BACKGROUND: Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis. METHODS: Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis. RESULTS: The lower detection limits ranged from 101 copies/microL to 5x101 copies/microL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent. CONCLUSIONS: The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens.
Acute Disease ; Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Meningitis/*diagnosis/microbiology/virology ; Middle Aged ; *Polymerase Chain Reaction ; RNA, Bacterial/cerebrospinal fluid ; RNA, Viral/cerebrospinal fluid ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Sequence Analysis, RNA

BACKGROUND: The enteroviruses are the most common etiologic agent of aseptic meningitis in adults and children. The incidence of enteroviral meningitis in childhood meningitis is up to 80%, but in adults is not known, worldwidely. In Korea, where tuberculosis is endemic, the rapid and accurate diagnostic method for enteroviral meningitis is required especially because early differential diagnosis of viral meningitis from tuberculous meningitis is very important. The aims of this study were the demonstration of enteroviruses from cerebrospinal fluid (CSF) of adult patients with aseptic meningitis by PCR/Southern hybridization and the verification of the usefulness of PCR/southern hybridization as a rapid diagnostic tool. METHODS: From July 1992 to June 1995, total 34 CSF samples (10 from children, 24 from adults) of patients with aseptic meningitis were studied. As a control group, 15 patients with tuberculous meningitis and 15 patients with bacterial meningitis were studied. Viral RNA was extracted from CSF, reverse transcriptied into cDNA and amplified. The PCR products were Southern hybridizied with enteroviruses-specific digoxigenin-labelled probe. RESULTS: 16/24(66.7%) samples of adult patients with aseptic meningitis were positive for enteroviruses, while in child patients with aseptic meningitis, 9/10(90%) samples were positive. And in one patient, PCR was positive from asymptomatic, onset-7th day CSF sample. CONCLUSION: Enteroviruses were the most common causative organisms of adult aseptic meningitis in Korea. And, this study showed the usefulness of PCR/Southern hybridization of enteroviruses from CSF for etiologic diagnosis of adult aseptic meningitis in subclinical, asymptomatic period.
Adult* ; Cerebrospinal Fluid* ; Child ; Diagnosis ; Diagnosis, Differential ; DNA, Complementary ; Enterovirus* ; Humans ; Incidence ; Korea ; Meningitis ; Meningitis, Aseptic* ; Meningitis, Bacterial ; Meningitis, Viral ; Polymerase Chain Reaction ; RNA, Viral ; Tuberculosis ; Tuberculosis, Meningeal

OBJECTIVETo establish a new method of multiplex semi-nested polymerase chain reaction (PCR) to detect pathogens in cerebrospinal fluid (CSF).

METHODSAccording to the analysis of the conservative and variable regions in bacterial 16S rRNA genes, we designed universal primers for all bacteria and specific primers for most gram-positive and gram-negative bacteria. All primers were added into the same reaction systems successively of a two-step PCR assay to amplify the different bacterial DNA in CSF, and the results were compared with common culture method with sensitivity and the specificity both detected at the same time.

RESULTSBoth gram-positive and gram-negative bacteria amplified DNA fragment about 1,032 bp after first-step amplification with universal primers. In the second step, specific fragments of 336 bp and 127 bp were amplified in gram-positive and gram-negative bacteria respectively besides fragments of 1,032 bp; The detection limit for E. coli was 8 cfu/ml. The comparison of 62 CSF samples detected by both multiplex semi-PCR and conventional culture method revealed sensitivity, specificity, positive and negative values of 93.8%, 95.7%, 88.2%, and 97.8% respectively for PCR.

CONCLUSIONThe result suggested that the multiplex semi-nested PCR we established was sensitive, specific and rapid method for clinical laboratory to detect pathogens in CSF.

Cerebrospinal Fluid ; microbiology ; DNA Primers ; genetics ; DNA Probes ; genetics ; DNA, Bacterial ; cerebrospinal fluid ; isolation & purification ; Escherichia coli ; genetics ; isolation & purification ; Gram-Negative Bacteria ; genetics ; isolation & purification ; Gram-Positive Bacteria ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Bacterial ; genetics ; RNA, Ribosomal, 16S ; genetics ; Sensitivity and Specificity ; Staphylococcus aureus ; genetics ; isolation & purification

Streptococcus suis infection is an emerging zoonosis in Asia. The most common disease manifestation is meningitis, which is often associated with hearing loss and cochleovestibular signs. S. suis infection in humans mainly occurs among risk groups that have frequent exposure to pigs or raw pork. Here, we report a case of S. suis meningitis in a 67-yr-old pig carcass handler, who presented with dizziness and sensorineural hearing loss followed by headaches. Gram-positive diplococci were isolated from cerebrospinal fluid (CSF) and blood cultures and showed gray-white colonies with alpha-hemolysis. S. suis was identified from CSF and blood cultures by using a Vitek 2 system (bioMerieux, France), API 20 STREP (bioMerieux), and performing 16S rRNA and tuf gene sequencing. Even after receiving antibiotic treatment, patients with S. suis infection frequently show complications such as hearing impairment and vestibular dysfunction. To the best of our knowledge, this is the first case of S. suis meningitis in Korea. Prevention through public health surveillance is recommended, especially for individuals who have occupational exposures to swine and raw pork.
Aged ; Animals ; Bacterial Proteins/genetics ; Blood/microbiology ; Cerebrospinal Fluid/microbiology ; Hearing Loss, Bilateral/complications/*diagnosis/microbiology ; Humans ; Male ; Meningitis, Bacterial/complications/*diagnosis/microbiology ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; Sequence Analysis, DNA ; Streptococcus suis/classification/genetics/*isolation & purification ; Swine ; Tomography, X-Ray Computed

No abstract available.
Aged ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Bacteremia/blood/*diagnosis/microbiology ; C-Reactive Protein/analysis ; Cholecystitis/blood/cerebrospinal fluid/microbiology ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus faecium/drug effects/isolation & purification/metabolism ; Humans ; Male ; Microbial Sensitivity Tests ; Microscopy, Electron, Scanning ; Ochrobactrum/drug effects/isolation & purification/*metabolism ; RNA, Ribosomal, 16S/analysis/genetics/metabolism ; Sequence Analysis, DNA ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization