Detection and Diagnosis Technologies for SARS-CoV-2

Detection and diagnosis platforms play key roles in early warning, outbreak control and exit strategy for any pandemic, and they are especially pertinent for the Coronavirus disease 2019 (COVID-19) pandemic. The challenges posed by the speed and extent of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) spread around the globe also offered unprecedented opportunities for the development and deployment of novel strategies and products - not only vaccines and therapeutics, but also diagnostics. This chapter provides a brief summary of the vast array of molecular, serological, cell-based and other diagnostic tools for the specific detection of SARS-CoV-2 infections and immune responses. The focus is on the principles and applications of each platform, while detailed protocols can be found in the cited references. © 2023 by World Scientific Publishing Co. Pte. Ltd.

CRISPR-based point-of-care plant disease diagnostics.

The coupling of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas RNA-programmable nucleases with nucleic acid detection platforms has brought radical changes to the field of disease diagnosis. Recently, Sánchez et al. developed a simple, rapid, highly sensitive, precise, and in-field deployable point-of-care (POC) and point-of-need (PON) molecular disease detection tool that can be used in diverse agricultural applications.

Current Advances in Paper-Based Biosensor Technologies for Rapid COVID-19 Diagnosis.

The global coronavirus disease 2019 (COVID-19) pandemic has had significant economic and social impacts on billions of people worldwide since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, in November 2019. Although polymerase chain reaction (PCR)-based technology serves as a robust test to detect SARS-CoV-2 in patients with COVID-19, there is a high demand for cost-effective, rapid, comfortable, and accurate point-of-care diagnostic tests in medical facilities. This review introduces the SARS-CoV-2 viral structure and diagnostic biomarkers derived from viral components. A comprehensive introduction of a paper-based diagnostic platform, including detection mechanisms for various target biomarkers and a COVID-19 commercial kit is presented. Intrinsic limitations related to the poor performance of currently developed paper-based devices and unresolved issues are discussed. Furthermore, we provide insight into novel paper-based diagnostic platforms integrated with advanced technologies such as nanotechnology, aptamers, surface-enhanced Raman spectroscopy (SERS), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. Finally, we discuss the prospects for the development of highly sensitive, accurate, cost-effective, and easy-to-use point-of-care COVID-19 diagnostic methods.

Lateral flow assay (LFA) in the diagnosis of COVID-19-associated pulmonary aspergillosis (CAPA): a single-center experience.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is seen during coronavirus-2019 (COVID-19), has been reported in different incidences, and is defined as COVID-19-associated pulmonary aspergillosis (CAPA). Detection of galactomannan antigen is an important diagnostic step in diagnosing IPA. Enzyme-linked immunoassay (ELISA) is the most frequently used method, and lateral flow assay (LFA) is increasingly used with high sensitivity and specificity for rapid diagnosis. The present study aimed to compare the sensitivity of LFA and ELISA in the diagnosis of CAPA in COVID-19 patients followed in our hospital's ICU for pandemic (ICU-P). METHODS: This study included patients with a diagnosis of COVID-19 cases confirmed by polymerase chain reaction and were followed up in ICU-P between August 2021 and February 2022 with acute respiratory failure. The diagnosis of CAPA was based on the European Confederation of Medical Mycology (ECMM) and the International Society for Human and Animal Mycology 2020 (ECMM/ ISHAM) guideline. Galactomannan levels were determined using LFA and ELISA in serum samples taken simultaneously from the patients. RESULTS: Out of the 174 patients followed in the ICU-P, 56 did not meet any criteria for CAPA and were excluded from the analysis. The rate of patients diagnosed with proven CAPA was 5.7% (10 patients). A statistically significant result was obtained with LFA for the cut-off value of 0.5 ODI in the diagnosis of CAPA (p < 0.001). The same significant statistical relationship was found for the cut-off value of 1.0 ODI for the ELISA (p < 0.01). The sensitivity of LFA was 80% (95% CI: 0.55-1.05, p < 0.05), specificity 94% (95% CI: 0.89-0.98, p < 0.05); PPV 53% (95% CI: 0.28-0.79, p > 0.05) and NPV was 98% (95% CI: 0.95-1.01, p < 0.05). The risk of death was 1.66 (HR: 1.66, 95% CI: 1.02-2.86, p < 0.05) times higher in patients with an LFA result of ≥ 0.5 ODI than those with < 0.5 (p < 0.05). CONCLUSIONS: It is reckoned that LFA can be used in future clinical practice, particularly given its effectiveness in patients with hematological malignancies and accuracy in diagnosing CAPA.

A Comparative Study of Nine SARS-CoV-2 IgG Lateral Flow Assays Using Both Post-Infection and Post-Vaccination Samples.

