摘要目的 探讨多重聚合酶链反应结合反向线性点杂交(multiplex polymerase chain reaction-based reverse line blot hybridization, mPCR/RLB)技术在检测新生儿化脓性脑膜炎病原及其耐药基因中的作用. 方法 回顾性收集2012年1月1日至2018年12月31日在首都医科大学附属北京儿童医院临床诊断为新生儿化脓性脑膜炎患儿(n=80)的病例资料及脑脊液标本(100份).入院首次采集脑脊液标本80份(使用抗生素前12份,使用抗生素后68份),另20份为治疗后复查脑脊液标本.脑脊液标本分别采用细菌培养和药敏鉴定,以及mPCR/RLB技术检测病原及其耐药情况,采用χ2检验比较2种方法的检测阳性率. 结果 (1)80例患儿入院首次脑脊液检测:mPCR/RLB 检测阳性率明显高于细菌培养 [26.3%(21/80)与 7.5%(6/80),χ2 =10.025, P=0.002].使用抗生素前的脑脊液标本mPCR/RLB检测阳性率与细菌培养差异无统计学意义(9/12与5/12,χ2 =1.543,P=0.214);使用抗生素后的脑脊液标本mPCR/RLB检测阳性率高于细菌培养[17.6%(12/68)与1.5%(1/68),χ2=13.176,P＜0.001].(2)复查脑脊液标本:细菌培养均阴性, mPCR/RLB阳性4例.该4例患儿首次脑脊液细菌培养阳性,mPCR/RLB结果亦阳性,且mPCR/RLB病原结果与细菌培养结果相符.(3)病原:细菌培养检出大肠埃希菌3例,B族链球菌2例,李斯特菌1例;mPCR/RLB技术在首次脑脊液标本中检出大肠埃希菌4例,B族链球菌5例,李斯特菌4例,脑膜炎奈瑟球菌4例,流感嗜血杆菌b型、革兰阴性菌和革兰阳性菌各1例,李斯特菌和流感嗜血杆菌b型混合感染1例.(4)耐药基因:1例大肠埃希菌药敏结果为超广谱β-内酰胺酶阳性.mPCR/RLB检出acrA阳性3例,acrB阳性2例,TetM阳性1例,CTX-M阳性1例(与药敏结果相符). 结论 mPCR/RLB与细菌培养相比阳性率高,一致性好,且可同时检测耐药基因,具有临床应用价值.

abstractsObjective To evaluate the performance of multiplex polymerase chain reaction-based reverse line blot hybridization (mPCR/RLB) in the detection of pathogens causing neonatal bacterial meningitis and associated drug resistance genes. Methods Clinical data and cerebrospinal fluid (CSF) samples were collected retrospectively from 80 cases diagnosed with neonatal bacterial meningitis in Beijing Children's Hospital from January 1, 2012 to December 31, 2018. A total of 100 CSF samples were obtained including 80 samples collected after admission (12 before and 68 after antibiotic treatment) and 20 recollected at follow-up. All CSF samples were analyzed by conventional culture, susceptibility test and mPCR/RLB. Differences in the detection of pathogens and drug resistance genes were analyzed by Chi-square test. Results (1) Among the 80 first-collected CSF samples, mPCR/RLB revealed significantly higher positive rate than conventional culture [26.3% (21/80) vs 7.5% (6/80), χ2=10.025, P=0.002]. No significant difference was showed between the two methods in analyzing the 12 samples collected before antibiotic therapy (9/12 vs 5/12, χ2=1.543, P=0.214), while the positive rate in 68 samples collected after antibiotic intervention detected by mPCR/RLB was obviously higher than that by conventional culture [17.6% (12/68) vs 1.5% (1/68), χ2=13.176, P<0.001]. (2) Conventional culture results of the 20 samples collected during follow-up were all negative, but four were positive using mPCR/RLB, which were also positive previously. Furthermore, the results of both methods in previous detections were identical. (3) According to the conventional culture results, the pathogens were Escherichia coli (three cases), Group B Streptococcus (two cases) and Listeria monocytogenes (one case), while mPCR/RLB detected Escherichia coli (four cases), Group B Streptococcus (five cases), Listeria monocytogenes (four cases), Neisseria meningitidis (four cases), Haemophilus influenzae b (one case), Gram-negative bacteria (one case), Gram-positive bacteria (one case), and Listeria monocytogenes and Haemophilus influenzae b coinfection (one case) in 80 first-collected CSF samples. (4) Antibiotic susceptibility test showed that one Escherichia coli strain produced extended spectrum beta-lactamases. Drug resistance gene detection by mPCR/RLB showed that acrA, acrB, CTX-M (consistent with antibiotic susseptibility test) and TetM genes were positive in three, two, one and one case, respectively. Conclusions mPCR/RLB is of great clinical value due to its higher detection rate and better accuracy compared with bacterial culture and can also detect drug resistance genes.