Since 2019, worldwide healthcare systems are being challenged by the COVID-19 pandemic, which is caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2). Because of the high infectiousness of the virus, it is important to test for it on a large scale, including with potential asymptomatic carriers [1
]. Optimizing test strategies during the SARS-CoV-2 pandemic is an important and challenging goal. Hence, we aimed to evaluate the LIAISON®
SARS-CoV-2 antigen assay (Ag assay) (DiaSorin), comparing its performance to real-time polymerase chain reaction (RT-PCR) for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs.
The LIAISON® SARS-CoV-2 Ag assay is a chemiluminescence sandwich-immunoassay (CLIA)-based technology for the determination of nucleocapsid antigen from SARS-CoV-2 samples in upper respiratory specimens. We used two different RT-PCRs: The RealStar® SARS-CoV-2 RT-PCR kit (Altona), which targets the SARS-CoV-2 genes S (spike) and E (envelope), and the Alinity m SARS-CoV-2 Assay (Abbott), which targets the SARS-CoV-2 genes RdRp (RNA dependent RNA polymerase) and N (nucleocapsid).
RT-PCR is actually considered the gold standard for the detection of SARS-CoV-2 [2
]. Nevertheless, there are some disadvantages with using RT-PCR. It is time-consuming, expensive, prone to contamination, and trained laboratory staff as well as specialized equipment are needed [1
]. In addition, during the pandemic, reagents have often been scarce or even unavailable [3
], and the demands on specialized laboratories (providing RT-PCR diagnostics) consequently increased. Hence, we evaluated whether using an antigen assay could be an adequate back up system for the RT-PCR. A benefit of using the antigen assay is the availability of the LIAISON®
in many laboratories, which could potentially expand our diagnostic capabilities. Previous studies have already compared different antigen rapid diagnostic tests with RT-PCR: Corman et al. [3
] showed that antigen rapid diagnostic tests were able to detect SARS-CoV-2 viral load of between 106
copies per swab.
The objective of our study was the comprehensive evaluation of the LIAISON® SARS-CoV-2 antigen assay for the detection of SARS-CoV-2 in respiratory tract samples, focusing on a head-to-head comparison with the gold-standard method, RT-PCR.
In this study, we compared the performance of the LIAISON®
SARS-CoV-2 antigen assay vs. the gold standard for SARS-CoV-2 detection, RT-PCR [2
]. Our cohort consisted of individuals tested by the Public Health Department of Essen. In case of outbreaks, this also included asymptomatic individuals. Unfortunately, we cannot provide precise information since none is available to us. The absence of such information, however, is not unusual for samples sent to a laboratory for SARS-CoV-2 testing, but it is still a limitation of the study.
A third of the samples with detectable SARS-CoV-2 RNA by RT-PCR were falsely classified as negative, with the antigen test taking the manufacturer’s recommended cut-off of 200 TCID50
/mL. The antigen test is, indeed, inferior to the RT-PCR in terms of sensitivity, which is not surprising. RT-PCR is considered the gold standard for SARS-CoV-2 detection. However, even the gold standard is not without disadvantages: it is expensive, time consuming, requires trained laboratory staff as well as specialized equipment [1
], and may not always be available (for example, due to lack of reagents) [3
However, no false positives were found, amounting to 100% specificity and 70% sensitivity. Antigen tests, especially antigen rapid diagnostic tests, are known to be less sensitive than RT-PCR [5
] and may be comparable or inferior in terms of sensitivity to other rapid detection methods, such as loop-mediated isothermal amplification assay [6
]. The WHO recommends the use of antigen tests with sensitivity and specificity greater than 80% and 97%, respectively [8
]. Using a ROC analysis, we identified different cut-offs, optimized for maximal sensitivity and/or a combination of sensitivity and specificity, and compared three of them. As expected, lowering the cut-off for the antigen assay increased sensitivity. In fact, the two lowest cut-offs increased sensitivity to over 90%. This is significantly higher than the sensitivity of various antigen rapid diagnostic tests, which range from 73% to 82%, as reported in a meta-analysis by Brümmer, et al. [5
], and may be as low as 62.5% in the case of asymptomatic subjects. However, the higher sensitivity, attained by lowering the cut-off, comes at the cost of decreased specificity. Using the cut-off between 22 and 23 TCID50
/mL was found to lead to false positive results for almost a fifth of the negative samples. The third and optimal cut-off (57.68 TCID50
/mL), calculated according to the maximal Youden Index, showed an acceptable combination of sensitivity (79%) and specificity (99%). This cut-off may be considered by the manufacturer. In addition, this cut-off roughly fulfilled the WHO standard for SARS-CoV-2 antigen tests [8
]. Recently, a Japanese group evaluated the performance of another quantitative, fully automated antigen assay with nasopharyngeal swabs. The Lumipulse®
SARS-CoV-2 Ag test (Fujirebio Inc., Tokyo, Japan) is also a chemiluminescence enzyme immunoassay, which tests the nucleocapsid antigen [9
]. They described a sensitivity of 91.7% and a specificity of 98.5% using an optimized cut-off. The specificity is roughly in line with our results, but the sensitivity is substantially higher. This may reflect the superiority of the Fujirebio assay compared to the LIAISON®
SARS-CoV-2 antigen assay. A head-to-head comparison of the two assays would be necessary to conclusively evaluate the point, since the two studies focused on different cohorts.
