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1.  Genome-Wide Sequencing of Cellular microRNAs Identifies a Combinatorial Expression Signature Diagnostic of Sepsis 
PLoS ONE  2013;8(10):e75918.
Rationale
Sepsis is a common cause of death in the intensive care unit with mortality up to 70% when accompanied by multiple organ dysfunction. Rapid diagnosis and the institution of appropriate antibiotic therapy and pressor support are therefore critical for survival. MicroRNAs are small non-coding RNAs that play an important role in the regulation of numerous cellular processes, including inflammation and immunity.
Objectives
We hypothesized changes in expression of microRNAs during sepsis may be of diagnostic value in the intensive care unit (ICU).
Methods
Massively parallel sequencing of microRNAs was utilised for screening microRNA candidates. Putative microRNAs were validated using quantitative real-time PCR (qRT-PCR). This study includes data from both a training cohort (UK) and an independent validation cohort (Sweden). A linear discriminant statistical model was employed to construct a diagnostic microRNA signature.
Results
A panel of known and novel microRNAs were detectable in the blood of patients with sepsis. After qRT-PCR validation, microRNA miR-150 and miR-4772-5p-iso were able to discriminate between patients who have systemic inflammatory response syndrome and patients with sepsis. This finding was also validated in independent cohort with an average diagnostic accuracy of 86%. Fractionating the cellular components of blood reveals miR-4772-5p-iso is expressed differentially in monocytes. Functional experiments using primary human monocytes demonstrate that it expressed in response to TLR ligation.
Conclusions
Taken together, these data provide a novel microRNA signature of sepsis that should allow rapid point-of-care diagnostic assessment of patients on ICU and also provide greater insight into the pathobiology of this severe disease.
doi:10.1371/journal.pone.0075918
PMCID: PMC3797812  PMID: 24146790
2.  Clustering of Antimicrobial Resistance Outbreaks Across Bacterial Species in the Intensive Care Unit 
Outbreaks of bacterial species occur for the majority of bacteria commonly identified in the intensive care unit. This study provides evidence for frequent temporal clustering of resistance outbreaks consistent with interspecies transmission of resistance elements.
Background There are frequent reports of intensive care unit (ICU) outbreaks due to transmission of particular antibiotic-resistant bacteria. Less is known about the burden of outbreaks of resistance due to horizontal transfer of mobile genetic elements between species. Moreover, the potential of existing statistical software as a preliminary means for detecting such events has never been assessed. This study uses a software package to determine the burden of species and resistance outbreaks in 2 adjacent ICUs and to look for evidence of clustering of resistance outbreaks consistent with interspecies transmission of resistance elements.
Methods A retrospective analysis of data from 2 adjacent 15-bed adult ICUs between 2002 and 2009 was undertaken. Detection of bacterial species-groups and resistance outbreaks was conducted using SaTScan and WHONet-SaTScan software. Resampling and permutation methods were applied to investigate temporal clustering of outbreaks.
Results Outbreaks occurred for 69% of bacterial species-groups (18/26), and resistance outbreaks were detected against 63% of antibiotics (10/16). Resistance outbreaks against 7 of 10 antibiotics were observed in multiple species-groups simultaneously and there was evidence of inter–species-group dependence for 4 of 7 antibiotics; background temporal changes in resistance did not explain the temporal aggregation of outbreaks in 3 of 7 antibiotics.
Conclusions Species outbreaks occurred for the majority of bacteria commonly identified in the ICU. There was evidence for frequent temporal clustering of resistance outbreaks consistent with interspecies transmission of resistance elements. Wider application of outbreak detection software combined with targeted sequencing of bacterial genomes is needed to understand the contribution of interspecies gene transfer to resistance emergence.
doi:10.1093/cid/cit192
PMCID: PMC3669527  PMID: 23549524
intensive care unit; outbreaks; antibiotic resistance
3.  An Association Between Bacterial Genotype Combined With a High-Vancomycin Minimum Inhibitory Concentration and Risk of Endocarditis in Methicillin-Resistant Staphylococcus aureus Bloodstream Infection 
Methicillin-resistant Staphylococcus aureus (MRSA) CC22 isolates with reduced vancomycin susceptibility and an increased association with endocarditis have emerged on a background of population vancomycin minimum inhibitory concentration creep and a successful MRSA control program.
