[Show abstract][Hide abstract] ABSTRACT: Background
Diarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease.
We use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age.
Our findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.
[Show abstract][Hide abstract] ABSTRACT: Although the prothrombin G20210A mutation has been implicated as a risk factor for venous thrombosis, its role in arterial ischemic stroke is unclear, particularly among young adults. To address this issue, we examined the association between prothrombin G20210A and ischemic stroke in a white case-control population and additionally performed a meta-analysis.
From the population-based Genetics of Early Onset Stroke (GEOS) study, we identified 397 individuals of European ancestry aged 15 to 49 years with first-ever ischemic stroke and 426 matched controls. Logistic regression was used to calculate odds ratios (ORs) in the entire population and for subgroups stratified by sex, age, oral contraceptive use, migraine, and smoking status. A meta-analysis of 17 case-control studies (n=2305 cases <55 years) was also performed with and without GEOS data.
Within GEOS, the association of the prothrombin G20210A mutation with ischemic stroke did not achieve statistical significance (OR=2.5; 95% confidence interval [CI]=0.9-6.5; P=0.07). However, among adults aged 15 to 42 years (younger than median age), cases were significantly more likely than controls to have the mutation (OR=5.9; 95% CI=1.2-28.1; P=0.03), whereas adults aged 42 to 49 years were not (OR=1.4; 95% CI=0.4-5.1; P=0.94). In our meta-analysis, the mutation was associated with significantly increased stroke risk in adults ≤55 years (OR=1.4; 95% CI=1.1-1.9; P=0.02), with significance increasing with addition of the GEOS results (OR=1.5; 95% CI=1.1-2.0; P=0.005).
The prothrombin G20210A mutation is associated with ischemic stroke in young adults and may have an even stronger association among those with earlier onset strokes. Our finding of a stronger association in the younger young adult population requires replication.
[Show abstract][Hide abstract] ABSTRACT: Cholera Cholera is still a major public health problem. The underlying bacterial pathogen Vibrio cholerae Vibrio cholerae (V. cholerae) is evolving and some of its mutations have set the stage for outbreaks outbreaks . After V. cholerae acquired the mobile elements VSP I & II, the El Tor pandemic began and spread across the tropics. The replacement of the O1 serotype encoding genes with the O139 encoding genes triggered an outbreak that swept across the Indian subcontinent. The sxt element generated a third selective sweep and most recently a fourth sweep was associated with the exchange of the El Tor ctx allele for a classical ctx allele in the El Tor background. In Kenya, variants of this fourth selective sweep have differentiated and become endemic residing in and emerging from environmental reservoirs. On a local level, studies in Bangladesh have revealed that outbreaks may arise from a nonrandom subset of the genetic lineages in the environment and as the population of the pathogen expands, many novel mutations may be found increasing the amount of genetic variation genetic variation , a phenomenon known as a founder flush. In Haiti, after the initial invasion and expansion of V. cholerae in 2010, a second outbreak occurred in the winter of 2011-2012 driven by natural selection of specific mutations.
Current topics in microbiology and immunology 01/2014; · 4.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe the identification of a novel picornavirus recovered from the fecal specimen of a child in The Gambia, provisionally named rosavirus 2. Comparison of the rosavirus 2 complete genome demonstrated 71.9% nucleotide identity to its closest relative rosavirus M-7, an unclassified picornavirus identified from rodent fecal material. A unique RNA structure was predicted in the 3' UTR of rosavirus 2 that was conserved with rosavirus M-7 and cadiciviruses. We detected rosavirus 2 in four pediatric fecal specimens (0.55% prevalence) in a Gambian diarrheal case-control cohort, but we did not detect it in a panel of 634 pediatric diarrheal stool specimens from the USA. There was no statistical evidence that rosavirus 2 was associated with diarrheal cases. This study broadens our understanding of unknown viruses present in children in developing country settings.
