Improving the efficiency and effectiveness of an industrial SARS-CoV-2 diagnostic facility.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing.

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods.

Effect of Serum on Electrochemical Detection of Bioassays Having Ag Nanoparticle Labels.

The effect of serum on electrochemical detection of bioassays having silver nanoparticle (AgNP) detection labels was investigated. Both a model assay and an antigen-specific sandwich bioassay for the heart failure marker NT-proBNP were examined. In both cases, the AgNP labels were conjugated to a detection antibody. Electrochemical detection was carried out using a galvanic exchange/anodic stripping voltammetry method in which Au3+ exchanges with AgNP labels. The assays were carried out using a paper-based electrode platform. The bioassays were exposed to different serum conditions prior to and during detection. There are three important outcomes reported in this article. First, both the model- and antigen-specific assays could be formed in undiluted serum with no detectable interferences from the serum components. Second, to achieve the maximum possible electrochemical signal, the highest percentage of serum that can remain in an assay buffer during electrochemical detection is 0.25% when no washing is performed. The assay results are rendered inaccurate when 0.50% or more of serum is present. Third, the factors inhibiting galvanic exchange in serum probably relate to surface adsorption of biomolecules onto the AgNP labels, chelation of Au3+ by serum components, or both. The results reported here provide general guidance for using metal NP labels for electrochemical assays in biofluids.

Silver Nanocubes as Electrochemical Labels for Bioassays.

Here, we report on the use of 40 ± 4 nm silver nanocubes (AgNCs) as electrochemical labels in bioassays. The model metalloimmunoassay combines galvanic exchange (GE) and anodic stripping voltammetry (ASV). The results show that a lower limit of detection is achieved by simply changing the shape of the Ag label yielding improved GE with AgNCs when compared to GE with spherical silver nanoparticles (sAgNPs). Specifically, during GE between electrogenerated Au3+ and the Ag labels, a thin shell of Au forms on the surface of the NP. This shell is more porous when GE proceeds on AgNCs compared to sAgNPs, and therefore, more exchange occurs when using AgNCs. ASV results show that the Ag collection efficiency (AgCE%) is increased by up to ∼57% when using AgNCs. When the electrochemical system is fully optimized, the limit of detection is 0.1 pM AgNCs, which is an order of magnitude lower than that of sAgNP labels.

Hybrid paper and 3D-printed microfluidic device for electrochemical detection of Ag nanoparticle labels.

In the present article we report a new hybrid microfluidic device (hyFlow) comprising a disposable paper electrode and a three-dimensional (3D) printed plastic chip for the electrochemical detection of a magnetic bead-silver nanoparticle (MB-AgNP) bioconjugate. This hybrid device evolved due to the difficulty of incorporating micron-scale MBs into paper-only fluidic devices. Specifically, paper fluidic devices can entrap MB-containing conjugates within their cellulose or nitrocellulose fiber matrix. The hyFlow system was designed to minimize such issues and transport MB conjugates more efficiently to the electrochemical detection zone of the device. The hyFlow system retains the benefit of fluid transport by pressure-driven flow, however, no pump is required for its operation. The hyFlow device is capable of detecting either pre-formed MB-AgNP conjugates or conjugates formed in situ. The detection limit of AgNPs using this device is 12 pM, which represents just 22 AgNPs per MB.

Electrochemical Detection of NT-proBNP Using a Metalloimmunoassay on a Paper Electrode Platform.

In this paper, we demonstrate an electrochemical method for detection of the heart failure biomarker, N-terminal prohormone brain natriuretic peptide (NT-proBNP). The approach is based on a paper electrode assembly and a metalloimmunoassay; it is intended for eventual integration into a home-use sensor. Sensing of NT-proBNP relies on the formation of a sandwich immunoassay and electrochemical quantification of silver nanoparticle (AgNP) labels attached to the detection antibodies (Abs). There are four important outcomes reported in this article. First, compared to physisorption of the detection Abs on the AgNP labels, a 27-fold increase in signal is observed when a heterobifunctional cross-linker is used to facilitate this labeling. Second, the assay is selective in that it does not cross-react with other cardiac natriuretic peptides. Third, the assay forms in undiluted human serum (though the electrochemical analysis is carried out in buffer). Finally, and most important, the assay is able to detect NT-proBNP at concentrations between 0.58 and 2.33 nM. This performance approaches the critical NT-proBNP concentration threshold often used by physicians for risk stratification purposes: ∼0.116 nM.

Lewy Body Dementia Association's Research Centers of Excellence Program: Inaugural Meeting Proceedings.

