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1.  Genetic Variation of Vibrio cholerae during Outbreaks, Bangladesh, 2010–2011 
Emerging Infectious Diseases  2014;20(1):54-60.
Most isolates are closely related, but genetic variation implies accelerated transmission of some lineages.
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.
PMCID: PMC3884724  PMID: 24377372
multilocus sequence analysis; infectious disease outbreaks; Vibrio cholerae; bacteria; Bangladesh; cholera; outbreak
2.  Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition 
Genome Biology  2014;15(6):R76.
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.
PMCID: PMC4072981  PMID: 24995464
3.  Robust methods for differential abundance analysis in marker gene surveys 
Nature methods  2013;10(12):1200-1202.
We introduce a novel methodology for differential abundance analysis in sparse high-throughput 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 under-sampling: a common feature of large-scale marker gene studies. We show, using simulated data and several published microbiota datasets, that metagenomeSeq outperforms the tools currently used in this field.
PMCID: PMC4010126  PMID: 24076764
4.  Illicit Drug Use and Risk for USA300 Methicillin-Resistant Staphylococcus aureus Infections with Bacteremia 
Emerging Infectious Diseases  2010;16(9):1419-1427.
PMCID: PMC3294968  PMID: 20735927
Staphylococcus aureus; antimicrobial resistance; community-acquired infections; bacteremia; bacterial typing techniques; electrophoresis; bacteria; illicit drug use; research
5.  Survey of Culture, GoldenGate Assay, Universal Biosensor Assay, and 16S rRNA Gene Sequencing as Alternative Methods of Bacterial Pathogen Detection 
Journal of Clinical Microbiology  2013;51(10):3263-3269.
Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding 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, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.
PMCID: PMC3811648  PMID: 23884998
6.  Discovery of STL polyomavirus, a polyomavirus of ancestral recombinant origin that encodes a unique T antigen by alternative splicing 
Virology  2012;436(2):295-303.
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.
PMCID: PMC3693558  PMID: 23276405
Polyomavirus; Virus discovery; Tumor antigen; Alternative splicing; Recombination
7.  Molecular Epidemiology of Geographically Dispersed Vibrio cholerae, Kenya, January 2009–May 2010 
Emerging Infectious Diseases  2012;18(6):925-931.
Isolates represent multiple genetic lineages, a finding consistent with multiple emergences from endemic reservoirs.
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.
PMCID: PMC3358164  PMID: 22607971
phenotypes; genotypes; Vibrio cholerae; cholera; characterization; molecular epidemiology; outbreaks; bacteria; Kenya
8.  Downregulated Kynurenine 3-Monooxygenase Gene Expression and Enzyme Activity in Schizophrenia and Genetic Association With Schizophrenia Endophenotypes 
Archives of general psychiatry  2011;68(7):10.1001/archgenpsychiatry.2011.71.
Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan degradation, is an antagonist at N-methyl-d-aspartate and α7 nicotinic acetylcholine receptors and modulates glutamate, dopamine, and acetylcholine signaling. Cortical kynurenic acid concentrations are elevated in the brain and cerebrospinal fluid of schizophrenia patients. The proximal cause may be an impairment of kynurenine 3-monooxygenase (KMO), a rate-limiting enzyme at the branching point of the kynurenine pathway.
To examine KMO messenger RNA expression and KMO enzyme activity in postmortem tissue from the frontal eye field (FEF; Brodmann area 6) obtained from schizophrenia individuals compared with healthy control individuals and to explore the relationship between KMO single-nucleotide polymorphisms and schizophrenia oculomotor endophenotypes.
Case-control postmortem and clinical study.
Maryland Brain Collection, outpatient clinics.
Postmortem specimens from schizophrenia patients (n=32) and control donors (n=32) and a clinical sample of schizophrenia patients (n=248) and healthy controls (n=228).
Main Outcome Measures
Comparison of quantitative KMO messenger RNA expression and KMO enzyme activity in postmortem FEF tissue between schizophrenia patients and controls and association of KMO single-nucleotide polymorphisms with messenger RNA expression in postmortem FEF and schizophrenia and oculomotor endophenotypes (ie, smooth pursuit eye movements and oculomotor delayed response).
In postmortem tissue, we found a significant and correlated reduction in KMO gene expression and KMO enzyme activity in the FEF in schizophrenia patients. In the clinical sample, KMO rs2275163 was not associated with a diagnosis of schizophrenia but showed modest effects on predictive pursuit and visuospatial working memory endophenotypes.
Our results provide converging lines of evidence implicating reduced KMO activity in the etiopathophysiology of schizophrenia and related neurocognitive deficits.
