scholarly journals A SISCAPA-based approach for detection of SARS-CoV-2 viral antigens from clinical samples

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Kiran K. Mangalaparthi ◽  
Sandip Chavan ◽  
Anil K. Madugundu ◽  
Santosh Renuse ◽  
Patrick M. Vanderboom ◽  
...  

AbstractSARS-CoV-2, a novel human coronavirus, has created a global disease burden infecting > 100 million humans in just over a year. RT-PCR is currently the predominant method of diagnosing this viral infection although a variety of tests to detect viral antigens have also been developed. In this study, we adopted a SISCAPA-based enrichment approach using anti-peptide antibodies generated against peptides from the nucleocapsid protein of SARS-CoV-2. We developed a targeted workflow in which nasopharyngeal swab samples were digested followed by enrichment of viral peptides using the anti-peptide antibodies and targeted parallel reaction monitoring (PRM) analysis using a high-resolution mass spectrometer. This workflow was applied to 41 RT-PCR-confirmed clinical SARS-CoV-2 positive nasopharyngeal swab samples and 30 negative samples. The workflow employed was highly specific as none of the target peptides were detected in negative samples. Further, the detected peptides showed a positive correlation with the viral loads as measured by RT-PCR Ct values. The SISCAPA-based platform described in the current study can serve as an alternative method for SARS-CoV-2 viral detection and can also be applied for detecting other microbial pathogens directly from clinical samples.

2020 ◽  
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  

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.


Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 658
Author(s):  
Melanie Fiedler ◽  
Caroline Holtkamp ◽  
Ulf Dittmer ◽  
Olympia E. Anastasiou

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.


Processes ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 1425
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  

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.


2020 ◽  
Vol 78 (8) ◽  
Author(s):  
Onya Opota ◽  
René Brouillet ◽  
Gilbert Greub ◽  
Katia Jaton

ABSTRACT Objectives:In order to cope with the rapid spread of the COVID-19 pandemic, we introduced on our in-house high-throughput molecular diagnostic platform (MDx Platform) a real-time reverse transcriptase PCR (RT-PCR) to detect the SARS-CoV-2 from any clinical specimens. The aim of this study was to compare the RT-PCR results obtain with the MDx Platform and the commercial assay cobas SARS-CoV-2 (Roche) on nasopharyngeal swab and other clinical specimens including sputum, bronchial aspirate, bronchoalveolar lavage and anal swabs. Methods: Samples received in our laboratory from patients suspected of COVID-19 (n = 262) were tested in parallel with our MDx platform SARS-CoV-2 PCR and with the cobas SARS-CoV-2 test. Results: The overall agreement between the two tests for all samples tested was 99.24% (260/262), which corresponded to agreements of 100% (178/178) on nasopharyngeal swabs, 95.45% (42/44) on lower respiratory tract specimen with discordant resultS obtained for very high cycle threshold (Ct) value and 100% (40/40) on anorectal swabs. The Ct values for nasopharyngeal swabs displayed an excellent correlation (R2 &gt; 96%) between both tests. Conclusions: The high agreements between the cobas SARS-CoV-2 test and the MDx platform supports the use of both methods for the diagnostic of COVID-19 on various clinical samples. Very few discrepant results may occur at very low viral load.


2022 ◽  
Vol 9 ◽  
Author(s):  
Manaf Alqahtani ◽  
Abdulkarim Abdulrahman ◽  
Fathi Mustafa ◽  
Abdulla I. Alawadhi ◽  
Batool Alalawi ◽  
...  

