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1.  What’s in a Name? Species-Wide Whole-Genome Sequencing Resolves Invasive and Noninvasive Lineages of Salmonella enterica Serotype Paratyphi B 
mBio  2016;7(4):e00527-16.
ABSTRACT
For 100 years, it has been obvious that Salmonella enterica strains sharing the serotype with the formula 1,4,[5],12:b:1,2—now known as Paratyphi B—can cause diseases ranging from serious systemic infections to self-limiting gastroenteritis. Despite considerable predicted diversity between strains carrying the common Paratyphi B serotype, there remain few methods that subdivide the group into groups that are congruent with their disease phenotypes. Paratyphi B therefore represents one of the canonical examples in Salmonella where serotyping combined with classical microbiological tests fails to provide clinically informative information. Here, we use genomics to provide the first high-resolution view of this serotype, placing it into a wider genomic context of the Salmonella enterica species. These analyses reveal why it has been impossible to subdivide this serotype based upon phenotypic and limited molecular approaches. By examining the genomic data in detail, we are able to identify common features that correlate with strains of clinical importance. The results presented here provide new diagnostic targets, as well as posing important new questions about the basis for the invasive disease phenotype observed in a subset of strains.
IMPORTANCE
Salmonella enterica strains carrying the serotype Paratyphi B have long been known to possess Jekyll and Hyde characteristics; some cause gastroenteritis, while others cause serious invasive disease. Understanding what makes up the population of strains carrying this serotype, as well as the source of their invasive disease, is a 100-year-old puzzle that we address here using genomics. Our analysis provides the first high-resolution view of this serotype, placing strains carrying serotype Paratyphi B into the wider genomic context of the Salmonella enterica species. This work reveals a history of disease dating back to the middle ages, caused by a group of distinct lineages with various abilities to cause invasive disease. By quantifying the key genomic differences between the invasive and noninvasive populations, we are able to identify key virulence-related targets that can form the basis of simple, rapid, point-of-care tests.
doi:10.1128/mBio.00527-16
PMCID: PMC4999539  PMID: 27555304
2.  Whole-Genome Sequencing for National Surveillance of Shiga Toxin–Producing Escherichia coli O157 
Whole-genome sequencing of Shiga toxin–producing Escherichia coli O157 isolates allowed identification of linked cases with unprecedented sensitivity and specificity, with twice as many clusters identified compared with current methods.
Background. National surveillance of gastrointestinal pathogens, such as Shiga toxin–producing Escherichia coli O157 (STEC O157), is key to rapidly identifying linked cases in the distributed food network to facilitate public health interventions. In this study, we used whole-genome sequencing (WGS) as a tool to inform national surveillance of STEC O157 in terms of identifying linked cases and clusters and guiding epidemiological investigation.
Methods. We retrospectively analyzed 334 isolates randomly sampled from 1002 strains of STEC O157 received by the Gastrointestinal Bacteria Reference Unit at Public Health England, Colindale, in 2012. The genetic distance between each isolate, as estimated by WGS, was calculated and phylogenetic methods were used to place strains in an evolutionary context.
Results. Estimates of linked clusters representing STEC O157 outbreaks in England and Wales increased by 2-fold when WGS was used instead of traditional typing techniques. The previously unidentified clusters were often widely geographically distributed and small in size. Phylogenetic analysis facilitated identification of temporally distinct cases sharing common exposures and delineating those that shared epidemiological and temporal links. Comparison with multi locus variable number tandem repeat analysis (MLVA) showed that although MLVA is as sensitive as WGS, WGS provides a more timely resolution to outbreak clustering.
Conclusions. WGS has come of age as a molecular typing tool to inform national surveillance of STEC O157; it can be used in real time to provide the highest strain-level resolution for outbreak investigation. WGS allows linked cases to be identified with unprecedented specificity and sensitivity that will facilitate targeted and appropriate public health investigations.
doi:10.1093/cid/civ318
PMCID: PMC4542925  PMID: 25888672
public health; whole-genome sequencing; Shiga toxin–producing Escherichia coli O157; national surveillance
3.  Neoadjuvant irinotecan, cisplatin, and concurrent radiation therapy with celecoxib for patients with locally advanced esophageal cancer 
BMC Cancer  2016;16:468.
Background
Patients with locally advanced esophageal cancer who are treated with trimodality therapy have a high recurrence rate. Preclinical evidence suggests that inhibition of cyclooxygenase 2 (COX2) increases the effectiveness of chemoradiation, and observational studies in humans suggest that COX-2 inhibition may reduce esophageal cancer risk. This trial tested the safety and efficacy of combining a COX2 inhibitor, celecoxib, with neoadjuvant irinotecan/cisplatin chemoradiation.
