Development of a rapid multiplexed assay for the direct screening of antimicrobial residues in raw milk

Antimicrobial residues found to be present in milk can have both health and economic impacts. For these reasons, the widespread routine testing of milk is required. Due to delays with sample handling and test scheduling, laboratory-based tests are not always suited for making decisions about raw material intake and product release, especially when samples require shipping to a central testing facility. Therefore, rapid on-site screening tests that can produce results within a matter of minutes are required to facilitate rapid intake and product release processes. Such tests must be simple for use by non-technical staff. There is increasing momentum towards the development and implementation of multiplexing tests that can detect a range of important antimicrobial residues simultaneously. A simple in situ multiplexed planar waveguide device that can simultaneously detect chloramphenicol, streptomycin and desfuroylceftiofur in raw dairy milk, without sample preparation, has been developed. Samples are simply mixed with antibody prior to an aliquot being passed through the detection cartridge for 5 min before reading on a field-deployable portable instrument. Multiplexed calibration curves were produced in both buffer and raw milk. Buffer curves, for chloramphenicol, streptomycin and desfuroylceftiofur, showed linear ranges (inhibitory concentration (IC)₂₀–IC₈₀) of 0.1–0.9, 3–129 and 12–26 ng/ml, whilst linear range in milk was 0.13–0.74, 11–376 and 2–12 ng/ml, respectively, thus meeting European legislated concentration requirements for both chloramphenicol and streptomycin, in milk, without the need for any sample preparation. Desfuroylceftiofur-contaminated samples require only simple sample dilution to bring positive samples within the range of quantification. Assay repeatability and reproducibility were lower than 12 coefficient of variation (%CV), whilst blank raw milk samples (n = 9) showed repeatability ranging between 4.2 and 8.1 %CV when measured on all three calibration curves.

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.

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.

Unique mitochondrial DNA lineages in Irish stickleback populations: cryptic refugium or rapid recolonization?

Repeated recolonization of freshwater environments following Pleistocene glaciations has played a major role in the evolution and adaptation of anadromous taxa. Located at the western fringe of Europe, Ireland and Britain were likely recolonized rapidly by anadromous fishes from the North Atlantic following the last glacial maximum (LGM). While the presence of unique mitochondrial haplotypes in Ireland suggests that a cryptic northern refugium may have played a role in recolonization, no explicit test of this hypothesis has been conducted. The three-spined stickleback is native and ubiquitous to aquatic ecosystems throughout Ireland, making it an excellent model species with which to examine the biogeographical history of anadromous fishes in the region. We used mitochondrial and microsatellite markers to examine the presence of divergent evolutionary lineages and to assess broad-scale patterns of geographical clustering among postglacially isolated populations. Our results confirm that Ireland is a region of secondary contact for divergent mitochondrial lineages and that endemic haplotypes occur in populations in Central and Southern Ireland. To test whether a putative Irish lineage arose from a cryptic Irish refugium, we used approximate Bayesian computation (ABC). However, we found no support for this hypothesis. Instead, the Irish lineage likely diverged from the European lineage as a result of postglacial isolation of freshwater populations by rising sea levels. These findings emphasize the need to rigorously test biogeographical hypothesis and contribute further evidence that postglacial processes may have shaped genetic diversity in temperate fauna.

Development and Validation of a Novel Lateral Flow Immunoassay (LFIA) for the Rapid Screening of Paralytic Shellfish Toxins (PSTs) from Shellfish Extracts

A single-step lateral flow immunoassay (LFIA) was developed and validated for the rapid screening of paralytic shellfish toxins (PSTs) from a variety of shellfish species, at concentrations relevant to regulatory limits of 800 μg STX-diHCl equivalents/kg shellfish meat. A simple aqueous extraction protocol was performed within several minutes from sample homogenate. The qualitative result was generated after a 5 min run time using a portable reader which removed subjectivity from data interpretation. The test was designed to generate noncompliant results with samples containing approximately 800 μg of STX-diHCl/kg. The cross-reactivities in relation to STX, expressed as mean ± SD, were as follows: NEO: 128.9% ± 29%; GTX1&4: 5.7% ± 1.5%; GTX2&3: 23.4% ± 10.4%; dcSTX: 55.6% ± 10.9%; dcNEO: 28.0% ± 8.9%; dcGTX2&3: 8.3% ± 2.7%; C1&C2: 3.1% ± 1.2%; GTX5: 23.3% ± 14.4% (n = 5 LFIA lots). There were no indications of matrix effects from the different samples evaluated (mussels, scallops, oysters, clams, cockles) nor interference from other shellfish toxins (domoic acid, okadaic acid group). Naturally contaminated sample evaluations showed no false negative results were generated from a variety of different samples and profiles (n = 23), in comparison to reference methods (MBA method 959.08, LC-FD method 2005.06). External laboratory evaluations of naturally contaminated samples (n = 39) indicated good correlation with reference methods (MBA, LC-FD). This is the first LFIA which has been shown, through rigorous validation, to have the ability to detect most major PSTs in a reliable manner and will be a huge benefit to both industry and regulators, who need to perform rapid and reliable testing to ensure shellfish are safe to eat.

