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

Multilocus molecular characterization of a Penicillium strain isolated from maize in Nigeria

[Excerpt] Aims: Access the phylogeny of Penicillium strain (MUM 14.07) within the P. sclerotiorum complex and the relationship with P. mallochii. [...

Mycotoxin contamination of Maize and Guinea corn from markets in Plateau State, Nigeria

Maize (Zea mays) and guinea corn (Sorghum bicolor) are major food items in Plateau state, Nigeria. A multistage sampling technique was used to select the markets and store/warehouses used for this study; sample collection employed a simple random sampling method from different sampling points within designated areas. A total of 18 representative samples were collected and analyzed for the following mycotoxins: aflatoxins (Aflatoxin B1 - AFB1, Aflatoxin B2 - AFB2, Aflatoxin G1 - AFG1 and Aflatoxin G2 - AFG2), fumonisins (Fumonisin B1 - FB1 and Fumonisin B2 - FB2 ) and cyclopiazonic acid (CPA). Out of 12 samples analyzed for Aflatoxins, AFB1 was detected in 5, AFB2 in 1, AFG1 in 1 and AFG2 in 6 samples respectively. The highest concentration of AFB1 and AFG2 were found in maize samples from Pankshin market. Only maize samples from Mangu market were contaminated with AFB2 and also harboured the lowest concentration of AFG2. AFG1 contamination occurred in only guinea corn from Shendam market. and FB1 was detected in all 18 samples analyzed. The mycotoxin CPA was not detected in any of the samples. Aflatoxins levels in analyzed samples were regarded as safe based on Nigerian and European Union maximum permissible levels of 4g/kg. With the exception of two samples, FB1 levels in analyzed maize samples were within European Union maximum permissible levels of 1,000 to 3000g/kg. The health and food safety implications of these results for the human and animal population are further discussed

Identification of mycotoxigenic fungi from grains in a Nigerian region using the modern polyphasic methodology

Mycotoxins are poisonous substances produced by fungi which contaminate agricultural commodities. Many foods and feeds can become contaminated with mycotoxins since they can form in commodities before harvest, during the time between harvesting and drying, and in storage. The food crops most often affected include maize, peanuts, sorghum, wheat, cocoa and tree nuts. Mycotoxins may also be carried over to animal products due to consumption of contaminated feed. Maize (Zea mays) and guinea corn (Sorghum bicolor) form a major staple of the study area and are high risk commodities for mycotoxigenic fungi and mycotoxin contamination. Multistage sampling technique was used to select the markets and store/warehouses used for this study; sample collection employed a simple random sampling method from different sampling points within designated areas. Identification of all fungal isolates was carried out using the modern polyphasic methodology for filamentous fungi identification. At the level of phenotypic approach, the mycotoxigenic fungi Aspergillus, Fusarium, and Penicillium were identified. These fungal isolates also produced the mycotoxins Aflatoxins B1 and B2, Fumonisin B1, Cyclopiazonic acid, Ochratoxin A and Ochratoxin alfa. Spectral analysis by MALDI-TOF MS identified the Aspergillus species as A. flavus, A. aculeatus, A. niger and A. tamarii. Work is currently on-going to complete fungal identification for all isolates down to species level using the genotypic approach. In view of the toxic effects of mycotoxin contamination, the isolation, identification and characterization of mycotoxigenic fungi from maize and guinea corn in the study area pose serious health risks for the human and animal population and also have implications for food safety and public health in Nigeria

Mycotoxicology Society of Nigeria Mycotoxicology, 2015, 2: 28-34 Mycotoxin Contamination of Maize and Guinea corn from Markets in Plateau State, Nigeria

ABSTRACT Maize (Zea mays) and guinea corn (Sorghum bicolor) are major food items in Plateau state, Nigeria. A multistage sampling technique was used to select the markets and store/warehouses used for this study; sample collection employed a simple random sampling method from different sampling points within designated areas. A total of 18 representative samples were collected and analyzed for the following mycotoxins: aflatoxins (Aflatoxin B 1 -AFB 1 , Aflatoxin B 2 -AFB 2 , Aflatoxin G 1 -AFG 1 and Aflatoxin G 2 -AFG 2 ), fumonisins (Fumonisin B 1 -FB 1 and Fumonisin B 2 -FB 2 ) and cyclopiazonic acid (CPA). Out of 12 samples analyzed for Aflatoxins, AFB 1 was detected in 5, AFB 2 in 1, AFG 1 in 1 and AFG 2 in 6 samples respectively. The highest concentration of AFB 1 and AFG 2 were found in maize samples from Pankshin market. Only maize samples from Mangu market were contaminated with AFB 2 and also harboured the lowest concentration of AFG 2. AFG 1 contamination occurred in only guinea corn from Shendam market. and FB 1 was detected in all 18 samples analyzed. The mycotoxin CPA was not detected in any of the samples. Aflatoxins levels in analyzed samples were regarded as safe based on Nigerian and European Union maximum permissible levels of 4µg/kg. With the exception of two samples, FB 1 levels in analyzed maize samples were within European Union maximum permissible levels of 1,000 to 3000µg/kg. The health and food safety implications of these results for the human and animal population are further discussed

Investigating the Use of Crude Atili Oil for Maintenance of Stock Fungal Cultures

Atili oil is obtained from the fruits of the Atili plant (Canarium schweinfurtii), a perennial tree commonly found in several parts of the country, especially the Northern and Southeastern Nigeria. In Pankshin local Government area of Plateau State, the oil extraction is done by several households both for commercial and domestic purposes. Atili oil obtained from Pankshin was incorporated in graded concentrations into Sabouraud’s dextrose agar to obtain 10%, 20%, 30%, 50% and 100% oil in SDA medium. A portion (4mm plug) of an actively growing fungus from a pure culture of each of the isolates of Microsporum ferrugineum, Trichosporon beigelii, Candida albicans, Trichophyton soudanense and Geotrichum candidum was placed on the SDA-oil medium and incubated at room temperature. The cultures were observed weekly, for several months, for growth and evaluated for viability by sub-culturing onto SDA devoid of oil. Controls were set up for each of the organisms and these served as the basis for comparison. The results of this work indicate a good prospect for maintenance of stock fungal cultures with Atili oil. The benefits of this finding to the laboratory mycologist in Nigeria are discussed.Key words: Canarium schweinfurtii, Atili plant, Pankshin, Sabouraud’s dextrose aga

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)                                                                                                                                   

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