Introduction. Antimicrobial resistance and bacterial virulence factors may increase the risk of hematogenous complications during methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI). This study reports on the impact of increasing vancomycin minimum inhibitory concentrations (V-MICs) and MRSA clone type on risk of hematogenous complications from MRSA BSI during implementation of an effective MRSA control program.
Methods. In sum, spa typing, staphylococcal cassette chromosome mec allotyping, and vancomycin and teicoplanin MICs were performed on 821 consecutive MRSA bloodstream isolates from 1999 to 2009. Prospectively collected data, including focus of infection, were available for 695 clinically significant cases. Logistic and multinomial logistic regression was used to determine the association between clone type, vancomycin MIC (V-MIC), and focus of infection.
Results. MRSA BSIs decreased by ∼90% during the 11 years. Typing placed isolates into 3 clonal complex (CC) groups that had different population median V-MICs (CC30, 0.5 μg/mL [n = 349]; CC22, 0.75 μg/mL [n = 272]; non-CC22/30, 1.5 μg/mL [n = 199]). There was a progressive increase in the proportion of isolates with a V-MIC above baseline median in each clonal group and a disproportionate fall in the clone group with lowest median V-MIC (CC30). In contrast, there were no increases in teicoplanin MICs. High V-MIC CC22 isolates (1.5–2 μg/mL) were strongly associated with endocarditis (odds ratio, 12; 95% confidence interval, 3.72–38.9) and with a septic metastasis after catheter-related BSI (odds ratio, 106; 95% confidence interval, 12.6–883) compared with other clone type/V-MIC combinations.
Conclusions. An interaction between clone type and V-MIC can influence the risk of endocarditis associated with MRSA BSI, implying involvement of both therapeutic and host-pathogen factors.
doi:10.1093/cid/cir858
PMCID: PMC3275756  PMID: 22186774
4.  Adjunctive rifampicin to reduce early mortality from Staphylococcus aureus bacteraemia (ARREST): study protocol for a randomised controlled trial 
Trials  2012;13:241.
Background
Staphylococcus aureus bacteraemia is a common and serious infection, with an associated mortality of ~25%. Once in the blood, S. aureus can disseminate to infect almost any organ, but bones, joints and heart valves are most frequently affected. Despite the infection’s severity, the evidence guiding optimal antibiotic therapy is weak: fewer than 1,500 patients have been included in 16 randomised controlled trials investigating S. aureus bacteraemia treatment. It is uncertain which antibiotics are most effective, their route of administration and duration, and whether antibiotic combinations are better than single agents. We hypothesise that adjunctive rifampicin, given in combination with a standard first-line antibiotic, will enhance killing of S. aureus early in the treatment course, sterilise infected foci and blood faster, and thereby reduce the risk of dissemination, metastatic infection and death. Our aim is to determine whether adjunctive rifampicin reduces all-cause mortality within 14 days and bacteriological failure or death within 12 weeks from randomisation.
Methods
We will perform a parallel group, randomised (1:1), blinded, placebo-controlled trial in NHS hospitals across the UK. Adults (≥18 years) with S. aureus (meticillin-susceptible or resistant) grown from at least one blood culture who have received ≤96 h of active antibiotic therapy for the current infection and do not have contraindications to the use of rifampicin will be eligible for inclusion. Participants will be randomised to adjunctive rifampicin (600-900mg/day; orally or intravenously) or placebo for the first 14 days of therapy in combination with standard single-agent antibiotic therapy. The co-primary outcome measures will be all-cause mortality up to 14 days from randomisation and bacteriological failure/death (all-cause) up to 12 weeks from randomisation. 940 patients will be recruited, providing >80% power to detect 45% and 30% reductions in the two co-primary endpoints of death by 14 days and bacteriological failure/death by 12 weeks respectively.
