Molecular detection and characterization of bacteria from CSF samples of patients with suspected cerebrospinal meningitis in parts of northern Nigeria using metagenomic DNA extracts

Background: The most commonly used approaches for detection and characterization of bacterial pathogens of meningitis in developing countries include culture, Gram stain, and latex agglutination. The positivity rate of culture is relatively low due to suboptimal storage and transportation conditions, culture practice, and/or antibiotic treatment administered before specimens are collected. Specimens that yield no growth in culture can still be analyzed using molecular methods, and metagenomic DNA (mDNA) extracted directly from clinical samples (CSF) can be used. We aimed to detect and characterize three major bacterial causes of cerebrospinal meningitis (CSM); Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae using mDNA extracted directly from CSF samples. Methodology: Metagenomic DNA templates were prepared directly from CSF specimens collected from 210 patients with suspected CSM. A multiplex Real Time PCR (mRT-PCR) using the ABI StepOne Plus Machine and Taqman Probe chemistry was used in the molecular detection, while serogroup/serotype-specific singleplex RT-PCR was used to characterize all positives samples. Results: Eighty-eight (41.9%) of the 210 samples were positive with the mRT-PCR assay for one or a combination of two of the three bacteria. Of these, 59 (67.1%) were N. meningitidis, 2 (2.3%) were H. influenzae, 3 (3.4%) were S. pneumoniae, 15 (17 %) had co-infections of N. meningitidis with H. influenzae, and 9 (10.2%) had co-infections of H. influenzae and S. pneumoniae. The serogroups of N. meningitidis encountered were A (13.5%), B (23%), C (8.1%), W135 (8.1%), X (5.4%), Y (32.4%), and non-groupable (9.5%). The serotypes of H. influenzae were Hia (3.8%), Hib (57.7%), Hic (3.85%), Hie (11.5%) and Hif (23.1%). The serotypes of S. pneumoniae were Wxy1 (8.3%), Wxy4 (33.3%), Wxy5 (50.0%), and Wxy9 (8.3%). Conclusion: Multiplex RT-PCR is a fast and accurate method for detecting and characterizing serogroups/serotypes of major bacteria implicated in CSM. Isolating DNA directly from CSF improves turnaround time, which will speed up patient care and management. Keywords: Cerebrospinal meningitis, metagenomic DNA, multiplex Real Time PCR, Northern Nigeria French title: Detection moleculaire et caracterisation de bacteries a partir d'echantillons de LCR de patients suspectes de meningite cerebrospinale dans certaines parties du nord du Nigeria a l'aide d'extraits d'ADN metagenomique Contexte: Les approches les plus couramment utilisees pour la detection et la caracterisation des agents pathogenes bacteriens de la meningite dans les pays en developpement comprennent la culture, la coloration de Gram et l'agglutination au latex. Le taux de positivite de la culture est relativement faible en raison des conditions de stockage et de transport sous-optimales, des pratiques de culture et/ou du traitement antibiotique administre avant le prelevement des echantillons. Les echantillons qui ne donnent pas de croissance en culture peuvent toujours etre analyses a l'aide de methodes moleculaires, et l'ADN metagenomique (ADNm) extrait directement d'echantillons cliniques (LCR) peut etre utilise. Nous visions a detecter et a caracteriser trois causes bacteriennes majeures de la meningite cerebrospinale (CSM); Neisseria meningitidis, Haemophilus influenzae et Streptococcus pneumoniae a l'aide d'ADNm extrait directement d'echantillons de LCR. Methodologie: Des matrices d'ADN metagenomique ont ete preparees directement a partir d'echantillons de LCR preleves sur 210 patients suspects de CSM. Une PCR multiplex en temps reel (mRT-PCR) utilisant la chimie de la machine ABI StepOne Plus et de la sonde Taqman a ete utilisee pour la detection moleculaire, tandis que la RT-PCR monoplex specifique au serogroupe/serotype a ete utilisee pour caracteriser tous les echantillons positifs. Resultats: Quatre-vingt-huit (41,9%) des 210 echantillons etaient positifs avec le test mRT-PCR pour une ou une combinaison de deux des trois bacteries. Parmi ceux-ci, 59 (67,1%) etaient N. meningitidis, 2 (2,3%) etaient H. influenzae, 3 (3,4%) etaient S. pneumoniae, 15 (17%) avaient des co-infections de N. meningitidis avec H. influenzae et 9 (10,2%) avaient des co-infections a H. influenzae et S. pneumoniae. Les serogroupes de N. meningitidis rencontres etaient A (13,5%), B (23%), C (8,1%), W135 (8,1%), X (5,4%), Y (32,4%) et non groupables (9,5%). Les serotypes de H. influenzae etaient Hia (3,8%), Hib (57,7%), Hic (3,85%), Hie (11,5%) et Hif (23,1%). Les serotypes de S. pneumoniae etaient Wxy1 (8,3%), Wxy4 (33,3%), Wxy5 (50,0%) et Wxy9 (8,3%). Conclusion: La RT-PCR multiplex est une methode rapide et precise de detection et de caracterisation des serogroupes/serotypes des principales bacteries impliquees dans le CSM. Isoler l'ADN directement du LCR ameliore le temps de traitement, ce qui accelerera les soins et la gestion des patients. Mots cles: meningite cerebro-spinale, ADN metagenomique, PCR multiplex en temps reel, nord du Nigeria