Methods
This single arm phase 2 trial combined irinotecan, cisplatin, and celecoxib with concurrent radiation therapy. Patients with stage IIA-IVA esophageal cancer received weekly cisplatin 30 mg/m2 plus irinotecan 65 mg/m2 on weeks 1, 2, 4, and 5 concurrently with 5040 cGy of radiation therapy. Celecoxib 400 mg was taken orally twice daily during chemoradiation, up to 1 week before surgery, and for 6 months following surgery.
Results
Forty patients were enrolled with stage IIa (30 %), stage IIb (20 %), stage III (22.5 %), and stage IVA (27.5 %) esophageal or gastroesophageal junction cancer (AJCC, 5th Edition). During chemoradiation, grade 3–4 treatment-related toxicity included dysphagia (20 %), anorexia (17.5 %), dehydration (17.5 %), nausea (15 %), neutropenia (12.5 %), diarrhea (10 %), fatigue (7.5 %), and febrile neutropenia (7.5 %). The pathological complete response rate was 32.5 %. The median progression free survival was 15.7 months and the median overall survival was 34.7 months. 15 % (n = 6) of patients treated on this study developed brain metastases.
Conclusions
The addition of celecoxib to neoadjuvant cisplatin-irinotecan chemoradiation was tolerable; however, overall survival appeared comparable to prior studies using neoadjuvant cisplatin-irinotecan chemoradiation alone. Further studies adding celecoxib to neoadjuvant chemoradiation in esophageal cancer are not warranted.
Trial registration
Clinicaltrials.gov: NCT00137852, registered August 29, 2005.
doi:10.1186/s12885-016-2485-9
PMCID: PMC4944495  PMID: 27412386
Esophageal cancer; Neoadjuvant therapy; Chemoradiation; Cyclooxygenase 2 inhibition
4.  Microevolution of Monophasic Salmonella Typhimurium during Epidemic, United Kingdom, 2005–2010 
Emerging Infectious Diseases  2016;22(4):617-624.
Microevolution resulted in considerable genotypic variation.
Microevolution associated with emergence and expansion of new epidemic clones of bacterial pathogens holds the key to epidemiologic success. To determine microevolution associated with monophasic Salmonella Typhimurium during an epidemic, we performed comparative whole-genome sequencing and phylogenomic analysis of isolates from the United Kingdom and Italy during 2005–2012. These isolates formed a single clade distinct from recent monophasic epidemic clones previously described from North America and Spain. The UK monophasic epidemic clones showed a novel genomic island encoding resistance to heavy metals and a composite transposon encoding antimicrobial drug resistance genes not present in other Salmonella Typhimurium isolates, which may have contributed to epidemiologic success. A remarkable amount of genotypic variation accumulated during clonal expansion that occurred during the epidemic, including multiple independent acquisitions of a novel prophage carrying the sopE gene and multiple deletion events affecting the phase II flagellin locus. This high level of microevolution may affect antigenicity, pathogenicity, and transmission.
doi:10.3201/eid2204.150531
PMCID: PMC4806966  PMID: 26982594
Salmonella; molecular epidemiology; monophasic; microevolution; heavy metal resistance; MDR; Salmonella Typhimurium; bacteria; United Kingdom; antimicrobial resistance
5.  Whole-miRNome profiling identifies prognostic serum miRNAs in esophageal adenocarcinoma: the influence of Helicobacter pylori infection status 
Carcinogenesis  2014;36(1):87-93.
Summary
The present study showed, for the first time, that aberrant cfmiRNAs may contribute to survival outcome of esophageal adenocarcinome (EA) and H. pylori infection status may modify the association between cfmiRNAs and EA survival.
Cell free circulating microRNAs (cfmiRNAs) have been recognized as robust and stable biomarkers of cancers. However, little is known about the prognostic significance of cfmiRNAs in esophageal adenocarcinoma (EA). In this study, we explored whether specific cfmiRNA profiles could predict EA prognosis and whether Helicobacter pylori (HP) infection status could influence the association between cfmiRNAs and EA survival outcome. We profiled 1075 miRNAs in pooled serum samples from 30 EA patients and 30 healthy controls. The most relevant cfmiRNAs were then assessed for their associations with EA survival in an independent cohort of 82 patients, using Log-rank test and multivariate Cox regression models. Quantitative real-time PCR (qRT-PCR) was used for cfmiRNA profiling. HP infection status was determined by immunoblotting assay. We identified a panel of 18 cfmiRNAs that could distinguish EA patients from healthy subjects (P = 3.0E–12). In overall analysis and in HP-positive subtype patients, no cfmiRNA was significantly associated with EA prognosis. In HP-negative patients, however, 15 cfmiRNAs were significantly associated with overall survival (OS) (all P < 0.05). A combined 2-cfmiRNA (low miR-3935 and high miR-4286) risk score was constructed; that showed greater risk for worse OS (HR = 2.22, P = 0.0019) than individual cfmiRNA alone. Patients with high-risk score had >10-fold increased risk of death than patients with low risk score (P = 0.0302; HR = 10.91; P = 0.0094). Our findings suggest that dysregulated cfmiRNAs may contribute to EA survival outcome and HP infection status may modify the association between cfmiRNAs and EA survival.
doi:10.1093/carcin/bgu228
PMCID: PMC4291048  PMID: 25381453
6.  Invasive Adenocarcinoma of the Lung is Associated with the Upper Lung Regions 
Objectives
We postulated that ventilation-perfusion (V/Q) relationships within the lung might influence where lung cancer occurs. To address this hypothesis we evaluated the location of lung adenocarcinoma, by both tumor lobe and superior-inferior regional distribution, and associated variables such as emphysema.
