Quantitative Proteomics of Clinically Relevant Drug-Metabolizing Enzymes and Drug Transporters and Their Intercorrelations in the Human Small Intestine.

The levels of drug-metabolizing enzymes (DMEs) and transporter proteins in the human intestine are pertinent to determine oral drug bioavailability. Despite the paucity of reports on such measurements, it is well recognized that these values are essential for translating in vitro data on drug metabolism and transport to predict drug disposition in gut wall. In the current study, clinically relevant DMEs [cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT)] and drug transporters were quantified in total mucosal protein preparations from the human jejunum (n = 4) and ileum (n = 12) using quantification concatemer-based targeted proteomics. In contrast to previous reports, UGT2B15 and organic anion-transporting polypeptide 1 (OATP1A2) were quantifiable in all our samples. Overall, no significant disparities in protein expression were observed between jejunum and ileum. Relative mRNA expression for drug transporters did not correlate with the abundance of their cognate protein, except for P-glycoprotein 1 (P-gp) and organic solute transporter subunit alpha (OST-α), highlighting the limitations of RNA as a surrogate for protein expression in dynamic tissues with high turnover. Intercorrelations were found within P450 [2C9-2C19 (P = 0.002, R 2 = 0.63), 2C9-2J2 (P = 0.004, R 2 = 0.40), 2D6-2J2 (P = 0.002, R 2 = 0.50)] and UGT [1A1-2B7 (P = 0.02, R 2 = 0.87)] family of enzymes. There were also correlations between P-gp and several other proteins [OST-α (P < 0.0001, R 2 = 0.77), UGT1A6 (P = 0.009, R 2 = 0.38), and CYP3A4 (P = 0.007, R 2 = 0.30)]. Incorporating such correlations into building virtual populations is crucial for obtaining plausible characteristics of simulated individuals. SIGNIFICANCE STATEMENT: A number of drug transporters were quantified for the first time in this study. Several intercorrelations of protein abundance were reported. mRNA expression levels proved to be a poor reflection of differences between individuals regarding the level of protein expression in gut. The reported abundance of drug-metabolizing enzymes and transporters and their intercorrelations will contribute to better predictions of oral drug bioavailability and drug-drug interactions by linking in vitro observations to potential outcomes through physiologically based pharmacokinetic models.

In Vitro-In Vivo Extrapolation Scaling Factors for Intestinal P-Glycoprotein and Breast Cancer Resistance Protein: Part I: A Cross-Laboratory Comparison of Transporter-Protein Abundances and Relative Expression Factors in Human Intestine and Caco-2 Cells.

Over the last 5 years the quantification of transporter-protein absolute abundances has dramatically increased in parallel to the expanded use of in vitro-in vivo extrapolation (IVIVE) and physiologically based pharmacokinetics (PBPK)-linked models, for decision-making in pharmaceutical company drug development pipelines and regulatory submissions. Although several research groups have developed laboratory-specific proteomic workflows, it is unclear if the large range of reported variability is founded on true interindividual variability or experimental variability resulting from sample preparation or the proteomic methodology used. To assess the potential for methodological bias on end-point abundance quantification, two independent laboratories, the University of Manchester (UoM) and Bertin Pharma (BPh), employing different proteomic workflows, quantified the absolute abundances of Na/K-ATPase, P-gp, and breast cancer resistance protein (BCRP) in the same set of biologic samples from human intestinal and Caco-2 cell membranes. Across all samples, P-gp abundances were significantly correlated (P = 0.04, Rs = 0.72) with a 2.4-fold higher abundance (P = 0.001) generated at UoM compared with BPh. There was a systematically higher BCRP abundance in Caco-2 cell samples quantified by BPh compared with UoM, but not in human intestinal samples. Consequently, a similar intestinal relative expression factor (REF), derived from distal jejunum and Caco-2 monolayer samples, between laboratories was found for P-gp. However, a 2-fold higher intestinal REF was generated by UoM (2.22) versus BPh (1.11). We demonstrate that differences in absolute protein abundance are evident between laboratories and they probably result from laboratory-specific methodologies relating to peptide choice.

In Vitro-In Vivo Extrapolation Scaling Factors for Intestinal P-glycoprotein and Breast Cancer Resistance Protein: Part II. The Impact of Cross-Laboratory Variations of Intestinal Transporter Relative Expression Factors on Predicted Drug Disposition.

