Showing papers by "Martin Antonio published in 2012"

Diagnostic Microbiologic Methods in the GEMS-1 Case/Control Study

Abstract: To understand the etiology of moderate-to-severe diarrhea among children in high mortality areas of sub-Saharan Africa and South Asia, we performed a comprehensive case/control study of children aged <5 years at 7 sites. Each site employed an identical case/control study design and each utilized a uniform comprehensive set of microbiological assays to identify the likely bacterial, viral and protozoal etiologies. The selected assays effected a balanced consideration of cost, robustness and performance, and all assays were performed at the study sites. Identification of bacterial pathogens employed streamlined conventional bacteriologic biochemical and serological algorithms. Diarrheagenic Escherichia coli were identified by application of a multiplex polymerase chain reaction assay for enterotoxigenic, enteroaggregative, and enteropathogenic E. coli. Rotavirus, adenovirus, Entamoeba histolytica, Giardia enterica, and Cryptosporidium species were detected by commercially available enzyme immunoassays on stool samples. Samples positive for adenovirus were further evaluated for adenovirus serotypes 40 and 41. We developed a novel multiplex assay to detect norovirus (types 1 and 2), astrovirus, and sapovirus. The portfolio of diagnostic assays used in the GEMS study can be broadly applied in developing countries seeking robust cost-effective methods for enteric pathogen detection.

Pre-vaccination nasopharyngeal pneumococcal carriage in a Nigerian population: epidemiology and population biology.

Abstract: BACKGROUND: Introduction of pneumococcal vaccines in Nigeria is a priority as part of the Accelerated Vaccine Introduction Initiative (AVI) of the Global Alliance for Vaccines and Immunisation (GAVI) However, country data on the burden of pneumococcal disease (IPD) is limited and coverage by available conjugate vaccines is unknown This study was carried out to describe the pre vaccination epidemiology and population biology of pneumococcal carriage in Nigeria METHODS: This was a cross sectional survey Nasopharyngeal swabs (NPS) were obtained from a population sample in 14 contiguous peri-urban Nigerian communities Data on demographic characteristics and risk factor for carriage were obtained from all study participants Pneumococci isolated from NPS were characterised by serotyping, antimicrobial susceptibility and Multi Locus Sequencing Typing (MLST) RESULTS: The prevalence of pneumococcal carriage was 525% Carriage was higher in children compared to adults (674% vs 26%), highest (≈90%) in infants aged <9 months and reduced significantly with increasing age (P<0001) Serotypes 19F (186%) and 6A (144%) were most predominant Potential vaccine coverage was 438%, 450% and 62% for PCV-7, PCV-10 and PCV-13 respectively There were 16 novel alleles, 72 different sequence types (STs) from the isolates and 3 Sequence Types (280, 310 and 5543) were associated with isolates of more than one serotype indicative of serotype switching Antimicrobial resistance was high for cotrimoxazole (93%) and tetracycline (84%), a third of isolates had intermediate resistance to penicillin Young age was the only risk factor significantly associated with carriage CONCLUSIONS: Pneumococcal carriage and serotype diversity is highly prevalent in Nigeria especially in infants Based on the coverage of serotypes in this study, PCV-13 is the obvious choice to reduce disease burden and prevalence of drug resistant pneumococci However, its use will require careful monitoring Our findings provide sound baseline data for impact assessment following vaccine introduction in Nigeria

Variation at the capsule locus, cps, of mistyped and non-typable Streptococcus pneumoniae isolates.

