Clinical evaluation of the Procleix SARS-CoV-2 assay, a sensitive, high-throughput test that runs on an automated system.
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ABSTRACT:
SUBMITTER: Sauleda S
PROVIDER: S-EPMC8464401 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for high-throughput screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC 50 values in the 4-21 µM range. The here described assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.
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Project description:As of July 22, 2020, more than 14.7 million infections of SARS-CoV-2, the virus responsible for Coronavirus Disease 2019 (COVID-19), have been confirmed globally. Serological assays are essential for community screening, assessing infection prevalence, aiding identification of infected patients, and enacting appropriate treatment and quarantine protocols in the battle against this rapidly expanding pandemic. Antibody detection by agglutination-PCR (ADAP) is a pure solution phase immunoassay that generates a PCR amplifiable signal when patient antibodies agglutinate DNA-barcoded antigen probes into a dense immune complex. Here, we present an ultrasensitive and high-throughput automated liquid biopsy assay based on the Hamilton Microlab ADAP STAR automated liquid-handling platform, which was developed and validated for the qualitative detection of total antibodies against spike protein 1 (S1) of SARS-CoV-2 that uses as little as 4 µL of serum. To assess the clinical performance of the ADAP assay, 57 PCR-confirmed COVID-19 patients and 223 control patients were tested. The assay showed a sensitivity of 98% (56/57) and a specificity of 99.55% (222/223). Notably, the SARS-CoV-2-negative control patients included individuals with other common coronaviral infections, such as CoV-NL63 and CoV-HKU, which did not cross-react. In addition to high performance, the hands-free automated workstation enabled high-throughput sample processing to reduce screening workload while helping to minimize analyst contact with biohazardous samples. Therefore, the ADAP STAR liquid-handling workstation can be used as a valuable tool to address the COVID-19 global pandemic.
| S-EPMC7441286 | biostudies-literature
Project description:RT-PCR is the gold standard to detect SARS-CoV-2, however, its capacity is limited. We evaluated an automated antigen detection (AAD) test, Elecsys SARS-CoV-2 Antigen (Roche, Germany), for detecting SARS-CoV-2. We compared the limit of detection (LOD) between AAD test, rapid antigen detection (RAD) test; SARS-CoV-2 Rapid Antigen Test (SD Biosensor, Korea), and in-house RT-PCR test. LOD results showed that the AAD test was 100 fold more sensitive than the RAD test, while the sensitivity of the AAD test was comparable to the RT-PCR test. The AAD test detected between 85.7% and 88.6% of RT-PCR-positive specimens collected from COVID-19 patients, false negative results were observed for specimens with Ct values >30. Although clinical sensitivity for the AAD test was not superior or comparable to the RT-PCR test in the present study, the AAD test may be an alternative to RT-PCR test in terms of turn-around time and throughput.
| S-EPMC8284070 | biostudies-literature
Project description:IntroductionMolecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to suffer from delays and shortages. Antigen tests have recently emerged as a viable alternative to detect patients with high viral loads, associated with elevated risk of transmission. While rapid lateral flow tests greatly improved accessibility of SARS-CoV-2 detection in critical areas, their manual nature limits scalability and suitability for large-scale testing schemes. The Elecsys® SARS-CoV-2 Antigen assay allows antigen immunoassays to be carried out on fully automated high-throughput serology platforms.MethodsA total of 3139 nasopharyngeal and oropharyngeal swabs were collected at 3 different testing sites in Germany. Swab samples were pre-characterized by reverse transcription real-time polymerase chain reaction (RT-qPCR) and consecutively subjected to the antigen immunoassay on either the cobas e 411 or cobas e 801 analyzer.ResultsOf the tested respiratory samples, 392 were PCR positive for SARS-CoV-2 RNA. Median concentration was 2.95 × 104 (interquartile range [IQR] 5.1 × 102-3.5 × 106) copies/ml. Overall sensitivity and specificity of the antigen immunoassay were 60.2% (95% confidence interval [CI] 55.2-65.1) and 99.9% (95% CI 99.6-100.0), respectively. A 93.7% (95% CI 89.7-96.5) sensitivity was achieved at a viral RNA concentration ≥ 104 copies/ml (~ cycle threshold [Ct] value < 29.9).ConclusionThe Elecsys SARS-CoV-2 Antigen assay reliably detected patient samples with viral loads ≥ 10,000 copies/ml. It thus represents a viable high-throughput alternative for screening of patients or in situations where PCR testing is not readily available.