Virology 01/2014; s 454–455:25–33. · 3.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cholera remains a major public health problem. To compare the relative contribution of strains from the environment with strains isolated from patients during outbreaks, we performed multilocus variable tandem repeat analyses on samples collected during the 2010 and 2011 outbreak seasons in 2 geographically distinct areas of Bangladesh. A total of 222 environmental and clinical isolates of V. cholerae O1 were systematically collected from Chhatak and Mathbaria. In Chhatak, 75 of 79 isolates were from the same clonal complex, in which extensive differentiation was found in a temporally consistent pattern of successive mutations at single loci. A total of 59 isolates were collected from 6 persons; most isolates from 1 person differed by sequential single-locus mutations. In Mathbaria, 60 of 84 isolates represented 2 separate clonal complexes. The small number of genetic lineages in isolates from patients, compared with those from the environment, is consistent with accelerated transmission of some strains among humans during an outbreak.
[Show abstract][Hide abstract] ABSTRACT: Recent evidence suggests that the abundance of enteric pathogens in the stool correlates with the presence of clinical diarrhea. We quantified the fecal pathogen after feeding enterotoxigenic E. coli (ETEC) strain H10407 to 30 adult volunteers. Stools were collected daily and examined using qualitative and quantitative (Q) culture. DNA was isolated and quantitative (Q)PCR targeting the heat-labile toxin (LT) gene was performed. Nine volunteers developed diarrhea. Among 131 stool specimens with complete data, pathogen abundance by QPCR was strongly correlated with Qculture, rho=0.61, p<0.0001. Receiver operating characteristic curve analysis comparing quantitative data against diarrhea status suggested cut-points, based on a maximum Youden Index, of 2.8x10(4) LT gene copies and 1.8x10(7) CFU. Based on these cut-points, QPCR had a sensitivity and specificity compared to diarrheal status of 0.75 and 0.87, respectively, and an OR of 20.0 (95% CI 5.7-70.2), whereas Qculture had a sensitivity and specificity of 0.73 and 0.91, respectively, and an OR of 28.6 (95% CI 7.7-106.6). Qculture had a sensitivity and specificity of 0.82 and 0.48, respectively and an OR of 4.4 (95% CI 1.2-16.0).The correlation between Qculture and QPCR was highest in diarrheal specimens and both quantitative methods demonstrated stronger association with diarrhea than qualitative culture. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: In a recent meta-analysis migraine was associated with a two-fold increase in stroke risk. While the mechanism driving this association is unknown, one intriguing hypothesis is that migraineurs are genetically predisposed to developing ischemic stroke. Mutations in the ATP1A2 gene are implicated in familial hemiplegic migraine type II and increase the severity of ischemic brain injury in animal models. To further explore these observations, we assessed the association between ATP1A2 polymorphisms, migraine, and the risk of ischemic stroke in participants of the Genetics of Early-Onset Stroke Study, a population-based case-control study of ischemic stroke among men and women aged 15-49. Using responses to a headache symptoms questionnaire, subjects were classified as having no migraine, or migraine with or without visual aura. Evaluating a total of 134 ATP1A2 polymorphisms genotyped using a combination of Illumina platforms (Cardiovascular Gene-centric 50 K SNP Array and HumanOmni1-Quad_v1-0_B Bead Chip), only one polymorphism (rs2070704) demonstrated a nominally significant association with stroke in an age-, gender-, ethnicity-adjusted model (OR = 0.83, 95% CI = 0.71-0.98, p = 0.025) and in a vascular risk factor model adjusting for age, gender, ethnicity, hypertension, diabetes, smoking, and myocardial infarction (OR = 0.74, 95% CI = 0.63-0.89, p = 0.001). Ethnicity-stratified analyses demonstrated a significant association for rs2070704 among African-Americans (OR = 0.68, 95% CI = 0.53-0.90, p = 0.005) but not Caucasians (OR = 0.82, 95% CI = 0.64-1.04, p = 0.107). These associations were unchanged when migraine subtypes were included as co-variates. We did not observe an association between ATP1A2 polymorphisms and migraine. While our results do not demonstrate a strong relationship between ATP1A2 polymorphisms and migraine associated stroke risk, the results are hypothesis generating and indicate that an association between ATP1A2 polymorphisms and stroke risk may exist. Additional studies are required.
