More than the Eye Can See: Shedding New Light on SARS-CoV-2 Lateral Flow Device-Based Immunoassays.
28 May 2021-ACS Applied Materials & Interfaces (American Chemical Society (ACS))-Vol. 13, Iss: 22, pp 25694-25700
TL;DR: In this paper, the authors show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a biosample.
Abstract: Containing the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented challenge due to high horizontal transmissivity and asymptomatic carriage rates. Lateral flow device (LFD) immunoassays were introduced in late 2020 to detect SARS-CoV-2 infection in asymptomatic or presymptomatic individuals rapidly. While LFD technologies have been used for over 60 years, their widespread use as a public health tool during a pandemic is unprecedented. By the end of 2020, data from studies into the efficacy of the LFDs emerged and showed these point-of-care devices to have very high specificity (ability to identify true negatives) but inadequate sensitivity with high false-negative rates. The low sensitivity (<50%) shown in several studies is a critical public health concern, as asymptomatic or presymptomatic carriers may wrongly be assumed to be noninfectious, posing a significant risk of further spread in the community. Here, we show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a biosample. We quantified significant immobilized antigen-antibody-labeled conjugate complexes within the LFDs visually scored as negative using high-sensitivity synchrotron X-ray fluorescence imaging. Correlating quantitative X-ray fluorescence measurements and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) determined numbers of viral copies, we identified that negatively scored samples could contain up to 100 PFU (equivalent here to â¼10â¯000 RNA copies/test). The study demonstrates where the shortcomings arise in many of the current direct-readout SARS-CoV-2 LFDs, namely, being a deficiency in the readout as opposed to the potential level of detection of the test, which is orders of magnitude higher. The present findings are of importance both to public health monitoring during the Coronavirus Disease 2019 (COVID-19) pandemic and to the rapid refinement of these tools for immediate and future applications.
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TL;DR: In this article, a dual-antigen sandwich structure using labeled/immobilized SARS-CoV-2 spike receptor binding domain antigen was developed, instead of general indirect antibody capturing approach, to reduce the false positive rate of GLFIA.
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TL;DR: In this paper , a dual-antigen sandwich structure using labeled/immobilized SARS-CoV-2 spike receptor binding domain antigen was developed, instead of general indirect antibody capturing approach, to reduce the false positive rate of GLFIA.
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TL;DR: This research uses mathematically derived visual logistics to interpret COVID-19 molecular and rapid antigen test (RAgT) performance, determine prevalence boundaries where risk exceeds expectations, and evaluate benefits of recursive testing along home, community, and emergency spatial care paths.
Abstract: This research uses mathematically derived visual logistics to interpret COVID-19 molecular and rapid antigen test (RAgT) performance, determine prevalence boundaries where risk exceeds expectations, and evaluate benefits of recursive testing along home, community, and emergency spatial care paths. Mathematica and open access software helped graph relationships, compare performance patterns, and perform recursive computations. Tiered sensitivity/specificity comprise: (T1) 90%/95%; (T2) 95%/97.5%; and (T3) 100%/≥99%, respectively. In emergency medicine, median RAgT performance peaks at 13.2% prevalence, then falls below T1, generating risky prevalence boundaries. RAgTs in pediatric ERs/EDs parallel this pattern with asymptomatic worse than symptomatic performance. In communities, RAgTs display large uncertainty with median prevalence boundary of 14.8% for 1/20 missed diagnoses, and at prevalence > 33.3–36.9% risk 10% false omissions for symptomatic subjects. Recursive testing improves home RAgT performance. Home molecular tests elevate performance above T1 but lack adequate validation. Widespread RAgT availability encourages self-testing. Asymptomatic RAgT and PCR-based saliva testing present the highest chance of missed diagnoses. Home testing twice, once just before mingling, and molecular-based self-testing, help avoid false omissions. Community and ER/ED RAgTs can identify contagiousness in low prevalence. Real-world trials of performance, cost-effectiveness, and public health impact could identify home molecular diagnostics as an optimal diagnostic portal.
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TL;DR: In this paper , a review of SARS-CoV-2-related cold chain food incidents and current methods for detecting SARS CoV2 was presented, where the needs, challenges and practicable countermeasures for SARSCoV2 detection, specifically for cold-chain foods and packaging, were underlined.
Abstract: The pandemic caused by SARS-CoV-2 has a huge impact on the global economy. SARS-CoV-2 could possibly and potentially be transmitted to humans through cold-chain foods and packaging (namely good-to-human), although it mainly depends on a human-to-human route. It is imperative to develop countermeasures to cope with the spread of viruses and fulfil effective surveillance of cold-chain foods and packaging. This review outlined SARS-CoV-2-related cold-chain food incidents and current methods for detecting SARS-CoV-2. Then the needs, challenges and practicable countermeasures for SARS-CoV-2 detection, specifically for cold-chain foods and packaging, were underlined. In fact, currently established detection methods for SARS-CoV-2 are mostly used for humans; thus, these may not be ideally applied to cold-chain foods directly. Therefore, it creates a need to develop novel methods and low-cost, automatic, mini-sized devices specifically for cold-chain foods and packaging. The review intended to draw people’s attention to the possible spread of SARS-CoV-2 with cold-chain foods and proposed perspectives for futuristic cold-chain foods monitoring during the pandemic.