The first Lewy Body Dementia Association (LBDA) Research Centers of Excellence (RCOE) Investigator's meeting was held on December 14, 2017, in New Orleans. The program was established to increase patient access to clinical experts on Lewy body dementia (LBD), which includes dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD), and to create a clinical trials-ready network. Four working groups (WG) were created to pursue the LBDA RCOE aims: (1) increase access to high-quality clinical care, (2) increase access to support for people living with LBD and their caregivers, (3) increase knowledge of LBD among medical and allied (or other) professionals, and (4) create infrastructure for a clinical trials-ready network as well as resources to advance the study of new therapeutics.

Managing Heart Failure at Home With Point-of-Care Diagnostics.

The objective of this paper is development of an inexpensive point-of-care sensor for detecting the primary heart failure marker peptide, NT-proBNP. The device technology is based on an antibody sandwich assay, but with three innovative aspects. First, chemical amplification is carried out via oxidation of silver nanoparticles (NPs) attached to signaling antibodies rather than by enzymatic amplification. The electrochemical method is faster and eliminates the need for long-term storage of enzymes. Second, the antibody sandwich is formed on mobile magnetic beads. This enhances the rate of mass transfer of the biomarker and the signaling antibody to the primary detection antibody, which is immobilized on the magnetic beads. Third, the sensor itself is fabricated on a paper platform with screen-printed electrodes. This coupled with assembly by simple paper folding, keeps the cost of the sensor low. Here, we report on two separate assays. The first is based on a simple biotin-streptavidin conjugate, which is a preliminary model for the antibody assay. The results indicate a detection limit of 2.1 pM of silver NPs and an assay time of 7 min. The actual NT-proBNP antibody assay takes somewhat longer, and the dynamic detection range is higher: 2.9-582 nM. On the basis of the results presented in this paper, we conclude that this inexpensive paper-based sensor represents a viable technology for point-of-care testing of NT-proBNP, but nevertheless several challenges remain prior to clinical implementation. These include attaining a lower detection limit and better reproducibility, and optimizing the device for human blood.

Quantitative electrochemical metalloimmunoassay for TFF3 in urine using a paper analytical device.

We report a paper-based assay platform for the detection of the kidney disease marker Trefoil Factor 3 (TFF3) in human urine. The sensor is based on a quantitative metalloimmunoassay that can determine TFF3 concentrations via electrochemical detection of environmentally stable silver nanoparticle (AgNP) labels attached to magnetic microbeads via a TFF3 immunosandwich. The paper electroanalytical device incorporates two preconcentration steps that make it possible to detect concentrations of TFF3 in human urine at the low end of the target TFF3 concentration range (0.03-7.0 µg mL(-1)). Importantly, the paper device provides a level of accuracy for TFF3 determination in human urine equivalent to that of a commercial kit. The paper sensor has a dynamic range of ∼2.5 orders of magnitude, only requires a simple, one-step incubation protocol, and is fast, requiring only 10 min to complete. The cost of the materials at the prototypic laboratory scale, excluding reagents, is just US$0.42.

Simple, sensitive, and quantitative electrochemical detection method for paper analytical devices.

We report a new type of paper analytical device that provides quantitative electrochemical output and detects concentrations as low as 767 fM. The model analyte is labeled with silver nanoparticles (AgNPs), which provide 250,000-fold amplification. AgNPs eliminate the need for enzymatic amplification, thereby improving device stability and response time. The use of magnetic beads to preconcentrate the AgNPs at the detection electrode further improves sensitivity. Response time is improved by incorporation of a hollow channel, which increases the flow rate in the device by a factor of 7 and facilitates the use of magnetic beads. A key reaction necessary for label detection is made possible by the presence of a slip layer, a fluidic switch that can be actuated by manually slipping a piece of paper. The design of the device is versatile and should be useful for detection of proteins, nucleic acids, and microbes.

The Boyd-McLeod procedure for tennis elbow: mid- to long-term results.