Relative expression factors (REFs) are used to scale in vitro transporter kinetic data via in vitro-in vivo extrapolation linked to physiologically based pharmacokinetic (IVIVE-PBPK) models to clinical observations. Primarily two techniques to quantify transporter protein expression are available, immunoblotting and liquid chromatography-tandem mass spectrometry. Literature-collated REFs ranged from 0.4 to 5.1 and 1.1 to 90 for intestinal P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), respectively. The impact of using human jejunum-Caco-2 REFs for P-gp (REFiP-gp) and BCRP (REFiBCRP), generated from the same samples and using different proteomic methodologies from independent laboratories, on PBPK outcomes was assessed. A 5-fold decrease in REFiP-gp for a single oral dose of digoxin resulted in a 1.19- and 1.31-fold higher plasma area under the curve and Cmax, respectively. All generated REFiP-gp values led to simulated digoxin Cmax values within observed ranges; however, combining kinetic data generated from a different laboratory with the 5-fold lower REFiP-gp could not recover a digoxin-rifampicin drug-drug interaction, emphasizing the necessity to obtain transporter-specific kinetic estimates and REFs from the same in vitro system. For a theoretical BCRP compound, with absorption taking place primarily in the jejunum, a decrease in the REFiBCRP from 2.22 (University of Manchester) to 1.11 (Bertin Pharma) promoted proximal intestinal absorption while delaying tmax 1.44-fold. Laboratory-specific differences in REF may lead to different IVIVE-PBPK outcomes. To understand the mechanisms underlying projected pharmacokinetic liabilities, it is important to assess the potential impact of bias on the generation of REFs on an interindividual basis within a target population.

Lost in centrifugation: accounting for transporter protein losses in quantitative targeted absolute proteomics.

In drug development, considerable efforts are made to extrapolate from in vitro and preclinical findings to predict human drug disposition by using in vitro-in vivo extrapolation (IVIVE) approaches. Use of IVIVE strategies linked with physiologically based pharmacokinetic (PBPK) modeling is widespread, and regulatory agencies are accepting and occasionally requesting model analysis to support licensing submissions. Recently, there has been a drive to improve PBPK models by characterizing the absolute abundance of enzymes, transporters, and receptors within mammalian tissues and in vitro experimental systems using quantitative targeted absolute proteomics (QTAP). The absolute abundance of proteins relevant to processes governing drug disposition provided by QTAP will enable IVIVE-PBPK to incorporate terms for the abundance of enzymes and transporters in target populations. However, most studies that report absolute abundances of enzymes and transporter proteins do so in enriched membrane fractions so as to increase the abundance per sample, and thus the assay's sensitivity, rather than measuring the expected lower abundance in the more biologically meaningful whole cells or tissues. This communication discusses the balance between protein enrichment and potential loss during the preparation of membrane fractions from whole cells or tissues. Accounting for losses with recovery factors throughout the fractionation procedure provides a means to correct for procedural losses, thereby enabling the scaling of protein abundance from subcellular fractions to whole-cell or organ abundances. PBPK models based on corrected abundances will more closely resemble biological systems and facilitate development of more meaningful IVIVE scaling factors, producing more accurate quantitative predictions of drug disposition.

Quantification of Proteins Involved in Intestinal Epithelial Handling of Xenobiotics.

The intestinal epithelium represents a natural barrier against harmful xenobiotics, while facilitating the uptake of nutrients and other substances. Understanding the interaction of chemicals with constituents of the intestinal epithelium and their fate in the body requires quantitative measurement of relevant proteins in in vitro systems and intestinal epithelium. Recent studies have highlighted the mismatch between messenger RNA (mRNA) and protein abundance for several drug-metabolizing enzymes and transporters in the highly dynamic environment of the intestinal epithelium; mRNA abundances cannot therefore be used as a proxy for protein abundances in the gut, necessitating direct measurements. The objective was to determine the expression of a wide range proteins pertinent to metabolism and disposition of chemicals and nutrients in the intestinal epithelium. Ileum and jejunum biopsy specimens were obtained from 16 patients undergoing gastrointestinal elective surgery. Mucosal fractions were prepared and analyzed using targeted and global proteomic approaches. A total of 29 enzymes, 32 transporters, 6 tight junction proteins, 2 adhesion proteins, 1 alkaline phosphatase, 1 thioredoxin, 5 markers, and 1 regulatory protein were quantified-60 for the first time. The global proteomic method identified a further 5,222 proteins, which are retained as an open database for interested parties to explore. This study significantly expands our knowledge of a wide array of proteins important for xenobiotic handling in the intestinal epithelium. Quantitative systems biology models will benefit from the novel systems data generated in the present study and the translation path offered for in vitro to in vivo translation.

