Molecular diagnosis of bacterial meningitis by multiplex real time PCR in Tunisian children

  • Sondes Haddad-Boubaker Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia
  • Marwa Lakhal Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia
  • Cyrine Fathallah Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia
  • Aida Bouafsoun Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia
  • Maher Kharrat University Tunis El Manar, Tunis, Tunisia
  • Monia Khemiri University Tunis El Manar, Tunis, Tunisia
  • Amel Kechrid Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia
  • Hanen Smaoui Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia
Keywords: N. meningitidis, S. pneumoniae, H. influenzae b, multiplex real-time PCR, bacterial meningitis, children

Abstract

Introduction: Bacterial meningitis is a medical emergency requiring a fast and reliable diagnosis. Molecular methods such as real-time PCR (rt-PCR) offer an attractive alternative. Thus, this study aims to establish multiplex rt-PCRs detecting N. meningitidis, S. pneumoniae and H. influenzae b from cerebrospinal fluid in Tunisian children beyond neonatal age.

Methodology: Using bioinformatic tools and experimentation, we validated the specificity and optimal criteria of PCRs for primers and probes of plyA (S. pneumoniae), ctrA and sodC (N. meningitidis) and bexA genes (H. influenzae b). We performed one multiplex RT-PCR for detection of S. pneumoniae and N. meningitidis targeting plyA and ctrA, sodC genes respectively, simultaneously with a singleplex RT-PCR for H. influenzae b. The sensitivity and specificity of our methods were assessed. Then, we tested our methods for 122 CSF samples collected from suspected meningitis cases between 2014 and 2016 in Bechir Hamza Children’s Hospital of Tunis.

Results: Our results have shown the sensitivity of the designed PCRs was up to 10-4 DNA dilution and the specificity was 100%. PCR evaluation has shown 51 positive samples: 38 of pneumococcal cases, 12 meningococcal cases, 1 case of H. influenzae b with 8.57% and 50% of supplementary positive cases rates respectively.

Conclusions: Our assay proved to be very sensitive, specific and rapid for bacterial meningitis diagnosis. In the recent context of Hib vaccination, the possibility of detecting S. pneumoniae and N. meningitidis separately constitute an attractive opportunity. Nevertheless, simultaneous detection of Hib remains relevant in specific clinical context and for epidemiologic study.

Author Biographies

Sondes Haddad-Boubaker, Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia

University Tunis El Manar, Faculty of Medicine, Bechir Hamza Children’s Hospital, Laboratory of Microbiology, 1006 Tunis, Tunisia

Assistant Professor

Marwa Lakhal, Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia

University Tunis El Manar, Faculty of Medicine, Bechir Hamza Children’s Hospital, Laboratory of Microbiology, 1006 Tunis, Tunisia

Master degree student

Cyrine Fathallah, Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia

University Tunis El Manar, Faculty of Medicine, Bechir Hamza Children’s Hospital, Laboratory of Microbiology, 1006 Tunis, Tunisia

PhD student

Aida Bouafsoun, Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia

University Tunis El Manar, Faculty of Medicine, Bechir Hamza Children’s Hospital, Laboratory of Microbiology, 1006 Tunis, Tunisia

Assistant Professor

Maher Kharrat, University Tunis El Manar, Tunis, Tunisia

University Tunis El Manar, Faculty of Medicine, Laboratory of Human Genetics, Tunis, Tunisia

Professor

Amel Kechrid, Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia

University Tunis El Manar, Faculty of Medicine, Bechir Hamza Children’s Hospital, Laboratory of Microbiology, 1006 Tunis, Tunisia

Professor

Hanen Smaoui, Béchir Hamza Children’s Hospital, University Tunis El Manar, Tunis, Tunisia

University Tunis El Manar, Faculty of Medicine, Bechir Hamza Children’s Hospital, Laboratory of Microbiology, 1006 Tunis, Tunisia

Professor

Published
2018-04-30
How to Cite
1.
Haddad-Boubaker S, Lakhal M, Fathallah C, Bouafsoun A, Kharrat M, Khemiri M, Kechrid A, Smaoui H (2018) Molecular diagnosis of bacterial meningitis by multiplex real time PCR in Tunisian children. J Infect Dev Ctries 12:235-243. doi: https://doi.org/10.3855/jidc.9650
Section
Original Articles