Virus concentrations of 106
copies/mL are almost always detected using the cut-off of 57.68 TCID50
/mL for the LIAISON®
SARS-CoV-2 antigen assay. Virus isolation is possible in only 20% of samples with virus concentrations within this range [10
]. In addition, Corman et al. [3
] suggested that a negative antigen test result obtained a week after the first symptoms is associated with loss of infectiousness. The LIAISON®
SARS-CoV-2 antigen assay could probably be useful on this aspect and could be the basis of lifting isolation in long-term hospitalized COVID-19 patients. However, more studies are necessary to correlate TCID50
and viral loads with absence of infectiousness in vitro. Hence, testing in cell culture might be an interesting additional investigation.
According to the manufacturer´s instructions for the LIAISON®
SARS-CoV-2 antigen assay, it is necessary to perform the sample deactivation within 12 h after taking the sample. Practical knowledge showed that this is usually impossible in a clinical daily routine. To overcome this problem, we evaluated antigen stability over time and found that the antigen measurement remains stable over three days. For other antigens, stability over time has been shown before, e.g., the stability of the hepatitis B surface antigen over 12 months stored at −20 °C [11
Apart from nasopharyngeal swab samples, which are most frequently used in the daily routine [12
], SARS-CoV-2 diagnostics includes the evaluation of other materials. Here, the LIAISON®
SARS-CoV-2 antigen assay performed suboptimally. It could not reliably detect SARS-CoV-2 in other materials, such as bronchoalveolar lavage, pharyngeal wash, or sputum, which is a limitation of this test in comparison to RT-PCR. Cross-reactivity toward other respiratory viruses was not observed. This underlines the reliability of the LIAISON®
SARS-CoV-2 antigen assay, since at the beginning of the pandemic, false positive results were generated by RT-PCR due to cross-reactivity [13
In conclusion, the LIAISON® SARS-CoV-2 antigen assay can reliably detect samples with high SARS-CoV-2 loads. Hence, the antigen assay might be a useful back-up system if performing RT-PCR is not an option (for example, due to lack of reagents). In addition, it might be a helpful tool for lifting the isolation of hospitalized patients.
4. Materials and Methods
We included 182 nasopharyngeal swabs for the detection of SARS-CoV-2, tested from 16 November to 21 December 2020 at the Institute for Virology, University Hospital Essen, in our study. Our cohort consisted of individuals tested by the Public Health Department of Essen. The sample collection was performed when patients informed the Public Health Department of their COVID-19-specific symptoms. Generally, this was briefly after the onset of symptoms. In case of outbreaks, this also included asymptomatic individuals.
The swab collection kits contained a viral transport medium, which was used for both the PCR and the detection of SARS-CoV-2 antigen, as described below.
Detection of SARS-CoV-2 RNA was performed using the RealStar® SARS-CoV-2 RT-PCR kit (Altona Diagnostics, Hamburg, Germany), which targets the SARS-CoV-2 genes S (spike) and E (envelope) for 155 samples and Alinity m SARS-CoV-2 Assay (Abbott, Wiesbaden, Germany), which targets the SARS-CoV-2 genes RdRp (RNA dependent RNA polymerase) and N (nucleocapsid) for 27 samples. For the Altona assay, the Ct values corresponding to the E-gene were taken into account for further analysis. Furthermore, we tested two SARS-CoV-2 standards provided by INSTAND (Berlin, Germany) of 106 and 107 SARS-CoV-2 copies/mL, which corresponded to Ct values of 23.11 (E-gene, Altona) and 22.42 (Alinity) and 20.18 (E-gene, Altona) and 18.67 (Alinity), respectively. We diluted the 106 standard and measured the Ct values corresponding to 105 and 104 copies/mL, amounting to Ct 26.3 and 31.4 (E-gene, Altona) and 25.1 and 28.74 (Alinity), respectively. Linear regression was performed for both series of values, and the viral load in copies/mL was calculated based on the equation deriving from it.
SARS-CoV-2 antigen detection was performed using the LIAISON®
SARS-CoV-2 Ag assay (Diasorin, Saluggia, Italy), a chemiluminescence sandwich-immunoassay (CLIA)-based technology for the determination of nucleocapsid antigen from SARS-CoV-2 samples in upper respiratory specimens. Samples positive for endemic coronaviruses (HCoV NL63, HCoV OC43, HCoV 229E, HCoV HKU), influenza A and B, RSV, and samples from patients with detectable EBV in blood were tested using the LIAISON SARS-CoV-2 Ag assay to evaluate cross-reactivity. The cut-off for sample positivity given by the manufacturer is 200 TCID50
/mL. The assay was performed according to the manufacturer´s instructions with two notable exceptions. For the inactivation of the samples, we mixed a 400 µL sample with a 400 µL inactivation buffer, instead of using 1 mL plus 1 mL. Many swab sample collection kits do not contain enough viral transport media to allow for the testing of the sample with the antigen assay, RT-PCR, and a potential retesting (for example, in case of inhibition or pipetting errors). The applied volume was within the range of the minimum sample volume given by the manufacturer (100 µL sample + 300 µL dead volume). Furthermore, we used samples taken up to 36 h before inactivation, since this reflects laboratory and clinical routine more accurately. The samples were cooled during transport and storage. The ethics committee of the medical faculty of the University of Duisburg-Essen approved the assessment of test samples for the improvement of diagnostic procedures (20-9512-BO). Statistical analysis was performed using SPSS software (v23, SPSS Inc., Chicago, IL, USA), GraphPad Prism 6.0 (GraphPad, CA, USA) and the platform VassarStats (http://vassarstats.net
(accessed on 23 March 2021). Two-tailed p
values less than 0.05 were considered to be statistically significant.