Potentiation of flagellin responses in gut epithelial cells by interferon-gamma is associated with STAT-independent regulation of MyD88 expression.

Flagellin acting via TLR5 (Toll-like receptor 5) is a key regulator of the host response to the gut microbial flora in both health and disease. The present study has investigated regulation of flagellin-TLR5 signalling in human colonocytes (HT29-19A) by IFNgamma (interferon-gamma), a cytokine released early in the inflammatory process which has multiple effects on gut epithelial function that may facilitate abnormal responses to enteric bacteria. Flagellin induced a dose-dependent secretion of chemokines CXCL8 and CCL2 in the human colonocyte line, HT29-19A. Exposure to IFNgamma did not induce chemokine secretion, but markedly potentiated responses to flagellin, increasing CXL8 gene expression and protein secretion by approx. 4-fold. Potentiation by IFNgamma was independent of changes in TLR5 and was associated with a rapid, sustained increase in expression of the downstream adaptor molecule MyD88 (myeloid differentiation factor 88). Knockdown of MyD88 expression using siRNA (small interfering RNA) abolished flagellin-dependent CXCL8 secretion and the potentiating effect of IFNgamma. Exposure of non-transformed mouse and human colonocytes to IFNgamma also increased MyD88 expression. STAT (signal transducer and activator of transcription) 1 knockdown and use of the broad-spectrum JAK (Janus kinase)-STAT inhibitor AG490 had no effect on IFNgamma-mediated up-regulation of MyD88. The findings of the present study suggest that IFNgamma sensitizes colonic epithelial cells to bacterial flagellin via a largely STAT-independent up-regulation of MyD88 expression leading to increased secretion of immunomodulatory factors. These results indicate that epithelial responses to flagellin are potentiated by IFNgamma, most likely mediated by increased MyD88 expression. The present study adds to our understanding of the spectrum of effects of this cytokine on gut epithelium that may contribute to bacterial-driven inflammation in the gut.

Bench-to-bedside review: the promise of rapid infection diagnosis during sepsis using polymerase chain reaction-based pathogen detection.

Early infection diagnosis as the cause of a patient's systemic inflammatory syndrome is an important facet of sepsis care bundles aimed at saving lives. Microbiological culture provides the main route for infection diagnosis but by its nature cannot provide time-critical results that can impact on early management. Consequently, broad-spectrum and high-potency antibiotics are essential during the immediate management of suspected sepsis in critical care but are associated with the development of drug-resistant organisms and superinfections. Established molecular laboratory techniques based on polymerase chain reaction (PCR) technology can detect pathogen DNA rapidly and have been developed for translation into a clinical diagnostic setting. In the setting of sepsis in critical care, emerging commercial systems are now available for the analysis of whole blood within hours, with the presumed aim of adoption into the current care bundles. In this review, we consider the importance of early infection diagnosis in sepsis, how this is limited by culture approaches and how the emerging PCR methods are showing promise in early clinical observational studies. The strengths and weaknesses of culture and PCR pathogen detection in whole-blood samples will be highlighted and recommendations made for urgent appropriately powered diagnostic validation studies in advance of clinical effectiveness trials before these emerging PCR pathogen detection techniques can be considered for adoption in clinical practice.

Characterisation of Escherichia coli strains involved in transcytosis across gut epithelial cells exposed to metabolic and inflammatory stress.

Translocation of normally non-pathogenic bacteria across the gut may drive inflammatory responses associated with sepsis and inflammatory bowel disease. Recent evidence suggests translocation may not be purely passive, but occurs via novel transcellular pathways activated in enterocytes by inflammatory and metabolic stress. The specificity of this pathway with respect to different E. coli strains and other bacterial species, and possible molecular determinants of the "translocating" phenotype have been investigated. Translocation of E. coli strains and other bacteria was studied across Caco-2 monolayers exposed to different forms of cellular stress. All bacteria, apart from the pathogen Shigella sonnei, exhibited low levels of translocation in untreated monolayers. However, following enterocyte stress, translocation of E. coli strains C25 and HBTEC-1 was markedly stimulated, accompanied by increased internalisation into enterocytes. C25 and HBTEC-1 were typed to ECOR group A and group D respectively. Pathoarray analysis showed both strains had profiles quite different to those predicted for typical ExPEC isolates, lacking many of the genes associated with pathogenicity, although they contained several ORFs in common with ExPEC isolates. These data suggest translocating E. coli strains associated with infections are not opportunistic ExPEC strains but may comprise a separate group of E. coli strains.

