scholarly journals Saliva Is a Valid Alternative to Nasopharyngeal Swab in Chemiluminescence-Based Assay for Detection of SARS-CoV-2 Antigen

2021 ◽  
Vol 10 (7) ◽  
pp. 1471
Author(s):  
Alessandra Amendola ◽  
Giuseppe Sberna ◽  
Eleonora Lalle ◽  
Francesca Colavita ◽  
Concetta Castilletti ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Clinical Performance ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Promising Alternative ◽  
Viral Loads ◽  
Healthy Donors ◽  
Antigen Assay ◽  
Highly Correlated

Diagnostic methods based on SARS-CoV-2 antigens detection are a promising alternative to SARS-CoV-2 RNA amplification. We evaluated the automated chemiluminescence-based Lumipulse® G SARS-CoV-2 Ag assay on saliva samples, using Simplexa™ COVID-19 Direct assay as a reference test. Analytical performance was established on a pool of healthy donors’ saliva samples spiked with the 2019-nCoV/Italy-INMI1 isolate, whereas clinical performance was assessed on fresh saliva specimens collected from hospitalized patients with suspect or confirmed COVID-19 diagnosis. The limit of detection (LOD) was 0.65 Log TCID50/mL, corresponding to 18,197 copies/mL of SARS-CoV-2 RNA. Antigen concentrations and SARS-CoV-2 RNA were highly correlated (r = 0.99; p < 0.0001). Substantial agreement (80.3%) and significant correlation (r = −0.675; p = 0.0006) were observed between Lumipulse® G assay results and Ct values on clinical samples, with 52.4% sensitivity and specificity 94.1%. Sensitivity exceeded 90.0% when calculated on samples with Ct < 25, and specificity was 100% when excluding samples from recovered patients with previous COVID-19 diagnosis. Overall, chemiluminescence-based antigen assay may be reliably applied to saliva samples to identify individuals with high viral loads, more likely to transmit the virus. However, the low positive predictive value in a context of low SARS-CoV-2 prevalence underscores the need for confirmatory testing in SARS-CoV-2 antigen-positive cases.

scholarly journals Microfluidic nano-scale qPCR enables ultra-sensitive detection of SARS-CoV-2

2020 ◽  
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
False Negative ◽  
False Negative Rate ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Viral Loads ◽  
Nano Scale ◽  
Negative Rate

Background: A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used standard RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. Methods: We implement a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification and nano-scale qPCR based on the Fluidigm 192.24 microfluidic chip. We validate the method using both positive controls and nasopharyngeal swab samples. Results: Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of the Fluidigm method by 1,000-fold, enabling detection below 1 copy/μl. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible detection of SARS-CoV-2 over five orders of magnitude (< 1 to 106 viral copies/μl). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/μl) in 17 samples with negative clinical diagnosis, indicating a potential false negative rate of 18.7% by clinical diagnostic procedures. Conclusion: The three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (< 1 viral copy/μl) and has the potential to reduce the false negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

scholarly journals Performance of the LIAISON® SARS-CoV-2 Antigen Assay vs. SARS-CoV-2-RT-PCR

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 658
Author(s):  
Melanie Fiedler ◽  
Caroline Holtkamp ◽  
Ulf Dittmer ◽  
Olympia E. Anastasiou
Keyword(s):  
Reference Method ◽  
Respiratory Viruses ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Viral Loads ◽  
Antigen Assay ◽  
Polymerase Chain

We aimed to evaluate the LIAISON® SARS-CoV-2 antigen assay (DiaSorin), comparing its performance to real-time polymerase chain reaction (RT-PCR) for the detection of SARS-CoV-2 RNA. 182 (110 PCR-positive and 72 PCR-negative) nasopharyngeal swab samples were taken for the detection of SARS-CoV-2. RT-PCR and antigen assay were performed using the same material. The sensitivity and specificity of the antigen assay were calculated for different cut-offs, with RT-PCR serving as the reference method. Stored clinical samples that were positive for other respiratory viruses were tested to evaluate cross-reactivity. One third (33/110, 30%) were falsely classified as negative, while no false positives were found using the 200 TCID50/mL cut-off for the SARS-CoV-2 antigen as proposed by the manufacturer. This corresponded to a sensitivity of 70% (60–78%) and a specificity of 100% (94–100%). Lowering the cut-off for positivity of the antigen assay to 22.79 or 57.68 TCID50/mL increased the sensitivity of the method, reaching a sensitivity of 92% (85–96%) vs. 79% (70–86%) and a specificity of 81% (69–89%) vs. 99% (91–100%), respectively. The antigen assay reliably detected samples with high SARS-CoV-2 viral loads (≥106 copies SARS-CoV-2/mL), while it cannot differentiate between negative and low positive samples. Cross-reactivity toward other respiratory viruses was not detected.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Virus detection and identification in minutes using single-particle imaging and deep learning