IntroductionThe best way to mitigate an outbreak besides mass vaccination is via early detection and isolation of infected cases. As such, a rapid, cost-effective test for the early detection of COVID-19 is required.MethodsThe study included 4,183 mildly symptomatic patients. A nasal and nasopharyngeal sample obtained from each patient was analyzed to determine the diagnostic ability of the rapid antigen detection test (RADT, nasal swab) in comparison with the current gold-standard (RT-PCR, nasopharyngeal swab).ResultsThe calculated sensitivity and specificity of the RADT was 82.1 and 99.1%, respectively. Kappa's coefficient of agreement between the RADT and RT-PCR was 0.859 (p &lt; 0.001). Stratified analysis showed that the sensitivity of the RADT improved significantly when lowering the cut-off RT-PCR Ct value to 24.ConclusionOur study's results support the potential use of nasal swab RADT as a screening tool in mildly symptomatic patients, especially in patients with higher viral loads.


2021 ◽  
Vol 2 (10) ◽  
pp. 929-938
Author(s):  
Khin Phyu Pyar ◽  
Khine Khine Su ◽  
Kyaw Wunna ◽  
Myo Thant ◽  
Kaung Myat ◽  
...  

Background: In COVID-19 pandemic, the diagnosis and treatment must be as early as possible to save the life of each patient. Moreover, screening of asymptomatic carriers, close contacts or healthy subjects must not be delay to prevent transmission to publics. For confirmation of diagnosis of SARS-CoV-2 infection, nasopharyngeal swab must be tested either by real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) tests or Rapid Antigen Test (RAT). RAT is faster, easier and cheaper; thus, it is suitable for health service in developing country. Objectives: The aim of this study was to assess the diagnostic accuracy of Roche SARS-CoV-2 Rapid Antigen Test (RAT) in diagnosing SARS-CoV-2 infection. Methods: Hospital based exploratory study was done in out-patient department and fever clinic, and molecular laboratory of No. (1) Defence Services General Hospital. Nasopharyngeal swabs were taken, and the Roche SARS- CoV-2 RAT was conducted in parallel with RT-PCR test (reference standard). Results: Among the 932 patients/subjects recruited, RT-PCR was positive in 468 individuals, corresponding to a prevalence of 50.2%. The RAT was positive in 363 patients (60.4%), false positive in 120 patients; it was negative in 569 individuals (39.6%), false negative in 225 patients. The overall sensitivity of the RAT was 51.9% (95% Confidence Interval [CI] 47.29-56.53) and, the specificity was 74.1% (95% CI 69.9-78.07); positive predictive value was 66.9% and negative predictive value was 60.5%. The sensitivity varied with Ct value; 78% in clinical samples with Ct values < 20, 57.5% in those with Ct values between 21 and 25, 41.8% in samples with Ct values between 26 and 30, and, 36.4% in samples with Ct value > 30. Conclusion: The accuracy of the SARS-CoV-2 Roche RAT in diagnosing SARS-CoV-2 infections was inferior to RT-PCR and manufacturer’s data. The sensitivity was with low Cycle threshold values < 20 which were inversely related to the viral load. RAT test should be used in association with clinical impression of physicians. In hospital setting especially in emergency department, the role of RAT should be reconsidered in those patients presenting with anosmia and some cases of dyspnoea, late symptoms in the course of disease, as the RAT results would be false negative. Other errors may arise if the operator for RAT has to handle more than recommended tests per hour especially in the peak of epidemics.


2020 ◽  
Author(s):  
John J. Schellenberg ◽  
Margaret Ormond ◽  
Yoav Keynan

AbstractThe current scale of public and private testing cannot be expected to meet the emerging need for higher levels of community-level and repeated screening of asymptomatic Canadians for SARS-CoV-2. Rapid point-of-care techniques are increasingly being deployed to fill the gap in screening levels required to identify undiagnosed individuals with high viral loads. However, rapid, point-of-care tests often have lower sensitivity in practice. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 has proven sensitive and specific and provides visual results in minutes. Using a commercially available kit for RT-LAMP and primer set targetting nucleocapsid (N) gene, we tested a blinded set of 101 archived nasopharyngeal (NP) swab samples with known RT-PCR results. RT-LAMP reactions were incubated at 65°C for 30 minutes, using heat-inactivated nasopharyngeal swab sample in viral transport medium, diluted tenfold in water, as input. RT-LAMP agreed with all RT-PCR defined negatives (N=51), and all positives with Ct less than 20 (N=24), 65% of positives with Ct between 20-30 (N=17), and no positives with Ct greater than 30 (N=9). RT-LAMP requires fewer and different core components, so may not compete directly with the mainline testing workflow, preserving precious central laboratory resources and gold standard tests for those with the greatest need. Careful messaging must be provided when using less-sensitive tests, so that people are not falsely reassured by negative results – “glass half empty” – in exchange for reliable detection of those with high levels of virus within an hour, using <$10 worth of chemicals – “glass half full”.