Early molecular and functional changes in colonic epithelium that precede increased gut permeability during colitis development in mdr1a(-/-) mice.

BACKGROUND: The early molecular changes preceding the onset of mucosal inflammation in colitis and their temporal relationship with gut permeability remain poorly defined. This study investigated functional and transcriptomic changes in mdr1a(-/-) mice lacking the intestinal transporter P-glycoprotein, which develop colitis spontaneously when exposed to normal enteric flora. METHODS: Mdr1a(-/-) mice were housed in specific pathogen-free conditions to slow colitis development and compared to congenic controls. Mucosal permeability and cytokine secretion were analyzed in ex vivo colon. Gene expression in colonic mucosal and epithelial preparations was analyzed by microarray and qPCR. Colonocyte responsiveness to bacterial antigens was measured in short-term culture. RESULTS: Colon from 4-5-week-old, disease-free mdr1a(-/-) mice was histologically normal with no evidence of increased permeability compared to controls. However, these tissues display a distinctive pattern of gene expression involving significant changes in a small number of genes. The majority of upregulated genes were associated with bacterial recognition and the ubiquitin-proteasome system and were gamma-interferon (IFN-gamma) responsive. Expression of the antiinflammatory factor pancreatitis-associated protein (PAP) and the related gene RegIIIgamma were markedly reduced. Colonocytes from 4-5-week mdr1a(-/-) exhibit similar transcriptomic changes, accompanied by higher basal chemokine secretion and increased responsiveness to LPS. Significant increases in colonic permeability were associated with older (12-16-week) mdr1a(-/-) mice displaying molecular and functional evidence of active inflammation. CONCLUSIONS: These studies show that early epithelial changes associated with altered responsiveness to bacteria precede increased permeability and mucosal inflammation in this model of colitis, highlighting the importance of P-glycoprotein in regulating interactions with the commensal microflora.

Compositional Changes in the Gut Mucus Microbiota Precede the Onset of Colitis-Induced Inflammation.

BACKGROUND: Inflammatory bowel disease (IBD) is associated with an inappropriate immune response to the gut microbiota. Notably, patients with IBD reportedly have alterations in fecal microbiota. However, the colonic microbiota occupies both the gut lumen and the mucus covering the epithelium. Thus, information about mucus-resident microbiota fails to be conveyed in the routine microbiota analyses of stool samples. Further, studies analyzing microbiota in IBD have mainly focused on stool samples taken after onset of inflammation. Our objective was to investigate both temporal and spatial changes in colonic microbiota communities preceding the onset of colitis. METHODS: We studied mucus and stool microbiota using a spontaneous model of colitis, the mdr1a mouse, and their respective wild-type littermate controls in a time series mode. RESULTS: Using this approach we have shown that microbial dysbiosis was evident in the mucus but not stools, with reduced abundance of Clostridiales evident in the mucus but not stools, of colitis-prone mice mdr1a mice 12 weeks before the onset of detectable inflammation. This altered microbial composition was coupled with a significantly thinner mucus layer. On emergence of inflammation, dysbiosis was evident in the stools and at this time point, the spatial segregation between microbiota and host tissue was also disrupted, correlating with worsened inflammation. Our results reveal that microbial dysbiosis is detectable before changes in the stools. Importantly, dysbiosis in the mucus layer preceded development of colitis. CONCLUSIONS: Our data reveal the importance of mucus sampling for understanding the underlying etiology of IBD and fundamental processes underlying disease progression.

Variability of bacterial translocation in the absence of intestinal mucosal damage following injury and the influence of interleukin-6.