2020 ◽  
Author(s):  
Nicolas Shiaelis ◽  
Alexander Tometzki ◽  
Leon Peto ◽  
Andrew McMahon ◽  
Christof Hepp ◽  
...  
Keyword(s):  
Neural Network ◽  
Deep Learning ◽  
Single Particle ◽  
Virus Detection ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Promising Alternative ◽  
Detection And Identification ◽  
Particle Imaging ◽  
Single Particle Imaging

AbstractThe increasing frequency and magnitude of viral outbreaks in recent decades, epitomized by the current COVID-19 pandemic, has resulted in an urgent need for rapid and sensitive viral diagnostic methods. Here, we present a methodology for virus detection and identification that uses a convolutional neural network to distinguish between microscopy images of single intact particles of different viruses. Our assay achieves labeling, imaging and virus identification in less than five minutes and does not require any lysis, purification or amplification steps. The trained neural network was able to differentiate SARS-CoV-2 from negative clinical samples, as well as from other common respiratory pathogens such as influenza and seasonal human coronaviruses, with high accuracy. Single-particle imaging combined with deep learning offers a promising alternative to traditional viral diagnostic methods, and has the potential for significant impact.

scholarly journals Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2

Processes ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 1425
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
False Negative ◽  
False Negative Rate ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Viral Loads ◽  
Nano Scale ◽  
Negative Rate

A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (<1 to 106 viral copies/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (<1 viral copy/µL) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

scholarly journals Triplex Hybridization-Based Nanosystem for the Rapid Screening of Pneumocystis Pneumonia in Clinical Samples

2020 ◽  
Vol 6 (4) ◽  
pp. 292
Author(s):  
Luis Pla ◽  
Anna Aviñó ◽  
Ramón Eritja ◽  
Alba Ruiz-Gaitán ◽  
Javier Pemán ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Pneumocystis Pneumonia ◽  
Rapid Screening ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Current Standard ◽  
Promising Alternative ◽  
Nanoporous Anodic Alumina ◽  
Dna Oligonucleotides ◽  
Risk Of Mortality

Pneumocystis pneumonia (PcP) is a disease produced by the opportunistic infection of the fungus Pneumocystis jirovecii. As delayed or unsuitable treatments increase the risk of mortality, the development of rapid and accurate diagnostic tools for PcP are of great importance. Unfortunately, current standard methods present severe limitations and are far from adequate. In this work, a time-competitive, sensitive and selective biosensor based on DNA-gated nanomaterials for the identification of P. jirovecii is presented. The biosensor consists of a nanoporous anodic alumina (NAA) scaffold which pores are filled with a dye reporter and capped with specific DNA oligonucleotides. In the presence of P. jirovecii genomic DNA, the gated biosensor is open, and the cargo is delivered to the solution where it is monitored through fluorescence spectroscopy. The use of capping oligonucleotides able to form duplex or triplex with P. jirovecii DNA is studied. The final diagnostic tool shows a limit of detection (LOD) of 1 nM of target complementary DNA and does not require previous amplification steps. The method was applied to identify DNA from P. jirovecii in unmodified bronchoalveolar lavage, nasopharyngeal aspirates, and sputum samples in 60 min. This is a promising alternative method for the routinely diagnosis of Pneumocystis pneumonia.

scholarly journals Verification and Validation of SARS-CoV-2 Assay Performance on the Abbott m2000 and Alinity m Systems

2021 ◽  
Author(s):  
Julie W. Hirschhorn ◽  
April Kegl ◽  
Tanisha Dickerson ◽  
W. Bailey Glen ◽  
Gang Xu ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Clinical Performance ◽  
Cycle Number ◽  
Percent Agreement ◽  
Assay Performance ◽  
Diagnostic Panel ◽  
Highly Correlated ◽  
Clinical Verification ◽  
Bridging Study ◽  
Bridging Studies