Materials and Methods
One hundred fifty-nine cases of invasive adenocarcinoma and adenocarcinoma with lepidic features were visually evaluated to identify lobar or regional tumor location. Regions were determined by automated division of the lungs into three equal volumes: (upper region, middle region, or lower region). Automated densitometry was used to measure radiographic emphysema.
Results
The majority of invasive adenocarcinomas occurred in the upper lobes (69%), with 94% of upper lobe adenocarcinomas occurring in the upper region of the lung. The distribution of adenocarcinoma, when classified as upper or lower lobe, was not different between invasive adenocarcinoma and adenocarcinoma with lepidic features (formerly bronchioloalveolar cell carcinoma, P=0.08). Regional distribution of tumor was significantly different between invasive adenocarcinoma and adenocarcinoma with lepidic features (P = 0.001). Logistic regression analysis with the outcome of invasive adenocarcinoma histology was used to adjust for confounders. Tumor region continued to be a significant predictor (OR 8.5, P=0.008, compared to lower region), whereas lobar location of tumor was not (P=0.09). In stratified analysis, smoking was not associated with region of invasive adenocarcinoma occurrence (p=0.089). There was no difference in total emphysema scores between invasive adenocarcinoma cases occurring in each of the three regions (P=0.155). There was also no difference in the distribution of region of adenocarcinoma occurrence between quartiles of emphysema (P=0.217).
Conclusion
Invasive adenocarcinoma of the lung is highly associated with the upper lung regions. This association is not related to smoking, history of COPD, or total emphysema. The regional distribution of invasive adenocarcinoma may be due to V/Q relationships or other local factors.
doi:10.1016/j.lungcan.2014.02.002
PMCID: PMC4004700  PMID: 24598367
Non-small cell lung cancer; tumor location; emphysema
7.  Insight into Shiga toxin genes encoded by Escherichia coli O157 from whole genome sequencing 
PeerJ  2015;3:e739.
The ability of Shiga toxin-producing Escherichia coli (STEC) to cause severe illness in humans is determined by multiple host factors and bacterial characteristics, including Shiga toxin (Stx) subtype. Given the link between Stx2a subtype and disease severity, we sought to identify the stx subtypes present in whole genome sequences (WGS) of 444 isolates of STEC O157. Difficulties in assembling the stx genes in some strains were overcome by using two complementary bioinformatics methods: mapping and de novo assembly. We compared the WGS analysis with the results obtained using a PCR approach and investigated the diversity within and between the subtypes. All strains of STEC O157 in this study had stx1a, stx2a or stx2c or a combination of these three genes. There was over 99% (442/444) concordance between PCR and WGS. When common source strains were excluded, 236/349 strains of STEC O157 had multiple copies of different Stx subtypes and 54 had multiple copies of the same Stx subtype. Of those strains harbouring multiple copies of the same Stx subtype, 33 had variants between the alleles while 21 had identical copies. Strains harbouring Stx2a only were most commonly found to have multiple alleles of the same subtype (42%). Both the PCR and WGS approach to stx subtyping provided a good level of sensitivity and specificity. In addition, the WGS data also showed there were a significant proportion of strains harbouring multiple alleles of the same Stx subtype associated with clinical disease in England.
doi:10.7717/peerj.739
PMCID: PMC4338798  PMID: 25737808
Stx; Genomics; Sequencing; O157; E. coli
8.  Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges 
Objective
To establish the relative importance of Salmonella enterica serovar Typhi with non-classical quinolone resistance.
Methods
Eight hundred and ninety-one isolates of S. Typhi, isolated between 2004 and 2011, were tested for antibiotic susceptibility determination using disc diffusion and E-test. The mechanisms of fluoroquinolone resistance were studied in a sub-set of the NALS (nalidixic acid susceptible) isolates by wave nucleic acid fragment analysis of PCR products from gyrA, gyrB, parC and parE and from the plasmid borne determinants: qnrA,B,S; aac(6′)-Ib-cr and qepA. To assess genetic relatedness multi-locus variable number tandem repeat analysis was carried out using five loci.
Results
Eighty isolates with a nalidixic acid MIC of <32 mg/L (NALS) and a ciprofloxacin MIC of >0.064 mg/L CIPI (ciprofloxacin reduced susceptibility) were found. In 36 NALS CIPI isolates two distinct genotypes were identified when compared with 16 susceptible controls: Group B (n = 34), mutation in gyrB at codon 464, NAL MIC of 3–12 mg/L and CIP MIC of 0.064–0.5 mg/L.; and Group C, mutation in gyrA at codon 83 (n = 2) NAL MIC of 16 mg/L and CIP MIC of 0.25–0.38 mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA.
