TY - JOUR AU - Haddad-Boubaker, Sondes AU - Lakhal, Marwa AU - Fathallah, Cyrine AU - Bouafsoun, Aida AU - Kharrat, Maher AU - Khemiri, Monia AU - Kechrid, Amel AU - Smaoui, Hanen PY - 2018/04/30 Y2 - 2024/03/29 TI - Molecular diagnosis of bacterial meningitis by multiplex real time PCR in Tunisian children JF - The Journal of Infection in Developing Countries JA - J Infect Dev Ctries VL - 12 IS - 04 SE - Original Articles DO - 10.3855/jidc.9650 UR - https://jidc.org/index.php/journal/article/view/31851632 SP - 235-243 AB - <p>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 <em>N. meningitidis</em>, <em>S. </em><em>pneumoniae </em>and <em>H. influenzae b</em> from cerebrospinal fluid in Tunisian children beyond neonatal age.</p><p>Methodology: Using bioinformatic tools and experimentation, we validated the specificity and optimal criteria of PCRs for primers and probes of <em>plyA</em> (<em>S. pneumoniae</em>), <em>ctrA </em>and <em>sodC</em> (<em>N. meningitidis</em>) and<em> bexA</em> genes (<em>H. influenzae b</em>). We performed one multiplex RT-PCR for detection of <em>S. pneumoniae </em>and<em> N. </em>meningitidis targeting <em>plyA</em> and <em>ctrA, sodC</em> genes respectively, simultaneously with a singleplex RT-PCR for <em>H. influenzae </em>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.</p><p>Results: Our results have shown the sensitivity of the designed PCRs was up to 10<sup>-4 </sup>DNA dilution and the specificity was 100%. PCR evaluation has shown 51 positive samples: 38 of pneumococcal cases, 12 meningococcal cases, 1 case of <em>H. influenzae</em> b with 8.57% and 50% of supplementary positive cases rates respectively.</p><p>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 <em>S. pneumoniae </em>and <em>N. meningitidis </em>separately constitute an attractive opportunity. Nevertheless, simultaneous detection of Hib remains relevant in specific clinical context and for epidemiologic study.</p> ER -