| S-EPMC8351226 | biostudies-literature
Project description:The ongoing SARS-CoV-2 pandemic and subsequent demand for viral testing worldwide has led to major issues in scaling the efforts of diagnostic labs and even in securing basic supplies for collection and processing of samples. This has in turn led to worldwide efforts by the scientific community to establish improved protocols that are cheaper, more scalable, and not as resource intensive. One such effort resulted in an assay called “Swab-Seq”, which was so named because it was originally developed to work with dry nasal swab samples, but is actually flexible in terms of the sample type it can accommodate for testing. The assay adapts the existing gold standard (RNA extracted from a nasopharyngeal (NP) swab that is subjected to quantitative reverse transcription polymerase chain reaction, “qRT-PCR”) to a next-generation sequencing readout. By pairing this modification with extraction-free sampling techniques it is possible to achieve high scalability at low cost per sample, and a reasonable turnaround time for reporting results. We evaluated the effectiveness of this assay both on samples collected from asymptomatic individuals using the traditional NP swab and using alternative extraction-free sampling techniques, including saliva and a saline mouth gargle protocol, and found the assay to be highly sensitive (comparable to the standard qRT-PCR assay), flexible (adaptable to saliva and gargle samples stored at room temperature up to a week), and scalable (easily accommodating hundreds of samples at a time). Continued development in the future will lead to more effective testing regimes that reduce the burden of transmissible respiratory infections on the global community.
2022-02-23 | GSE176224 | GEO
Project description:Background and aimsImmediate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for preventing the spread of coronavirus disease 2019 (COVID-19). The LabTurbo AIO 48 system is an automated platform that allows nucleic acid extraction and sample analysis on the same instrument, producing faster results without affecting their accuracy. We aimed to independently evaluate the LabTurbo AIO 48 (all-in-one system) for SARS-CoV-2 detection.Materials and methodsComparative limit of detection (LOD) was assessed on both the LabTurbo AIO 48 and current standard detection system based on real-time reverse transcriptase polymerase chain reaction (RT-PCR), using SARS-CoV-2 RNA control. Additional 125 primary clinical samples were assessed using both the protocols in parallel.ResultsThe turnaround time from sample to results for 48 samples analyzed on LabTurbo AIO 48 was approximately 2.5 h, whereas that analyzed using the in-house RT-PCR protocol was 4.8 h. LabTurbo AIO 48 also demonstrated higher sensitivity than our reference RT-PCR assay, with a LOD of 9.4 copies/reaction. The overall percentage agreement between both the methods for 125 samples was 100%.ConclusionLabTurbo AIO 48 is a robust detection option for SARS-CoV-2, allowing faster results and, consequently, aiding in better control and prevention of COVID-19.
| S-EPMC7836538 | biostudies-literature
Project description:The COVID-19 pandemic caused by the new SARS-CoV-2 coronavirus has imposed severe challenges on laboratories in their effort to achieve sufficient diagnostic testing capability for identifying infected individuals. In this study, we report the analytical and clinical performance characteristics of a new, high-throughput, fully automated nucleic acid amplification test system for the detection of SARS-CoV-2. The assay utilizes target capture, transcription-mediated amplification, and acridinium ester-labeled probe chemistry on the automated Panther system to directly amplify and detect two separate target sequences in the open reading frame 1ab (ORF1ab) region of the SARS-CoV-2 RNA genome. The probit 95% limit of detection of the assay was determined to be 0.004 50% tissue culture infective dose (TCID50)/ml using inactivated virus and 25 copies/ml (c/ml) using synthetic in vitro transcript RNA targets. Analytical sensitivity (100% detection) was confirmed to be 83 to 194 c/ml using three commercially available SARS-CoV-2 nucleic acid controls. No cross-reactivity or interference was observed with testing of six related human coronaviruses, as well as 24 other viral, fungal, and bacterial pathogens, at high titers. Clinical nasopharyngeal swab specimen testing (n = 140) showed 100%, 98.7%, and 99.3% positive, negative, and overall agreement, respectively, with a validated reverse transcription-PCR nucleic acid amplification test (NAAT) for SARS-CoV-2 RNA. These results provide validation evidence for a sensitive and specific method for pandemic-scale automated molecular diagnostic testing for SARS-CoV-2.
