Clinical Validation of GenBody COVID-19 Ag, Nasal and Nasopharyngeal Rapid Antigen Tests for Detection of SARS-CoV-2 in European Adult Population.

Accurate and rapid identification of COVID-19 is critical for effective patient treatment and disease outcomes, as well as the prevention of SARS-CoV-2 transmission. Rapid antigen tests (RATs) for identifying SARS-CoV-2 are simpler, faster and less expensive than molecular assays. Any new product to be considered a medical device is subject to evaluation and data analysis to verify the in vitro diagnostic ability to achieve its intended purpose. Clinical validation of such a test is a prerequisite before clinical application. This study was a clinical validation on adult Europeans of GenBody COVID-19 Ag, nasal and nasopharyngeal RATs. A set of 103 positive and 301 negative from nose and nasopharynx samples confirmed by RT-qPCR were examined. The tests were safe to use and showed 100% specificity in both specimens, and high sensitivity of 94.17% (95%CI 87.75% to 97.83%) and 97.09% (95%CI 91.72% to 99.4%), respectively. The parameters were significantly better for samples with higher virus loads (the highest for CT ≤ 25). The GenBody COVID-19 Ag RATs are inexpensive (compared to RT-qPCR), reliable and rapid with high sensitivity and specificity, making them suitable for diagnosis and timely isolation and treatment of COVID-19 patients, contributing to the better control of virus spread.

Assessing a New Prescreening Score for the Simplified Evaluation of the Clinical Quality and Relevance of eHealth Apps: Instrument Validation Study.

BACKGROUND: In 2020, more than 250 eHealth solutions were added to app stores each day, or 90,000 in the year; however, the vast majority of these solutions have not undergone clinical validation, their quality is unknown, and the user does not know if they are effective and safe. We sought to develop a simple prescreening scoring method that would assess the quality and clinical relevance of each app. We designed this tool with 3 health care stakeholder groups in mind: eHealth solution designers seeking to evaluate a potential competitor or their own tool, investors considering a fundraising candidate, and a hospital clinician or IT department wishing to evaluate a current or potential eHealth solution. OBJECTIVE: We built and tested a novel prescreening scoring tool (the Medical Digital Solution scoring tool). The tool, which consists of 26 questions that enable the quick assessment and comparison of the clinical relevance and quality of eHealth apps, was tested on 68 eHealth solutions. METHODS: The Medical Digital Solution scoring tool is based on the 2021 evaluation criteria of the French National Health Authority, the 2022 European Society of Medical Oncology recommendations, and other provided scores. We built the scoring tool with patient association and eHealth experts and submitted it to eHealth app creators, who evaluated their apps via the web-based form in January 2022. After completing the evaluation criteria, their apps obtained an overall score and 4 categories of subscores. These criteria evaluated the type of solution and domain, the solution's targeted population size, the level of clinical assessment, and information about the provider. RESULTS: In total, 68 eHealth solutions were evaluated with the scoring tool. Oncology apps (22%, 20/90) and general health solutions (23%, 21/90) were the most represented. Of the 68 apps, 32 (47%) were involved in remote monitoring by health professionals. Regarding clinical outcomes, 5% (9/169) of the apps assessed overall survival. Randomized studies had been conducted for 21% (23/110) of the apps to assess their benefit. Of the 68 providers, 38 (56%) declared the objective of obtaining reimbursement, and 7 (18%) out of the 38 solutions seeking reimbursement were assessed as having a high probability of reimbursement. The median global score was 11.2 (range 4.7-17.4) out of 20 and the distribution of the scores followed a normal distribution pattern (Shapiro-Wilk test: P=.33). CONCLUSIONS: This multidomain prescreening scoring tool is simple, fast, and can be deployed on a large scale to initiate an assessment of the clinical relevance and quality of a clinical eHealth app. This simple tool can help a decision-maker determine which aspects of the app require further analysis and improvement.

