Comparison of Allplex (TM) 2019-nCoV and TaqPath (TM) COVID-19 Assays

The clinical presentation of COVID-19 is non-specific, and to improve and limit the spread of the SARS-CoV-2 virus, an accurate diagnosis with a robust method is needed. A total of 500 nasopharyngeal swab specimens were tested for SARS-CoV-2. Of these, 184 samples were found to be positive with Allplex (TM) 2019-nCoV Assay, which is fully automated. All the positive samples were retested with TaqPath (TM) COVID-19 CE-IVD RT-PCR Kit (after this, referred to as TaqPath (TM) COVID-19), semi-automated. The comparison of RT-qPCR for SARS-CoV-2 genes target points shows only one target point in common, the N gene. Therefore, the N gene was used to compare both assays. We noticed different Ct values between the tests. Therefore, samples were divided into four groups depending to the Ct value results: (1) Ct < 25, (2) Ct 25-30, (3) Ct 30-35, (4) Ct > 35. TaqPath (TM) COVID-19 Kit reconfirmed the results obtained from Allplex (TM) 2019-nCoV Assay. In conclusion, both the Allplex (TM) 2019-nCoV assay and TaqPath (TM) COVID-19 tests accurately confirm the diagnosis of SARS-CoV-2 infection. Even if TaqPath (TM) COVID-19 has a semi-automated workflow, it does not introduce bias in the diagnostic screening of SARS-CoV-2, and it supports the indirect identification of variants of concern to undergo sequencing.

Evaluation of two RT-PCR screening assays for identifying SARS-CoV-2 variants.

BACKGROUND: The recent emergence of new SARS CoV-2 variants (variants of concern, VOC) that spread rapidly and may lead to immune escape has emphasized the urgent need to monitor and control their spread. METHODS: We analyzed 2018 SARS-CoV-2 positive specimens collected between February 9 and March 22, 2021 using the Thermofisher® TaqPath™ COVID-19 CE-IVD RT-PCR kit (TaqPath) and the ID solutions® ID™ SARS-CoV-2/UK/SA Variant Triplex RT-PCR (ID triplex) assay to screen for VOCs. RESULTS: The ID triplex assay identified 62.8% of them as VOCs: 61.8% B.1.1.7 and 0.9% B.1.351/P.1. The agreement between the ID triplex results for B.1.1.7 and the TaqPath S gene target failure (SGTF)/ S gene target late detection (SGTL) profile for this variant agreed very well (k = 0.86). A low virus load was the main cause of discrepancies. Sequencing discordant results with both assays indicated that the TaqPath assay detected the B.1.1.7 lineage slightly better. Both assays suggested that the virus loads of B.1.1.7 variants were significantly higher than those of non-B.1.1.7 strains. Only 10/20 B1.351/P.1 strains detected with the ID triplex assay were confirmed by sequencing. CONCLUSIONS: We conclude that the SGTF/SGTL profiles identified using the TaqPath assay and ID triplex results are suitable for detecting the B.1.1.7 lineage. The ID triplex assay, which rapidly determines all three current VOCs simultaneously, could be a valuable tool for limiting virus spread by supporting contact-tracing and isolation.

Comparative evaluation of the Thermo fisher TaqPath™ COVID-19 combo kit with the Cepheid Xpert® Xpress SARS-CoV-2 assay for detecting SARS-CoV-2 in nasopharyngeal specimens.