[Show abstract][Hide abstract] ABSTRACT: SUMMARY Transmission of Staphylococcus aureus colonization in community-based populations is not well understood. We sought to describe the molecular epidemiology of S. aureus colonization in the Old Order Amish. The study was a prospective, observational study of healthy adults and their same-sex siblings who were cultured from the anterior nares twice. S. aureus isolates were characterized using spa typing. Overall, 40% (159/398) of the study population was colonized with S. aureus. There were 84 spa types with the most abundant spa types being t012 (13%) and t021 (7%). There was no clustering of spa types within sibling groups; however, there was clustering within households. There were 111 S. aureus-colonized participant pairs living within the same household. Of these, 47% had concordant spa types. The diversity of spa types across a relatively isolated, genetically homogenous population with a similar lifestyle is striking. Taken together this suggests that S. aureus transmission is a local phenomenon limited to very close contact.
Epidemiology and Infection 11/2013; · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: de Araujo for help with capturing and dissecting bats. We thank Kathryn Holmes and Thomas M. Yuill for critically reviewing this manuscript and advising and facilitating this collaboration.
[Show abstract][Hide abstract] ABSTRACT: We introduce a methodology to assess differential abundance in sparse high-throughput microbial marker-gene survey data. Our approach, implemented in the metagenomeSeq Bioconductor package, relies on a novel normalization technique and a statistical model that accounts for undersampling-a common feature of large-scale marker-gene studies. Using simulated data and several published microbiota data sets, we show that metagenomeSeq outperforms the tools currently used in this field.
[Show abstract][Hide abstract] ABSTRACT: Cultivation based assays combined with PCR or ELISA based methods to detect virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya and Bangladesh. The children were identified as either controls or cases with moderate to severe diarrhea using currently accepted clinical protocols. 3,610 stool samples were tested by established clinical culture techniques; 3,179 DNA samples by the Universal Biosensor® (Ibis Biosciences, Inc.); 1,466 DNA samples by GoldenGate® (Illumina, Inc.); and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected differing proportions of samples testing positive for each of seven enteric pathogens- enteroaggregative Escherichia coli (EAEC), enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), Shigella, Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. Comparisons among detection methods included the frequency of positive stools and kappa values to make pair wise comparisons. Overall, standard culture methods detected Shigella, EPEC, ETEC and EAEC in a smaller proportion of the samples than either of the methods based on detection of the virulence genes from DNA in whole stool. The GoldenGate® method revealed the greatest agreement with the most other methods. Agreement among methods was higher in cases than controls. New molecular technologies have high potential for highly sensitive identification of bacterial diarrhea pathogens.
Journal of clinical microbiology 07/2013; · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Estimates of Shigella prevalence are limited by suboptimal sensitivity of current methods. We used a quantitative (Q)PCR assay to detect Shigella in stools of 3,533 children aged less than 59 -months from The Gambia, Mali, Kenya and Bangladesh, with or without moderate to severe diarrhea (MSD). We compared the results of conventional culture to those of QPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cut point to be 2.9x10E4 ipaH gene copies per 100 ng of stool DNA for Set 1 (N=877). One-hundred and fifty-eight (18%) samples specimens yielded greater than 2.9x10E4 ipaH gene copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with a value of ≥2.9x10E4 have 5.6 times higher odds of having diarrhea compared to those with values <2.9x10E4 (95%CI 3.7-8.5; p<0.0001). Nearly identical results were found using an independent set of samples. QPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture within both samples. Among a subset (N=2,874) comprising MSD cases, and age-, gender- and location-matched controls, the fraction of MSD cases attributable to Shigella infection increased from 9.6% (N=129) for culture to 17.6% (N=262) for QPCR employing our cut-point. We suggest that QPCR with a cut-point of approximately 1.4×10E4 become the new reference standard for the detection and diagnosis of Shigellosis in children in low-income countries. Acceptance of a new standard would substantially increase the fraction of MSD attributable to Shigella.