Discussion
This pragmatic trial addresses the long-standing hypothesis that adjunctive rifampicin improves outcome from S. aureus bacteraemia through enhanced early bacterial killing. If proven correct, it will provide a paradigm through which further improvements in outcome from S. aureus bacteraemia can be explored.
Trial registration
Current Controlled Trial ISRCTN 37666216
doi:10.1186/1745-6215-13-241
PMCID: PMC3557210  PMID: 23249501
Staphylococcus aureus; Bacteraemia; Rifampicin; Mortality
5.  How Representative Are Research Tissue Biobanks of the Local Populations? Experience of the Infectious Diseases Biobank at King's College, London, UK 
Biopreservation and Biobanking  2011;9(3):287-288.
Biobanks have a primary responsibility to collect tissues that are a true reflection of their local population and thereby promote translational research, which is applicable to the community. The Infectious Diseases BioBank (IDB) at King's College London is located in the southeast of the city, an area that is ethnically diverse. Transplantation programs have frequently reported a low rate of donation among some ethnic minorities. To determine whether patients who volunteered peripheral venous blood samples to the IDB were representative of the local community, we compared local government demographic data to characteristics of patients who have donated to the IDB. There was a good match between these statistics, indicating that the IDB's volunteer population of human immunodeficiency virus patients was similar to local demographics.
doi:10.1089/bio.2011.0015
PMCID: PMC3178420  PMID: 21977243
6.  Quantifying Type-Specific Reproduction Numbers for Nosocomial Pathogens: Evidence for Heightened Transmission of an Asian Sequence Type 239 MRSA Clone 
PLoS Computational Biology  2012;8(4):e1002454.
An important determinant of a pathogen's success is the rate at which it is transmitted from infected to susceptible hosts. Although there are anecdotal reports that methicillin-resistant Staphylococcus aureus (MRSA) clones vary in their transmissibility in hospital settings, attempts to quantify such variation are lacking for common subtypes, as are methods for addressing this question using routinely-collected MRSA screening data in endemic settings. Here we present a method to quantify the time-varying transmissibility of different subtypes of common bacterial nosocomial pathogens using routine surveillance data. The method adapts approaches for estimating reproduction numbers based on the probabilistic reconstruction of epidemic trees, but uses relative hazards rather than serial intervals to assign probabilities to different sources for observed transmission events. The method is applied to data collected as part of a retrospective observational study of a concurrent MRSA outbreak in the United Kingdom with dominant endemic MRSA clones (ST22 and ST36) and an Asian ST239 MRSA strain (ST239-TW) in two linked adult intensive care units, and compared with an approach based on a fully parametric transmission model. The results provide support for the hypothesis that the clones responded differently to an infection control measure based on the use of topical antiseptics, which was more effective at reducing transmission of endemic clones. They also suggest that in one of the two ICUs patients colonized or infected with the ST239-TW MRSA clone had consistently higher risks of transmitting MRSA to patients free of MRSA. These findings represent some of the first quantitative evidence of enhanced transmissibility of a pandemic MRSA lineage, and highlight the potential value of tailoring hospital infection control measures to specific pathogen subtypes.
Author Summary
Different strains of hospital pathogens may differ in their ability to spread between patients and respond differently to control measures. Attempts to quantify such between-strain variation are lacking in high prevalence settings. We analysed data from concurrent outbreaks with different MRSA strains in two adult intensive care units. MRSA is usually carried by patients asymptomatically, and most of our data came from routine screening swabs used to detect such carriage. We divided strains into two groups: common United Kingdom strains and strains from a type often found in Southeast Asia. We developed a new method to estimate how transmission changes over time and compared results with those from an adaptation of a previously described approach. An advantage of the new method is that it makes weaker assumptions about the process generating the data. The methods gave broadly similar results: the introduction of daily antiseptic bodywashes for all patients was the only intervention associated with a substantial fall in transmission, but this intervention was less effective for the Asian strain. This work should be useful for assessing the between-strain variation in the transmission of other hospital pathogens, and for assessing the impact of interventions on patient-to-patient transmission.
doi:10.1371/journal.pcbi.1002454
PMCID: PMC3325179  PMID: 22511854
7.  Evolution of MRSA During Hospital Transmission and Intercontinental Spread 
Science (New York, N.Y.)  2010;327(5964):469-474.