Bacterial translocation and intestinal mucosal damage have been reported as potentially clinically important sequelae of injury. Evidence that endogenous interleukin-6 (IL-6) is able to protect against infection, and that orally administered IL-6 could prevent bacterial translocation and mucosal damage following haemorrhage, led us to evaluate the impact of injury on the intestinal mucosa and the role of endogenous IL-6. Normal and IL-6-deficient (IL-6-/-) mice were subjected to haemorrhage of increasing severity, hind limb ischaemia, or both. Mucosal integrity and bacterial translocation to the liver, spleen and mesenteric lymph nodes (MLN) were examined after 16 h. Bacterial translocation to each of these tissues was observed in unoperated animals. The more severe haemorrhage procedures, and hind limb ischaemia, increased bacterial translocation to the liver significantly in most experiments with normal mice. The IL-6-/- mice survived the most severe haemorrhage procedure less well (p = 0.0015), although increased bacterial translocation was not seen. There was no clear evidence of mucosal damage, or bacterial translocation to spleen and mesenteric lymph nodes, in either normal or IL-6-/- mice. Intestinal IgA concentrations were the same in IL-6-/- mice and controls. These data demonstrate that increased bacterial translocation can be observed following severe injury, but that neither bacterial translocation nor severe injury are inevitably associated with morphological damage to the intestinal mucosa, and endogenous IL-6 is more likely to promote bacterial translocation than protect the gut.

Breast Cancer Resistance Protein Abundance, but Not mRNA Expression, Correlates With Estrone-3-Sulfate Transport in Caco-2.

Transporter mRNA and protein expression data are used to extrapolate in vitro transporter kinetics to in vivo drug disposition predictions. Breast cancer resistance protein (BCRP) possesses broad substrate specificity; therefore, understanding BCRP expression-activity relationships are necessary for the translation to in vivo. Bidirectional transport of estrone-3-sulfate (E-3-S), a BCRP probe, was evaluated with respect to relative BCRP mRNA expression and absolute protein abundance in 10- and 29-day cultured Caco-2 cells. BCRP mRNA expression was quantified by real-time PCR against a housekeeper gene, Cyclophilin A. The BCRP protein abundance in total membrane fractions was quantified by targeted proteomics, and [(3)H]-E-3-S bidirectional transport was determined in the presence or absence of Ko143, a potent BCRP inhibitor. BCRP mRNA expression was 1.5-fold higher in 29- versus 10-day cultured cells (n = 3), whereas a 2.4-fold lower (p < 0.001) BCRP protein abundance was observed in 29- versus 10-day cultured cells (1.28 ± 0.33 and 3.06 ± 0.22 fmol/µg protein, n = 6, respectively). This correlated to a 2.45-fold lower (p < 0.01) efflux ratio for E-3-S in 29- versus 10-day cultured cells (8.97 ± 2.51 and 3.32 ± 0.66, n = 6, respectively). Caco-2 cell BCRP protein abundance, but not mRNA levels, correlates with BCRP activity, suggesting that extrapolation strategies incorporating BCRP protein abundance-activity relationships may be more successful.

Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR.

BACKGROUND: More than two decades after its discovery, contaminant microbial DNA in PCR reagents continues to impact the sensitivity and integrity of broad-range PCR diagnostic techniques. This is particularly relevant to their use in the setting of human sepsis, where a successful diagnostic on blood samples needs to combine universal bacterial detection with sensitivity to 1-2 genome copies, because low levels of a broad range of bacteria are implicated. RESULTS: We investigated the efficacy of ethidium monoazide (EMA) and propidium monoazide (PMA) treatment as emerging methods for the decontamination of PCR reagents. Both treatments were able to inactivate contaminating microbial DNA but only at concentrations that considerably affected assay sensitivity. Increasing amplicon length improved EMA/PMA decontamination efficiency but at the cost of assay sensitivity. The same was true for UV exposure as an alternative decontamination strategy, likely due to damage sustained by oligonucleotide primers which were a significant source of contamination. However, a simple combination strategy with UV-treated PCR reagents paired with EMA-treated primers produced an assay capable of two genome copy detection and a <5% contamination rate. This decontamination strategy could have important utility in developing improved pan-bacterial assays for rapid diagnosis of low pathogen burden conditions such as in the blood of patients with suspected blood stream infection.

Diagnostic accuracy of SeptiFast multi-pathogen real-time PCR in the setting of suspected healthcare-associated bloodstream infection.