Abstract: The capsule polysaccharide locus (cps) is the site of the capsule biosynthesis gene cluster in encapsulated Streptococcus pneumoniae A set of pneumococcal samples and non-pneumococcal streptococci from Denmark, the Gambia, the Netherlands, Thailand, the UK and the USA were sequenced at the cps locus to elucidate serologically mistyped or non-typable isolates We identified a novel serotype 33B/33C mosaic capsule cluster and previously unseen serotype 22F capsule genes, disrupted and deleted cps clusters, the presence of aliB and nspA genes that are unrelated to capsule production, and similar genes in the non-pneumococcal samples These data provide greater understanding of diversity at a locus which is crucial to the antigenic diversity of the pathogen and current vaccine strategies

The Genome of Mycobacterium Africanum West African 2 Reveals a Lineage-Specific Locus and Genome Erosion Common to the M. tuberculosis Complex

Abstract: M. africanum West African 2 constitutes an ancient lineage of the M. tuberculosis complex that commonly causes human tuberculosis in West Africa and has an attenuated phenotype relative to M. tuberculosis.In search of candidate genes underlying these differences, the genome of M. africanum West African 2 was sequenced using classical capillary sequencing techniques. Our findings reveal a unique sequence, RD900, that was independently lost during the evolution of two important lineages within the complex: the "modern" M. tuberculosis group and the lineage leading to M. bovis. Closely related to M. bovis and other animal strains within the M. tuberculosis complex, M. africanum West African 2 shares an abundance of pseudogenes with M. bovis but also with M. africanum West African clade 1. Comparison with other strains of the M. tuberculosis complex revealed pseudogenes events in all the known lineages pointing toward ongoing genome erosion likely due to increased genetic drift and relaxed selection linked to serial transmission-bottlenecks and an intracellular lifestyle.The genomic differences identified between M. africanum West African 2 and the other strains of the Mycobacterium tuberculosis complex may explain its attenuated phenotype, and pave the way for targeted experiments to elucidate the phenotypic characteristic of M. africanum. Moreover, availability of the whole genome data allows for verification of conservation of targets used for the next generation of diagnostics and vaccines, in order to ensure similar efficacy in West Africa.

Effect of Age and Vaccination With a Pneumococcal Conjugate Vaccine on the Density of Pneumococcal Nasopharyngeal Carriage

Abstract: Background. This study evaluated the impact of age and pneumococcal vaccination on the density of pneu-mococcal nasopharyngeal carriage.Methods. A cluster-randomized trial was conducted in rural Gambia. In 11 villages (the vaccine group), allresidents received 7-valent pneumococcal conjugate vaccine (PCV-7), while in another 10 villages (the controlgroup), only children <30 months old or born during the study period received PCV-7. Cross-sectional surveys(CSSs) were conducted to collect nasopharyngeal swabs before vaccination (baseline CSS) and 4, 12, and 22months after vaccination. Pneumococcal density was defined using a semiquantitative classification (range, 1–4)among colonized individuals. An age-trend analysis of density was conducted using data from the baseline CSS.Mean pneumococcal density was compared in CSSs conducted before and after vaccination.Results. Mean bacterial density among colonized individuals in the baseline CSS was 2.57 for vaccine-type(VT) and non–vaccine-type (NVT) pneumococci; it decreased with age (P<.001 for VT and NVT). There was adecrease in the density of VT carriage following vaccination in individuals older than 5 years (from 2.44 to 1.88;P=.001) and in younger individuals (from 2.57 to 2.11; P=.070) in the vaccinated villages. Similar decreases indensity were observed with NVT within vaccinated and control villages. No significant differences were foundbetween vaccinated and control villages in the postvaccination comparisons for either VT or NVT.Conclusions. A high density of carriage among young subjects might partly explain why children are moreefficient than adults in pneumococcal transmission. PCV-7 vaccination lowered the density of VT and of NVTpneumococcal carriage in the before-after vaccination analysis.Clinical Trials Registration. ISRCTN51695599.Prevention of pneumococcal disease is a public healthpriority [1]. Nasopharyngeal pneumococcal carriageplays an important role in the pathogenesis of invasivepneumococcal disease (IPD) and pneumococcal trans-mission. The effectiveness of pneumococcal conjugatevaccines (PCVs) in preventing disease due to vaccine-serotype (VT) pneumococci is due in large measure totheir impact on nasopharyngeal carriage in both vacci-nees and their contacts [2–5]. Such a reduction issometimes accompanied by an increase in carriage ofnon–vaccine-serotype (NVT) pneumococci [2–7].How this increase in NVT pneumococcal carriage isbrought about is still not fully understood. Eliminationof VT pneumococci from the nasopharynx might openup an ecological niche that can be colonized by NVT

Highly accurate diagnosis of pleural tuberculosis by immunological analysis of the pleural effusion.

