Development and Clinical Evaluation of an Immunochromatography-Based Rapid Antigen Test (GenBody™ COVAG025) for COVID-19 Diagnosis

Antigen tests for SARS-CoV-2 diagnosis are simpler and faster than their molecular counterparts. Clinical validation of such tests is a prerequisite before their field applications. We developed and clinically evaluated an immunochromatographic immunoassay, GenBody™ COVAG025, for the rapid detection of SARS-CoV-2 nucleocapsid (NP) antigen in two different clinical studies. Retrospectively, 130 residual nasopharyngeal swabs transferred in viral transport medium (VTM), pre-examined for COVID-19 through emergency use authorization (EUA)-approved real-time RT-PCR assay and tested with GenBody™ COVAG025, revealed a sensitivity and specificity of 90.00% (27/30; 95% CI: 73.47% to 97.89%) and 98.00% (98/100; 95% CI: 92.96% to 99.76%), respectively, fulfilling WHO guidelines. Subsequently, the prospective examination of 200 symptomatic and asymptomatic nasopharyngeal swabs, collected on site and tested with GenBody™ COVAG025 and EUA-approved real-time RT-PCR assay simultaneously, revealed a significantly higher sensitivity and specificity of 94.00% (94/100; 95% CI: 87.40% to 97.77%) and 100.00% (100/100; 95% CI: 96.38% to 100.00%), respectively. Clinical sensitivity and specificity were significantly high for samples with Ct values ≤ 30 as well as within 3 days of symptom onset, justifying its dependency on the viral load. Thus, it is assumed this can help with the accurate diagnosis and timely isolation and treatment of patients with COVID-19, contributing to better control of the global pandemic.

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Evaluation of the performance of the COVID-19 rapid antigen tests in a tertiary level hospital in Bangladesh.

10.21203/rs.3.rs-1056525/v1 ◽

2021 ◽

Author(s):

Mohammad Jahidur Rahman Khan ◽

Md. Shahadat Hossain ◽

Samshad Jahan Shumu ◽

Md. Selim Reza ◽

Farzana Mim ◽

...

Keyword(s):

Viral Load ◽

Real Time ◽

Sensitivity And Specificity ◽

Detection Rate ◽

High Sensitivity ◽

Rt Pcr ◽

College Hospital ◽

Medical College ◽

Qrt Pcr ◽

Antigen Tests

Abstract Background: While the COVID-19 pandemic is a worldwide crisis, tests with high sensitivity and specificity are essential for identifying and managing COVID-19 patients. Globally, several rapid antigen tests RATs for COVID-19 have been developed, but their clinical efficacy has not been well established. This study aimed to evaluate the performance of several rapid antigen tests (RATs) to diagnose SARS-CoV-2 infection.Methods: This prospective observational study was conducted at Shaheed Suhrawardy Medical College hospital from February 2021 to April 2021 in Dhaka, Bangladesh. This study included the patients admitted in this hospital at the COVID-19 isolation unit or referred from the triage facility of the outdoor department of this hospital suspected as COVID-19 case. Two nasopharyngeal samples were collected simultaneously. one sample was used on the spot for the RAT. The other was sent to the adjacent Shaheed Suhrawardy Medical College COVID-19 RT-PCR laboratory for real-time reverse transcription-polymerase chain reaction (qRT-PCR). The performance of the RAT was evaluated using the results of qRT-PCR as a reference.Results: A total of 223 patients were included in this study, and the real-time RT-PCR detected SARS-CoV-2 in 84 (37.7%) patients. Of these 84 patients, 9 (10.7%) were asymptomatic. The overall sensitivity and specificity of RATs were 78.6% and 99.3%, respectively. The sensitivity was 81.3% in symptomatic cases and 55.6% in asymptomatic cases. False-negatives were observed in 18 patients, 3 of whom were asymptomatic and had a low viral load (cycle threshold (Ct) > 30). The detection rate of RATs was 100% when the Ct value was up to 24. The detection rate was 42.3% when the Ct was >29. The detection rate of RATs was 92.3% when the onset of symptoms was within three days. The detection rate was 33.3% when the onset of symptoms was >7 days.Conclusions: RATs for COVID-19 used in this study delivered an acceptable performance in patients with high viral load and within the first week of the onset of symptoms. They can be used as a supplementary method to RT-PCR for the diagnosis of COVID-19 patients.

