title: Employment of broad range 16S rDNA PCR and sequencing in the detection of aetiological agents of meningitis.
abstract: 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.
authors: Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast, BT9 7AD, UK.
authors: Jiru
authors: J
authors: Xu
authors: John E
authors: JE
authors: Moore
authors: Beverley C
authors: BC
authors: Millar
authors: Hugh
authors: H
authors: Webb
authors: Michael D
authors: MD
authors: Shields
authors: Colin E
authors: CE
authors: Goldsmith
mesh_terms: D000150:Acinetobacter / Q000235:genetics / Q000302:isolation & purification; D001769:Blood / Q000382:microbiology; D002555:Cerebrospinal Fluid / Q000382:microbiology; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000235:genetics*; D004866:Equipment Contamination; D006801:Humans; D016920:Meningitis, Bacterial / Q000175:diagnosis* / Q000382:microbiology*; D009345:Neisseria meningitidis / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D011549:Pseudomonas / Q000235:genetics / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013291:Streptococcus / Q000235:genetics / Q000302:isolation & purification
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S