Abstract: Pleural TB is notoriously difficult to diagnose due to its paucibacillary nature yet it is the most common cause of pleural effusions in TB endemic countries such as The Gambia. We identified both cellular and soluble biomarkers in the pleural fluid that allowed highly accurate diagnosis of pleural TB compared to peripheral blood markers. Multi-plex cytokine analysis on unstimulated pleural fluid showed that IP-10 resulted in a positive likelihood ratio (LR) of 9.6 versus 2.8 for IFN-γ; a combination of IP-10, IL-6 and IL-10 resulted in an AUC of 0.96 and positive LR of 10. A striking finding was the significantly higher proportion of PPD-specific IFN-γ+TNF-α+ cell population (PPD-IGTA) in the pleural fluid compared to peripheral blood of TB subjects. Presence of this pleural PPD-IGTA population resulted in 95% correct classification of pleural TB disease with a sensitivity of 95% and specificity of 100%. These data suggest that analysis of the site of infection provides superior diagnostic accuracy compared to peripheral blood for pleural TB, likely due to the sequestration of effector cells at this acute stage of disease.

Genome Analysis of a Highly Virulent Serotype 1 Strain of Streptococcus pneumoniae from West Africa

Abstract: Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and bacteremia, estimated to cause 2 million deaths annually. The majority of pneumococcal mortality occurs in developing countries, with serotype 1 a leading cause in these areas. To begin to better understand the larger impact that serotype 1 strains have in developing countries, we characterized virulence and genetic content of PNI0373, a serotype 1 strain from a diseased patient in The Gambia. PNI0373 and another African serotype 1 strain showed high virulence in a mouse intraperitoneal challenge model, with 20% survival at a dose of 1 cfu. The PNI0373 genome sequence was similar in structure to other pneumococci, with the exception of a 100 kb inversion. PNI0373 showed only15 lineage specific CDS when compared to the pan-genome of pneumococcus. However analysis of non-core orthologs of pneumococcal genomes, showed serotype 1 strains to be closely related. Three regions were found to be serotype 1 associated and likely products of horizontal gene transfer. A detailed inventory of known virulence factors showed that some functions associated with colonization were absent, consistent with the observation that carriage of this highly virulent serotype is unusual. The African serotype 1 strains thus appear to be closely related to each other and different from other pneumococci despite similar genetic content.

Chrysomya putoria, a Putative Vector of Diarrheal Diseases

Abstract: BACKGROUND: Chrysomya spp are common blowflies in Africa, Asia and parts of South America and some species can reproduce in prodigious numbers in pit latrines. Because of their strong association with human feces and their synanthropic nature, we examined whether these flies are likely to be vectors of diarrheal pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Flies were sampled using exit traps placed over the drop holes of latrines in Gambian villages. Odor-baited fly traps were used to determine the relative attractiveness of different breeding and feeding media. The presence of bacteria on flies was confirmed by culture and bacterial DNA identified using PCR. A median of 7.00 flies/latrine/day (IQR?=?0.0-25.25) was collected, of which 95% were Chrysomya spp, and of these nearly all were Chrysomya putoria (99%). More flies were collected from traps with feces from young children (median?=?3.0, IQR?=?1.75-10.75) and dogs (median?=?1.50, IQR?=?0.0-13.25) than from herbivores (median?=?0.0, IQR?=?0.0-0.0; goat, horse, cow and calf; p<0.001). Flies were strongly attracted to raw meat (median?=?44.5, IQR?=?26.25-143.00) compared with fish (median?=?0.0, IQR?=?0.0-19.75, ns), cooked and uncooked rice, and mangoes (median?=?0.0, IQR?=?0.0-0.0; p<0.001). Escherichia coli were cultured from the surface of 21% (15/72 agar plates) of Chrysomya spp and 10% of these were enterotoxigenic. Enteroaggregative E. coli were identified by PCR in 2% of homogenized Chrysomya spp, Shigella spp in 1.4% and Salmonella spp in 0.6% of samples. CONCLUSIONS/SIGNIFICANCE: The large numbers of C. putoria that can emerge from pit latrines, the presence of enteric pathogens on flies, and their strong attraction to raw meat and fish suggests these flies may be common vectors of diarrheal diseases in Africa.