PURPOSE: SeptiFast is a real-time PCR assay which targets ribosomal DNA sequences of bacteria and fungi, enabling detection and identification of the commonest pathogens in blood within a few hours, including those acquired in healthcare settings. We report here the first detailed assessment of SeptiFast that focuses on healthcare-associated bloodstream infections which develop during routine critical care. METHODS: This was a prospective multicentre study designed to compare the clinical diagnostic accuracy of SeptiFast versus microbiological culture and independent clinical adjudication. This Phase III diagnostic study was performed in an adequately sized cohort of adult patients who developed new signs of suspected bloodstream infection while receiving routine critical care. RESULTS: Of 1,006 new episodes of suspected bloodstream infection in 853 patients, 922 (92 %) of these episodes in 795 patients met the inclusion criteria of the study. Patients had been exposed to a median of 8 days (interquartile range 4-16) of hospital care and had received high levels of organ support and recent antibiotic exposure. The SeptiFast test, when compared with bloodstream infection at the species/genus level, had a greater specificity [0.86, 95 % confidence interval (CI) 0.83-0.88] than sensitivity (0.50, 95 % CI 0.39-0.61). There was a low prevalence of blood culture-proven pathogens (9.2 %, 95 % CI 7.4-11.2 %), and the post-test probabilities of both a positive (26.3 %, 95 % CI 19.8-33.7 %) and a negative SeptiFast test (5.6 %, 95 % CI 4.1-7.4 %) indicated potential limitations of this technology in diagnosing bloodstream infection. CONCLUSION: When compared with blood culture, SeptiFast is likely to have limited utility for the diagnosis of healthcare-associated bloodstream infection in critical care patients despite its potential to deliver results more rapidly.

Accuracy of LightCycler(®) SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review and meta-analysis.

PURPOSE: There is an urgent need to develop diagnostic tests to improve the detection of pathogens causing life-threatening infection (sepsis). SeptiFast is a CE-marked multi-pathogen real-time PCR system capable of detecting DNA sequences of bacteria and fungi present in blood samples within a few hours. We report here a systematic review and meta-analysis of diagnostic accuracy studies of SeptiFast in the setting of suspected sepsis. METHODS: A comprehensive search strategy was developed to identify studies that compared SeptiFast with blood culture in suspected sepsis. Methodological quality was assessed using QUADAS. Heterogeneity of studies was investigated using a coupled forest plot of sensitivity and specificity and a scatter plot in receiver operator characteristic space. Bivariate model method was used to estimate summary sensitivity and specificity. RESULTS: From 41 phase III diagnostic accuracy studies, summary sensitivity and specificity for SeptiFast compared with blood culture were 0.68 (95 % CI 0.63-0.73) and 0.86 (95 % CI 0.84-0.89) respectively. Study quality was judged to be variable with important deficiencies overall in design and reporting that could impact on derived diagnostic accuracy metrics. CONCLUSIONS: SeptiFast appears to have higher specificity than sensitivity, but deficiencies in study quality are likely to render this body of work unreliable. Based on the evidence presented here, it remains difficult to make firm recommendations about the likely clinical utility of SeptiFast in the setting of suspected sepsis.

Rapid detection of health-care-associated bloodstream infection in critical care using multipathogen real-time polymerase chain reaction technology: a diagnostic accuracy study and systematic review.