Assessment of the Diagnostic Performance of a Novel SARS-CoV-2 Antigen Sealing Tube Test Strip (Colloidal Gold) as Point-of-Care Surveillance Test.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant outbreaks have highlighted the need of antigen-detecting rapid diagnostic tests (Ag-RDTs) that can be used at the point-of-care (POC). Although many Ag-RDTs have been approved for SARS-CoV-2 detection, studies demonstrating the clinical performance of Ag-RDTs against variants of concern, especially the new Omicron variant, are limited. The aim of this study was to evaluate the diagnostic sensitivity and specificity of the AMAZING COVID-19 Antigen Sealing Tube Test Strip (Colloidal Gold) in 584 early symptomatic and asymptomatic participants (age range 0-90 years). The performance of this Ag-RDT was assessed by comparing its results with reverse transcription RT-PCR (rRT-PCR). One hundred twenty positive samples were also analyzed with rRT-PCR to discriminate Omicron and Delta/Kappa variants (72.50% Omicron; 27.50% Delta/Kappa). Overall, the Ag-RDT showed high positive and negative percent values of 92.52% (95% CI, 86.61-95.95%) and 98.05% (95% CI, 96.41-98.95%), respectively, as well as an overall diagnostic accuracy of 96.92% (95% CI, 95.17-98.16%). Taken together, these data indicate that this inexpensive and simple-to-use Ag-RDT presents excellent analytical performance and can reliably detect Omicron and Delta/Kappa variants.

Clinical validation of two immunochromatographic SARS-CoV-2 antigen tests in near hospital facilities.

INTRODUCTION: Rapid antigen tests to detect SARS-CoV-2 virus need to be validated. The purpose of clinical validation is to place the test into the everyday working process in health care institutions. METHODOLOGY: The clinical validation of Alltest Covid19 antigen test (Alltest, China) and Vivadiag Pro SARS- CoV-2 antigen tests (Vivacheck, China) started in four Slovenian health care institutions in December as a point-of-care test. Institutions compared the results of antigen tests to Seegene Allplex™ 2019-nCoV rt-PCR assay (SeeGene, South Korea) and Cobas 6800 SARS CoV-2 rt-PCR (Roche, USA). RESULTS: Sensitivity (90.6%, 95% CI = 84.94%-94.36%) and specificity (100%, 95% CI = 99.41%-100%) of Vivadiag Pro SARS CoV-2 Ag test were observed. While validating Alltest Covid19 Ag assay we got similar results (sensitivity 94.37%, 95% CI = 89.20% - 97.54%), specificity 100% (95% CI = 98.83% - 100%). CONCLUSIONS: Vivadiag Pro SARS CoV-2 Ag test and Alltest Covid19 test proved to be a good screening tool to detect SARS-CoV-2. The accurate information about the patient's status was available almost immediately and there was no need to wait for rt-PCR results. We could prevent further spread of the SARS-CoV-2 in primary care and hospital settings.

Rapid detection of multiple SARS-CoV-2 variants of concern by PAM-targeting mutations.

SARS-CoV-2 variants of concern (VOCs) that increase transmission or disease severity or reduce diagnostic or vaccine efficacy continue to emerge across the world. Current methods available to rapidly detect these can be resource intensive and thus sub-optimal for large-scale deployment needed during a pandemic response. Here, we describe a CRISPR-based assay that detects mutations in spike gene CRISPR PAM motif or seed regions to identify a pan-specific VOC single-nucleotide polymorphism (SNP)) ((D614G) and Alpha- and Delta-specific (S982A and D950N) SNPs. This assay exhibits good diagnostic sensitivity and strain specificity with nasal swabs and is designed for use in laboratory and point-of-care settings. This should enable rapid, high-throughput VOC identification required for surveillance and characterization efforts to inform clinical and public health decisions. Furthermore, the assay can be adapted to target similar SNPs associated with emerging SARS-CoV-2 VOCs, or other rapidly evolving viruses.

Optimal Insertion Depth for Nasal Mid-Turbinate and Nasopharyngeal Swabs.

Millions of people are tested for COVID-19 daily during the pandemic, and a lack of evidence to guide optimal nasal swab testing can increase the risk of false-negative test results. This study aimed to determine the optimal insertion depth for nasal mid-turbinate and nasopharyngeal swabs. The measurements were made with a flexible endoscope during the collection of clinical specimens with a nasopharyngeal swab at a public COVID-19 test center in Copenhagen, Denmark. Participants were volunteer adults undergoing a nasopharyngeal SARS-CoV-2 rapid antigen test. All 109 participants (100%) completed the endoscopic measurements; 52 (48%) women; 103 (94%) white; mean age 34.39 (SD, 13.2) years; and mean height 176.7 (SD, 9.29) cm. The mean swab length to the posterior nasopharyngeal wall was 9.40 (SD, 0.64) cm. The mean endoscopic distance to the anterior and posterior end of the inferior turbinate was 1.95 (SD, 0.61) cm and 6.39 (SD, 0.62) cm, respectively. The mean depth to nasal mid-turbinate was calculated as 4.17 (SD, 0.48) cm. The optimal depths of insertion for nasal mid-turbinate swabs are underestimated in current guidelines compared with our findings. This study provides clinical evidence to guide the performance of anatomically correct nasal and nasopharyngeal swab specimen collection for virus testing.