BACKGROUND: Tennis elbow is a common condition that usually responds to conservative measures. In refractory cases, surgical intervention is indicated. A plethora of surgical techniques have been described. We report the mid- to long-term outcomes of the Boyd-McLeod procedure for refractory tennis elbow. METHODS: A retrospective analysis and current review of patients that had undergone the Boyd-McLeod procedure over a 12-year period was undertaken. Demographics, time to discharge, length of follow-up and outcome scores were collected. RESULTS: Seventy patients underwent surgery. Mean time to discharge was 15.35 weeks, with 88% successful outcomes. Fifty-four patients were available for current follow-up at mean of 5.52 years (range 1.17 years to 11.49 years). Range of motion in all patients was unchanged. There were no revision procedures. Mean (SD) Mayo Elbow Performance Score was 90.85 (13.11), with 75.5% returning a good or excellent score and 24.5% a fair outcome. The mean (SD) Oxford Elbow Score was 44.04 (6.92); mean (SD) pain score was 89.5 (17.58); mean (SD) function score was 95.34 (9.59) and mean (SD) socio-psychological score was 91.50 (17.01). Overall, 83% of patients had an Oxford Elbow Score of 43 or greater, suggesting excellent outcome. CONCLUSIONS: We show that the Boyd-McLeod procedure is an excellent option over both the short- and long-term for refractory tennis elbow.

Ultra-rapid screening for substance-use disorders: the Alcohol, Smoking and Substance Involvement Screening Test (ASSIST-Lite).

BACKGROUND: The Alcohol, Smoking and Substance Involvement Screening Test (ASSIST 3.0; index test) is a structured interview for alcohol, tobacco, cannabis, stimulants, sedatives and opioid use disorders in general medical settings. Perceived administration time deters routine use. This study releases a short-form: the ASSIST-Lite. METHODS: Diagnostic accuracy study among 2082 adults recruited from general medical (70%) and specialist mental health/addiction treatment services (22%). Current DSM-IV substance dependence (MINI International Neuropsychiatric Interview) and moderate-severe tobacco dependence (Fagerstrom Nicotine Dependence Test) were reference standards. Exploratory factor and item-response theory models re-calibrated ordinal test items. Items for the ASSIST-Lite were selected by diagnostic accuracy evaluation (area under the receiver-operating characteristic [AUC] curve [≤0.7]), sensitivity, specificity, positive and negative predictive values [PVP, NVP], kappa, likelihood ratios [LR+, LR-], and clinical utility index [CU+, CU-]). RESULTS: For each substance an item pair was selected (AUC [0.8-1.0], sensitivity [0.8-1.0], specificity [0.7-0.8], PVP [0.8-1.0], NVP [0.7-1.0], kappa [0.5-0.9], LR+ [2.5-5.9], LR- [0.0-0.2], CU+ [0.7-0.9], and CU- [0.5-0.8]). Gender, age and recruitment setting (specialist mental health versus general medical) did not moderate accuracy, with the exception of opioids (AUC <0.7, participants ≥59 years). Male opioid users had more severe substance involvement scores that females (differential item functioning analysis, P=0.00). There was no evidence of differential accuracy between countries (AUC range, 0.8-1.0). CONCLUSION: The ASSIST-Lite is an ultra-rapid screener which has been optimised for general medical settings. Optionally, a criterion question can be added to capture hazardous drinking, and to capture use of another type of mood-altering substance.

Abnormality of mismatch negativity in response to tone omission in dyslexic adults.

Evidence of abnormalities in the perception of rapidly presented sounds in dyslexia has been interpreted as evidence of a prolonged time window within which sounds can influence the perception of temporally surrounding sounds. We recorded the magnetic mismatch negativity (MMNm) to infrequent tone omissions in a group of six dyslexic adults and six IQ and age-matched controls. An MMNm is only elicited in response to a complete stimulus omission when successive inputs fall within the temporal window of integration (stimulus onset asynchrony (SOA) approximately 160 ms). No MMNm responses were recorded in either experimental group when stimuli were presented at SOAs falling just outside the temporal window of integration (SOA = 175 ms). However, while presentation rates of 100 ms resulted in MMNm responses for all control participants, the same stimulus omissions elicited an MMNm response in only one of the six dyslexic participants. These results cannot support the hypothesis of a prolonged time window of integration, but rather indicate auditory grouping deficits in the dyslexic population.

Comments on 'The dyslexia ecosystem': a reply to Nicolson.

The central issue facing the dyslexia community, and the underlying theme of Nicolson's 'The Dyslexia Ecosystem' (Nicolson, 2002, Dyslexia, 8, 55-66), is how we can best translate what we know about this particular developmental disorder into practice to give each child the greatest opportunity of acquiring the enabling skill of literacy. To achieve this, and notwithstanding Nicolson's caveat on this point, we have to consider how we can best move from our sphere of expertise to a greater sphere of influence, both as individuals and as a community of research practitioners. In our response, we first consider aspects of Nicolson's general analysis of 'The Dyslexia Ecosystem' and then examine some of the specific objectives that have been proposed.