Conclusion
The use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India.
doi:10.1016/j.bdq.2015.01.003
PMCID: PMC5121207  PMID: 27896141
Salmonella Typhi; Decreased ciprofloxacin susceptibility; DHPLC; gyrB mutation; VNTR
9.  Enteroaggregative Escherichia coli Have Evolved Independently as Distinct Complexes within the E. coli Population with Varying Ability to Cause Disease 
PLoS ONE  2014;9(11):e112967.
Enteroaggregative E. coli (EAEC) is an established diarrhoeagenic pathotype. The association with virulence gene content and ability to cause disease has been studied but little is known about the population structure of EAEC and how this pathotype evolved. Analysis by Multi Locus Sequence Typing of 564 EAEC isolates from cases and controls in Bangladesh, Nigeria and the UK spanning the past 29 years, revealed multiple successful lineages of EAEC. The population structure of EAEC indicates some clusters are statistically associated with disease or carriage, further highlighting the heterogeneous nature of this group of organisms. Different clusters have evolved independently as a result of both mutational and recombination events; the EAEC phenotype is distributed throughout the population of E. coli.
doi:10.1371/journal.pone.0112967
PMCID: PMC4240581  PMID: 25415318
10.  Evidence of Evolving Extraintestinal Enteroaggregative Escherichia coli ST38 Clone 
Emerging Infectious Diseases  2014;20(11):1935-1937.
doi:10.3201/eid2011.131845
PMCID: PMC4214294  PMID: 25340736
Escherichia coli; enteroaggregative; ESBL; extended-spectrum β-lactamase; extraintestinal; ST38; diarrheagenic E. coli; multiple drug resistance; Germany; the Netherlands; United Kingdom; bacteria
11.  Risk factor analysis for the recurrence of resected solitary fibrous tumours of the pleura: a 33-year experience and proposal for a scoring system† 
OBJECTIVES
Surveillance after resection of solitary fibrous tumours of the pleura (SFTP) remains undefined. This study reviews our experience with surgical treatment of SFTP to determine the specific risk factors to predict recurrence.
METHODS
A retrospective review of 59 patients surgically treated for SFTP during the years 1977–2010 was conducted. Clinico-pathological factors for recurrence were analysed by Kaplan–Meier and Cox proportional hazard methods.
RESULTS
The mean age was 57 ± 14 years. There were 32 (54%) men. Among 32 (54%) symptomatic patients, chest pain (22%), cough (19%) and dyspnoea (17%) were most frequent. The mean tumour size was 7.3 ± 6.7 cm, and 14 patients had SFTPs larger than 10 cm. An SFTP was pedunculated in 38 (67%) cases and had a visceral origin in 40 (68%). Paraneoplastic syndromes were observed in 3 (5%) patients. On histopathologic analysis, 4 (7%) presented ≥4 mitosis/10 high-power fields (HPFs), 8 (15%) atypia, 14 (24%) hypercellularity and 6 (10%) necrosis. After a mean follow-up of 8.8 ± 7.0 years, we observed 8 (14%) recurrences; median time to recurrence was 6 years (range 2–16 years). Two (3%) patients received adjuvant therapy. We constructed a predictive score for recurrence by assigning one point to each of the six variables: parietal (vs visceral) pleural origin, sessile (vs pedunculated) morphology, size >10 cm (vs <10 cm), the presence of hypercellularity, necrosis and mitotic activity ≥4/HPF (vs <4). A score of ≥3 best predicted recurrence (sensitivity: 100%, specificity: 92%, area under receiver operating characteristic curve = 0.966, P < 0.0001). With a score of ≥3, recurrence-free survival was 80%, 69, 23 and 23% at 3, 5, 10 and 15 years, whereas a score of <3 was 100% up to 15 years. Our scoring system was superior in predicting malignant behaviour and recurrence compared with England's criteria or de Perrot staging.
CONCLUSIONS
The proposed scoring system is simple, easily obtained from existing pathological description and reliably predicts recurrence in this patient population harbouring SFTP. The SFTP score may stratify patient risk and guide postoperative surveillance. We recommend validation in additional clinical series.
doi:10.1093/ejcts/ezs629
PMCID: PMC3681536  PMID: 23233072
Solitary fibrous tumour; Pleura; Recurrence
13.  An Investigation of the Diversity of Strains of Enteroaggregative Escherichia coli Isolated from Cases Associated with a Large Multi-Pathogen Foodborne Outbreak in the UK 
PLoS ONE  2014;9(5):e98103.
Following a large outbreak of foodborne gastrointestinal (GI) disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75%) of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC) group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx) genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.
doi:10.1371/journal.pone.0098103
PMCID: PMC4028294  PMID: 24844597
14.  Short And Long-Term Outcomes After Esophagectomy For Cancer In Elderly Patients 
The Annals of thoracic surgery  2013;95(5):1741-1748.