We verified the analytical performance of the Abbott RealTime SARS-CoV-2 assay on the m2000 system and compared its clinical performance to the CDC 2019-nCoV Real-Time PCR Diagnostic Panel and the ThermoFisher TaqPath RT-PCR COVID-19 kit. We also performed a bridging study comparing the RealTime SARS-CoV-2 assay with the new Abbott Alinity m SARS-CoV-2 assay. A number of standards, reference materials, and commercially available controls were used for the analytical verification to confirm the limit of detection, linearity, and reproducibility. We used nasopharyngeal (NP) swab specimens collected in saline for the clinical verification and bridging studies. Overall, we found 91.2% positive percent agreement (PPA) (95% CI 76.2 to 98.14%) and a 100% negative percent agreement (NPA) (95% CI 97.97 to 100%) between the results of the RealTime SARS-CoV-2 and CDC tests with 217 NP specimens (P=0.13). We found a PPA of 100% (95% CI 90.26 to 100%) and a NPA of 95.15% (95% CI 83.47 to 99.4%) between the results of the RealTime and TaqPath tests with 77 NP specimens (P=0.24). Finally, we tested 203 NP swab specimens for SARS-CoV-2 on the m2000 on the Alinity m systems. The PPA and NPA were 92.2% (95% CI, 85.3 to 96.59%) and 92% (95% PI, 84.8 to 96.5%), respectively (P=0.4). Although cycle number (Cn) values obtained for the concordant positive samples were highly correlated (R2, 0.95), the Cn values were on average 14.14 higher on the Alinity m system due to the unread cycles with the RealTime SARS-CoV-2 assay.

scholarly journals 369. Alternative Workflow for SARS-CoV-2 Testing Using a Heat Lysis Protocol for Respiratory Specimens

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S287-S287
Author(s):  
Meghna Yadav ◽  
Tiffany Martinez ◽  
Isabel Regoli ◽  
Osvaldo Hernandez ◽  
Phuong Le ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Influenza A ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Extraction System ◽  
Nasopharyngeal Swab ◽  
Biomedical Sciences ◽  
Direct Extraction ◽  
Computational Molecular Biology ◽  
Extraction Protocol

Abstract Background The SARS-CoV-2 pandemic has demonstrated the need for streamlined workflows in high-throughput testing. In extraction-based testing, limited extraction reagents and required proprietary instrumentation may pose a bottleneck for labs. As a solution, ChromaCode developed a Direct Extraction protocol for the HDPCR™ SARS-CoV-2 Assay, distributed in accordance with the guidance on Policy for Coronavirus Disease-2019 Tests During the Public Health Emergency, Section IV.C., which allows for the processing of specimens without an extraction system. In lieu of an extraction system, the Direct Extraction protocol uses a thermal cycler to lyse and inactivate specimens which are directly added to the Polymerase Chain Reaction (PCR). Methods The Limit of Detection (LoD), Clinical Performance, and effect of Interfering Substances was determined for the Direct Extraction protocol. The LoD was established on 6 PCR platforms with dilutions of inactivated SARS-CoV-2 virus spiked into residual, negative nasopharyngeal swab (NPS) matrix. Clinical performance was assessed with 48 positive and 50 negative frozen retrospective samples using the Direct Extraction protocol compared to an external Emergency Use Authorized (EUA) comparator assays (cobas® Liat® SARS-CoV-2 & Influenza A/B assay and the Hologic Panther Fusion® SARS-CoV-2 Assay respectively) on three PCR platforms. The Direct Extraction protocol was evaluated for performance in the presence of 13 potentially interfering substances that can be present in a respiratory specimen. Results The LoD of the Direct Extraction protocol ranges from 1000 – 3000 genomic equivalents (GE)/mL. The clinical performance of the assay was 95.8% positive agreement (95% CI of 84.6% - 99.3%) and 100% negative agreement (95% CI of 90.9% - 100% or 91.1% – 100%) across all three PCR platforms tested. The viral target was detected at 3X LoD for all interferents tested. Conclusion The Direct Extraction protocol of ChromaCode’s SARS-CoV-2 Assay is a sensitive test that eliminates the need for sample extraction and performs very well against traditional extraction-based workflows. The inclusion of this protocol can reduce costs, reliance on extraction systems, and time associated with extraction-based protocols. Disclosures Meghna Yadav, Ph.D. Molecular Biology, ChromaCode Inc. (Employee, Shareholder) Tiffany Martinez, n/a, ChromaCode (Employee, Shareholder) Isabel Regoli, MS, Bioinformatics, ChromaCode (Employee, Shareholder) Osvaldo Hernandez, B.S., Molecular Biology, ChromaCode (Employee, Shareholder) Phuong Le, B.S., Biochemistry, ChromaCode (Employee, Shareholder) Heather Carolan, Masters, Computational Molecular Biology, ChromaCode (Employee, Shareholder) Brad Brown, Ph.D Biomedical Sciences, ChromaCode (Employee, Shareholder) Karen Menge, Ph.D. Biochemistry, ChromaCode (Employee, Shareholder)ChromaCode (Employee, Shareholder)

scholarly journals Correlation of SARS-CoV-2 nucleocapsid antigen and RNA concentrations in nasopharyngeal samples from children and adults using an ultrasensitive and quantitative antigen assay