PMCID: PMC3855543  PMID: 21727251
9.  Quantitative PCR for Detection of Shigella Improves Ascertainment of Shigella Burden in Children with Moderate-to-Severe Diarrhea in Low-Income Countries 
Journal of Clinical Microbiology  2013;51(6):1740-1746.
Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 104 ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 104 ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥2.9 × 104 ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 104 ipaH copies (95% confidence interval, 3.7 to 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 in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 104 ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.
PMCID: PMC3716050  PMID: 23536399
10.  Diarrheagenic Pathogens in Polymicrobial Infections 
Emerging Infectious Diseases  2011;17(4):606-611.
During systematic active surveillance of the causes of diarrhea in patients admitted to the Infectious Diseases and Beliaghata General Hospital in Kolkata, India, we looked for 26 known gastrointestinal pathogens in fecal samples from 2,748 patients. Samples from about one-third (29%) of the patients contained multiple pathogens. Polymicrobial infections frequently contained Vibrio cholerae O1 and rotavirus. When these agents were present, some co-infecting agents were found significantly less often (p = 10–5 to 10–33), some were detected significantly more often (p = 10–5 to 10–26), and others were detected equally as often as when V. cholerae O1 or rotavirus was absent. When data were stratified by patient age and season, many nonrandom associations remained statistically significant. The causes and effects of these nonrandom associations remain unknown.
PMCID: PMC3377398  PMID: 21470448
Diarrhea; Vibrio infections; rotavirus; Vibrio cholerae; coinfection; bacteria; viruses; parasites; research
11.  Vibrio cholerae O1 El Tor and O139 Bengal Strains Carrying ctxBET, Bangladesh 
Emerging Infectious Diseases  2013;19(10):1713-1715.
PMCID: PMC3810759  PMID: 24050113
Vibrio cholerae; ctxBET; ctxBCL; altered El Tor; prototype El Tor; antibiotic resistance; PFGE; pulsed-field gel electrophoresis; bacteria
12.  A small core predatory genome in the divergent marine Bacteriovorax marinus SJ and the terrestrial Bdellovibrio bacteriovorus 
The ISME journal  2012;7(1):148-160.
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 B. bacteriovorus HD100. Based on their predatory lifestyle, Bacteriovorax were originally designated 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, attaches, penetrates through the cell wall to form a bdelloplast, in which it replicates, as shown using microscopy.
Bacteriovorax is distinct, since 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 shared geneset Bacteriovorax and Bdellovibrio that is not conserved amongst 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 key indicators of predatory-specific processes required for prey entry. The hit locus from Bd. bacteriovorus is implicated in the switch from predatory to prey/host-independent (HI) 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 genera.
PMCID: PMC3526173  PMID: 22955231
13.  Seasonal Cholera from Multiple Small Outbreaks, Rural Bangladesh 
Emerging Infectious Diseases  2008;14(5):831-833.
Clinical and environmental Vibrio cholerae organisms collected from February 2004 through April 2005 were systematically isolated from 2 rural Bangladeshi locales. Their genetic relatedness was evaluated at 5 loci that contained a variable number of tandem repeats (VNTR). The observed minimal overlap in VNTR patterns between the 2 communities was consistent with sequential, small outbreaks from local sources.
PMCID: PMC2600222  PMID: 18439375
Cholera; variable number of tandem repeats; epidemiology; outbreaks; genetic variation; dispatch
14.  Prolonged Colonization with the Methicillin-Resistant Staphylococcus aureus Strain USA300 among Residents of Extended Care Facilities 
We performed a retrospective cohort study (n = 129) to assess whether residents of extended care facilities who were initially colonized or infected with the methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 were less likely to have prolonged colonization than were residents colonized or infected with other MRSA strains. We found no difference in prolonged colonization (adjusted odds ratio, 1.1 [95% confidence interval, 0.5–2.4]).
PMCID: PMC3677585  PMID: 20569116
15.  Molecular Epidemiology of O139 Vibrio cholerae: Mutation, Lateral Gene Transfer, and Founder Flush 
Emerging Infectious Diseases  2003;9(7):810-814.
Vibrio cholerae in O-group 139 was first isolated in 1992 and by 1993 had been found throughout the Indian subcontinent. This epidemic expansion probably resulted from a single source after a lateral gene transfer (LGT) event that changed the serotype of an epidemic V. cholerae O1 El Tor strain to O139. However, some studies found substantial genetic diversity, perhaps caused by multiple origins. To further explore the relatedness of O139 strains, we analyzed nine sequenced loci from 96 isolates from patients at the Infectious Diseases Hospital, Calcutta, from 1992 to 2000. We found 64 novel alleles distributed among 51 sequence types. LGT events produced three times the number of nucleotide changes compared to mutation. In contrast to the traditional concept of epidemic spread of a homogeneous clone, the establishment of variant alleles generated by LGT during the rapid expansion of a clonal bacterial population may be a paradigm in infections and epidemics.
