culture. Among them, pbp2b can be used as a recommended gene to screen for S. pneumoniae infection and to predict the extent of penicillin resistance to a certain extent.