Evaluation of the artus® Prep&Amp UM RT-PCR for detection of SARS-CoV-2 from nasopharyngeal swabs without prior nucleic acid eluate extraction.

Here we describe a retrospective clinical evaluation of the QIAGEN artus® SARS-CoV-2 Prep&Amp UM RT-PCR assay that detects SARS-CoV-2 RNA without the need for a nucleic acid eluate extraction procedure. Using Roche SARS-CoV-2 RT-PCR on the cobas® 8800 platform as a reference standard, a total of 225 confirmed SARS-CoV-2 positive and 320 negative nasopharyngeal swabs in viral transport media, were used to evaluate the artus® assay. Using the RT-PCR cycle threshold as a semi-quantitative marker of viral load, an assessment of over 370,000 SARS-CoV-2 RT-PCR positive results was used in the design of the reference positive specimen cohort. The viral load of all reference positive specimens used in the evaluation was a unique and accurate representation of the range and levels of SARS-CoV-2 positivity observed over a 13-month period of the COVID-19 pandemic. The artus® RT-PCR detects the presence of SARS-CoV-2 RNA, an internal control, and the human RNase P gene to ensure specimen quality. The diagnostic sensitivity of artus® was 92.89% with a specificity of 100%. To assess the analytical sensitivity, a limit of detection was performed using the 1st WHO NIBSC SARS-CoV-2 international standard, recording a 95% LOD of 1.1 × 103 IU/ml. The total invalid rate of specimens was 7.34% due to a lack of detectable RNase P (Ct >35). The artus® SARS-CoV-2 Prep&Amp UM RT-PCR assay is a new rapid RT-PCR assay, which may be considered to produce acceptable levels of diagnostic sensitivity and specificity whilst potentially halving the laboratory processing time.

Evaluation of Four Strategies for SARS-CoV-2 Detection: Characteristics and Prospects.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed an enormous burden on the global public health system and has had disastrous socioeconomic consequences. Currently, single sampling tests, 20-in-1 pooling tests, nucleic acid point-of-care tests (POCTs), and rapid antigen tests are implemented in different scenarios to detect SARS-CoV-2, but a comprehensive evaluation of them is scarce and remains to be explored. In this study, 3 SARS-CoV-2 inactivated cell culture supernatants were used to evaluate the analytical performance of these strategies. Additionally, 5 recombinant SARS-CoV-2 nucleocapsid (N) proteins were also used for rapid antigen tests. For the wild-type (WT), Delta, and Omicron strains, the lowest inactivated virus concentrations to achieve 100% detection rates of single sampling tests ranged between 1.28 × 102 to 1.02 × 103, 1.28 × 102 to 4.10 × 103, and 1.28 × 102 to 2.05 × 103 copies/mL. The 20-in-1 pooling tests ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL. The nucleic acid POCTs were all 1.42 × 103 copies/mL. The rapid antigen tests ranged between 2.84 × 105 to 7.14 × 106, 8.68 × 104 to 7.14 × 106, and 1.12 × 105 to 3.57 × 106 copies/mL. For the WT, Delta AY.2, Delta AY.1/AY.3, Omicron BA.1, and Omicron BA.2 recombinant N proteins, the lowest concentrations to achieve 100% detection rates of rapid antigen tests ranged between 3.47 to 142.86, 1.74 to 142.86, 3.47 to 142.86, 3.47 to 142.86, and 5.68-142.86 ng/mL, respectively. This study provided helpful insights into the scientific deployment of tests and recommended the full-scale consideration of the testing purpose, resource availability, cost performance, result rapidity, and accuracy to facilitate a profound pathway toward the long-term surveillance of coronavirus disease 2019 (COVID-19). IMPORTANCE In the study, we reported an evaluation of 4 detection strategies implemented in different scenarios for SARS-CoV-2 detection: single sampling tests, 20-in-1 pooling tests, nucleic acid point-of-care tests, and rapid antigen tests. 3 SARS-CoV-2-inactivated SARS-CoV-2 cell culture supernatants and 5 recombinant SARS-CoV-2 nucleocapsid proteins were used for evaluation. In this analysis, we found that for the WT, Delta, and Omicron supernatants, the lowest concentrations to achieve 100% detection rates of single sampling tests ranged between 1.28 × 102 to 1.02 × 103, 1.28 × 102 to 4.10 × 103, and 1.28 × 102 to 2.05 × 103 copies/mL. The 20-in-1 pooling tests ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL. The nucleic acid POCTs were all 1.42 × 103 copies/mL. The rapid antigen tests ranged between 2.84 × 105 to 7.14 × 106, 8.68 × 104 to 7.14 × 106, and 1.12 × 105 to 3.57 × 106 copies/mL. For the WT, Delta AY.2, Delta AY.1/AY.3, Omicron BA.1, and Omicron BA.2 recombinant N proteins, the lowest concentrations to achieve 100% detection rates of rapid antigen tests ranged between 3.47 to 142.86, 1.74 to 142.86, 3.47 to 142.86, 3.47 to 142.86, and 5.68 to 142.86 ng/mL, respectively.

