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

Indoor air mycoflora of residential dwellings in Jos metropolis

Background: The quality of air in the environment where one lives or works can have potential effects on human health. There are strong indications that in many parts of the world, our homes, schools and workplaces are heavily contaminated with air-borne molds and other biological contaminants. Objectives: This study was carried out to assess the level of fungal contamination of indoor air, health related experiences of residents, and the prevalent fungi species in the homes. Methods: The investigation was done between May 2005 and January 2006, using structured questionnaires and the agar plate exposure. 150 houses from 14 locations were examined. Results: 380 fungi belonging to 10 species were isolated, Chaetomium globosum (17%), Aspergillus fumigatus (14%), Stachybotrys alternans (14%) and Alternaria alternata (14%) being the predominant isolates. Conclusion: The indoor air quality of residential dwellings in Jos is poor. Rate of isolation of fungi was not significantly different in the wet and dry periods of the year and residential density affected the occurrence of fungal contaminants. Residents are displeased with fungal presence in their homes and the associated health implications. There is need for proper attention to the quality of the indoor environment.Key words: indoor, fungi, residential, dwelling

FUNGAL INFECTIONS IN JOS: A 9-YEAR STUDY

The prevalent mycoses and their aetiologic agents were studied in 1,287 patients in Jos and environs. 917 (71.3%) of the study population were infected. A male preponderance was observed and the age groups 11-20 and 21 –30 were predominantly infected. The most frequently isolated aetiologic agent was Candida spp and the commonest site of infection was the trunk. Unusual dissemination of lesions caused by Trichosporon beigelii infections was observed. The seasonal occurrence of mycoses and the effect on prevailing aetiologic fungi was studied. (Af J Clinical & Exp Microbiology: 2003 4(2): 2-10)                                                                                                                                   

Molecular characterization of environmental Cryptococcus neoformans VNII isolates in Jos, Plateau State, Nigeria = Caractérisation moléculaire de souches de Cryptococcus neoformans VNII isolés dans l’environnement à Jos, état du Plateau, Nigeria

Cryptococcus neoformans and Cryptococcus gattii are encapsulated yeasts able to cause fatal neurological infections in both human and other mammals. Cryptococcosis is the most common fungal infection of the central nervous system and has a huge burden in sub-Saharan Africa and South East Asia. Bird excreta are considered an environmental reservoir for C. neoformans in urban areas, therefore a study aimed at isolating and characterizing this yeast is important in disease management. In this study, one hundred samples of pigeon droppings were collected in Jos, Plateau State, Nigeria. C. neoformans was isolated from three samples and initially identified using standard phenotypic and biochemical tests. Molecular analysis revealed that all three isolates belonged to C. neoformans genotype VNII, mating type \u3b1 and were assigned to the sequence type ST43 by multilocus sequence typing analysis. This study reports, for the first time, the molecular characterization of C. neoformans in Nigeria, where little is still known about the environmental distribution of the genotypes, serotypes and mating types of this important human pathogen.Cryptococcus neoformans et Cryptococcus gattii sont des levures encapsul\ue9es capables de causer des infections neurologiques mortelles chez l\u2019homme et d\u2019autres mammif\ue8res. La cryptococcose est l\u2019infection fongique la plus commune du syst\ue8me nerveux central avec une incidence \ue9lev\ue9e en Afrique subsaharienne et en Asie du Sud-Est. Les excr\ue9ments d\u2019oiseaux sont consid\ue9r\ue9s comme le r\ue9servoir environnemental pour C. neoformans dans les zones urbaines ; par cons\ue9quent, une \ue9tude visant \ue0 isoler et caract\ue9riser cette levure est utile pour la prise en charge de la maladie. Dans cette \ue9tude, une centaine d\u2019\ue9chantillons de fientes de pigeons a \ue9t\ue9 recueillie \ue0 Jos, Plateau State, au Nigeria. C. neoformans a \ue9t\ue9 isol\ue9 \ue0 partir de trois \ue9chantillons et initialement identifi\ue9s \ue0 l\u2019aide de tests ph\ue9notypique et biochimiques. L\u2019analyse mol\ue9culaire, par multilocus sequence typing, a r\ue9v\ue9l\ue9 que les trois souches appartenaient au type mol\ue9culaire VNII, mating type \u3b1 et au g\ue9notype ST43 par analyse multilocus sequence typing. Cette \ue9tude rend compte, pour la premi\ue8re fois au Nigeria, de la caract\ue9risation mol\ue9culaire de souches environnementales de C. neoformans, o\uf9 peu est encore connu \ue0 propos de la diffusion dans l\u2019environnement des g\ue9notypes, s\ue9rotypes et mating types de cet important pathog\ue8ne humain