Multiplex polymerase chain reaction-based reverse line blot hybridization to detect pathogens causing neonatal bacterial meningitis and relevant drug resistance genes

Abstract

Objective 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. Key words: Meningitis, bacterial; beta-Lactamases; Multiplex polymerase chain reaction; Nucleic acid hybridization; Drug resistance, bacterial; Infant, newborn