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Rapid SARS-CoV-2 antigen detection assay in comparison with real-time RT-PCR assay for laboratory diagnosis of COVID-19 in Thailand

Virology Journal ◽

10.1186/s12985-020-01452-5 ◽

2020 ◽

Vol 17 (1) ◽

Cited By ~ 3

Author(s):

Chutikarn Chaimayo ◽

Bualan Kaewnaphan ◽

Nattaya Tanlieng ◽

Niracha Athipanyasilp ◽

Rujipas Sirijatuphat ◽

...

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

False Negative ◽

Laboratory Diagnosis ◽

Antigen Detection ◽

Rt Pcr ◽

Pcr Assay ◽

Screening Assay ◽

The Real ◽

Pcr Test

Abstract Background The Coronavirus disease 2019 (COVID-19) pandemic continues to spread across the world. Hence, there is an urgent need for rapid, simple, and accurate tests to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Performance characteristics of the rapid SARS-CoV-2 antigen detection test should be evaluated and compared with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) test for diagnosis of COVID-19 cases. Methods The rapid SARS-CoV-2 antigen detection test, Standard™ Q COVID-19 Ag kit (SD Biosensor®, Republic of Korea), was compared with the real-time RT-PCR test, Allplex™ 2019-nCoV Assay (Seegene®, Korea) for detection of SARS-CoV-2 in respiratory specimens. Four hundred fifty-four respiratory samples (mainly nasopharyngeal and throat swabs) were obtained from COVID-19 suspected cases and contact individuals, including pre-operative patients at Siriraj Hospital, Bangkok, Thailand during March–May 2020. Results Of 454 respiratory samples, 60 (13.2%) were positive, and 394 (86.8%) were negative for SARS-CoV-2 RNA by real-time RT-PCR assay. The duration from onset to laboratory test in COVID-19 suspected cases and contact individuals ranged from 0 to 14 days with a median of 3 days. The rapid SARS-CoV-2 antigen detection test’s sensitivity and specificity were 98.33% (95% CI, 91.06–99.96%) and 98.73% (95% CI, 97.06–99.59%), respectively. One false negative test result was from a sample with a high real-time RT-PCR cycle threshold (Ct), while five false positive test results were from specimens of pre-operative patients. Conclusions The rapid assay for SARS-CoV-2 antigen detection showed comparable sensitivity and specificity with the real-time RT-PCR assay. Thus, there is a potential use of this rapid and simple SARS-CoV-2 antigen detection test as a screening assay.

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Multi-site Evaluation of SARS-CoV-2 Spike Mutation Detection Using a Multiplex Real-time RT-PCR Assay

10.1101/2021.05.05.21254713 ◽

2021 ◽

Author(s):

Carolin Bier ◽

Anke Edelmann ◽

Kathrin Theil ◽

Rolf Schwarzer ◽

Maria Deichner ◽

...

Keyword(s):

Real Time ◽

Limit Of Detection ◽

False Negative ◽

Analytical Sensitivity ◽

Test Results ◽

Rt Pcr ◽

Pcr Assay ◽

Clinical Specimens ◽

Negative Results ◽

Global Pandemic

Background. SARS-CoV-2 causes COVID-19, which can be fatal and is responsible for a global pandemic. Variants with increased transmissibility or the potential to evade immunity have emerged and represent a threat to global pandemic control. Variants of concern (VOC) can be identified by sequencing of viral RNA, or by more rapid methods for detection of subsets of signature mutations. Methods. We developed a multiplex, real-time RT-PCR assay (cobas SARS-CoV-2 Variant Set 1) for the qualitative detection and differentiation of three key SARS-CoV-2 mutations in the viral spike protein: del 69-70, E484K and N501Y. Analytical sensitivity and accuracy were evaluated at three testing sites using clinical specimens from patients infected with SARS-CoV-2 variants belonging to several different lineages, including B.1.1.7, B.1.351, and P.1. Results. The limit of detection for E484K was between 180 and 620 IU/mL for the three different isolates tested. For N501Y, the LOD was between 270 and 720 IU/mL (five isolates), while for del 69-70, it was 80 - 92 IU/mL (two isolates). Valid test results were obtained with all clinical specimens that were positive using routine diagnostic tests. Compared to sequencing (Sanger and next-generation), test results were 100% concordant at all three loci; no false positive or false negative results were observed. Conclusions. Data collected at three independent laboratories indicates excellent performance and concordance of cobas SARS-CoV-2 Variant Set 1 with sequencing. New sets of primers and probes that target additional loci can be rapidly deployed in response to the identification of other emerging variants.

