A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid

  • Idrissa Diawara Faculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, Morocco
  • Khalid Katfy University Hospital Centre Ibn Rochd, Casablanca, Morocco
  • Khalid Zerouali Faculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, Morocco
  • Houria Belabbes Faculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, Morocco
  • Naima Elmdaghri Faculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, Morocco
Keywords: Neisseria meningitidis, Streptococcus pneumoniae, meningitis, RT-PCR, melting curve analysis

Abstract

Introduction: Acute bacterial meningitis is one of the most severe infectious diseases. Rapid, accurate, and inexpensive diagnosis of bacterial meningitis is crucial for patient management. This study describes a duplex real-time (RT) PCR assay for detection of Neisseria meningitidis and Streptococcus pneumoniae in the cerebrospinal fluid (CSF) for meningitis diagnosis using SYBR Green-based RT-PCR method coupled with melting curve analysis.

Methodology: We used SYBR Green-based RT-PCR method coupled with melting curve analysis to detect S. pneumoniae and N. meningitidis in CSF samples. The sensitivity, specificity, and limit of detection were determined. The gold standard for routine tests of CSF analysis is direct examination, culture, and/or latex agglutination. The assay was evaluated on 132 CSF samples to measure clinical sensitivity.

Results: A duplex RT-PCR assay for N. meningitidis and S. pneumoniae detection in CSF was evaluated. Two peaks at different melting temperatures (87.5°C and 85.5°C) for N. meningitidis and S. pneumoniae, respectively, were obtained. The sensitivity of RT-PCR was 100% (95% confidence limits [CI] = 82.4–100) for N. meningitidis and 100% (95% CI = 85.1–100) for S. pneumoniae. Specificity was the same (100%) for the bacteria (95% CI = 88.6–100). The percentage of cases accurately diagnosed with meningitis caused by N. meningitidis and S. pneumoniae increased to 50.7% and 28.6%, respectively, when RT-PCR was added to the standard microbiologic methods.

Conclusions: Duplex RT-PCR and melting curve analysis with SYBR Green is an inexpensive, sensitive, and specific method to rapidly diagnose bacterial meningitis. Accurate identification of the bacterial causative agents will improve patient management and epidemiological investigations.

Author Biographies

Idrissa Diawara, Faculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, Morocco

Phd student in Microbiology,

Department of Microbiology, Faculty of Medicine and Pharmacy

Khalid Katfy, University Hospital Centre Ibn Rochd, Casablanca, Morocco
Bacteriology, Virology and Hospital Hygiene Laboratory, University Hospital Centre Ibn Rochd
Khalid Zerouali, Faculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, Morocco

Professor of Microbiology,

Department of Microbiology, Faculty of Medicine and Pharmacy

Houria Belabbes, Faculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, Morocco
Professor of Microbiology,
Department of Microbiology, Faculty of Medicine and Pharmacy
Naima Elmdaghri, Faculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, Morocco
Professor of Microbiology,
Department of Microbiology, Faculty of Medicine and Pharmacy
Published
2016-01-31
How to Cite
1.
Diawara I, Katfy K, Zerouali K, Belabbes H, Elmdaghri N (2016) A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid. J Infect Dev Ctries 10:53-61. doi: 10.3855/jidc.5647
Section
Original Articles