PMCID: PMC3023423  PMID: 12890320
cholera; evolution; base sequence; DNA; bacterial; evolution; molecular sequence data; research
16.  Evidence of Missense Mutations on the Neuregulin 1 Gene Affecting Function of Prepulse Inhibition 
Biological psychiatry  2007;63(1):17-23.
Neuregulin 1 (NRG1) is one of the leading candidate genes in schizophrenia. Rodents with NRG1 knock-out showed significantly impaired prepulse inhibition (PPI) in the original report linking NRG1 to schizophrenia (Stefansson et al 2002). PPI is a widely used surrogate measure of psychosis in animal models and is considered a schizophrenia endophenotype. We hypothesized that if NRG1 influences PPI in rodents, then it should have a similar effect on PPI in humans.
We examined the potential neurophysiological effects of two nonsynonymous single nucleotide polymorphisms (SNPs) located on NRG1 (rs3924999 and rs10503929) on PPI. Genotyping were completed in 430 unrelated individuals, including 244 schizophrenia cases and 186 controls. PPI was available in a subgroup of 113 cases and 63 controls.
Rs3924999 genotype was significantly associated with PPI (p=0.003): PPI was lowest in the subjects who were homozygous for the minor allele A/A carriers, intermediate in A/G carriers, and highest in homozygous major alleles G/G carriers. The associations persisted within cases (p=0.02) and controls (p=0.02) analyzed separately. An additive model suggested that rs3924999 alone contributes to 7.9% of the PPI variance. In contrast, rs10503929 genotype was not associated with PPI (p=0.85). Schizophrenia patients had reduced PPI compared to control subjects (p=0.04). Neither SNP was associated with schizophrenia (all p>0.37). However, schizophrenia patients with abnormal PPI may be associated with rs3924999 (p=0.05).
A missense mutation on rs3924999 of the neuregulin 1 gene may have a functional effect on prepulse inhibition in both schizophrenia and healthy control populations.
PMCID: PMC3569848  PMID: 17631867
schizophrenia; glutamatergic; psychosis; endophenotype; nonsynonymous; polymorphism; PPI; NRG; NMDA; SNP
17.  Polymorphisms in migraine-associated gene, atp1a2, and ischemic stroke risk in a biracial population: the genetics of early onset stroke study 
SpringerPlus  2013;2:46.
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.
PMCID: PMC3582818  PMID: 23459313
Headache; Migraine; Stroke; Genetics; ATP1A2; Young
18.  Identification of Coli Surface Antigen 23, a Novel Adhesin of Enterotoxigenic Escherichia coli 
Infection and Immunity  2012;80(8):2791-2801.
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.
PMCID: PMC3434557  PMID: 22645287
19.  A small predatory core genome in the divergent marine Bacteriovorax marinus SJ and the terrestrial Bdellovibrio bacteriovorus 
The ISME Journal  2012;7(1):148-160.
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.
PMCID: PMC3526173  PMID: 22955231
Bacteriovorax; Bdellovibrio; genome sequence; BALO; subtractive hybridization; host-interaction locus
20.  USA300 Methicillin-Resistant Staphylococcus aureus Bacteremia and the Risk of Severe Sepsis: Is USA300 MRSA Associated with More Severe Infections? 
USA300 methicillin-resistant Staphylococcus aureus (MRSA) is increasing as a cause of severe community-associated bacteremic infections. We assessed severe sepsis in response to infection in patients with USA300 MRSA compared to non-USA300 MRSA bacteremia.
A cohort study was conducted from 1997–2008 comparing sepsis in response to infection in 271 patients with MRSA bacteremia from four VA hospitals.
Sixty-seven (25%) patients with MRSA bacteremia were USA300 MRSA; 204 (75%) were non-USA300 MRSA. The proportion of MRSA bacteremia caused by USA300 MRSA increased over time (χ2 p<0.0001). Adjusting for age and nosocomial infection, patients with USA300 MRSA bacteremia were more likely to have severe sepsis or septic shock in response to infection than patients with non-USA300 MRSA bacteremia (adjusted Relative Risk=1.82; 95% CI: 1.16–2.87; p=0.01).
This suggests that patients with USA300 MRSA are more likely to develop severe sepsis in response to their infection, which could be due to host or bacterial differences.
PMCID: PMC3118841  PMID: 21558047
21.  A CHRNA5 Allele Related to Nicotine Addiction and Schizophrenia 
Genes, brain, and behavior  2011;10(5):530-535.