2021 ◽  
Vol 10 (7) ◽  
pp. 1471
Author(s):  
Alessandra Amendola ◽  
Giuseppe Sberna ◽  
Eleonora Lalle ◽  
Francesca Colavita ◽  
Concetta Castilletti ◽  
...  

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.


PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10801
Author(s):  
Lorena Porte ◽  
Paulette Legarraga ◽  
Mirentxu Iruretagoyena ◽  
Valeska Vollrath ◽  
Gabriel Pizarro ◽  
...  

Background Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) is currently the only recommended diagnostic method for SARS-CoV-2. However, rapid immunoassays for SARS-CoV-2 antigen could significantly reduce the COVID-19 burden currently weighing on laboratories around the world. Methods We evaluated the performance of two rapid fluorescence immunoassays (FIAs), SOFIA SARS Antigen FIA (Quidel Corporation, San Diego, CA, USA) and STANDARD F COVID-19 Ag FIA (SD Biosensor Inc., Gyeonggi-do, Republic of Korea), which use an automated reader. The study used 64 RT-PCR characterized clinical samples (32 positive; 32 negative), which consisted of nasopharyngeal swabs in universal transport medium. Results Of the 32 positive specimens, all from patients within 5 days of symptom onset, the Quidel and SD Biosensor assays detected 30 (93.8%) and 29 (90.6%) samples, respectively. Among the 27 samples with high viral loads (Ct ≤ 25), the two tests had a sensitivity of 100%. Specificity was 96.9% for both kits. Conclusion The high performance of the evaluated FIAs indicates a potential use as rapid and PCR-independent tools for COVID-19 diagnosis in early stages of infection. The excellent sensitivity to detect cases with viral loads above ~106 copies/mL (Ct values ≤ 25), the estimated threshold of contagiousness, suggests that the assays might serve to rapidly identify infective individuals.


2009 ◽  
Vol 55 (12) ◽  
pp. 1375-1380 ◽  
Author(s):  
Safaa Lamhoujeb ◽  
Hugues Charest ◽  
Ismail Fliss ◽  
Solange Ngazoa ◽  
Julie Jean

To improve the sensitivity and efficiency of the real-time nucleic acid sequence based amplification (NASBA) assay targeting the open reading frame 1–2 (ORF1–ORF2) junction of the norovirus (NoV) genome, a selection of clinical samples were analyzed. The assay results were compared with those of TaqMan and conventional reverse transcription PCR (RT-PCR) and a commercial enzyme-linked immunoassay (ELISA) for the specific detection of GII NoV in 96 fecal samples. Based on end-point dilution, the two real-time assays had similar sensitivities (0.01 particle detectable units), two log10cycles greater than that of conventional RT-PCR. GII NoV was detected in 88.54% of the samples by real-time NASBA, in 86.46% by TaqMan RT-PCR, in 81.25% by conventional RT-PCR, and in 65.7% by ELISA. The two real-time assays were in agreement for 88.5% of the samples. These results demonstrate that real-time NASBA with a molecular beacon probe is highly sensitive, accurate, and specific for NoV detection in clinical samples. Applying this technique to samples with complex matrix and low viral loads, such as food and environmental samples, could be useful for the detection of NoVs and will improve the prevention of NoV outbreaks.


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