A Point-of-Care Device for Fully Automated, Fast and Sensitive Protein Quantification via qPCR.

This paper presents a fully automated point-of-care device for protein quantification using short-DNA aptamers, where no manual sample preparation is needed. The device is based on our novel aptamer-based methodology combined with real-time polymerase chain reaction (qPCR), which we employ for very sensitive protein quantification. DNA amplification through qPCR, sensing and real-time data processing are seamlessly integrated into a point-of-care device equipped with a disposable cartridge for automated sample preparation. The system's modular nature allows for easy assembly, adjustment and expansion towards a variety of biomarkers for applications in disease diagnostics and personalised medicine. Alongside the device description, we also present a new algorithm, which we named PeakFluo, to perform automated and real-time quantification of proteins. PeakFluo achieves better linearity than proprietary software from a commercially available qPCR machine, and it allows for early detection of the amplification signal. Additionally, we propose an alternative way to use the proposed device beyond the quantitative reading, which can provide clinically relevant advice. We demonstrate how a convolutional neural network algorithm trained on qPCR images can classify samples into high/low concentration classes. This method can help classify obese patients from their leptin values to optimise weight loss therapies in clinical settings.

Assessing a novel, lab-free, point-of-care test for SARS-CoV-2 (CovidNudge): a diagnostic accuracy study.

BACKGROUND: Access to rapid diagnosis is key to the control and management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Laboratory RT-PCR testing is the current standard of care but usually requires a centralised laboratory and significant infrastructure. We describe our diagnostic accuracy assessment of a novel, rapid point-of-care real time RT-PCR CovidNudge test, which requires no laboratory handling or sample pre-processing. METHODS: Between April and May, 2020, we obtained two nasopharyngeal swab samples from individuals in three hospitals in London and Oxford (UK). Samples were collected from three groups: self-referred health-care workers with suspected COVID-19; patients attending emergency departments with suspected COVID-19; and hospital inpatient admissions with or without suspected COVID-19. For the CovidNudge test, nasopharyngeal swabs were inserted directly into a cartridge which contains all reagents and components required for RT-PCR reactions, including multiple technical replicates of seven SARS-CoV-2 gene targets (rdrp1, rdrp2, e-gene, n-gene, n1, n2 and n3) and human ribonuclease P (RNaseP) as sample adequacy control. Swab samples were tested in parallel using the CovidNudge platform, and with standard laboratory RT-PCR using swabs in viral transport medium for processing in a central laboratory. The primary analysis was to compare the sensitivity and specificity of the point-of-care CovidNudge test with laboratory-based testing. FINDINGS: We obtained 386 paired samples: 280 (73%) from self-referred health-care workers, 15 (4%) from patients in the emergency department, and 91 (23%) hospital inpatient admissions. Of the 386 paired samples, 67 tested positive on the CovidNudge point-of-care platform and 71 with standard laboratory RT-PCR. The overall sensitivity of the point-of-care test compared with laboratory-based testing was 94% (95% CI 86-98) with an overall specificity of 100% (99-100). The sensitivity of the test varied by group (self-referred healthcare workers 94% [95% CI 85-98]; patients in the emergency department 100% [48-100]; and hospital inpatient admissions 100% [29-100]). Specificity was consistent between groups (self-referred health-care workers 100% [95% CI 98-100]; patients in the emergency department 100% [69-100]; and hospital inpatient admissions 100% [96-100]). Point of care testing performance was similar during a period of high background prevalence of laboratory positive tests (25% [95% 20-31] in April, 2020) and low prevalence (3% [95% 1-9] in inpatient screening). Amplification of viral nucleocapsid (n1, n2, and n3) and envelope protein gene (e-gene) were most sensitive for detection of spiked SARS-CoV-2 RNA. INTERPRETATION: The CovidNudge platform was a sensitive, specific, and rapid point of care test for the presence of SARS-CoV-2 without laboratory handling or sample pre-processing. The device, which has been implemented in UK hospitals since May, 2020, could enable rapid decisions for clinical care and testing programmes. FUNDING: National Institute of Health Research (NIHR) Imperial Biomedical Research Centre, NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Oxford University in partnership with Public Health England, NIHR Biomedical Research Centre Oxford, and DnaNudge.

Pooled sputum to optimise the efficiency and utility of rapid, point-of-care molecular SARS-CoV-2 testing.

BACKGROUND: As SARS-CoV-2 testing expands, particularly to widespread asymptomatic testing, high sensitivity point-of-care PCR platforms may optimise potential benefits from pooling multiple patients' samples. METHOD: We tested patients and asymptomatic citizens for SARS-CoV-2, exploring the efficiency and utility of CovidNudge (i) for detection in individuals' sputum (compared to nasopharyngeal swabs), (ii) for detection in pooled sputum samples, and (iii) by modelling roll out scenarios for pooled sputum testing. RESULTS: Across 295 paired samples, we find no difference (p = 0.1236) in signal strength for sputum (mean amplified replicates (MAR) 25.2, standard deviation (SD) 14.2, range 0-60) compared to nasopharyngeal swabs (MAR 27.8, SD 12.4, range 6-56). At 10-sample pool size we find some drop in absolute strength of signal (individual sputum MAR 42.1, SD 11.8, range 13-60 vs. pooled sputum MAR 25.3, SD 14.6, range 1-54; p < 0.0001), but only marginal drop in sensitivity (51/53,96%). We determine a limit of detection of 250 copies/ml for an individual test, rising only four-fold to 1000copies/ml for a 10-sample pool. We find optimal pooled testing efficiency to be a 12-3-1-sample model, yet as prevalence increases, pool size should decrease; at 5% prevalence to maintain a 75% probability of negative first test, 5-sample pools are optimal. CONCLUSION: We describe for the first time the use of sequentially dipped sputum samples for rapid pooled point of care SARS-CoV-2 PCR testing. The potential to screen asymptomatic cohorts rapidly, at the point-of-care, with PCR, offers the potential to quickly identify and isolate positive individuals within a population "bubble".