Journal of clinical microbiology 03/2013; · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The predatory Bacteriovorax are Gram-negative bacteria ubiquitous in saltwater systems that prey upon other Gram-negative bacteria in a similar manner to the related genus Bdellovibrio. Among the phylogenetically defined clusters of Bacteriovorax, cluster V has only been isolated from estuaries suggesting that it may be a distinct estuarine phylotype. To assess this hypothesis, the spatial and temporal distribution of cluster V and other Bacteriovorax phylogenetic assemblages along the salinity gradient of Chesapeake Bay were determined. Cluster V was expected to be found in significantly greater numbers in low to moderate salinity waters compared to high salinity areas. The analyses of water and sediment samples from sites in the bay revealed cluster V to be present at the lower salinity and not high salinity sites, consistent with it being an estuarine phylotype. Cluster IV had a similar distribution pattern and may also be specifically adapted to estuaries. While the distribution of clusters V and IV were similar for salinity, they were distinct on temperature gradients, being found in cooler and in warmer temperatures, respectively. The differentiation of phylotype populations along the salinity and temporal gradients in Chesapeake Bay revealed distinct niches inhabited by different phylotypes of Bacteriovorax and unique estuarine phylotypes.
[Show abstract][Hide abstract] ABSTRACT: The family Polyomaviridae is comprised of circular double-stranded DNA viruses, several of which are associated with diseases, including cancer, in immunocompromised patients. Here we describe a novel polyomavirus recovered from the fecal microbiota of a child in Malawi, provisionally named STL polyomavirus (STLPyV). We detected STLPyV in clinical stool specimens from USA and The Gambia at up to 1% frequency. Complete genome comparisons of two STLPyV strains demonstrated 5.2% nucleotide divergence. Alternative splicing of the STLPyV early region yielded a unique form of T antigen, which we named 229T, in addition to the expected large and small T antigens. STLPyV has a mosaic genome and shares an ancestral recombinant origin with MWPyV. The discovery of STLPyV highlights a novel alternative splicing strategy and advances our understanding of the complex evolutionary history of polyomaviruses.
[Show abstract][Hide abstract] ABSTRACT: Bacteriovorax marinus SJ is a predatory delta-proteobacterium isolated from a marine environment. The genome sequence of this strain provides an interesting contrast to that of the terrestrial predatory bacterium Bdellovibrio bacteriovorus HD100. Based on their predatory lifestyle, Bacteriovorax were originally designated as members of the genus Bdellovibrio but subsequently were re-assigned to a new genus and family based on genetic and phenotypic differences. B. marinus attaches to Gram-negative bacteria, penetrates through the cell wall to form a bdelloplast, in which it replicates, as shown using microscopy. Bacteriovorax is distinct, as it shares only 30% of its gene products with its closest sequenced relatives. Remarkably, 34% of predicted genes over 500 nt in length were completely unique with no significant matches in the databases. As expected, Bacteriovorax shares several characteristic loci with the other delta-proteobacteria. A geneset shared between Bacteriovorax and Bdellovibrio that is not conserved among other delta-proteobacteria such as Myxobacteria (which destroy prey bacteria externally via lysis), or the non-predatory Desulfo-bacteria and Geobacter species was identified. These 291 gene orthologues common to both Bacteriovorax and Bdellovibrio may be the key indicators of host-interaction predatory-specific processes required for prey entry. The locus from Bdellovibrio bacteriovorus is implicated in the switch from predatory to prey/host-independent growth. Although the locus is conserved in B. marinus, the sequence has only limited similarity. The results of this study advance understanding of both the similarities and differences between Bdellovibrio and Bacteriovorax and confirm the distant relationship between the two and their separation into different families.The ISME Journal advance online publication, 6 September 2012; doi:10.1038/ismej.2012.90.