PURPOSE: With over 50 SARS-CoV-2 gene amplification assays that have been EUA cleared with minimal experimental validation performed, it is likely that not all of these assays are comparable in their ability to detect SARS-CoV-2 in clinical specimens. Thermo Fisher Scientific is a relatively new company in the molecular diagnostics field and the purpose of this study was to compare the performance of the Thermo Fisher TaqPath™ Combo Kit with an established test, the Cepheid Xpert® Xpress SARS-CoV-2 assay, for its ability to detect SARS-CoV-2 in nasopharyngeal specimens. METHODS: A total of 300 randomly selected nasopharyngeal specimens were evaluated and tested by the TaqPath and GeneXpert assays. Discordant test specimens were arbitrated by performing an alternative PCR assay and Sanger sequencing. RESULTS: The TaqPath assay had a 96.7 and 99.6% positive and negative agreement respectively when compared to the Xpert Xpress test. However, after test arbitration, the three discordant specimens were arbitrated in favor of the TaqPath assay producing a positive and negative percent agreement of 100% for the TaqPath Combo Kit while the Xpress SARS-CoV-2 assay had a positive and negative percent agreement of 98.3 and 99.2% respectively. CONCLUSIONS: The TaqPath Combo Kit is a high complexity assay that compares favorably with the Xpert Xpress test and can be reliably used for the detection of SARS-CoV-2 in nasopharyngeal specimens.

S-Variant SARS-CoV-2 Lineage B1.1.7 Is Associated With Significantly Higher Viral Load in Samples Tested by TaqPath Polymerase Chain Reaction.

A SARS-CoV-2 variant B1.1.7 containing mutation Δ69/70 has spread rapidly in the United Kingdom and shows an identifiable profile in ThermoFisher TaqPath RT-qPCR, S gene target failure (SGTF). We analyzed recent test data for trends and significance. Linked cycle threshold (Ct) values for respiratory samples showed that a low Ct for ORF1ab and N were clearly associated with SGTF. Significantly more SGTF samples had higher inferred viral loads between 1×107 and 1×108. Our conclusion is that patients whose samples exhibit the SGTF profile are more likely to have high viral loads, which may explain higher infectivity and rapidity of spread.

Specificity of SARS-CoV-2 Real-Time PCR Improved by Deep Learning Analysis.

Real-time PCR (RT-PCR) is widely used to diagnose human pathogens. RT-PCR data are traditionally analyzed by estimating the threshold cycle (CT ) at which the fluorescence signal produced by emission of a probe crosses a baseline level. Current models used to estimate the CT value are based on approximations that do not adequately account for the stochastic variations of the fluorescence signal that is detected during RT-PCR. Less common deviations become more apparent as the sample size increases, as is the case in the current SARS-CoV-2 pandemic. In this work, we employ a method independent of CT value to interpret RT-PCR data. In this novel approach, we built and trained a deep learning model, qPCRdeepNet, to analyze the fluorescent readings obtained during RT-PCR. We describe how this model can be deployed as a quality assurance tool to monitor result interpretation in real time. The model's performance with the TaqPath COVID19 Combo Kit assay, widely used for SARS-CoV-2 detection, is described. This model can be applied broadly for the primary interpretation of RT-PCR assays and potentially replace the CT interpretive paradigm.

Clinical and Epidemiologic Evaluation of Inconclusive COVID-19 PCR Results Using a Quantitative Algorithm.

OBJECTIVES: The inconclusive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR) result causes confusion and delay for infection prevention precautions and patient management. We aimed to develop a quantitative algorithm to assess and interpret these inconclusive results. METHODS: We created a score-based algorithm by combining laboratory, clinical, and epidemiologic data to evaluate 69 cases with inconclusive coronavirus disease 2019 (COVID-19) PCR results from the Centers for Disease Control and Prevention (CDC) assay (18 cases) and the TaqPath assay (51 cases). RESULTS: We determined 5 (28%) of 18 (CDC assay) and 20 (39%) of 51 (TaqPath assay) cases to be false positive. Lowering the cycle threshold cutoff from 40 to 37 in the TaqPath assay resulted in a dramatic reduction of the false-positive rate to 14%. We also showed testing of asymptomatic individuals is associated with a significantly higher probability of having a false-positive result. CONCLUSIONS: A substantial percentage of inconclusive SARS-CoV-2 PCR results can be false positive, especially among asymptomatic patients. The quantitative algorithm we created was shown to be effective and could provide a useful tool for clinicians and hospital epidemiologists to interpret inconclusive COVID-19 PCR results and provide clinical guidance when additional PCR or antibody test results are available.