Background
As worldwide life expectancy rises, the number of candidates for surgical treatment of esophageal cancer over 70 years will increase. This study aims to examine outcomes after esophagectomy in elderly patients.
Methods
Retrospective review of 474 patients undergoing esophagectomy for cancer during 2002–2011. 334 (70.5%) patients were <70 years old (group A), 124 (26.2%) 70–79 years (group B) and 16 (3.4%) ≥80 years (group C). We analyzed the effect of age on outcome variables including overall and disease specific survival.
Results
Major morbidity was observed to occur in 115 (35.6%) patients of group A, 58 (47.9%) of group B and 10 (62.5%) of group C (p=0.010). Mortality, both 30- and 90-day was observed in 2(0.6%) and 7(2.2%) of group A, 4(3.2%) and 7 (6.1%) of group B, and 1(6.3%) and 2(14.3%) of group C, respectively (p=0.032 and p=0.013). Anastomotic leak was observed in 16(4.8%) patients of group A, 6(4.8%) of group B and 0(0%) of group C (p=0.685). Anastomotic stricture (defined by the need for ≥2 dilations) was observed in 76(22.8%) of group A, 13(10.5%) of group B and 1(6.3%) of group C (p=0.005). Five-year overall and disease specific survival was 64.8% and 72.4% for group A, 41.7% and 53.4% for group B, 49.2% and 49.2% for group C patients (p=0.0006), respectively.
Conclusions
Esophagectomy should be carefully considered in patients 70–79 years old and can be justified with low mortality. Outcomes in octogenarians are worse suggesting esophagectomy be considered on a case by case basis. Stricture rate is inversely associated to age.
doi:10.1016/j.athoracsur.2013.01.084
PMCID: PMC3732120  PMID: 23500043
Esophagus; Esophageal cancer; Esophageal surgery; Outcomes; Statistics-regression analysis
15.  Epidemiological Evidence That Garden Birds Are a Source of Human Salmonellosis in England and Wales 
PLoS ONE  2014;9(2):e88968.
The importance of wild bird populations as a reservoir of zoonotic pathogens is well established. Salmonellosis is a frequently diagnosed infectious cause of mortality of garden birds in England and Wales, predominantly caused by Salmonella enterica subspecies enterica serovar Typhimurium definitive phage types 40, 56(v) and 160. In Britain, these phage types are considered highly host-adapted with a high degree of genetic similarity amongst isolates, and in some instances are clonal. Pulsed field gel electrophoresis, however, demonstrated minimal variation amongst matched DT40 and DT56(v) isolates derived from passerine and human incidents of salmonellosis across England in 2000–2007. Also, during the period 1993–2012, similar temporal and spatial trends of infection with these S. Typhimurium phage types occurred in both the British garden bird and human populations; 1.6% of all S. Typhimurium (0.2% of all Salmonella) isolates from humans in England and Wales over the period 2000–2010. These findings support the hypothesis that garden birds act as the primary reservoir of infection for these zoonotic bacteria. Most passerine salmonellosis outbreaks identified occurred at and around feeding stations, which are likely sites of public exposure to sick or dead garden birds and their faeces. We, therefore, advise the public to practise routine personal hygiene measures when feeding wild birds and especially when handling sick wild birds.
doi:10.1371/journal.pone.0088968
PMCID: PMC3935841  PMID: 24586464
16.  Risk factors for the development of severe typhoid fever in Vietnam 
Background
Typhoid fever is a systemic infection caused by the bacterium Salmonella enterica serovar Typhi. Age, sex, prolonged duration of illness, and infection with an antimicrobial resistant organism have been proposed risk factors for the development of severe disease or fatality in typhoid fever.
Methods
We analysed clinical data from 581 patients consecutively admitted with culture confirmed typhoid fever to two hospitals in Vietnam during two periods in 1993–1995 and 1997–1999. These periods spanned a change in the antimicrobial resistance phenotypes of the infecting organisms i.e. fully susceptible to standard antimicrobials, resistance to chloramphenicol, ampicillin and trimethoprim-sulphamethoxazole (multidrug resistant, MDR), and intermediate susceptibility to ciprofloxacin (nalidixic acid resistant). Age, sex, duration of illness prior to admission, hospital location and the presence of MDR or intermediate ciprofloxacin susceptibility in the infecting organism were examined by logistic regression analysis to identify factors independently associated with severe typhoid at the time of hospital admission.