Multisite Clinical Validation of Isothermal Amplification-Based SARS-CoV-2 Detection Assays Using Different Sampling Strategies.

Isothermal amplification-based tests have been introduced as rapid, low-cost, and simple alternatives to real-time reverse transcriptase PCR (RT-PCR) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. The clinical performance of two isothermal amplification-based tests (Atila Biosystems iAMP coronavirus disease of 2019 [COVID-19] detection test and OptiGene COVID-19 direct plus RT-loop-mediated isothermal amplification [LAMP] test) was compared with that of clinical RT-PCR assays using different sampling strategies. A total of 1,378 participants were tested across 4 study sites. Compared with standard of care RT-PCR testing, the overall sensitivity and specificity of the Atila iAMP test for detection of SARS-CoV-2 were 76.2% and 94.9%, respectively, and increased to 88.8% and 89.5%, respectively, after exclusion of an outlier study site. Sensitivity varied based on the anatomic site from which the sample was collected. Sensitivity for nasopharyngeal sampling was 65.4% (range across study sites, 52.8% to 79.8%), for midturbinate was 88.2%, for saliva was 55.1% (range across study sites, 42.9% to 77.8%), and for anterior nares was 66.7% (range across study sites, 63.6% to 76.5%). The specificity for these anatomic collection sites ranged from 96.7% to 100%. Sensitivity improved in symptomatic patients (overall, 82.7%) and those with a higher viral load (overall, 92.4% for cycle threshold [CT] of ≤25). Sensitivity and specificity of the OptiGene direct plus RT-LAMP test, which was conducted at a single study site, were 25.5% and 100%, respectively. The Atila iAMP COVID test with midturbinate sampling is a rapid, low-cost assay for detecting SARS-CoV-2, especially in symptomatic patients and those with a high viral load, and could be used to reduce the risk of SARS-CoV-2 transmission in clinical settings. Variation of performance between study sites highlights the need for site-specific clinical validation of these assays before clinical adoption. IMPORTANCE Numerous SARS-CoV-2 detection assays have been developed and introduced into the market under emergency use authorizations (EUAs). EUAs are granted primarily based on small studies of analytic sensitivity and specificity with limited clinical validations. A thorough clinical performance evaluation of SARS-CoV-2 assays is important to understand the strengths, limitations, and specific applications of these assays. In this first large-scale multicentric study, we evaluated the clinical performance and operational characteristics of two isothermal amplification-based SARS-CoV-2 tests, namely, (i) iAMP COVID-19 detection test (Atila BioSystems, USA) and (ii) COVID-19 direct plus RT-LAMP test (OptiGene Ltd., UK), compared with those of clinical RT-PCR tests using different sampling strategies (i.e., nasopharyngeal, self-sampled anterior nares, self-sampled midturbinate, and saliva). An important specific use for these isothermal amplification-based, rapid, low-cost, and easy-to-perform SARS-CoV-2 assays is to allow for a safer return to preventive clinical encounters, such as cancer screening, particularly in low- and middle-income countries that have low SARS-CoV-2 vaccination rates.

Longitudinal evaluation of laboratory-based serological assays for SARS-CoV-2 antibody detection.

Serological assays for SARS-CoV-2 infection are now widely available for use in diagnostic laboratories. Limited data are available on the performance characteristics in different settings, and at time periods remote from the initial infection. Validation of the Abbott (Architect SARS-CoV-2 IgG), DiaSorin (Liaison SARS-CoV-2 S1/S2 IgG) and Roche (Cobas Elecsys Anti-SARS-CoV-2) assays was undertaken utilising 217 serum samples from 131 participants up to 7 months following COVID-19 infection. The Abbott and DiaSorin assays were implemented into routine laboratory workflow, with outcomes reported for 2764 clinical specimens. Sensitivity and specificity were concordant with the range reported by the manufacturers for all assays. Sensitivity across the convalescent period was highest for the Roche at 95.2-100% (95% CI 81.0-100%), then the DiaSorin at 88.1-100% (95% CI 76.0-100%), followed by the Abbott 68.2-100% (95% CI 53.4-100%). Sensitivity of the Abbott assay fell from approximately 5 months; on this assay paired serum samples for 45 participants showed a significant drop in the signal-to-cut-off ratio and 10 sero-reversion events. When used in clinical practice, all samples testing positive by both DiaSorin and Abbott assays were confirmed as true positive results. In this low prevalence setting, despite high laboratory specificity, the positive predictive value of a single positive assay was low. Comprehensive validation of serological assays is necessary to determine the optimal assay for each diagnostic setting. In this low prevalence setting we found implementation of two assays with different antibody targets maximised sensitivity and specificity, with confirmatory testing necessary for any sample which was positive in only one assay.

