Low rates of Pseudomonas aeruginosa misidentification in isolates from cystic fibrosis patients

Pseudomonas aeruginosa is an important cause of pulmonary infection in cystic fibrosis (CF). Its correct identification ensures effective patient management and infection control strategies. However, little is known about how often CF sputum isolates are falsely identified as P. aeruginosa. We used P. aeruginosa-specific duplex real-time PCR assays to determine if 2,267 P. aeruginosa sputum isolates from 561 CF patients were correctly identified by 17 Australian clinical microbiology laboratories. Misidentified isolates underwent further phenotypic tests, amplified rRNA gene restriction analysis, and partial 16S rRNA gene sequence analysis. Participating laboratories were surveyed on how they identified P. aeruginosa from CF sputum. Overall, 2,214 (97.7%) isolates from 531 (94.7%) CF patients were correctly identified as P. aeruginosa. Further testing with the API 20NE kit correctly identified only 34 (59%) of the misidentified isolates. Twelve (40%) patients had previously grown the misidentified species in their sputum. Achromobacter xylosoxidans (n = 21), Stenotrophomonas maltophilia (n = 15), and Inquilinus limosus (n = 4) were the species most commonly misidentified as P. aeruginosa. Overall, there were very low rates of P. aeruginosa misidentification among isolates from a broad cross section of Australian CF patients. Additional improvements are possible by undertaking a culture history review, noting colonial morphology, and performing stringent oxidase, DNase, and colistin susceptibility testing for all presumptive P. aeruginosa isolates. Isolates exhibiting atypical phenotypic features should be evaluated further by additional phenotypic or genotypic identification techniques.

Char oxidation of torrefied biomass at high temperatures and high heating rates

The char oxidation of a torrefied biomass and its parent material was carried out in an isothermal plug flow reactor (IPFR), which is able to rapidly heat the biomass particles to a maximum temperature of 1400 °C at a heating rate of 104 °C/s, similar to the real conditions found in power plant furnaces. During each char oxidation test, the residues of biomass particles were collected and analyzed to determine the weight loss based on the ash tracer method. According to the experimental results, it can be concluded that chars produced from a torrefied biomass are less reactive than the ones produced, under the same conditions, from its raw material. The apparent kinetics of the torrefied biomass and its parent material are determined by minimizing the difference between the modeled and the experimental results. The predicted weight loss during char oxidation, using the determined kinetics, agrees well with experimental results

Rapid quantification of microRNAs in plasma using a fast real time PCR system

The ability to rapidly detect circulating small RNAs, in particular microRNAs (miRNAs), would further increase their already established potential as biomarkers in a range of conditions. One rate-limiting factor is the time taken to perform quantitative real time PCR amplification. We therefore evaluated the ability of a novel thermal cycler to perform this step in less than 10 minutes. Quantitative PCR was performed on an xxpress® thermal cycler (BJS Biotechnologies, Perivale, UK), which employs a resistive heating system and forced air cooling to achieve thermal ramp rates of 10 °C/s, and a conventional peltier-controlled LightCycler 480 system (Roche, Basel, Switzerland) ramping at 4.8 °C/s. The threshold cycle (Ct) for detection of 18S rDNA from a standard genomic DNA sample was significantly more variable across the block (F-test, p=2.4x10-25) for the xxpress (20.01±0.47SD) than the LightCycler (19.87±0.04SD). RNA was extracted from human plasma, reverse transcribed and a panel of miRNAs amplified and detected using SYBR green (Kapa Biosystems, Wilmington, Ma, USA). The sensitivity of both systems was broadly comparable and both detected a panel of miRNAs reliably and indicated similar relative abundances. The xxpress thermal cycler facilitates rapid qPCR detection of small RNAs and brings point-of care diagnostics based upon circulating miRNAs a step closer to reality.

The effects of transportation stress and barnacle fouling on predation rates of starfish (Asterias rubens) on mussels (Mytilus edulis).

The starfish, Asterias rubens, is widely distributed throughout the northern hemisphere and is an important predator on benthic mussel (Mytilus edulis) beds. Whilst several studies have examined how the size of individuals determines this predator–prey relationship, less is known about how the physiological condition of the prey (mussels) and the extent of their fouling may alter these relationships. Such issues are of particular interest to those working within the benthic mussel cultivation industry to inform best management practice and to help minimise losses during the aquaculture process. The potential role of starfish in the removal of epibiotic barnacles from mussels, the presence of which increases processing costs within the industry, is also of interest. We tested whether stressing mussels by aerial exposure for 48 h and whether the extent of barnacle fouling on mussels affected the feeding rates of three different size classes of starfish feeding on two different size classes of mussels. Feeding rates on stressed and unstressed mussels were similar for each starfish–mussel size combination. Barnacle fouling reduced the feeding rate of medium-sized starfish on larger-sized mussels. We also observed starfish, of all size classes, preying directly on the epibiotic barnacles on mussels, however, feeding rates were low and considered unlikely to reduce the extent of fouling on mussels. Our findings show that the predator–prey relationship between starfish and mussels does not differ between unstressed mussels and those experimentally stressed by aerial exposure for 48 h so that this level of stress is unlikely to affect predation rates by A. rubens following relaying in commercial operations. Whilst barnacle fouling suppressed predation rates in one of our experimental treatments, it does not appear that fouling by barnacles would provide a significant refuge from predation for the majority of mussels in benthic aquaculture stocks. Instead we found the size relationship between starfish and mussels was more important in determining predation rates. Starfish are also unlikely to help reduce barnacle fouling on cultured mussels by preying solely on fouling barnacles and the need to control starfish predation during culture remains.

