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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. [ABSTRACT FROM AUTHOR]

Background: Following an increase in the practice of starting antimicrobial therapy prior to clinical sample collection, the ability to confirm pathogenic microorganisms of bacterial meningitis has decreased by approximately 30%. Culture results may be false negative when fastidious or culture-resistant bacteria are involved or when patient samples are obtained after antimicrobial therapy has started. Molecular diagnosis using PCR can be performed directly on clinical samples after metagenomic DNA (mDNA) extraction not requiring live organisms for a positive result. The specific objectives of this study are to perform mDNA extraction directly from cerebrospinal fluids (CSF) using appropriate spin column method, and to determine the quality of the mDNA elute. Methodology: Cerebrospinal fluid specimens were collected from 210 patients with suspected acute cerebrospinal meningitis (CSM) in the Federal Capital Territory and some States in Northern Nigeria during the 2017 and 2018 outbreak seasons. Metagenomic DNA was extracted from approximately 200µL of CSF specimens using the Qiagen QIAamp(R) DNA Mini kit specific for bacterial agents only. DNA quality check was performed on all DNA elutes using fluorometric, spectrophotometric and agarose gel electrophoresis methods. Results: Of the 210 CSF samples analyzed microbiologically, Gram reaction was positive in 94 cases (44.8 %) but only 17 (8.1 %) were culture positive for two of the three major bacterial causes of meningitis. One hundred and eighty (85.7%) samples had DNA concentrations ≥ 0.005 ng/µL, 55 (30.6 %) of these had DNA purity (A260/A280) of ≥ 1.7, 103 (57.2%) had purity value between 1.0 - 1.69, 14 (7.8%) had value of 0.57 - 0.99, and 8 (4.4%) failed purity evaluation with value of 0.00 at A260/A280. Conclusion: The essence of mDNA extraction is multipurpose. A multiplex PCR can be performed on the extracted mDNA to interrogate the presence of microbial pathogens of interest using specific primers and probes (when applicable). Quality mDNA from CSF samples will ensure successful qPCR results for rapid and accurate detection of bacterial pathogens in meningitis. This will eliminate the challenges associated with traditional culture methods. [ABSTRACT FROM AUTHOR]