Rapid Diagnosis and Discrimination of Bacterial Meningitis in Children Using Gram Probe Real-Time Polymerase Chain Reaction

Abstract

In this study, we developed a method of simultaneous detection and discrimination of bacteria in cerebrospinal fluid (CSF) with gram probe real-time polymerase chain reaction (PCR). Our results showed 25 clinical strains representing 13 gram-positive and 12 gram-negative bacterial species. They were identified correctly with the corresponding gram probe. The standard curve showed that the amplification efficiency of templates with different concentrations of bacteria was almost the same with a potential detection limit of 10 colony-forming units/mL. A total of 482 children who were clinically suspected of bacterial meningitis were included in this study. A total of 1.0 mL of CSF was collected from every child and was subjected to gram probe–based PCR (GP-PCR), CSF culture, and CSF routine analysis. The positive rate of the GP-PCR array was (32/482, 6.64%) significantly higher than that of CSF culture (23/482, 4.77%). GP-PCR was proved to be an excellent technique for rapid and accurate diagnosis and discrimination of bacterial meningitis, and hence its use as a diagnostic tool in future seems very promising.

Keywords

bacterial meningitis gram probe real-time PCR children

Get full access to this article

View all access options for this article.

References

Mahmoudi

Zandi

Pourakbari

Ashtiani

Mamishi

. Acute bacterial meningitis among children admitted into an Iranian referral children’s hospital. Jpn J Infect Dis. 2013;66:503-506.

Chen

Wang

Zeng

. The clinical diagnostic significance of cerebrospinal fluid d-lactate for bacterial meningitis. Clin Chim Acta. 2012;413:1512-1515.

Ben

Kung

Chang

Feng

Hsieh

. Rapid diagnosis of bacterial meningitis using a microarray. J Formos Med Assoc. 2008;107:448-453.

Chen

Duan

Cai

Shang

. Rapid diagnosis of sepsis and bacterial meningitis in children with real-time fluorescent quantitative polymerase chain reaction amplification in the bacterial 16S rRNA gene. Clin Pediatr (Phila). 2009;48:641-647.

Duan

Shang

. Rapid diagnosis of bacterial meningitis in children with fluorescence quantitative polymerase chain reaction amplification in the bacterial 16S rRNA gene. Eur J Pediatr. 2009;168:211-216.

Taha

Fox

. Quality assessed nonculture techniques for detection and typing of meningococci. FEMS Microbiol Rev. 2007;31:37-42.

Janner

Probert

McClure

. Ribosomal DNA assay of culture-negative Streptococcus pneumoniae meningitis. Pediatr Neurol. 2007;37:55-58.

Pandit

Kumar

Karunasagar

. Diagnosis of partially treated culture-negative bacterial meningitis using 16S rRNA universal primers and restriction endonuclease digestion. J Med Microbiol. 2005;54(pt 6):539-542.

Møller

. Detection of Neisseria meningitidis in cerebrospinal fluid using a multiplex PCR and the Luminex detection technology. Methods Mol Biol. 2012;799:37-53.

10.

Zhu

Wang

Wen

. Development of a multiplex PCR assay for detection and genogrouping of Neisseria meningitidis. J Clin Microbiol. 2012;50:46-51.

11.

Deutch

Møller

Ostergaard

. Combined assay for two-hour identification of Streptococcus pneumoniae and Neisseria meningitidis and concomitant detection of 16S ribosomal DNA in cerebrospinal fluid by real-time PCR. Scand J Infect Dis. 2008;40:607-614.

12.

Picazo

Contreras

Ríos

. Rapid diagnosis of invasive pneumococcal disease in pediatric population. J Microbiol Methods. 2013;93:116-120.

13.

Chen

. Gram stain-specific-probe-based real-time PCR for diagnosis and discrimination of bacterial neonatal sepsis. J Clin Microbiol. 2008;46:2613-2619.