Current methods for differentiating isolates of predominant lineages of pathogenic bacteria often do not provide sufficient resolution to define precise relationships. Here, we describe a high-throughput genomics approach that provides a high-resolution view of the epidemiology and microevolution of a dominant strain of methicillin-resistant Staphylococcus aureus (MRSA). This approach reveals the global geographic structure within the lineage, its intercontinental transmission through four decades, and the potential to trace person-to-person transmission within a hospital environment. The ability to interrogate and resolve bacterial populations is applicable to a range of infectious diseases, as well as microbial ecology.
doi:10.1126/science.1182395
PMCID: PMC2821690  PMID: 20093474
8.  Clinical Application of Real-Time PCR to Screening Critically Ill and Emergency-Care Surgical Patients for Methicillin-Resistant Staphylococcus aureus: a Quantitative Analytical Study▿ †  
Journal of Clinical Microbiology  2009;47(12):4102-4108.
The clinical utility of real-time PCR screening assays for methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) colonization is constrained by the predictive values of their results: as MRSA prevalence falls, the assay's positive predictive value (PPV) drops, and a rising proportion of positive PCR assays will not be confirmed by culture. We provide a quantitative analysis of universal PCR screening of critical care and emergency surgical patients using the BD GeneOhm MRSA PCR system, involving 3,294 assays over six months. A total of 248 PCR assays (7.7%) were positive; however, 88 failed to be confirmed by culture, giving a PPV of 65%. Multivariate analysis was performed to compare PCR-positive culture-positive (P+C+) and PCR-positive culture-negative (P+C−) assays. P+C− results were positively associated with a history of methicillin-sensitive Staphylococcus aureus infection or colonization (odds ratio [OR], 3.15; 95% confidence interval [CI], 1.32 to 7.54) and high PCR thresholds of signal intensity, indicative of a low concentration of target DNA (OR, 1.19 per cycle; 95% CI, 1.11 to 1.26). P+C− results were negatively associated with a history of MRSA infection or colonization (OR, 0.19; 95% CI, 0.09 to 0.42) and male sex (OR, 0.40; 95% CI, 0.20 to 0.81). P+C+ patients were significantly more likely to have subsequent positive MRSA culture assays and microbiological evidence of clinical MRSA infection. The risk of subsequent MRSA infection in P+C− patients was not significantly different from that in case-matched PCR-negative controls. We conclude that, given the low PPV and poor correlation between a PCR-positive assay and the clinical outcome, it would be prudent to await culture confirmation before altering infection control measures on the basis of a positive PCR result.
doi:10.1128/JCM.01332-09
PMCID: PMC2786683  PMID: 19846648
9.  Genome Sequence of a Recently Emerged, Highly Transmissible, Multi-Antibiotic- and Antiseptic-Resistant Variant of Methicillin-Resistant Staphylococcus aureus, Sequence Type 239 (TW)▿ † 
Journal of Bacteriology  2009;192(3):888-892.
The 3.1-Mb genome of an outbreak methicillin-resistant Staphylococcus aureus (MRSA) strain (TW20) contains evidence of recently acquired DNA, including two large regions (635 kb and 127 kb). The strain is resistant to a wide range of antibiotics, antiseptics, and heavy metals due to resistance genes encoded on mobile genetic elements and also mutations in housekeeping genes.
doi:10.1128/JB.01255-09
PMCID: PMC2812470  PMID: 19948800
10.  Human Monocytes Kill Shigella flexneri but Then Die by Apoptosis Associated with Suppression of Proinflammatory Cytokine Production  
Infection and Immunity  2002;70(7):3833-3842.