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Low level SARS-CoV-2 RNA detected in plasma samples from a cohort of Nigerians: Implications for blood transfusion

PLoS ONE ◽

10.1371/journal.pone.0252611 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252611

Author(s):

Azuka Patrick Okwuraiwe ◽

Chika Kingsley Onwuamah ◽

Joseph Ojonugwa Shaibu ◽

Samuel Olufemi Amoo ◽

Fehintola Anthonia Ige ◽

...

Keyword(s):

Blood Transfusion ◽

Real Time ◽

Sensitivity And Specificity ◽

Viral Rna ◽

Global Pandemic ◽

Viral Transport Medium ◽

Nasal Swabs ◽

Polymerase Chain ◽

Study Participants ◽

Manual Extraction

The present global pandemic triggered by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has lingered for over a year in its devastating effects. Diagnosis of coronavirus disease 2019 (COVID-19) is currently established with a polymerase chain reaction (PCR) test by means of oropharyngeal-, nasopharyngeal-, anal-swabs, sputum and blood plasma. However, oral and nasal swabs are more commonly used. This study, therefore, assessed sensitivity and specificity of plasma as a diagnostic in comparison with a combination of oral and nasal swab samples, and the implications for blood transfusion. Oropharyngeal (OP) and nasopharyngeal (NP) swab samples were obtained from 125 individuals suspected to have COVID-19 and stored in viral transport medium (VTM) tubes. Ten millilitres of blood samples in EDTA were also obtained by venepuncture and spun to obtain plasma. Viral RNA was obtained from both swabs and plasma by manual extraction with Qiagen QIAamp viral RNA Mini Kit. Detection was done using a real time fluorescent RT-qPCR BGI kit, on a QuantStudio 3 real-time PCR instrument. Average age of study participants was 41 years, with 74 (59.2%) being male. Out of the 125 individuals tested for COVID-19, 75 (60%) were positive by OP/NP swab. However, only 6 (4.8%) had a positive plasma result for COVID-19 with median Ct value of 32.4. Sensitivity and specificity of RT-PCR SARS-CoV-2 test using plasma was 8% and 100% respectively. There was no false positive recorded, but 69 (55.2%) false negatives were obtained by plasma. SARS-CoV-2 viral RNA was detected, albeit low (4.8%) in plasma. Plasma is likely not a suitable biological sample to diagnose acute SARS-CoV-2 infection. The implication of transfusing blood in this era of COVID-19 needs further investigations.

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Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status

Journal of Clinical Microbiology ◽

10.1128/jcm.00855-17 ◽

2017 ◽

Vol 55 (11) ◽

pp. 3201-3209 ◽

Cited By ~ 11

Author(s):

Sumudu R. Perera ◽

Nurul H. Khan ◽

Irene Martin ◽

Ali Taheri ◽

Rajinder P. Parti ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Genomic Dna ◽

Rt Pcr ◽

Pcr Assay ◽

Content Type ◽

Simultaneous Identification ◽

Susceptibility Status

ABSTRACTA real-time PCR (RT-PCR) assay was designed for the simultaneous identification ofNeisseria gonorrhoeaeand its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) ingyrAofN. gonorrhoeae. The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254N. gonorrhoeaeisolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non-N. gonorrhoeaespecies isolates. The performance of the primers was validated using genomic DNA from 100 differentN. gonorrhoeaeisolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non-N. gonorrhoeaeisolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of theN. gonorrhoeaeisolates and 23 non-N. gonorrhoeaeisolates. These primers detectedN. gonorrhoeaeand its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification ofN. gonorrhoeaeand its ciprofloxacin susceptibility status.