Characterisation of novel strains of multiply antibiotic-resistant Salmonella recovered from poultry in Southern Senegal

Abstract: Introduction: Non-typhoidal Salmonella (NTS) contamination in poultry and poultry products is a major cause of food-borne disease in humans. This study presents the molecular epidemiology of NTS isolated from poultry in Senegal. Methodology: A total of 261 NTS recovered from broiler farms, chicken carcasses and street vendors were characterized using random amplification of polymorphic DNA (RAPD) and multilocus sequence typing (MLST) techniques. Results: We observed 20 distinct RAPD profiles corresponding to 18 different serotypes. Strains from each of these 20 groups were further analysed using MLST. Consequently, 12 new MLST alleles and 17 new sequence types were discovered. Three sequence types ( S . Kentucky ST198, S . Agona ST13 and S. Istanbul ST33) have previously been described in Senegal and other countries, suggesting that these clones are geographically widely distributed and are circulating in a wide range of hosts. Nine clones showed multi-resistance to the most commonly used antibiotics in both humans and animals. However, a novel multi-resistant clone of S . Kentucky ST832 was found. Conclusion: This study gives new insights into the genetic diversity of NTS in Senegal. Molecular tools remain essential to improve our understanding of the epidemiology of NTS by tracking the sources of infection and/or contamination.

Comparative phylogenomics of Streptococcus pneumoniae isolated from invasive disease and nasopharyngeal carriage from West Africans

Abstract: We applied comparative phylogenomics (whole genome comparisons of microbes using DNA microarrays combined with Bayesian-based phylogenies) to investigate S. pneumoniae isolates from West Africa, with the aim of providing insights into the pathogenicity and other features related to the biology of the organism. The strains investigated comprised a well defined collection of 58 invasive and carriage isolates that were sequenced typed and included eight different S. pneumoniae serotypes (1, 3, 5, 6A, 11, 14, 19 F and 23 F) of varying invasive disease potential. The core genome of the isolates was estimated to be 38% and was mainly represented by gene functional categories associated with housekeeping functions. Comparison of the gene content of invasive and carriage isolates identified at least eleven potential genes that may be important in virulence including surface proteins, transport proteins, transcription factors and hypothetical proteins. Thirteen accessory regions (ARs) were also identified and did not show any loci association with the eleven virulence genes. Intraclonal diversity (isolates of the same serotype and MLST but expressing different patterns of ARs) was observed among some clones including ST 1233 (serotype 5), ST 3404 (serotype 5) and ST 3321 (serotype 14). A constructed phylogenetic tree of the isolates showed a high level of heterogeneity consistent with the frequent S. pneumoniae recombination. Despite this, a homogeneous clustering of all the serotype 1 strains was observed. Comparative phylogenomics of invasive and carriage S. pneumoniae isolates identified a number of putative virulence determinants that may be important in the progression of S. pneumoniae from the carriage phase to invasive disease. Virulence determinants that contribute to S. pneumoniae pathogenicity are likely to be distributed randomly throughout its genome rather than being clustered in dedicated loci or islands. Compared to other S. pneumoniae serotypes, serotype 1 appears most genetically uniform.

Pathogen Genomics and the Potential for Understanding Diseases in the Developing World

Abstract: Approximately 46% and 32% of deaths among children under five globally occur in sub-Saharan Africa and South Asia, respectively. Over 80% of the 4.2 million child deaths in Africa are caused by infectious diseases, sharply contrasted to Europe where 39% of the 0.15 million child deaths are attributable to infectious diseases (Fig. 5.1) (Black et al. 2010). Hence, despite the remarkable public health advancements in hygiene, sanitation, antimicrobial drugs and vaccine strategies of the twenty-first century, the burden of infectious diseases remains unacceptably high in the developing world.