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TL;DR: In this article , a phage-based lateral flow (LFA) was used to detect SARS-CoV-2 nucleoprotein in nasal swab extract using a FluorChem gel documentation system.
Abstract: The COVID-19 pandemic has highlighted the urgent need for sensitive, affordable, and widely accessible testing at the point of care. Here we demonstrate a new, universal LFA platform technology using M13 phage conjugated with antibodies and HRP enzymes that offers high analytical sensitivity and excellent performance in a complex clinical matrix. We also report its complete integration into a sensitive chemiluminescence-based smartphone-readable lateral flow assay for the detection of SARS-CoV-2 nucleoprotein. We screened 84 anti-nucleoprotein monoclonal antibody pairs in phage LFA and identified an antibody pair that gave an LoD of 25 pg mL-1 nucleoprotein in nasal swab extract using a FluorChem gel documentation system and 100 pg mL-1 when the test was imaged and analyzed by an in-house-developed smartphone reader. The smartphone-read LFA signals for positive clinical samples tested (N = 15, with known Ct) were statistically different (p < 0.001) from signals for negative clinical samples (N = 11). The phage LFA technology combined with smartphone chemiluminescence imaging can enable the timely development of ultrasensitive, affordable point-of-care testing platforms for SARS-CoV-2 and beyond.
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References
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1,595 citations
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TL;DR: SARS-CoV-2 Vero cell infectivity was only observed for RT-PCR Ct < 24 and STT < 8 days, suggesting Infectivity of patients with Ct >24 and duration of symptoms >8 days may be low.
Abstract: BACKGROUND: Reverse-transcription polymerase chain reaction (RT-PCR) has become the primary method to diagnose viral diseases, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RT-PCR detects RNA, not infectious virus; thus, its ability to determine duration of infectivity of patients is limited. Infectivity is a critical determinant in informing public health guidelines/interventions. Our goal was to determine the relationship between E gene SARS-CoV-2 RT-PCR cycle threshold (Ct) values from respiratory samples, symptom onset to test (STT), and infectivity in cell culture. METHODS: In this retrospective cross-sectional study, we took SARS-CoV-2 RT-PCR-confirmed positive samples and determined their ability to infect Vero cell lines. RESULTS: Ninety RT-PCR SARS-CoV-2-positive samples were incubated on Vero cells. Twenty-six samples (28.9%) demonstrated viral growth. Median tissue culture infectious dose/mL was 1780 (interquartile range, 282-8511). There was no growth in samples with a Ctâ
>â
24 or STTâ
>â
8 days. Multivariate logistic regression using positive viral culture as a binary predictor variable, STT, and Ct demonstrated an odds ratio (OR) for positive viral culture of 0.64 (95% confidence interval [CI], .49-.84; Pâ
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24. CONCLUSIONS: SARS-CoV-2 Vero cell infectivity was only observed for RT-PCR Ctâ
â
24 and duration of symptomsâ
>â
8 days may be low. This information can inform public health policy and guide clinical, infection control, and occupational health decisions. Further studies of larger size are needed.
1,020 citations
TL;DR: Correlation between successful isolation of virus in cell culture and Ct value of quantitative RT-PCR targeting E gene suggests that patients with Ct above 33–34 using the RT- PCR system are not contagious and thus can be discharged from hospital care or strict confinement for non-hospitalized patients.
Abstract: In a preliminary clinical study, we observed that the combination of hydroxychloroquine and azithromycin was effective against SARS-CoV-2 by shortening the duration of viral load in Covid-19 patients. It is of paramount importance to define when a treated patient can be considered as no longer contagious. Correlation between successful isolation of virus in cell culture and Ct value of quantitative RT-PCR targeting E gene suggests that patients with Ct above 33-34 using our RT-PCR system are not contagious and thus can be discharged from hospital care or strict confinement for non-hospitalized patients.
748 citations
TL;DR: Probability of culturing virus declines to 8% in samples with Ct > 35 and to 6% 10 days after onset and it is similar in asymptomatic and symptomatic persons.
Abstract: Severe acute respiratory syndrome coronavirus 2 viral load in the upper respiratory tract peaks around symptom onset and infectious virus persists for 10 days in mild-to-moderate coronavirus disease (n = 324 samples analysed). RT-PCR cycle threshold (Ct) values correlate strongly with cultivable virus. Probability of culturing virus declines to 8% in samples with Ct > 35 and to 6% 10 days after onset; it is similar in asymptomatic and symptomatic persons. Asymptomatic persons represent a source of transmissible virus.
734 citations