BACKGROUND: There is growing interest in the potential utility of real-time polymerase chain reaction (PCR) in diagnosing bloodstream infection by detecting pathogen deoxyribonucleic acid (DNA) in blood samples within a few hours. SeptiFast (Roche Diagnostics GmBH, Mannheim, Germany) is a multipathogen probe-based system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection. As background to this study, we report a systematic review of Phase III diagnostic accuracy studies of SeptiFast, which reveals uncertainty about its likely clinical utility based on widespread evidence of deficiencies in study design and reporting with a high risk of bias. OBJECTIVE: Determine the accuracy of SeptiFast real-time PCR for the detection of health-care-associated bloodstream infection, against standard microbiological culture. DESIGN: Prospective multicentre Phase III clinical diagnostic accuracy study using the standards for the reporting of diagnostic accuracy studies criteria. SETTING: Critical care departments within NHS hospitals in the north-west of England. PARTICIPANTS: Adult patients requiring blood culture (BC) when developing new signs of systemic inflammation. MAIN OUTCOME MEASURES: SeptiFast real-time PCR results at species/genus level compared with microbiological culture in association with independent adjudication of infection. Metrics of diagnostic accuracy were derived including sensitivity, specificity, likelihood ratios and predictive values, with their 95% confidence intervals (CIs). Latent class analysis was used to explore the diagnostic performance of culture as a reference standard. RESULTS: Of 1006 new patient episodes of systemic inflammation in 853 patients, 922 (92%) met the inclusion criteria and provided sufficient information for analysis. Index test assay failure occurred on 69 (7%) occasions. Adult patients had been exposed to a median of 8 days (interquartile range 4-16 days) of hospital care, had high levels of organ support activities and recent antibiotic exposure. SeptiFast real-time PCR, when compared with culture-proven bloodstream infection at species/genus level, had better specificity (85.8%, 95% CI 83.3% to 88.1%) than sensitivity (50%, 95% CI 39.1% to 60.8%). When compared with pooled diagnostic metrics derived from our systematic review, our clinical study revealed lower test accuracy of SeptiFast real-time PCR, mainly as a result of low diagnostic sensitivity. There was a low prevalence of BC-proven pathogens in these patients (9.2%, 95% CI 7.4% to 11.2%) such that the post-test probabilities of both a positive (26.3%, 95% CI 19.8% to 33.7%) and a negative SeptiFast test (5.6%, 95% CI 4.1% to 7.4%) indicate the potential limitations of this technology in the diagnosis of bloodstream infection. However, latent class analysis indicates that BC has a low sensitivity, questioning its relevance as a reference test in this setting. Using this analysis approach, the sensitivity of the SeptiFast test was low but also appeared significantly better than BC. Blood samples identified as positive by either culture or SeptiFast real-time PCR were associated with a high probability (> 95%) of infection, indicating higher diagnostic rule-in utility than was apparent using conventional analyses of diagnostic accuracy. CONCLUSION: SeptiFast real-time PCR on blood samples may have rapid rule-in utility for the diagnosis of health-care-associated bloodstream infection but the lack of sensitivity is a significant limiting factor. Innovations aimed at improved diagnostic sensitivity of real-time PCR in this setting are urgently required. Future work recommendations include technology developments to improve the efficiency of pathogen DNA extraction and the capacity to detect a much broader range of pathogens and drug resistance genes and the application of new statistical approaches able to more reliably assess test performance in situation where the reference standard (e.g. blood culture in the setting of high antimicrobial use) is prone to error. STUDY REGISTRATION: The systematic review is registered as PROSPERO CRD42011001289. FUNDING: The National Institute for Health Research Health Technology Assessment programme. Professor Daniel McAuley and Professor Gavin D Perkins contributed to the systematic review through their funded roles as codirectors of the Intensive Care Foundation (UK).

Increased susceptibility to Trichuris muris infection and exacerbation of colitis in Mdr1a-/- mice.

AIM: To investigate the influence of Trichuris muris (T. muris) infection in a mouse model of genetic susceptibility to inflammatory bowel disease, Mdr1a-/-. METHODS: Mdr1a-/- mice were housed under specific pathogen free conditions to slow the development of colitis and compared to congenic FVB controls. Mice were infected with approximately 200 embryonated ova from T. muris and assessed for worm burden and histological and functional markers of gut inflammation on day 19 post infection. RESULTS: Mdr1a-/- mice exhibited a marked increase in susceptibility to T. muris infection with a 10-fold increase in colonic worm count by day 19 pi compared to FVB controls. Prior to infection, Mdr1a-/- exhibited low-level mucosal inflammation with evidence of an enhanced Th1 environment. T. muris infection accelerated the progression of colitis in Mdr1a-/- as evidenced by marked increases in several indicators including histological damage score, mucosal CD⁺ T-cell and DC infiltration and dramatically increased production of pro-inflammatory cytokines. CONCLUSION: These data provide further evidence of the complex interaction between T. muris and an inflammatory bowel disease (IBD)-susceptible host which may have relevance to the application of helminth therapy in the treatment of human IBD.

Combined molecular gram typing and high-resolution melting analysis for rapid identification of a syndromic panel of bacteria responsible for sepsis-associated bloodstream infection.

