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

Sondes Haddad-Boubaker,Cyrine Fathallah,Aida Bouafsoun,Monia Khemiri,Amel Kechrid,Marwa Lakhal,Hanen Smaoui,Maher Kharrat

doi:10.3855/jidc.9650

Sondes Haddad-Boubaker, Cyrine Fathallah + Show 6 more

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https://doi.org/10.3855/jidc.9650

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Abstract

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. 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. 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. 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.

Highlights

Bacterial meningitis is a medical emergency requiring a fast and reliable diagnosis
The analysis results showed that conventional microbiological methods detected among the 122 cerebrospinal fluid (CSF) samples, 31.15% of S. pneumoniae, 9.02% of N. meningitidis and 0.82% of Haemophilus influenzae b (Hib) meningitis cases, whereas the designed RT-PCR assays increased the rate of detection for pneumococcal by 8.57% and meningococcal cases by 50%
By comparing conventional methods to real-time PCR (rt-PCR) results, we found that difference in detection was significant especially for the N. meningitidis

Summary

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. The main diagnosis is the examination of the cerebrospinal fluid (CSF) [2]; it includes CSF culture, the “gold standard” method, CSF count cell and CSF staining and immunological diagnosis such as latex agglutination test and immunochromatography test [5,6] These methods have shown limits in assessing bacterial meningitis diagnosis resulting from low bacterial titre in the CSF, long incubation period in bacterial culture, cross reaction in immunological methods and mostly antibiotic pretreatment prior to clinical sample collection [1,3,6,7]

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