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: Détection moléculaire et caractérisation de bactéries à partir d'échantillons de LCR de patients suspectés de méningite cérébrospinale dans certaines parties du nord du Nigéria à l'aide d'extraits d'ADN métagénomique   Contexte: Les approches les plus couramment utilisées pour la détection et la caractérisation des agents pathogènes bactériens de la méningite dans les pays en développement comprennent la culture, la coloration de Gram et l'agglutination au latex. Le taux de positivité 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 administré avant le prélèvement des échantillons. Les échantillons qui ne donnent pas de croissance en culture peuvent toujours être analysés à l'aide de méthodes moléculaires, et l'ADN métagénomique (ADNm) extrait directement d'échantillons cliniques (LCR) peut être utilisé. Nous visions à détecter et à caractériser trois causes bactériennes majeures de la méningite cérébrospinale (CSM); Neisseria meningitidis, Haemophilus influenzae et Streptococcus pneumoniae à l'aide d'ADNm extrait directement d'échantillons de LCR. Méthodologie: Des matrices d'ADN métagénomique ont été préparées directement à partir d'échantillons de LCR prélevés sur 210 patients suspects de CSM. Une PCR multiplex en temps réel (mRT-PCR) utilisant la chimie de la machine ABI StepOne Plus et de la sonde Taqman a été utilisée pour la détection moléculaire, tandis que la RT-PCR monoplex spécifique au sérogroupe/sérotype a été utilisée pour caractériser tous les échantillons positifs. Résultats: Quatre-vingt-huit (41,9%) des 210 échantillons étaient positifs avec le test mRT-PCR pour une ou une combinaison de deux des trois bactéries. Parmi ceux-ci, 59 (67,1%) étaient N. meningitidis, 2 (2,3%) étaient H. influenzae, 3 (3,4%) étaient S. pneumoniae, 15 (17%) avaient des co-infections de N. meningitidis avec H. influenzae et 9 (10,2%) avaient des co-infections à H. influenzae et S. pneumoniae. Les sérogroupes de N. meningitidis rencontrés étaient A (13,5%), B (23%), C (8,1%), W135 (8,1%), X (5,4%), Y (32,4%) et non groupables (9,5%). Les sérotypes de H. influenzae étaient Hia (3,8%), Hib (57,7%), Hic (3,85%), Hie (11,5%) et Hif (23,1%). Les sérotypes de S. pneumoniae étaient Wxy1 (8,3%), Wxy4 (33,3%), Wxy5 (50,0%) et Wxy9 (8,3%). Conclusion: La RT-PCR multiplex est une méthode rapide et précise de détection et de caractérisation des sérogroupes/sérotypes des principales bactéries impliquées dans le CSM. Isoler l'ADN directement du LCR améliore le temps de traitement, ce qui accélérera les soins et la gestion des patients. Mots clés: méningite cérébro-spinale, ADN métagénomique, PCR multiplex en temps réel, nord du Nigéri

Quality of metagenomic DNA extracted for molecular identification of microorganisms from CSF samples of patients with suspected cerebrospinal meningitis in northern Nigeria

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. Keywords: Meningitis, CSF, DNA Quality Check, Fluorometr

Methicilin-resistant Staphylococcus aureus (MRSA) at Jos University Teaching Hospital.

A prospective surveillance of Methicillin resistant staphylococcus aureus (MRSA) was carried out at Jos University Teaching Hospital, Nigeria, over a one year period. This study highlights the continuos importance of MRSA in causing both hospital and to a less extent community acquired infections. Out of the 180 consecutive isolates of S. aureus tested, 758 (43%) were found to be methicillin resistant, 81% (63 isolates) of the MRSA were from hospital in-patients while 19% (15 isolates) were from out-patients. The highest rate of methicillin resistance (81%) was found in surgical wound infections while the special care baby unity (SCBU) service recorded 4%. 85% of the MRSA were sensitive to Ofloxacilin while 46% were sensitive to peflacine. Most MRSA isolates were multiply resistant to Augumentin, centriaxone and ceftazidime, thus confirming the nosocomial nature of the isolates. Vancomycin and teicoplanin are not locally available and so ofloxacillin is the drug of choice. This study has demonstrated a high prevalence of MRSA in our hospital, which definitely plays a significant role in hospital acquired inflections. In conclusion, the relatively high prevalence of MRSA in this study has shown that there is a “limited” level of infection control activity in our hospital. (Af. J. of Clinical and Experimental Microbiology: 2003 4(1): 52-55

STUDIES ON DIABETIC FOOT ULCERS IN PATIENTS AT JOS UNIVERSITY TEACHING HOSPITAL, NIGERIA

An epidemiologcal and microbiological studies of diabetic foot ulcers were carried out in our hospital, with a view to reducing the amputation and mortality rate associated with the disease. Wound swabs from 38 Diabetes Mellitus (DM) foot ulcer patients were investigated using culture methods for both strict aerobes and anaerobes. The bacterial isolates were subjected to antibiotic susceptibility tests using the disc diffusion method. Baseline biochemical and haematological analysis were also carried out. The prevalence of the disease was stratified in relation to some clinical and laboratory parameters, gender, age, educational and occupational status of the patients. The prevalence of the disease was 24.7%, with amputation and mortality rates of 18.4% and 15.8% respectively. Only 13% had DM for less than 1 year, while 53% for more than 10 years. 28.9% have regular shoe-wearing habits. Duration of healing ranged from 2 weeks to 24 weeks (mean = 2.7months). 31% of the patients with marked periosteal reaction had lower extremity amputation or died before amputation could be done. Staphylococus aureus (31%), Proteus spp (16%), Pseudomonas aeruginosa (10%), Klebsiella spp (6%), Peptococcus spp (6%), Bacteroides fragilis (3%), Streptococcus pyogenes (3%), Escherichia coli (3%), Candida albicans (3%), Streptococcus viridans (1%), Flavobacterium spp (1.5%) and Bacteroides melaninogenicus (1%) were isolated. Most of the bacteria isolates were sensitive to pefloxacin. Our results demonstrate a very high rate of diabetic foot ulcer with the corresponding high rate of amputation and mortality. A multi-disciplinary approach to the management of DM foot ulcers is advocated. Efforts should be made to carry out cultures of samples from refractory ulcers to rule out yeast colonization, which if not treated will delay wound healing. Key words: Diabetic foot ulcers, Microbial and antimicrobial surveillance, Refractory ulcers. (Af J Clinical & Exp Microbiology: 2003 4(2): 52-61