Serum 25-hydroxyvitamin D measurement: Comparative evaluation of three automated immunoassays.

OBJECTIVES: the different analytical methods for measurement of serum 25-hydroxyvitamin D (25(OH)D) are not yet fully harmonized and no consensus exists on a threshold of 25(OH)D defining a deficiency status. In this study, we compared the results from the assays of serum 25(OH)D performed with three different methods to evaluate the presence of potential biases and how much these biases can influence the assignment of patients to specific 25(OH)D deficiency/sufficiency categories. DESIGN AND METHODS: Liaison 25(OH) Vitamin D Total (DiaSorin Liaison XL), Elecsys Vitamin D Total II (Roche Elecsys) and Lumipulse G25(OH) Vitamin D (Fujirebio Lumipulse G1200) were used. Methods comparability was established performing Passing-Bablok regression and Bland-Altman analysis to prove whether the differences found were lower than the preliminarily pre-established maximum acceptable bias. RESULTS: all Passing-Bablok regressions exhibited the presence of a proportional and constant systematic error. Bland-Altman analysis revealed biases well above the maximum acceptable bias, so the 25(OH)D concentrations measured were not comparable. To evaluate whether the three methods had the same ability to classify patients into different categories of vitamin D levels, we categorized results obtained by each method in reference classes. Lumipulse categorized most patients into the class with the lowest 25(OH)D concentrations (<20 ng/mL) whereas Elecsys ranked the lowest number. CONCLUSIONS: Liaison XL and Elecsys have shown good accuracy compared to Lumipulse in measuring 25(OH)D levels. Nevertheless, the assays were not interchangeable due to the lack of comparability of results as well as to the disagreement in classification of hormone deficiency or sufficiency.

Use of Immunochromatographic SARS-CoV-2 Antigen Testing in Eight Long-Term Care Facilities for the Elderly.

The clinical validation of the NADAL COVID-19 antigen test (Nal von Minden, Moers, Germany) started in eight Slovenian long-term health care facilities in October 2020. The purpose of clinical validation is to implement the test into the everyday working process in long-term care (LTC) facilities and demonstrate how it can be used to mitigate the spread of the virus in these environments. The facilities compared the results of antigen tests to the results obtained using Cobas 6800 SARS-CoV-2 real-time reverse transcription polymerase chain reaction (RT-PCR) (Roche, USA). Sensitivity (86.96%, 95% CI: 66.41-97.23%) and specificity (88.24%, 95% CI: 80.35-93.77%) of the NADAL COVID-19 antigen test were good. Rapid antigen testing served well for early detection of infection and helped to prevent and control spread of the SARS Cov2 in six out of eight LTCs. Moreover, mini-outbreaks were quickly resolved in all six LTCs. Locally validated immunochromatographic SARS-CoV-2 antigen testing can be used to contain the spread of the virus in LTCs. Antigen tests also deliver accurate information very quickly if used early with a low threshold. The NADAL COVID-19 antigen test proved to be a good screening tool to detect SARS-COV-2 in LTCs.

Clinical Application of a New SARS-CoV-2 Antigen Detection Kit (Colloidal Gold) in the Detection of COVID-19.

The precise diagnosis of COVID-19 is of outmost importance in order to effectively treat patients and prevent SARS-CoV-2 transmission. Herein, we evaluated the sensitivity and specificity of the COVID-19 Antigen Detection Kit (Colloidal Gold-CG) compared with PCR in nasopharyngeal and nasal samples. A total of 114 positive and 244 negative nasopharyngeal specimens confirmed by PCR were used in this comparative study. When the PCR positive Cycle Threshold (Ct) value was ≤25, CG sensitivity was 100%. When the PCR positive Ct value was ≤33, CG sensitivity was 99%. When the PCR positive Ct value was ≤40, CG sensitivity was 89.47%. Regarding nasal swabs, a total of 109 positive and 250 negative specimens confirmed by PCR were used. When the PCR positive Ct value was ≤25, CG sensitivity was 100%. When the PCR positive Ct value was ≤33, CG sensitivity was 96.12%. When the PCR positive Ct value was ≤37, CG sensitivity was 91.74%. Specificity was above 99% regardless of the Ct value of PCR positivity for both nasopharyngeal and nasal specimens. Overall, the CG showed high sensitivity and specificity when the PCR Ct value was less than 33. Therefore, CG can be used for screening early in the disease course. Confirmatory PCR is essential when a false negative result is suspected.