Analytical sensitivity of COVID-19 rapid antigen tests: A case for a robust reference standard.

Aggressive diagnostic testing remains an indispensable strategy for health and aged care facilities to prevent the transmission of SARS-CoV-2 in vulnerable populations. The preferred diagnostic platform has shifted towards COVID-19 rapid antigen tests (RATs) to identify the most infectious individuals. As such, RATs are being manufactured faster than at any other time in our history yet lack the relevant quantitative analytics required to inform on absolute analytical sensitivity enabling manufacturers to maintain high batch-to-batch reproducibility, and end-users to accurately compare brands for decision making. Here, we describe a novel reference standard to measure and compare the analytical sensitivity of RATs using a recombinant GFP-tagged nucleocapsid protein (NP-GFP). Importantly, we show that the GFP tag does not interfere with NP detection and provides several advantages affording streamlined protein expression and purification in high yields as well as faster, cheaper and more sensitive quality control measures for post-production assessment of protein solubility and stability. Ten commercial COVID-19 RATs were evaluated and ranked using NP-GFP as a reference standard. Analytical sensitivity data of the selected devices as determined with NP-GFP did not correlate with those reported by the manufacturers using the median tissue culture infectious dose (TCID50) assay. Of note, TCID50 discordance has been previously reported. Taken together, our results highlight an urgent need for a reliable reference standard for evaluation and benchmarking of the analytical sensitivity of RAT devices. NP-GFP is a promising candidate as a reference standard that will ensure that RAT performance is accurately communicated to healthcare providers and the public.

Analytic and Clinical Performance of Major Commercial Severe Acute Respiratory Syndrome Coronavirus 2 Molecular Assays in the United States.

The rapid development of commercially available molecular assays in response to the COVID-19 pandemic has been essential in identifying positive cases and guiding state and national response plans. With over 200 SARS-CoV-2 molecular tests having received emergency use authorization by the US Food and Drug Administration, numerous studies have been conducted to evaluate these methods and compare their analytical and clinical performance. By applying the lessons learned from the rapid development of molecular assays in response to the COVID-19 pandemic, the diagnostic industry will be better prepared to respond to future outbreaks of novel infectious diseases.

Comparison of analytical sensitivity of SARS-CoV-2 molecular detection kits.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has had a significant impact on global public health systems, making nucleic acid detection an important tool in epidemic prevention and control. Detection kits based on real-time reverse transcriptase PCR (rRT-PCR) have been used widely in clinics, but their analytical sensitivity (limit of detection, LOD) remains controversial. Moreover, there is limited research evaluating the analytical sensitivity of other molecular detection kits. METHODS: In this study, armored ribonucleic acid reference materials developed in-house were used to evaluate the analytical sensitivity of SARS-CoV-2 detection kits approved by the National Medical Products Administration. These were based on rRT-PCR and other molecular detection assays. RESULTS: The percentage retesting required with rRT-PCR kits was as follows: 0%, 7.69%, 15.38%, and 23.08% for samples with concentrations ranging from 50 000 to 781 copies/ml. In total, 93% of rRT-PCR kits had a LOD <1000 copies/ml. Only one kit had an LOD >1000 copies/ml. The LOD of other molecular detection kits ranged from 68 to 2264 copies/ml. CONCLUSIONS: The study findings can help pharmaceutical companies optimize and improve detection kits, guide laboratories in selecting kits, and assist medical workers in their daily work.

Morning SARS-CoV-2 Testing Yields Better Detection of Infection Due to Higher Viral Loads in Saliva and Nasal Swabs upon Waking.