[Show abstract][Hide abstract] ABSTRACT: Numerous outbreaks of cholera have occurred in Kenya since 1971. To more fully understand the epidemiology of cholera in Kenya, we analyzed the genetic relationships among 170 Vibrio cholerae O1 isolates at 5 loci containing variable tandem repeats. The isolates were collected during January 2009-May 2010 from various geographic areas throughout the country. The isolates grouped genetically into 5 clonal complexes, each comprising a series of genotypes that differed by an allelic change at a single locus. No obvious correlation between the geographic locations of the isolates and their genotypes was observed. Nevertheless, geographic differentiation of the clonal complexes occurred. Our analyses showed that multiple genetic lineages of V. cholerae were simultaneously infecting persons in Kenya. This finding is consistent with the simultaneous emergence of multiple distinct genetic lineages of V. cholerae from endemic environmental reservoirs rather than recent introduction and spread by travelers.
[Show abstract][Hide abstract] ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains.
Infection and immunity 05/2012; 80(8):2791-801. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (~2 lanes Illumina 76 bp PE) and high human DNA contamination (up to ~90%) we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.
PLoS ONE 01/2012; 7(6):e37919. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genetic architecture of ischemic stroke is complex and is likely to include rare or low frequency variants with high penetrance and large effect sizes. Such variants are likely to provide important insights into disease pathogenesis compared to common variants with small effect sizes. Because a significant portion of human functional variation may derive from the protein-coding portion of genes we undertook a pilot study to identify variation across the human exome (i.e., the coding exons across the entire human genome) in 10 ischemic stroke cases. Our efforts focused on evaluating the feasibility and identifying the difficulties in this type of research as it applies to ischemic stroke. The cases included 8 African-Americans and 2 Caucasians selected on the basis of similar stroke subtypes and by implementing a case selection algorithm that emphasized the genetic contribution of stroke risk. Following construction of paired-end sequencing libraries, all predicted human exons in each sample were captured and sequenced. Sequencing generated an average of 25.5 million read pairs (75 bp×2) and 3.8 Gbp per sample. After passing quality filters, screening the exomes against dbSNP demonstrated an average of 2839 novel SNPs among African-Americans and 1105 among Caucasians. In an aggregate analysis, 48 genes were identified to have at least one rare variant across all stroke cases. One gene, CSN3, identified by screening our prior GWAS results in conjunction with our exome results, was found to contain an interesting coding polymorphism as well as containing excess rare variation as compared with the other genes evaluated. In conclusion, while rare coding variants may predispose to the risk of ischemic stroke, this fact has yet to be definitively proven. Our study demonstrates the complexities of such research and highlights that while exome data can be obtained, the optimal analytical methods have yet to be determined.
PLoS ONE 01/2012; 7(4):e35591. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Factor V Leiden (FVL) has been associated with ischemic stroke in children but not in adults. Although the FVL mutation is associated with increased risk for venous thrombosis, its association with ischemic stroke in young adults remains uncertain. Therefore, we examined the association between FVL and ischemic stroke in participants of the Genetics of Early Onset Stroke (GEOS) study. METHODS: A population-based case control study identified 354 women and 476 men 15 to 49 years of age with first-ever ischemic stroke and 907 controls. Participant-specific data included vascular risk factors, FVL genotype and, for cases, the ischemic stroke subtype by modified Trial of ORG 10172 in Acute Stroke criteria. Logistic regression was used to calculate odds ratios for the entire population and for subgroups stratified by risk factors and ischemic stroke subtype. RESULTS: The frequency of the FVL mutation was similar between ischemic stroke patients (3.6%; 95% confidence interval [CI] 2.5%-5.1%) and nonstroke controls (3.8%; 95% CI 2.7%-5.2%). This frequency did not change significantly when cases were restricted to patients with stroke of undetermined etiology (4.1%; 95% CI 2.6%-6.4%). CONCLUSIONS: Among young adults, we found no evidence for an association between FVL and either all ischemic stroke or the subgroup with stroke of undetermined etiology.
Journal of stroke and cerebrovascular diseases: the official journal of National Stroke Association 11/2011;