Effective diagnosis and treatment of bloodstream infections are often hampered by a lack of time-critical information from blood cultures. Molecular techniques aimed at the detection of circulating pathogen DNA have the potential to dramatically improve the timeliness of infection diagnosis. Our aim in this study was to establish a rapid, low-cost PCR approach using high-resolution melting analysis to identify a syndromic panel of 21 pathogens responsible for most bloodstream bacterial infections encountered in critical care environments. A broad-range, real-time PCR technique that combines primers for molecular Gram classification and high-resolution melting analysis in a single run was established. The differentiation of bacterial species was achieved using a multiparameter, decision-tree approach that was based on Gram type, grouping according to melting temperature, and sequential comparisons of melting profiles against multiple reference organisms. A preliminary validation study was undertaken by blinded analysis of 53 consecutive bloodstream isolates from a clinical microbiology laboratory. Fifty isolates contained organisms that were present in the panel, and 96% of these were identified correctly at the genus or species level. A correct Gram classification was reported for all 53 isolates. This technique shows promise as a cost-effective tool for the timely identification of bloodstream pathogens, allowing clinicians to make informed decisions on appropriate antibiotic therapies at an earlier stage.

Accuracy of LightCycler(R) SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review protocol.

UNLABELLED: Background There is growing interest in the potential utility of molecular diagnostics in improving the detection of life-threatening infection (sepsis). LightCycler® SeptiFast is a multipathogen probe-based real-time PCR system targeting DNA sequences of bacteria and fungi present in blood samples within a few hours. We report here the protocol of the first systematic review of published clinical diagnostic accuracy studies of this technology when compared with blood culture in the setting of suspected sepsis. Methods/design Data sources: the Cochrane Database of Systematic Reviews, the Database of Abstracts of Reviews of Effects (DARE), the Health Technology Assessment Database (HTA), the NHS Economic Evaluation Database (NHSEED), The Cochrane Library, MEDLINE, EMBASE, ISI Web of Science, BIOSIS Previews, MEDION and the Aggressive Research Intelligence Facility Database (ARIF). STUDY SELECTION: diagnostic accuracy studies that compare the real-time PCR technology with standard culture results performed on a patient's blood sample during the management of sepsis. DATA EXTRACTION: three reviewers, working independently, will determine the level of evidence, methodological quality and a standard data set relating to demographics and diagnostic accuracy metrics for each study. Statistical analysis/data synthesis: heterogeneity of studies will be investigated using a coupled forest plot of sensitivity and specificity and a scatter plot in Receiver Operator Characteristic (ROC) space. Bivariate model method will be used to estimate summary sensitivity and specificity. The authors will investigate reporting biases using funnel plots based on effective sample size and regression tests of asymmetry. Subgroup analyses are planned for adults, children and infection setting (hospital vs community) if sufficient data are uncovered. Dissemination Recommendations will be made to the Department of Health (as part of an open-access HTA report) as to whether the real-time PCR technology has sufficient clinical diagnostic accuracy potential to move forward to efficacy testing during the provision of routine clinical care. Registration PROSPERO-NIHR Prospective Register of Systematic Reviews (CRD42011001289).

Investigation of systemic and mesenteric inflammatory signaling and gut-derived endothelial toxicity in patients undergoing high-risk abdominal aortic surgery.

Evidence from animal models of trauma and hemorrhage has suggested that the gut plays an active role in the pathogenesis of systemic inflammatory responses and multiple organ dysfunction syndrome. The aim of the present study was to seek evidence for gut-derived signals in man in a group of eight patients undergoing elective abdominal aortic reconstruction, a procedure that is associated with sterile tissue injury, controlled colonic ischemia as a consequence of aortic cross-clamping, and a significant risk of developing systemic inflammation and multiple organ dysfunction syndrome. Despite the presence of a marked systemic inflammatory response (IL-6, IL-18, monocyte chemoattractant protein 1, and IL-8) and a gut-derived inflammatory signal (IL-6), we could find no evidence that gut-derived pathogen DNA was present in the central or mesenteric circulation, and we could find no evidence that either central or mesenteric plasma samples could induce apoptotic or necrotic cell death in human umbilical vein endothelial cells in vitro. Similarly, we could find no evidence of adhesion molecule upregulation in the endothelial monolayers exposed to central or mesenteric plasma sampled at any time point during surgery. There was, however, evidence of an increase in the expression of RAGE (receptor for advanced glycation end products) by endothelial cells following exposure to mesenteric venous, but not central, plasma sampled during maximum ischemia. In conclusion, during sterile tissue injury and controlled colonic ischemia-reperfusion in man, there is a marked systemic proinflammatory response, which is in part gut derived, in the absence of evidence for the presence of toxic endothelial factors or gut-derived microorganisms in the central or mesenteric circulations.