Realist synthesis: Critical care rapid response systems.

Background Rapid Response Systems (RRS) consist of four interrelated and interdependent components; an event detection and trigger mechanism, a response strategy, a governance structure and process improvement system. These multiple components of the RRS pose problems in evaluation as the intervention is complex and cannot be evaluated using a traditional systematic review. Complex interventions in healthcare aimed at changing service delivery and related behaviour of health professionals require a different approach to summarising the evidence. Realist synthesis is such an approach to reviewing research evidence on complex interventions to provide an explanatory analysis of how and why an intervention works or doesn’t work in practice. The core principle is to make explicit the underlying assumptions about how an intervention is suppose to work (ie programme theory) and then use this theory to guide evaluation. Methods A realist synthesis process was used to explain those factors that enable or constrain the success of RRS programmes. Results The findings from the review include the articulation of the RRS programme theories, evaluation of whether these theories are supported or refuted by the research evidence and an evaluation of evidence to explain the underlying reasons why RRS works or doesn’t work in practice. Rival conjectured RRS programme theories were identified to explain the constraining factors regarding implementation of RRS in practice. These programme theories are presented using a logic model to highlight all the components which impact or influence the delivery of RRS programmes in the practice setting. The evidence from the realist synthesis provided the foundation for the development of hypothesis to test and refine the theories in the subsequent stages of the Realist Evaluation PhD study [1]. This information will be useful in providing evidence and direction for strategic and service planning of acute care to improve patient safety in hospital. References: McGaughey J, Blackwood B, O’Halloran P, Trinder T. J. & Porter S. (2010) Realistic Evaluation of Early Warning Systems and the Acute Life-threatening Events – Recognition and Treatment training course for early recognition and management of deteriorating ward-based patients: research protocol. Journal of Advanced Nursing 66 (4), 923-932.

Development and Validation of a Lateral Flow Immunoassay for the Rapid Screening of Okadaic Acid and All Dinophysis Toxins from Shellfish Extracts

A single-step lateral flow immunoassay was developed and validated to detect okadaic acid (OA) and dinophysis toxins (DTXs), which cause diarrhetic shellfish poisoning. The performance characteristics of the test were investigated, in comparison to reference methods (liquid chromatography tandem mass spectrometry and/or bioassay), using both spiked and naturally contaminated shellfish. A portable reader was used to generate a qualitative result, indicating the absence or presence of OA-group toxins, at concentrations relevant to the maximum permitted level (MPL). Sample homogenates could be screened in 20 min (including extraction and assay time) for the presence of free toxins (OA, DTX1, DTX2). DTX3 detection could be included with the addition of a hydrolysis procedure. No matrix effects were observed from the species evaluated (mussels, scallops, oysters, and clams). Results from naturally contaminated samples (n = 72) indicated no false compliant results and no false noncompliant results at <50% MPL. Thus, the development of a new low-cost but highly effective tool for monitoring a range of important phycotoxins has been demonstrated.

Do spectral gamma ray data really reflect humid-arid palaeoclimates? A test from the Palaeogene Interbasaltic weathered horizons at the Giant’s Causeway, N.Ireland

Since 1999, the rapid, inexpensive and non-destructive use of Th/K and Th/U ratios from spectral gamma ray measurements have been used as a proxy for changes in palaeo-hinterland weathering. This model is tested here by analysis of in situ palaeoweathering horizons where clay mineral contents are well-known. A residual palaeoweathered horizon of Palaeogene laterite (developed on basalt) has been logged at 14 locations across N. Ireland using spectral gamma ray detectors. The results are compared to published elemental and mineralogical data. While the model of K and U loss during the early stages of weathering to smectite and kaolinite is supported, the formation (during progressively more advanced weathering) of gibbsite and iron oxides has reversed the predicted pattern and caused U and Th retention in the weathering profile. The severity (duration, humidity) of weathering and palaeoweathering may be estimated using Th/K ratios as a proxy. The use of Th/U ratios is more problematic should detrital gibbsite (or similar clays) or iron oxides be detected. Mineralogical analysis is needed in order to evaluate the hosts for K, U and Th: nonetheless, the spectral gamma ray machine offers a real-time, inexpensive and effective tool for the preliminary or conjunctive assessment of degrees of weathering or palaeoweathering.