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Clinical sensitivity and specificity of a real-time PCR assay forCampylobacter fetussubspvenerealisin preputial samples from bulls

American Journal of Veterinary Research ◽

10.2460/ajvr.75.9.851 ◽

2014 ◽

Vol 75 (9) ◽

pp. 851-860 ◽

Cited By ~ 5

Author(s):

Alvaro García Guerra ◽

Bonnie Chaban ◽

Janet E. Hill ◽

Cheryl L. Waldner ◽

Steven H. Hendrick

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Pcr Assay ◽

Clinical Sensitivity

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Extraction Free Rapid Detection of SARS-CoV-2 from Oropharyngeal/Nasopharyngeal Swabs by Real Time PCR

JOURNAL FOR CLINICAL AND DIAGNOSTIC RESEARCH ◽

10.7860/jcdr/2021/50193.15401 ◽

2021 ◽

Author(s):

Parul Sinha ◽

Sandeep Gupta ◽

Megha Gupta ◽

Dinesh Kumar Jain ◽

Malvika Sharma ◽

...

Keyword(s):

Real Time ◽

Extraction Method ◽

Rna Extraction ◽

Molecular Testing ◽

Acid Extraction ◽

Rt Pcr ◽

Transport Medium ◽

Viral Transport ◽

Viral Transport Medium ◽

Medium Sample

Introduction: The emergence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic has been troublesome particularly for developing countries that lack infrastructure and capacities to produce the kits locally. Simplification of the method can increase diagnostic efficiency which can benefit patients and help in infection control, consequently saving time and lives. Aim: To evaluate the diagnostic value of four methods (that omit extraction step) for detection of SARS-CoV-2 against the traditional extraction method. Materials and Methods: This was a cross-sectional analysis for evaluating diagnostic accuracy of four methods for detection of SARS-CoV-2 by real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR), conducted in the Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India, in October 2020. Ninety four SARS-CoV-2 RT-PCR positive samples and 20 negative samples were taken for this study. Automated extraction system was used for Ribonucleic Acid (RNA) extraction and four different approaches were compared to the traditional extraction method for detection of SARS-CoV-2 by RT-PCR. Data was entered and analysed using Statistical Package for the Social Sciences (SPSS) statistical software version 24.0. Results: The automated RNA extraction method was compared to the method of direct addition of samples with (Heat processed Direct Viral transport medium Sample (HDVS)) and without heating (Direct Viral transport medium Sample (DVS)), directs addition of diluted (1:5) sample with (Heat processed diluted VTM sample (HdVS)) and without heating (Diluted VTM sample (dVS)) as well as after addition of Proteinse K (PK) to the diluted samples that came either negative/invalid. Out of four methods, the HdVS method gave the best results, considering extraction with Perkin Elmer as standard, this method showed sensitivity of 96.74%, specificity of 100%. Conclusion: In current pandemic, molecular testing is critically challenged by the limited supplies of reagents of nucleic acid extraction alternative method like diluting and heating of Viral Transport Media (VTM) samples and using them directly as elutes serve as an easy, fast and inexpensive alternative.

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Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

Future Microbiology ◽

10.2217/fmb-2020-0094 ◽

2020 ◽

Vol 15 (15) ◽

pp. 1483-1487

Author(s):

Nikhil S Sahajpal ◽

Ashis K Mondal ◽

Allan Njau ◽

Sudha Ananth ◽

Kimya Jones ◽

...

Keyword(s):

Supply Chain ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Sample Collection ◽

Rt Pcr ◽

Pcr Assay ◽

Short Supply ◽

Viral Transport ◽

3D Printed ◽

Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

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Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638721994810 ◽

2021 ◽

pp. 104063872199481

Author(s):

Yixin Xiao ◽

Fan Yang ◽

Fumin Liu ◽

Hangping Yao ◽

Nanping Wu ◽

...

Keyword(s):

Avian Influenza ◽

Real Time ◽

Influenza A ◽

Minor Groove Binder ◽

Rt Pcr ◽

Influenza A Viruses ◽

Pcr Assay ◽

Infectious Dose ◽

The Matrix ◽

Taqman Minor Groove Binder

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID50)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.

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