Optimizing specimen collection methods to achieve the most reliable SARS-CoV-2 detection for a given diagnostic sensitivity would improve testing and minimize COVID-19 outbreaks. From September 2020 to April 2021, we performed a household-transmission study in which participants self-collected specimens every morning and evening throughout acute SARS-CoV-2 infection. Seventy mildly symptomatic participants collected saliva, and of those, 29 also collected nasal swab specimens. Viral load was quantified in 1,194 saliva and 661 nasal swab specimens using a high-analytical-sensitivity reverse transcription-quantitative PCR (RT-qPCR) assay. Viral loads in both saliva and nasal swab specimens were significantly higher in morning-collected specimens than in evening-collected specimens after symptom onset. This aspect of the biology of SARS-CoV-2 infection has implications for diagnostic testing. We infer that morning collection would have resulted in significantly improved detection and that this advantage would be most pronounced for tests with low to moderate analytical sensitivity. Collecting specimens for COVID-19 testing in the morning offers a simple and low-cost improvement to clinical diagnostic sensitivity of low- to moderate-analytical-sensitivity tests. IMPORTANCE Our findings suggest that collecting saliva and nasal swab specimens in the morning immediately after waking yields higher SARS-CoV-2 viral loads than collection later in the day. The higher viral loads from morning specimen collection are predicted to significantly improve detection of SARS-CoV-2 in symptomatic individuals, particularly when using moderate- to low-analytical-sensitivity COVID-19 diagnostic tests, such as rapid antigen tests.

A nationwide analytical and clinical evaluation of 44 rapid antigen tests for SARS-CoV-2 compared to RT-qPCR.

BACKGROUND: The SARS-CoV-2 pandemic has resulted in massive testing by Rapid Antigen Tests (RAT) without solid independent data regarding clinical performance being available. Thus, decision on purchase of a specific RAT may rely on manufacturer-provided data and limited peer-reviewed data. METHODS: This study consists of two parts. In the retrospective analytical part, 33 RAT from 25 manufacturers were compared to RT-PCR on 100 negative and 204 positive deep oropharyngeal cavity samples divided into four groups based on RT-PCR Cq levels. In the prospective clinical part, nearly 200 individuals positive for SARS-CoV-2 and nearly 200 individuals negative for SARS-CoV-2 by routine RT-PCR testing were retested within 72 h for each of 44 included RAT from 26 manufacturers applying RT-PCR as the reference method. RESULTS: The overall analytical sensitivity differed significantly between the 33 included RAT; from 2.5% (95% CI 0.5-4.8) to 42% (95% CI 35-49). All RAT presented analytical specificities of 100%. Likewise, the overall clinical sensitivity varied significantly between the 44 included RAT; from 2.5% (95% CI 0.5-4.8) to 94% (95% CI 91-97). All RAT presented clinical specificities between 98 and 100%. CONCLUSION: The study presents analytical as well as clinical performance data for 44 commercially available RAT compared to the same RT-PCR test. The study enables identification of individual RAT that has significantly higher sensitivity than other included RAT and may aid decision makers in selecting between the included RAT. FUNDING: The study was funded by a participant fee for each test and the Danish Regions.

Comparison between the analytical sensitivity and clinical performance of two cobas SARS-CoV-2 tests based on high-throughput and point-of-care systems.

Objectives: This study examined analytical sensitivity, specificity, and the clinical performance in detecting SARS-CoV-2 of the Cobas SARS-CoV-2 Test based on the high-throughput Cobas 6800 system and the Cobas SARS-CoV-2 & Flu A/B Test based on the point-of-care cobas Liat system. Methods: The commercial reagents containing SARS-CoV-2 RNA subgenomes were diluted for assessing the sensitivity of the RT-qPCR assay. 385 nasopharyngeal swab specimens taken from contacts of COVID-19 cases were tested for the SARS-CoV-2 detection with both Cobas SARS-CoV-2 Tests. Results: In analytical sensitivity assays, the Cobas SARS-CoV-2 & Flu A/B Test on the Liat system had a lower limit of detection (12.5-25 copies/mL) than the cobas SARS-CoV-2 Test on the cobas 6800 system (25-50 copies/mL). In clinical performance assays, the cobas SARS-CoV-2 Test demonstrated 89.36% (42 out of 47) PPA (positive percent agreement) and 98.82% (334 out of 338) NPA (negative percent agreement) compared to the results of the Cobas SARS-CoV-2 & Flu A/B test. Among five discordant specimens, four had the positive result of the cobas SARS-CoV-2 test, but the negative result of the cobas SARS-CoV-2 & Flu A/B Test. Moreover, these discordant specimens had the Ct values of greater than 33 for the cobas SARS-CoV-2 Test, implying a very small number of virions in the samples. Remarkably, four specimens with a presumptive positive result of the cobas SARS-CoV-2 test had been confirmed by the Cobas SARS-CoV-2 & Flu A/B Test. Next, the scatter plots of the Ct values showed a highly positive correlation between cobas SARS-CoV-2 & Flu A/B Test and the cobas SARS-CoV-2 Test (R-squared value = 0.954-0.962). Conclusions: Both SARS-CoV2 tests of the cobas 6800 and Liat systems produce reliable high throughput and point-of-care assays respectively for the early virus detection and the personal care decision-making during COVID-19 pandemic.