IMMUNO-COV v2.0: Development and Validation of a High-Throughput Clinical Assay for Measuring SARS-CoV-2-Neutralizing Antibody Titers.

Neutralizing antibodies are key determinants of protection from future infection, yet well-validated high-throughput assays for measuring titers of SARS-CoV-2-neutralizing antibodies are not generally available. Here, we describe the development and validation of IMMUNO-COV v2.0, a scalable surrogate virus assay, which titrates antibodies that block infection of Vero-ACE2 cells by a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins (VSV-SARS2-Fluc). Antibody titers, calculated using a standard curve consisting of stepped concentrations of SARS-CoV-2 spike monoclonal antibody, correlated closely (P < 0.0001) with titers obtained from a gold standard 50% plaque-reduction neutralization test (PRNT50%) performed using a clinical isolate of SARS-CoV-2. IMMUNO-COV v2.0 was comprehensively validated using data acquired from 242 assay runs performed over 7 days by five analysts, utilizing two separate virus lots, and 176 blood samples. Assay performance was acceptable for clinical use in human serum and plasma based on parameters including linearity, dynamic range, limit of blank and limit of detection, dilutional linearity and parallelism, precision, clinical agreement, matrix equivalence, clinical specificity and sensitivity, and robustness. Sufficient VSV-SARS2-Fluc virus reagent has been banked to test 5 million clinical samples. Notably, a significant drop in IMMUNO-COV v2.0 neutralizing antibody titers was observed over a 6-month period in people recovered from SARS-CoV-2 infection. Together, our results demonstrate the feasibility and utility of IMMUNO-COV v2.0 for measuring SARS-CoV-2-neutralizing antibodies in vaccinated individuals and those recovering from natural infections. Such monitoring can be used to better understand what levels of neutralizing antibodies are required for protection from SARS-CoV-2 and what booster dosing schedules are needed to sustain vaccine-induced immunity. IMPORTANCE Since its emergence at the end of 2019, SARS-CoV-2, the causative agent of COVID-19, has caused over 100 million infections and 2.4 million deaths worldwide. Recently, countries have begun administering approved COVID-19 vaccines, which elicit strong immune responses and prevent disease in most vaccinated individuals. A key component of the protective immune response is the production of neutralizing antibodies capable of preventing future SARS-CoV-2 infection. Yet, fundamental questions remain regarding the longevity of neutralizing antibody responses following infection or vaccination and the level of neutralizing antibodies required to confer protection. Our work is significant as it describes the development and validation of a scalable clinical assay that measures SARS-CoV-2-neutraling antibody titers. We have critical virus reagent to test over 5 million samples, making our assay well suited for widespread monitoring of SARS-CoV-2-neutralizing antibodies, which can in turn be used to inform vaccine dosing schedules and answer fundamental questions regarding SARS-CoV-2 immunity.

Clinical Validation of a SARS-CoV-2 Real-Time Reverse Transcription PCR Assay Targeting the Nucleocapsid Gene.

BACKGROUND: Detection of SARS-CoV-2 viral RNA is important for the diagnosis and management of COVID-19. METHODS: We present a clinical validation of a reverse transcription PCR (RT-PCR) assay for the SARS-CoV-2 nucleocapsid (N1) gene. Off-board lysis on an automated nucleic acid extraction system was optimized with endemic coronaviruses (OC43 and NL63). Genomic RNA and SARS-CoV-2 RNA in a recombinant viral protein coat were used as control materials and compared for recovery from nucleic acid extraction. RESULTS: Nucleic acid extraction showed decreased recovery of endemic Coronavirus in vitro transcribed RNA (NL63) compared with attenuated virus (OC43). SARS-CoV-2 RNA had more reliable recovery from extraction through amplification than genomic RNA. Recovery of genomic RNA was improved by combining lysis buffer with clinical matrix before adding RNA. The RT-PCR assay demonstrated 100% in silico sensitivity and specificity. The accuracy across samples was 100% (75 of 75). Precision studies showed 100% intra-run, inter-run, and inter-technologist concordance. The limit of detection was 264 copies per milliliter (estimated 5 copies per reaction; 35.56 mean threshold cycle value). CONCLUSIONS: This SARS-CoV-2 assay demonstrates appropriate characteristics for use under an Emergency Use Authorization. Endemic coronavirus controls were useful in optimizing the extraction procedure. In the absence of live or attenuated virus, recombinant virus in a protein coat is an appropriate control specimen type for assay validation during a pandemic.