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.

Game-­theoretic Resource Allocation with Real-­time Probabilistic Surveillance Information

Game-theoretic security resource allocation problems have generated significant interest in the area of designing and developing security systems. These approaches traditionally utilize the Stackelberg game model for security resource scheduling in order to improve the protection of critical assets. The basic assumption in Stackelberg games is that a defender will act first, then an attacker will choose their best response after observing the defender’s strategy commitment (e.g., protecting a specific asset). Thus, it requires an attacker’s full or partial observation of a defender’s strategy. This assumption is unrealistic in real-time threat recognition and prevention. In this paper, we propose a new solution concept (i.e., a method to predict how a game will be played) for deriving the defender’s optimal strategy based on the principle of acceptable costs of minimax regret. Moreover, we demonstrate the advantages of this solution concept by analyzing its properties.

Retrieving Regions of Interest for User Exploration

We consider an application scenario where points of interest (PoIs) each have a web presence and where a web user wants to iden- tify a region that contains relevant PoIs that are relevant to a set of keywords, e.g., in preparation for deciding where to go to conve- niently explore the PoIs. Motivated by this, we propose the length- constrained maximum-sum region (LCMSR) query that returns a spatial-network region that is located within a general region of in- terest, that does not exceed a given size constraint, and that best matches query keywords. Such a query maximizes the total weight of the PoIs in it w.r.t. the query keywords. We show that it is NP- hard to answer this query. We develop an approximation algorithm with a (5 + ǫ) approximation ratio utilizing a technique that scales node weights into integers. We also propose a more efficient heuris- tic algorithm and a greedy algorithm. Empirical studies on real data offer detailed insight into the accuracy of the proposed algorithms and show that the proposed algorithms are capable of computingresults efficiently and effectively.

Indexing and matching trajectories under inconsistent sampling rates

Quantifying the similarity between two trajectories is a fundamental operation in analysis of spatio-temporal databases. While a number of distance functions exist, the recent shift in the dynamics of the trajectory generation procedure violates one of their core assumptions; a consistent and uniform sampling rate. In this paper, we formulate a robust distance function called Edit Distance with Projections (EDwP) to match trajectories under inconsistent and variable sampling rates through dynamic interpolation. This is achieved by deploying the idea of projections that goes beyond matching only the sampled points while aligning trajectories. To enable efficient trajectory retrievals using EDwP, we design an index structure called TrajTree. TrajTree derives its pruning power by employing the unique combination of bounding boxes with Lipschitz embedding. Extensive experiments on real trajectory databases demonstrate EDwP to be up to 5 times more accurate than the state-of-the-art distance functions. Additionally, TrajTree increases the efficiency of trajectory retrievals by up to an order of magnitude over existing techniques.

Protocol for a pilot randomised controlled trial of an intervention to increase the use of traffic light food labelling in UK shoppers (the FLICC trial)

Background: Traffic light labelling of foods—a system that incorporates a colour-coded assessment of the level of total fat, saturated fat, sugar and salt on the front of packaged foods—has been recommended by the UK Government and is currently in use or being phased in by many UK manufacturers and retailers. This paper describes a protocol for a pilot randomised controlled trial of an intervention designed to increase the use of traffic light labelling during real-life food purchase decisions.

Methods/design: The objectives of this two-arm randomised controlled pilot trial are to assess recruitment, retention and data completion rates, to generate potential effect size estimates to inform sample size calculations for the main trial and to assess the feasibility of conducting such a trial. Participants will be recruited by email from a loyalty card database of a UK supermarket chain. Eligible participants will be over 18 and regular shoppers who frequently purchase ready meals or pizzas. The intervention is informed by a review of previous interventions encouraging the use of nutrition labelling and the broader behaviour change literature. It is designed to impact on mechanisms affecting belief and behavioural intention formation as well as those associated with planning and goal setting and the adoption and maintenance of the behaviour of interest, namely traffic light label use during purchases of ready meals and pizzas. Data will be collected using electronic sales data via supermarket loyalty cards and web-based questionnaires and will be used to estimate the effect of the intervention on the nutrition profile of purchased ready meals and pizzas and the behavioural mechanisms associated with label use. Data collection will take place over 48 weeks. A process evaluation including semi-structured interviews and web analytics will be conducted to assess feasibility of a full trial.

Discussion: The design of the pilot trial allows for efficient recruitment and data collection. The intervention could be generalised to a wider population if shown to be feasible in the main trial.

On the context-dependent scaling of consumer feeding rates

The stability of consumer-resource systems can depend on the form of feeding interactions (i.e. functional responses). Size-based models predict interactions - and thus stability - based on consumer-resource size ratios. However, little is known about how interaction contexts (e.g. simple or complex habitats) might alter scaling relationships. Addressing this, we experimentally measured interactions between a large size range of aquatic predators (4-6400 mg over 1347 feeding trials) and an invasive prey that transitions among habitats: from the water column (3D interactions) to simple and complex benthic substrates (2D interactions). Simple and complex substrates mediated successive reductions in capture rates - particularly around the unimodal optimum - and promoted prey population stability in model simulations. Many real consumer-resource systems transition between 2D and 3D interactions, and along complexity gradients. Thus, Context-Dependent Scaling (CDS) of feeding interactions could represent an unrecognised aspect of food webs, and quantifying the extent of CDS might enhance predictive ecology.

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR.

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10(5) polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.