2021 ◽  
Author(s):  
Nira R. Pollock ◽  
Timothy J. Savage ◽  
Hanna Wardell ◽  
Rose A. Lee ◽  
Anu Mathew ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Rapid Diagnostic Tests ◽  
Clinical Samples ◽  
New Approach ◽  
Antigen Assay ◽  
Adults And Children ◽  
Electrochemiluminescence Immunoassay ◽  
Insight Into

Background: Diagnosis of COVID-19 by PCR offers high sensitivity, but the utility of detecting samples with high cycle threshold (Ct) values remains controversial. Currently available rapid diagnostic tests (RDTs) for SARS-CoV-2 nucleocapsid antigens (Ag) have sensitivity well below PCR. The correlation of Ag and RNA quantities in clinical nasopharyngeal (NP) samples is unknown. Methods: An ultrasensitive, quantitative electrochemiluminescence immunoassay for SARS-CoV-2 nucleocapsid (the MSD® S-PLEX® CoV-2 N assay) was used to measure Ag in clinical NP samples from adults and children previously tested by PCR. Results: The S-PLEX Ag assay had a limit of detection (LOD) of 0.16 pg/mL and a cutoff of 0.32 pg/mL. Ag concentrations measured in clinical NP samples (collected in 3.0 mL media) ranged from less than 160 fg/mL to 2.7 ug/mL. Log-transformed Ag concentrations correlated tightly with Ct values. In 35 adult and 101 pediatric PCR-positive samples, sensitivity was 91% (95% CI, 77-98%) and 79% (70-87%), respectively. In samples with Ct ≤ 35, sensitivity was 100% (88-100%) and 96% (88-99%), respectively. In 50 adult and 40 pediatric PCR-negative specimens, specificity was 100% (93-100%) and 98% (87-100%), respectively. Conclusions: Nucleocapsid concentrations in clinical NP samples span 8 orders of magnitude and correlate closely with RNA concentrations (Ct values). The S-PLEX Ag assay had 96-100% sensitivity in samples from children and adults with Ct values ≤ 35, and 98-100% specificity. These results clarify Ag concentration distributions in clinical samples, providing insight into the performance of Ag RDTs and offering a new approach to diagnosis of COVID-19.

scholarly journals Clinical Validation of Automated and Rapid mariPOC SARS-CoV-2 Antigen Test

2021 ◽  
Author(s):  
Juha M. Koskinen ◽  
Petri Antikainen ◽  
Kristina Hotakainen ◽  
Anu Haveri ◽  
Niina Ikonen ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
High Capacity ◽  
Nasopharyngeal Swab ◽  
Analytical Sensitivity ◽  
Clinical Sensitivity ◽  
Qrt Pcr ◽  
Antigen Tests ◽  
Novel Coronavirus

ABSTRACTNovel SARS coronavirus causing COVID-19 was recognized in late 2019. Diagnostics was quickly ramped up worldwide based on the detection of viral RNA. Based on the scientific knowledge for pre-existing coronaviruses, it was expected that the RNA of this novel coronavirus will be detected at significant rates from symptomatic and asymptomatic individuals due to existence of non-infectious RNA. To increase the efficacy of diagnostics, surveillance, screening and pandemic control, rapid methods, such as antigen tests, are needed for decentralized testing and to assess infectiousness. The objectives were to verify analytical sensitivity and specificity, and assess the clinical sensitivity, specificity and usability of a novel automated mariPOC SARS-CoV-2 test based on sophisticated optical laser technology detecting viral structure proteins. Analytical performance was verified using bacterial and viral preparations. Clinical performance of the test was evaluated against qRT-PCR in a retrospective study with nasopharyngeal swab specimens (N=211) collected from symptomatic patients suspected of acute SARS-CoV-2 infections. Sensitivity and specificity of the mariPOC test were 92.3% (12/13) and 100.0% (198/198), respectively. The test’s limit of detection was 22 PFU/test and it had no cross-reactions with the tested respiratory microbes. Our study shows that the mariPOC can detect infectious individuals already in 20 minutes while clinical sensitivity close to qRT-PCR is achieved in two hours or less. The test targets conserved epitopes of SARS-CoV-2 nucleoprotein, making it robust against strain variations. The new test is a promising and versatile tool for syndromic testing of symptomatic cases and for high capacity infection control screening.

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