| S-EPMC7512162 | biostudies-literature
Project description:Introduction:To evaluate the automated cartridge-based PCR approach ARIES SARS-CoV-2 Assay targeting the ORF-sequence and the N-gene of SARS-CoV-2. Methods:In line with the suggestions by Rabenau and colleagues, the automated ARIES SARS-CoV-2 Assay was challenged with strongly positive samples, weakly positive samples and negative samples. Further, intra-assay and inter-assay precision as well as the limit-of-detection (lod) were defined with quantified target RNA and DNA. The Cepheid Xpert Xpress SARS-Cov-2 Assay was used as gold standard. Results:Concordance between the ARIES assay and the Cepheid assay was 100% for strongly positive samples and for negative samples, respectively. For weakly positive samples as confirmed applying the Cepheid assay, a relevant minority of 4 out of 15 samples (26.7%) went undetected by the ARIES assay. Intra- and inter-assay precision were satisfactory, while the lod was in the 103 DNA copies/reaction-range, in the 103 virus copies/reaction-range, or in the 103-104 free RNA copies/reaction-range in our hands. Conclusions:The automated ARIES assay shows comparable test characteristics as the Cepheid assay focusing on strongly positive and negative samples but a slightly reduced sensitivity with weakly positive samples. Decisions on diagnostic use should include considerations on the lod.
| S-EPMC7592518 | biostudies-literature
Project description:High-throughput serological tests that can detect neutralizing antibodies against SARS-CoV-2 are desirable for serosurveillance and vaccine efficacy evaluation. Although the conventional neutralization test (cVNT) remains the gold standard to confirm the presence of neutralizing antibodies in sera, the test is too labour-intensive for massive screening programs and less reproducible as live virus and cell culture is involved. Here, we performed an independent evaluation of a commercially available surrogate virus neutralization test (sVNT, GenScript cPass™) that can be done without biosafety level 3 containment in less than 2 h. When using the cVNT and a Luminex multiplex immunoassay (MIA) as reference, the sVNT obtained a sensitivity of 94 % (CI 90-96 %) on a panel of 317 immune sera that were obtained from hospitalized and mild COVID-19 cases from Belgium and a sensitivity of 88 % (CI 81-93 %) on a panel of 184 healthcare workers from the Democratic Republic of Congo. We also found strong antibody titer correlations (rs>0.8) among the different techniques used. In conclusion, our evaluation suggests that the sVNT could be a powerful tool to monitor/detect neutralising antibodies in cohort and population studies. The technique could be especially useful for vaccine evaluation studies in sub-Saharan Africa where the basic infrastructure to perform cVNTs is lacking.
| S-EPMC8253660 | biostudies-literature
Project description:Sensitive serological assays are needed to provide valuable information about acute and past viral infections. For example, detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies could serve as the basis for an "immunity passport" that would enable individuals to travel internationally. Here, utilizing a novel Magnetic Modulation Biosensing (MMB) system and the receptor-binding domain of the SARS-CoV-2 spike protein, we demonstrate a highly sensitive and specific anti-SARS-CoV-2 IgG serological assay. Using anti-SARS-CoV-2 IgG antibodies, RT-qPCR SARS-CoV-2-positive and healthy patients' samples, and vaccinees' samples, we compare the MMB-based SARS-CoV-2 IgG assay's analytical and clinical sensitivities to those of the enzyme-linked immunosorbent assay (ELISA). Compared with ELISA, the MMB-based assay has an ~6-fold lower limit of detection (129 ng/L vs. 817 ng/L), and it detects an increase in the IgG concentration much earlier after vaccination. Using 85 RT-qPCR SARS-CoV-2-positive samples and 79 -negative samples, the MMB-based assay demonstrated similar clinical specificity (98% vs. 99%) and sensitivity (93% vs. 92%) to the ELISA test, but with a much faster turnaround time (45 min vs. 245 min). The high analytical and clinical sensitivity, short turnaround time, and simplicity of the MMB-based assay makes it a preferred method for antibody detection.
| S-EPMC8773989 | biostudies-literature