Results
The prevalence of severe typhoid was 15.5% (90/581) and included: gastrointestinal bleeding (43; 7.4%); hepatitis (29; 5.0%); encephalopathy (16; 2.8%); myocarditis (12; 2.1%); intestinal perforation (6; 1.0%); haemodynamic shock (5; 0.9%), and death (3; 0.5%). Severe disease was more common with increasing age, in those with a longer duration of illness and in patients infected with an organism exhibiting intermediate susceptibility to ciprofloxacin. Notably an MDR phenotype was not associated with severe disease. Severe disease was independently associated with infection with an organism with an intermediate susceptibility to ciprofloxacin (AOR 1.90; 95% CI 1.18-3.07; p = 0.009) and male sex (AOR 1.61 (1.00-2.57; p = 0.035).
Conclusions
In this group of patients hospitalised with typhoid fever infection with an organism with intermediate susceptibility to ciprofloxacin was independently associated with disease severity. During this period many patients were being treated with fluoroquinolones prior to hospital admission. Ciprofloxacin and ofloxacin should be used with caution in patients infected with S. Typhi that have intermediate susceptibility to ciprofloxacin.
doi:10.1186/1471-2334-14-73
PMCID: PMC3923984  PMID: 24512443
Salmonella enterica serovar Typhi; Severe typhoid; Antimicrobial resistance; Multidrug resistance; Intermediate ciprofloxacin susceptibility
17.  Immunological monitoring to prevent and treat sepsis 
Critical Care  2013;17(1):109.
The clinical, human and economic burden associated with sepsis is huge. Initiatives such as the Surviving Sepsis Campaign aim to effectively reduce risk of death from severe sepsis and septic shock. Nonetheless, although substantial benefits raised from the implementation of this campaign have been obtained, much work remains if we are to realise the full potential promised by this strategy. A deeper understanding of the processes leading to sepsis is necessary before we can design an effective suite of interventions. Dysregulation of the immune response to infection is acknowledged to contribute to the pathogenesis of the disease. Production of both proinflammatory and immunosuppressive cytokines is observed from the very first hours following diagnosis. In addition, hypogammaglobulinemia is often present in patients with septic shock. Moreover, levels of IgG, IgM and IgA at diagnosis correlate directly with survival. In turn, nonsurvivors have lower levels of C4 (a protein of the complement system) than the survivors. Natural killer cell counts and function also seem to have an important role in this disease. HLA-DR in the surface of monocytes and counts of CD4+CD25+ T-regulatory cells in blood could also be useful biomarkers for sepsis. At the genomic level, repression of networks corresponding to major histocompatibility complex antigen presentation is observed in septic shock. In consequence, cumulative evidence supports the potential role of immunological monitoring to guide measures to prevent or treat sepsis in a personalised and timely manner (early antibiotic administration, immunoglobulin replacement, immunomodulation). In conclusion, although diffuse and limited, current available information supports the development of large comprehensive studies aimed to urgently evaluate immunological monitoring as a tool to prevent sepsis, guide its treatment and, as a consequence, diminish the morbidity and mortality associated with this severe condition.
doi:10.1186/cc11922
PMCID: PMC4057291  PMID: 23351425
18.  Evaluating the Use of Multilocus Variable Number Tandem Repeat Analysis of Shiga Toxin-Producing Escherichia coli O157 as a Routine Public Health Tool in England 
PLoS ONE  2014;9(1):e85901.
Multilocus variable number tandem repeat analysis (MLVA) provides microbiological support for investigations of clusters of cases of infection with Shiga toxin-producing E. coli (STEC) O157. All confirmed STEC O157 isolated in England and submitted to the Gastrointestinal Bacteria Reference Unit (GBRU) during a six month period were typed using MLVA, with the aim of assessing the impact of this approach on epidemiological investigations. Of 539 cases investigated, 341 (76%) had unique (>2 single locus variants) MLVA profiles, 12% of profiles occurred more than once due to known household transmission and 12% of profiles occurred as part of 41 clusters, 21 of which were previously identified through routine public health investigation of cases. The remaining 20 clusters were not previously detected and STEC enhanced surveillance data for associated cases were retrospectively reviewed for epidemiological links including shared exposures, geography and/or time. Additional evidence of a link between cases was found in twelve clusters. Compared to phage typing, the number of sporadic cases was reduced from 69% to 41% and the diversity index for MLVA was 0.996 versus 0.782 for phage typing. Using MLVA generates more data on the spatial and temporal dispersion of cases, better defining the epidemiology of STEC infection than phage typing. The increased detection of clusters through MLVA typing highlights the challenges to health protection practices, providing a forerunner to the advent of whole genome sequencing as a diagnostic tool.
doi:10.1371/journal.pone.0085901
PMCID: PMC3895024  PMID: 24465775
19.  Complete Genome Sequence of the Campylobacter coli Clinical Isolate 15-537360 
Genome Announcements  2013;1(6):e01056-13.