An Environment for Rapid Derivatives Design and Experimentation

In the highly competitive world of modern finance, new derivatives are continually required to take advantage of changes in financial markets, and to hedge businesses against new risks. The research described in this paper aims to accelerate the development and pricing of new derivatives in two different ways. Firstly, new derivatives can be specified mathematically within a general framework, enabling new mathematical formulae to be specified rather than just new parameter settings. This Generic Pricing Engine (GPE) is expressively powerful enough to specify a wide range of stand¬ard pricing engines. Secondly, the associated price simulation using the Monte Carlo method is accelerated using GPU or multicore hardware. The parallel implementation (in OpenCL) is automatically derived from the mathematical description of the derivative. As a test, for a Basket Option Pricing Engine (BOPE) generated using the GPE, on the largest problem size, an NVidia GPU runs the generated pricing engine at 45 times the speed of a sequential, specific hand-coded implementation of the same BOPE. Thus a user can more rapidly devise, simulate and experiment with new derivatives without actual programming.

Sepsis: the LightCycler SeptiFast Test MGRADE®, SepsiTest™ and IRIDICA BAC BSI assay for rapidly identifying bloodstream bacteria and fungi – a systematic review and economic evaluation

Background: Sepsis can lead to multiple organ failure and death. Timely and appropriate treatment can reduce in-hospital mortality and morbidity. Objectives: To determine the clinical effectiveness and cost-effectiveness of three tests [LightCycler SeptiFast Test MGRADE® (Roche Diagnostics, Risch-Rotkreuz, Switzerland); SepsiTest™ (Molzym Molecular Diagnostics, Bremen, Germany); and the IRIDICA BAC BSI assay (Abbott Diagnostics, Lake Forest, IL, USA)] for the rapid identification of bloodstream bacteria and fungi in patients with suspected sepsis compared with standard practice (blood culture with or without matrix-absorbed laser desorption/ionisation time-offlight mass spectrometry). Data sources: Thirteen electronic databases (including MEDLINE, EMBASE and The Cochrane Library) were searched from January 2006 to May 2015 and supplemented by hand-searching relevant articles. Review methods: A systematic review and meta-analysis of effectiveness studies were conducted. A review of published economic analyses was undertaken and a de novo health economic model was constructed. A decision tree was used to estimate the costs and quality-adjusted life-years (QALYs) associated with each test; all other parameters were estimated from published sources. The model was populated with evidence from the systematic review or individual studies, if this was considered more appropriate (base case 1). In a secondary analysis, estimates (based on experience and opinion) from seven clinicians regarding the benefits of earlier test results were sought (base case 2). A NHS and Personal Social Services perspective was taken, and costs and benefits were discounted at 3.5% per annum. Scenario analyses were used to assess uncertainty. Results: For the review of diagnostic test accuracy, 62 studies of varying methodological quality were included. A meta-analysis of 54 studies comparing SeptiFast with blood culture found that SeptiFast had an estimated summary specificity of 0.86 [95% credible interval (CrI) 0.84 to 0.89] and sensitivity of 0.65 (95% CrI 0.60 to 0.71). Four studies comparing SepsiTest with blood culture found that SepsiTest had an estimated summary specificity of 0.86 (95% CrI 0.78 to 0.92) and sensitivity of 0.48 (95% CrI 0.21 to 0.74), and four studies comparing IRIDICA with blood culture found that IRIDICA had an estimated summary specificity of 0.84 (95% CrI 0.71 to 0.92) and sensitivity of 0.81 (95% CrI 0.69 to 0.90). Owing to the deficiencies in study quality for all interventions, diagnostic accuracy data should be treated with caution. No randomised clinical trial evidence was identified that indicated that any of the tests significantly improved key patient outcomes, such as mortality or duration in an intensive care unit or hospital. Base case 1 estimated that none of the three tests provided a benefit to patients compared with standard practice and thus all tests were dominated. In contrast, in base case 2 it was estimated that all cost per QALY-gained values were below £20,000; the IRIDICA BAC BSI assay had the highest estimated incremental net benefit, but results from base case 2 should be treated with caution as these are not evidence based. Limitations: Robust data to accurately assess the clinical effectiveness and cost-effectiveness of the interventions are currently unavailable. Conclusions: The clinical effectiveness and cost-effectiveness of the interventions cannot be reliably determined with the current evidence base. Appropriate studies, which allow information from the tests to be implemented in clinical practice, are required.