BACKGROUND: Since the SARS-CoV-2 pandemic, lateral flow assays (LFA) detecting specific antibodies have entered the market in abundance. Despite being CE-IVD-labeled, the antigenic compounds of the assays are often unknown, the performance characteristics provided by the manufacturer are often incomplete, and the samples used to obtain the data are not detailed. OBJECTIVE: To perform a comparative evaluation of nine lateral flow assays to detect IgG responses against SARS-CoV-2. For the evaluation, a carefully designed serum panel containing post-infection samples and post-vaccination (both mRNA vaccine and inactivated virus vaccine) samples was used. RESULTS: The sensitivity of the assays overall ranged from 9 to 90.3% and the specificity ranged from 94.2 to 100%. Spike protein-containing assays performed generally better than the assays with only nucleocapsid protein. The sensitivity of some assays was higher on post-infection samples, while other assays had a higher sensitivity to post-vaccination samples. CONCLUSION: A comparative approach in the verification of LFAs with an adequately designed serum panel enabled the identification of the antigens used in the assays. Sensitivities differed between post-infection and post-vaccination samples, depending on the assays used. This demonstrates that the verification of assays must be performed with samples representative of the intended use of the assay.

Aspergillus Lateral Flow Assay with Digital Reader for the Diagnosis of COVID-19-Associated Pulmonary Aspergillosis (CAPA): a Multicenter Study.

This multicenter study evaluated the IMMY Aspergillus Galactomannan Lateral Flow Assay (LFA) with automated reader for diagnosis of pulmonary aspergillosis in patients with COVID-19-associated acute respiratory failure (ARF) requiring intensive care unit (ICU) admission between 03/2020 and 04/2021. A total of 196 respiratory samples and 148 serum samples (n = 344) from 238 patients were retrospectively included, with a maximum of one of each sample type per patient. Cases were retrospectively classified for COVID-19-associated pulmonary aspergillosis (CAPA) status following the 2020 consensus criteria, with the exclusion of LFA results as a mycological criterion. At the 1.0 cutoff, sensitivity of LFA for CAPA (proven/probable/possible) was 52%, 80% and 81%, and specificity was 98%, 88% and 67%, for bronchoalveolar lavage fluid (BALF), nondirected bronchoalveolar lavage (NBL), and tracheal aspiration (TA), respectively. At the 0.5 manufacturer's cutoff, sensitivity was 72%, 90% and 100%, and specificity was 79%, 83% and 44%, for BALF, NBL and TA, respectively. When combining all respiratory samples, the receiver operating characteristic (ROC) area under the curve (AUC) was 0.823, versus 0.754, 0.890 and 0.814 for BALF, NBL and TA, respectively. Sensitivity and specificity of serum LFA were 20% and 93%, respectively, at the 0.5 ODI cutoff. Overall, the Aspergillus Galactomannan LFA showed good performances for CAPA diagnosis, when used from respiratory samples at the 1.0 cutoff, while sensitivity from serum was limited, linked to weak invasiveness during CAPA. As some false-positive results can occur, isolated results slightly above the recommended cutoff should lead to further mycological investigations.

Lateral flow-based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use.

The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.

Identification and Engineering of Aptamers for Theranostic Application in Human Health and Disorders.

An aptamer is a short sequence of synthetic oligonucleotides which bind to their cognate target, specifically while maintaining similar or higher sensitivity compared to an antibody. The in-vitro selection of an aptamer, applying a conjoining approach of chemistry and molecular biology, is referred as Systematic Evolution of Ligands by Exponential enrichment (SELEX). These initial products of SELEX are further modified chemically in an attempt to make them stable in biofluid, avoiding nuclease digestion and renal clearance. While the modification is incorporated, enough care should be taken to maintain its sensitivity and specificity. These modifications and several improvisations have widened the window frame of aptamer applications that are currently not only restricted to in-vitro systems, but have also been used in molecular imaging for disease pathology and treatment. In the food industry, it has been used as sensor for detection of different diseases and fungal infections. In this review, we have discussed a brief history of its journey, along with applications where its role as a therapeutic plus diagnostic (theranostic) tool has been demonstrated. We have also highlighted the potential aptamer-mediated strategies for molecular targeting of COVID-19. Finally, the review focused on its future prospective in immunotherapy, as well as in identification of novel biomarkers in stem cells and also in single cell proteomics (scProteomics) to study intra or inter-tumor heterogeneity at the protein level. Small size, chemical synthesis, low batch variation, cost effectiveness, long shelf life and low immunogenicity provide advantages to the aptamer over the antibody. These physical and chemical properties of aptamers render them as a strong biomedical tool for theranostic purposes over the existing ones. The significance of aptamers in human health was the key finding of this review.

Serum Lateral Flow assay with digital reader for the diagnosis of invasive pulmonary aspergillosis: A two-centre mixed cohort study.