Campylobacter coli strain 15-537360 was originally isolated in 2001 from a 42-year-old patient with gastroenteritis. Here, we report its complete genome sequence, which comprises a 1.7-Mbp chromosome and a 29-kbp conjugative cryptic plasmid. This is the first complete genome sequence of a clinical isolate of C. coli.
doi:10.1128/genomeA.01056-13
PMCID: PMC3861437  PMID: 24336384
20.  In Vivo Imaging of Tracheal Epithelial Cells in Mice during Airway Regeneration 
Many human lung diseases, such as asthma, chronic obstructive pulmonary disease, bronchiolitis obliterans, and cystic fibrosis, are characterized by changes in the cellular composition and architecture of the airway epithelium. Intravital fluorescence microscopy has emerged as a powerful approach in mechanistic studies of diseases, but it has been difficult to apply this tool for in vivo respiratory cell biology in animals in a minimally invasive manner. Here, we describe a novel miniature side-view confocal probe capable of visualizing the epithelium in the mouse trachea in vivo at a single-cell resolution. We performed serial real-time endotracheal fluorescence microscopy in live transgenic reporter mice to view the three major cell types of the large airways, namely, basal cells, Clara cells, and ciliated cells. As a proof-of-concept demonstration, we monitored the regeneration of Clara cells over 18 days after a sulfur dioxide injury. Our results show that in vivo tracheal microscopy offers a new approach in the study of altered, regenerating, or metaplastic airways in animal models of lung diseases.
doi:10.1165/rcmb.2012-0164OC
PMCID: PMC3547097  PMID: 22984086
in vivo fluorescence microscopy; mouse imaging; epithelial regeneration
21.  Genomic Characterisation of Invasive Non-Typhoidal Salmonella enterica Subspecies enterica Serovar Bovismorbificans Isolates from Malawi 
Background
Invasive Non-typhoidal Salmonella (iNTS) are an important cause of bacteraemia in children and HIV-infected adults in sub-Saharan Africa. Previous research has shown that iNTS strains exhibit a pattern of gene loss that resembles that of host adapted serovars such as Salmonella Typhi and Paratyphi A. Salmonella enterica serovar Bovismorbificans was a common serovar in Malawi between 1997 and 2004.
Methodology
We sequenced the genomes of 14 Malawian bacteraemia and four veterinary isolates from the UK, to identify genomic variations and signs of host adaptation in the Malawian strains.
Principal Findings
Whole genome phylogeny of invasive and veterinary S. Bovismorbificans isolates showed that the isolates are highly related, belonging to the most common international S. Bovismorbificans Sequence Type, ST142, in contrast to the findings for S. Typhimurium, where a distinct Sequence Type, ST313, is associated with invasive disease in sub-Saharan Africa. Although genome degradation through pseudogene formation was observed in ST142 isolates, there were no clear overlaps with the patterns of gene loss seen in iNTS ST313 isolates previously described from Malawi, and no clear distinction between S. Bovismorbificans isolates from Malawi and the UK.
The only defining differences between S. Bovismorbificans bacteraemia and veterinary isolates were prophage-related regions and the carriage of a S. Bovismorbificans virulence plasmid (pVIRBov).
Conclusions
iNTS S. Bovismorbificans isolates, unlike iNTS S. Typhiumrium isolates, are only distinguished from those circulating elsewhere by differences in the mobile genome. It is likely that these strains have entered a susceptible population and are able to take advantage of this niche. There are tentative signs of convergent evolution to a more human adapted iNTS variant. Considering its importance in causing disease in this region, S. Bovismorbificans may be at the beginning of this process, providing a reference against which to compare changes that may become fixed in future lineages in sub-Saharan Africa.
Author Summary
Bacteraemia and meningitis caused by non-typhoidal Salmonella (including serovars Typhimurium, Enteritidis and Bovismorbificans) are a serious health issue in sub-Saharan Africa, particularly in young children and HIV-infected adults. Previous work has indicated that a distinct S. Typhimurium sequence type, ST313, has evolved and spread in these countries, and may be more human-adapted than isolates found in the developed world. We therefore investigated the genomes of Salmonella enterica serovar Bovismorbificans bacteraemia isolates from Malawi and compared them to genomes of veterinary S. Bovismorbificans isolates from the UK using Next Generation Sequencing Technology and subsequent genomic comparisons to establish if there is a genetic basis for this increase in invasive disease observed among African NTS. Contrary to the previous findings for S. Typhimurium, where a distinct ST is found only in sub-Saharan Africa, we discovered that the S. Bovismorbificans isolates from Malawi belong to the most common ST of the serovar and the genome is highly conserved across all sequenced isolates. The major differences between UK veterinary and African human isolates were due to prophage regions inserted into the genomes of African isolates, coupled with a higher prevalence of a virulence plasmid compared to the UK isolates.
doi:10.1371/journal.pntd.0002557
PMCID: PMC3828162  PMID: 24244782
22.  Mechanical Ventilation and Air Leaks After Lung Biopsy for Acute Respiratory Distress Syndrome 
The Annals of thoracic surgery  2006;82(1):10.1016/j.athoracsur.2006.02.022.
Background
Open lung biopsy in acute respiratory distress syndrome (ARDS) may provide a specific etiology and change clinical management, yet concerns about complications remain. Persistent air leak is the most common postoperative complication. Risk factors in this setting are not known.