Shigella flexneri infection of human macrophages is followed by rapid bacterial escape into the cytosol and secretion of IpaB, which activates caspase-1 to mediate cell death and release of mature interleukin (IL)-1β. Here we report a different outcome following infection of human peripheral blood monocytes. S. flexneri infects monocytes inefficiently in the absence of complement and, following complement-dependent uptake, cannot escape the endosomal compartment. Consequently, bacteria are killed within the first 60 min in the absence of monocyte cell death, as demonstrated by immunofluorescence and electron microscopy and enumeration of colonies in a gentamicin protection assay. Despite early bacterial death, wild-type S. flexneri influenced the subsequent monocyte proinflammatory cytokine response and cell fate. Infection with wild-type S. flexneri resulted in IpaB-dependent suppression of IL-1β, tumor necrosis factor alpha, and IL-6 compared with that of plasmid-cured avirulent S. flexneri-infected cells. Furthermore, over the following 6 to 8 h, virulent S. flexneri-infected monocytes died by apoptosis whereas avirulent infected monocytes died by necrosis. Together, these results imply that monocytes migrating into the inflammatory site during the early stages of shigellosis kill S. flexneri but that during bacterial uptake, they receive virulence signals from S. flexneri which induce delayed apoptosis associated with suppression of the proinflammatory cytokine response to bacterial phagocytosis. This delayed apoptosis may have important effects on the ordered initiation of the innate immune response, leading to the excessive inflammatory response characteristic of shigellosis.
doi:10.1128/IAI.70.7.3833-3842.2002
PMCID: PMC128053  PMID: 12065527
11.  Screening, isolation, and decolonisation strategies in the control of meticillin resistant Staphylococcus aureus in intensive care units: cost effectiveness evaluation 
Objective To assess the cost effectiveness of screening, isolation, and decolonisation strategies in the control of meticillin resistant Staphylococcus aureus (MRSA) in intensive care units.
Design Economic evaluation based on a dynamic transmission model.
Setting England and Wales.
Population Theoretical population of patients on an intensive care unit.
Main outcome measures Infections, deaths, costs, quality adjusted life years (QALYs), incremental cost effectiveness ratios for alternative strategies, and net monetary benefits.
Results All decolonisation strategies improved health outcomes and reduced costs. Although universal decolonisation (regardless of MRSA status) was the most cost effective in the short term, strategies using screening to target MRSA carriers may be preferred owing to the reduced risk of selecting for resistance. Among such targeted strategies, universal admission and weekly screening with polymerase chain reaction coupled with decolonisation using nasal mupirocin was the most cost effective. This finding was robust to the size of intensive care units, prevalence of MRSA on admission, proportion of patients classified as high risk, and precise value of willingness to pay for health benefits. All strategies using isolation but not decolonisation improved health outcomes but costs were increased. When the prevalence of MRSA on admission to the intensive care unit was 5% and the willingness to pay per QALY gained was between £20 000 (€23 000; $32 000) and £30 000, the best such strategy was to isolate only those patients at high risk of carrying MRSA (either pre-emptively or after identification by admission and weekly screening for MRSA using chromogenic agar). Universal admission and weekly screening using polymerase chain reaction based detection of MRSA coupled with isolation was unlikely to be cost effective unless prevalence was high (10% of patients colonised with MRSA on admission).
Conclusions MRSA control strategies that use decolonisation are likely to be cost saving in an intensive care unit setting provided resistance is lacking, and combining universal screening using polymerase chain reaction with decolonisation is likely to represent good value for money if untargeted decolonisation is considered unacceptable. In intensive care units where decolonisation is not implemented, evidence is insufficient to support universal screening for MRSA outside high prevalence settings.
doi:10.1136/bmj.d5694
PMCID: PMC3188660  PMID: 21980062

Results 1-11 (11)