Schizophrenia and nicotine addiction are both highly heritable phenotypes. Because individuals with schizophrenia have a higher rate of smoking than those in the general population, one could hypothesize that genes associated with smoking might be over-represented in schizophrenia and thus help explain their increased smoking incidence. Although a number of genes have been proposed to explain the increased smoking risk in schizophrenia, none of them have been consistently linked to smoking and schizophrenia and thus difficult to explain the increased smoking in schizophrenia. A functional smoking-related nicotinic acetylcholine receptor α5 subunit gene (CHRNA5) nonsynonymous SNP rs16969968 (Asp398Asn) has recently been discovered and replicated. As such, we tested whether this variant contributes to smoking in schizophrenia in a sample of 313 schizophrenia patients and 525 controls. The Asp398Asn risk allele is significantly associated with smoking severity independently in schizophrenia patient smokers (p=0.001) and in control smokers (p=0.029). Furthermore, the same risk allele is significantly associated with schizophrenia in both Caucasian (p=0.022) and African American (p=0.006) nonsmoker schizophrenia patients compared to control nonsmokers. Intriguing, this SNP was not significantly associated with smoking status (smokers vs. nonsmokers) in either schizophrenia patients or controls. Therefore, our study identifies a genetic variant that is simultaneously linked to smoking and schizophrenia in the same cohort, but whether and how this SNP contributes to the increased smoking prevalence in schizophrenia patients requires additional studies.
PMCID: PMC3126887  PMID: 21418140
smoking; nicotine addiction; schizophrenia; nAChR; alpha5; comorbidity
22.  Return of Chloroquine-Susceptible Falciparum Malaria in Malawi Was a Reexpansion of Diverse Susceptible Parasites 
The Journal of infectious diseases  2010;202(5):801-808.
The spread of drug-resistant Plasmodium falciparum malaria has been a major impediment to malaria control and threatens prospects for elimination. We recently demonstrated the return of chloroquine-susceptible malaria in Malawi after chloroquine use was abandoned. In this study, we trace the origins of chloroquine-resistant and chloroquine-susceptible parasites in Malawi by sequencing the P. falciparum chloroquine resistance transporter gene (pfcrt) and by genotyping microsatellites flanking this gene in isolates from infections that occurred in Malawi from 1992 through 2005. Malaria parasites from 2005 harbored the expected wild-type pfcrt haplotype associated with chloroquine susceptibility and have maintained high levels of diversity without linkage disequilibrium, which suggests that the return of chloroquine susceptibility is not the result of a back mutation in a formerly resistant parasite or a new selective sweep. Chloroquine-susceptible parasites that predominate in Malawi likely represent a reexpansion of the susceptible parasites that survived in the population despite widespread drug pressure in the region.
PMCID: PMC3380613  PMID: 20662717
23.  Deep Sequencing of the Oral Microbiome Reveals Signatures of Periodontal Disease 
PLoS ONE  2012;7(6):e37919.
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.
PMCID: PMC3366996  PMID: 22675498
24.  Rare Variants in Ischemic Stroke: An Exome Pilot Study 
PLoS ONE  2012;7(4):e35591.
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.
PMCID: PMC3334983  PMID: 22536414
25.  The Role of Patient-to-Patient Transmission in the Acquisition of Imipenem-Resistant Pseudomonas aeruginosa Colonization in the Intensive Care Unit 
The Journal of Infectious Diseases  2009;200(6):900-905.
Imipenem-resistant Pseudomonas aeruginosa (IRPA) is an emerging problem. The causal role of antibiotic selective pressure versus patient-to-patient transmission has not been assessed using a large cohort.
Patients who were admitted to the medical and surgical intensive care units (ICUs) at the University of Maryland Medical Center from 2001 through 2006 had multiple perianal culture samples collected. Using pulsed-field gel electrophoresis (PFGE), the number of patients who acquired IRPA as a result of patient-to-patient transmission was determined. We also analyzed a subset of patients who had a previous surveillance culture that grew an imipenem-susceptible P. aeruginosa (ISPA) and a subsequent culture that grew IRPA.
Our cohort consisted of 7071 patients. Three hundred patients were colonized with IRPA. 151 patients had positive culture findings at ICU admission, and 149 patients acquired an IRPA. Among the patients who acquired IRPA, 46 (31%) had a PFGE pattern similar to that for another isolate, and 38 (26%) were found to be colonized with an ISPA on the basis of earlier culture results. Of the 38-patient subset, 28 (74%) had identical PFGE patterns.
Our data showed that, of those cases of IRPA acquisition, 46 (31%) were defined as cases of patient-to-patient transmission, and 28 (19%) were cases of acquisition by the patients’ endogenous flora.
PMCID: PMC3312466  PMID: 19673646

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