An approach to the identification of the pathogens of bacterial meningitis by the polymerase chain reaction

Hall, Lucinda M. C.; Duke, B.; Urwin, Gillian

doi:10.1007/bf01590946

Cited by 46 publications

(31 citation statements)

References 11 publications

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“…Previous studies assessing the use of PCR-based strategies for the diagnosis of meningococcal meningitis in blood and CSF [e.g., 3, 5±8] have resolved the PCR ampli®cation products on agarose gels [5] or have relied on detection and identi®cation by ELISA [3] or DNA sequencing [7,8]. The present study investigated the possibility of making the PCR technique more accessible to routine diagnostic laboratories by the use of a simple and cheap immunoassay device for detection of PCR products.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of a PCR-immunoassay technique for detection of Neisseria meningitidis in cerebrospinal fluid and peripheral blood

Seward

Towner

2000

Journal of Medical Microbiology

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Section: Discussionmentioning

confidence: 99%

Evaluation of a PCR-immunoassay technique for detection of Neisseria meningitidis in cerebrospinal fluid and peripheral blood

Seward

Towner

2000

Journal of Medical Microbiology

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“…Several studies have demonstrated the usefulness of eubacterial broad-range PCRs for the diagnosis of bacterial meningitis (3,6,13,14,23). However, most published PCR protocols were either time-consuming or did not facilitate species diagnosis of the bacterial pathogens.…”

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confidence: 99%

Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

et al. 2005

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Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy-and culture-positive samples (n ‫؍‬ 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy-and culture-negative samples (n ‫؍‬ 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.

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“…Varying degrees of success have been realized using PCR-based assays for detecting S. pneumoniae with primers specific to repetitive regions or to genes encoding rRNA (7,9,12), pneumolysin (18,19,23), or autolysin (5,14,18). Autolysin and pneumolysin, which are encoded by the lytA and ply genes, respectively, represent potential targets for the specific detection of S. pneumoniae.…”

mentioning

confidence: 99%

Loop-Mediated Isothermal Amplification Method Targeting the lytA Gene for Detection of Streptococcus pneumoniae

Seki

Yamashita

Torigoe³

et al. 2005

J Clin Microbiol

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It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulencefactor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.Streptococcus pneumoniae is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media (2). While traditional antimicrobial therapy has proven to be an effective treatment, the emergence of antimicrobial-resistant strains has resulted in an increasing number of cases (10). The isolation and identification of pneumococcus is complicated by contamination with alpha-hemolytic streptococci belonging to the normal flora. Pneumococcal detection using classical techniques, including growth-based assays, colonial morphology, optochin susceptibility, bile solubility, and serology, can be time-consuming and can produce equivocal results. Sensitive and specific assays that can be completed promptly in the clinical laboratory are essential for early diagnosis and effective antibiotic therapy. Molecular assays are inherently valuable because detection can be achieved with enhanced sensitivity and specificity and detection is not diminished with nonviable organisms.The molecular methods that have been used to date (3, 6, 17) are expensive and complicated to perform. Varying degrees of success have been realized using PCR-based assays for detecting S. pneumoniae with primers specific to repetitive regions or to genes encoding rRNA (7, 9, 12), pneumolysin (18,19,23), or autolysin (5, 14, 18). Autolysin and pneumolysin, which are encoded by the lytA and ply genes, respectively, represent potential targets for the specific detection of S. pneumoniae. Both genes are essential for pathogenesis and are well-characterized virulence markers (1). The restricted allelic variation of lytA (4, 21) makes this gene an ideal target for specific identification in clinical and epidemiological studies. For example, the gene may allow the differentiation of S. pneumoniae from the genotypically similar species Streptococcus mitis and...

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An approach to the identification of the pathogens of bacterial meningitis by the polymerase chain reaction

Cited by 46 publications

References 11 publications

Evaluation of a PCR-immunoassay technique for detection of Neisseria meningitidis in cerebrospinal fluid and peripheral blood

Evaluation of a PCR-immunoassay technique for detection of Neisseria meningitidis in cerebrospinal fluid and peripheral blood

Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

Loop-Mediated Isothermal Amplification Method Targeting the lytA Gene for Detection of Streptococcus pneumoniae

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