BACKGROUND: Detection of galactomannan (GM) from bronchoalveolar lavage fluid (BALF) or serum is broadly used for diagnosis of invasive aspergillosis (IA), although the sensitivity of GM from serum is lower in non-neutropenic patients. We evaluated the Aspergillus galactomannan Lateral Flow assay (LFA) with digital readout from serum in a mixed cohort of patients. METHODS: We performed a retrospective two-centre study evaluating the LFA from serum of patients with clinical suspicion of IA obtained between 2015 and 2021 at the University of California San Diego and the Medical University of Graz. The sensitivity and specificity was calculated for proven/probable aspergillosis versus no aspergillosis. Correlation with same-sample GM was calculated using Spearman correlation analysis and kappa statistics. RESULTS: In total, 122 serum samples from 122 patients were analysed, including proven IA (n = 1), probable IA or coronavirus-associated pulmonary aspergillosis (CAPA) (n = 27), and no IA/CAPA/non-classifiable (n = 94). At a 0.5 ODI cut-off, the sensitivity and specificity of the LFA was 78.6% and 80.5%. Spearman correlation analysis showed a strong correlation between serum LFA ODI and serum GM ODI (ρ 0.459, p < .0001). Kappa was 0.611 when both LFA and GM were used with a 0.5 ODI cut-off, showing substantial agreement (p < .001). DISCUSSION: The LFA with digital read out from serum showed good performance for the diagnosis of probable/proven aspergillosis, with substantial agreement to GM from serum. Like the LFA from BALF, the LFA from serum may serve as a more rapid test compared to conventional GM, particularly in settings where GM is not readily available.

pyPOCQuant - A tool to automatically quantify Point-Of-Care Tests from images.

Lateral flow Point-Of-Care Tests (POCTs) are a valuable tool for rapidly detecting pathogens and the associated immune response in humans and animals. In the context of the SARS-CoV-2 pandemic, they offer rapid on-site diagnostics and can relieve centralized laboratory testing sites, thus freeing resources that can be focused on especially vulnerable groups. However, visual interpretation of the POCT test lines is subjective, error prone and only qualitative. Here we present pyPOCQuant, an open-source tool implemented in Python 3 that can robustly and reproducibly analyze POCTs from digital images and return an unbiased and quantitative measurement of the POCT test lines.

Dissolvable sugar barriers to enhance the sensitivity of nitrocellulose membrane lateral flow assay for COVID-19 nucleic acid.

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.

Diagnostic performance of a SARS-CoV-2 rapid antigen test in a large, Norwegian cohort.

BACKGROUND: Rapid antigen tests (RATs) may be included in national strategies for handling the SARS-CoV-2 pandemic, as they provide test results rapidly, are easily performed outside laboratories, and enable immediate contract tracing. However, before implementation further clinical evaluation of test sensitivity is warranted. OBJECTIVES: To examine the performance of Abbott's Panbio™ COVID-19 Ag Rapid Test Device for SARS-CoV-2 testing in a low to medium prevalence setting in Norway. STUDY DESIGN: A prospective study comparing the results of the Panbio RAT with PCR in 4857 parallel samples collected at a SARS-CoV-2 test station in Oslo, and from COVID-19 outbreaks in six Norwegian municipalities. RESULTS: A total of 4857 cases were included in the study; 3991 and 866 cases from the test station and the outbreak municipalities, respectively. The prevalence at the test station in Oslo was 6.3 %, and the overall sensitivity of the RAT was 74 %. Increased sensitivity was observed in patients who experienced symptoms (79 %) and when considering samples with viral loads above estimated level of infectivity (84 %), while it was lower in asymptomatic persons (55 %). In the outbreak municipalities, the overall prevalence was 6.9 %, and the total sensitivity of the RAT was 70 %. CONCLUSIONS: Our results indicate that the test correctly identified most infectious individuals. Nevertheless, the sensitivity is considerably lower than for PCR, and it is important that the limitations of the test are kept in mind in the follow-up of tested individuals.

Recent advances in sensitive surface-enhanced Raman scattering-based lateral flow assay platforms for point-of-care diagnostics of infectious diseases.

This review reports the recent advances in surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) platforms for the diagnosis of infectious diseases. As observed through the recent infection outbreaks of COVID-19 worldwide, a timely diagnosis of the disease is critical for preventing the spread of a disease and to ensure epidemic preparedness. In this regard, an innovative point-of-care diagnostic method is essential. Recently, SERS-based assay platforms have received increasing attention in medical communities owing to their high sensitivity and multiplex detection capability. In contrast, LFAs provide a user-friendly and easily accessible sensing platform. Thus, the combination of LFAs with a SERS detection system provides a new diagnostic modality for accurate and rapid diagnoses of infectious diseases. In this context, we briefly discuss the recent application of LFA platforms for the POC diagnosis of SARS-CoV-2. Thereafter, we focus on the recent advances in SERS-based LFA platforms for the early diagnosis of infectious diseases and their applicability for the rapid diagnosis of SARS-CoV-2. Finally, the key issues that need to be addressed to accelerate the clinical translation of SERS-based LFA platforms from the research laboratory to the bedside are discussed.