Methods
We performed a retrospective analysis of 53 patients who underwent open lung biopsy for clinical ARDS (based on American European Consensus Conference criteria) between 1989 and 2000.
Results
Sixteen patients (30.2%) developed an air leak lasting more than 7 days or died with an air leak. Univariate analyses showed no significant correlation with age, gender, sex, corticosteroid use, diabetes, immunocompromised status, or pathologic diagnosis. A lower risk of air leak was associated with lower peak airway pressure and tidal volume, use of pressure-cycled ventilation, and use of an endoscopic stapling device. In multivariate analyses, only peak airway pressure remained a significant predictor. The risk of prolonged air leak was reduced by 42% (95% confidence interval [CI: 17% to 60%]) for every 5 cm H2O reduction in peak airway pressure.
Conclusions
The use of a lung-protective ventilatory strategy that limits peak airway pressures is strongly associated with a reduced risk of postoperative air leak after open lung biopsy in ARDS. Using such a strategy may allow physicians to obtain information from open lung biopsy to make therapeutic decisions without undue harm to ARDS patients.
doi:10.1016/j.athoracsur.2006.02.022
PMCID: PMC3822769  PMID: 16798226
23.  Comparative Analysis of ESBL-Positive Escherichia coli Isolates from Animals and Humans from the UK, The Netherlands and Germany 
PLoS ONE  2013;8(9):e75392.
The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring blaCTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both blaCTX-M-group-1 and blaOXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, blaOXA-1-like and blaCTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.
doi:10.1371/journal.pone.0075392
PMCID: PMC3784421  PMID: 24086522
24.  MTHFR Polymorphisms, Folate Intake, and Carcinogen DNA Adducts in the Lung 
The methylenetetrahydrofolate reductase (MTHFR) genes and folate in one-carbon metabolism are essential for DNA methylation and synthesis. However, their role in carcinogen DNA damage in target lung tissue, a dosimeter for cancer risk, is not known. Our study aimed to investigate the association between genetic and nutritional one-carbon metabolism factors and DNA adducts in target lung. Data on 135 lung cancer cases from the Massachusetts General Hospital were studied. Genotyping was completed for MTHFR C677T (rs1801133) and A1298C (rs1801131). Information on dietary intake for one-carbon related micronutrients, folate and other B vitamin, was derived from a validated food frequency questionnaire. DNA adducts in lung were measured by 32P-postlabeling. After adjusting for potential confounders, DNA adduct levels in lung significantly increased by 69.2% [95% confidence interval (CI), 5.5% to 171.5%] for the MTHFR 1298AC+CC genotype. The high risk group, combining the A1298C (AC+CC) plus C677T (CT+TT) genotypes, had significantly enhanced levels of lung adducts by 210.7% (95% CI, 21.4% to 695.2%) in contrast to the A1298C (AA) plus C677T (CC) genotypes. Elevation of DNA adduct was pronounced - 111.3% (95% CI, −3.0 to 360.5%) among 1298AC+CC patients who consumed the lowest level of folate intake as compared with 1298AA individuals with highest tertile of intake. These results indicate that DNA adducts levels are influenced by MTHFR polymorphisms and low folate consumption, suggesting an important role of genetic and nutritional factors in protecting DNA damage from lung carcinogen in at-risk populations.
doi:10.1002/ijc.27338
PMCID: PMC3293105  PMID: 22052259
MTHFR; folate; genetic polymorphisms; DNA adducts; one carbon metabolism
25.  Public Health Value of Next-Generation DNA Sequencing of Enterohemorrhagic Escherichia coli Isolates from an Outbreak 
Journal of Clinical Microbiology  2013;51(1):232-237.
In 2009, an outbreak of enterohemorrhagic Escherichia coli (EHEC) on an open farm infected 93 persons, and approximately 22% of these individuals developed hemolytic-uremic syndrome (HUS). Genome sequencing was used to investigate outbreak-derived animal and human EHEC isolates. Phylogeny based on the whole-genome sequence was used to place outbreak isolates in the context of the overall E. coli species and the O157:H7 sequence type 11 (ST11) subgroup. Four informative single nucleotide polymorphisms (SNPs) were identified and used to design an assay to type 122 other outbreak isolates. The SNP phylogeny demonstrated that the outbreak strain was from a lineage distinct from previously reported O157:H7 ST11 EHEC and was not a member of the hypervirulent clade 8. The strain harbored determinants for two Stx2 verotoxins and other putative virulence factors. When linked to the epidemiological information, the sequence data indicate that gross contamination of a single outbreak strain occurred across the farm prior to the first clinical report of HUS. The most likely explanation for these results is that a single successful strain of EHEC spread from a single introduction through the farm by clonal expansion and that contamination of the environment (including the possible colonization of several animals) led ultimately to human cases.
doi:10.1128/JCM.01696-12
PMCID: PMC3536255  PMID: 23135946

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