Xu Jiru, Moore John E, Millar Beverley C, Webb Hugh, Shields Michael D, Goldsmith Colin E
Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast, BT9 7AD, UK.
New Microbiol. 2005 Apr;28(2):135-43.
To employ partial 16S rDNA PCR and automated sequencing technique to identify non-culturable causal agents of bacterial meningitis, 73 peripheral blood samples and 413 culture-negative and eight culture-positive CSF clinical specimens from patients with suspected acute meningitis were examined for the presence of bacterial genomic DNA employing broad range 16S rDNA PCR followed by sequencing of the amplicons. In blood samples, 63/73 specimens were PCR positive (86.3%) and after direct sequencing of the PCR amplicons, only 12.7% (8/63) gave clear sequencing results and 55/63 (87.3%) were mixed with more than one organism detected. The mixed PCR amplicons were separated by using PAGE and mixed amplicons from 29/55 (52.7%) specimens were successfully identified through sequencing. Of the CSF samples, 8/8 culture-positive samples were also PCR positive and 45/413 (10.9%) of culture-negative gave a strong PCR signal and 88/413 (21.3%) specimens yielded a weak PCR signal. The remaining 280 culture-negative specimens were also PCR negative. Nested PCR was set up for the 88 weak positive samples and yielded 72/88 (81.8%) strong positives, with the remainder failing to amplify 133/413 (32.2%) culture-negative samples were PCR positive. In this study, the most common bacteria identified from blood specimens were Neisseria meningitidis, 13/63 (20.6%); Streptococcus spp, 5/63 (7.9%); Acinetobacter spp and Pseudomonas spp 4/63 (6.3%). From culture-negative CSF, the pattern was different in that Staphylococcus spp (13/58, 22.4%), Neisseria meningitidis (9/58, 15.52%) and Pseudomonas spp (8/58, 14.79%), were the most frequent. Overall, 16S rRNA broad-range PCR combined with direct DNA sequencing is a valuable molecular tool to aid with the detection as well as identification of non-culturable aetiological agents of acute bacterial meningitis and can augment cultural methods in the diagnosis of central nervous system infections in patients who have been treated with antibiotics. However, this study demonstrates that contamination is an important complication of the molecular assay, which should be attempted to be eliminated through careful laboratory controls. Hence there should be careful interpretation of any molecular finding, in tandem with other laboratory findings, such as culture, immunological and biochemical markers, and the clinical scenario of the patient.
为采用部分16S rDNA聚合酶链反应（PCR）和自动测序技术鉴定细菌性脑膜炎不可培养的病原体，对73份外周血样本以及413份来自疑似急性脑膜炎患者的脑脊液（CSF）临床标本（413份培养阴性，8份培养阳性）进行检测，利用广谱16S rDNA PCR检测细菌基因组DNA的存在情况，随后对扩增产物进行测序。在血样中，63/73份标本PCR呈阳性（86.3%），对PCR扩增产物进行直接测序后，只有12.7%（8/63）得到清晰的测序结果，55/63（87.3%）检测到多种微生物混合存在。通过聚丙烯酰胺凝胶电泳（PAGE）分离混合的PCR扩增产物，29/55（52.7%）份标本的混合扩增产物经测序成功鉴定。在脑脊液样本中，8/8份培养阳性样本PCR也呈阳性，45/413（10.9%）份培养阴性样本给出强PCR信号，88/413（21.3%）份标本产生弱PCR信号。其余280份培养阴性标本PCR也为阴性。对88份弱阳性样本进行巢式PCR，72/88（81.8%）呈强阳性，其余未能扩增。133/413（32.2%）份培养阴性样本PCR呈阳性。在本研究中，血标本中鉴定出的最常见细菌为脑膜炎奈瑟菌，13/63（20.6%）；链球菌属，5/63（7.9%）；不动杆菌属和假单胞菌属，4/63（6.3%）。在培养阴性的脑脊液中，模式有所不同，葡萄球菌属（13/58，22.4%）、脑膜炎奈瑟菌（9/58，15.52%）和假单胞菌属（8/58，14.79%）最为常见。总体而言，16S rRNA广谱PCR结合直接DNA测序是一种有价值的分子工具，有助于检测和鉴定急性细菌性脑膜炎不可培养的病原体，并且可以在诊断接受过抗生素治疗患者的中枢神经系统感染时补充培养方法。然而，本研究表明污染是分子检测的一个重要并发症，应通过严格的实验室控制予以消除。因此，应结合其他实验室检查结果，如培养、免疫学和生化标志物以及患者的临床情况，谨慎解读任何分子检测结果。