Impact of SARS-CoV-2 Variants on the Analytical Sensitivity of rRT-PCR Assays.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility, pathogenicity, and immune escape ability have ravaged many countries and regions, which has brought substantial challenges to pandemic prevention and control. Real-time reverse transcriptase PCR (rRT-PCR) is widely used for SARS-CoV-2 detection but may be limited by the continuous evolution of the virus. However, the sensitivity of Chinese commercial rRT-PCR kits to critical SARS-CoV-2 variants remains unknown. In this study, contrived MS2 virus-like particles were used as reference materials to evaluate the analytical sensitivity of Daan, BioGerm, EasyDiagnosis, Liferiver, and Sansure kits when detecting six important variants (Alpha, Beta, Gamma, Delta, Omicron, and Fin-796H). The Beta and Delta variants adversely affected the analytical sensitivity of the BioGerm ORF1ab gene assay (9.52% versus 42.96%, P = 0.014, and 14.29% versus 42.96%, P = 0.040, respectively), whereas the N gene assay completely failed in terms of the Fin-796H variant. The Gamma and Fin-796H variants impeded the PCR amplification efficiency for the Sansure ORF1ab gene assay (33.33% versus 66.67%, P = 0.031, and 66.67% versus 95.24%, P = 0.040, respectively), and the Delta variant compromised the E gene assay (52.38% versus 85.71%, P = 0.019). The Alpha and Omicron variants had no significant effect on the kits. This study highlights the necessity of identifying the potential effect of viral mutations on the efficacy and sensitivity of clinical detection assays. It can also provide helpful insights regarding the development and optimization of diagnostic assays and aid the strategic management of the ongoing pandemic.

Analytical sensitivity comparison of 14 conventional and three rapid RT-PCR assays for SARS-CoV-2 detection.

Recent reports have compared the analytical sensitivities of some SARS-CoV-2 RT-PCR assays, but differences in the viral materials used for these evaluations made comprehensive conclusions difficult. We carried out a direct comparison of the analytical sensitivities of 14 conventional and three rapid RT-PCR assays for the detection of SARS-CoV-2. The comparison was performed utilizing a certified reference material for SARS-CoV-2 RNA that was serially two-fold diluted in RNA storage solution. Our results show that the analytical sensitivities of the 17 assays varied within an 8-fold range (100-800 copies/mL). Moreover, a trend with some rapid assays yielding slightly higher analytical sensitivities (2- to 4-fold) compared with conventional assays was observed. We conclude that most of the RT-PCR assays can be used for routine COVID-19 diagnosis, but some assays with the poorest analytical sensitivities may lead to false-negative results when used to identify asymptomatic individuals who can carry a low viral load but still be infectious. These findings should be kept in mind when selecting high-sensitivity and rapid assays.

Laboratory evaluation of SARS-CoV-2 in the COVID-19 pandemic.

Laboratory evaluation of SARS-CoV-2 involves the detection of viral nucleic acid, viral protein antigens, and the antibody response. Molecular detection of SARS-CoV-2 is the only diagnostic test currently available in acutely or recently infected individuals. In contrast, serological testing is typically performed once viral RNA has been cleared and symptoms have resolved. This leads to some confusion among clinicians as to which test to order and when each is appropriate. While SARS-CoV-2 assays can suffer from poor sensitivity, all FDA authorized assays to date are intended to be qualitative. Serological tests have multiple assay formats, detect various classes of immunoglobulins, and have a distinct role in seroprevalence studies; however, the association with long-term protection remains unclear. Both molecular and serological testing for SARS-CoV-2 have complementary roles in patient management, and we highlight the challenges faced by clinicians and laboratorians alike in the evaluation and interpretation of the currently available laboratory assays.