Analysis of diagnostic performance and factors causing nonspecific reactions in SARS-CoV-2 rapid antigen detection tests.

INTRODUCTION: Early diagnosis and appropriate infection control are important to prevent the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we aimed to assess the diagnostic performance of SARS-CoV-2 rapid antigen detection (RAD) tests and the factors that cause nonspecific reactions. METHODS: Nasopharyngeal swab specimens (n = 100), sputum specimens (n = 10), and lithium-heparin plasma samples (n = 100) were collected. We evaluated Espline®SARS-CoV-2 (Espline) and SARS-CoV-2 Rapid Antigen Test that also known as STANDARD Q® (STANDARD Q), with reverse transcription-polymerase chain reaction (RT-PCR) and Lumipulse® Presto SARS-CoV-2 Ag as reference tests. In addition, we investigated the effects of inadequate pretreatment methods and five potential causes of nonspecific reactions. RESULTS: The sensitivities of Espline and STANDARD Q were 60% and 57%, respectively, and their specificity was 100%. It was confirmed that the judgment line for the positive insufficiently mixed specimens was faint. A false-positive result was observed with STANDARD Q when sputum was used as a specimen to investigate judgment the effect of viscosity. CONCLUSIONS: Espline and STANDARD Q show good sensitivity for specimens with Ct values less than 25, but specimens collected within 9 days of symptom onset may still give false negatives. The test should be performed carefully, and the results should be judged comprehensively, taking into account clinical symptoms and patient background.

Analytical performance of four rapid molecular testing for SARS-CoV-2

Various reagents and equipment for testing SARS-CoV-2 infections have been developed, particularly rapid molecular tests based on polymerase chain reaction (PCR).We evaluated the analytical performance of four rapid molecular tests for SARS-CoV-2. We used 56 nasopharyngeal swabs from patients with confirmed SARS-CoV-2 infection;36 diagnosed as positive by the Ampdirect™ 2019-nCoV Detection Kit (Shimadzu assay) were considered as true-positive samples.The sensitivity of Cobas® Liat SARS-CoV-2 and Flu A/B (Cobas) was the highest among the four molecular test kits. The limit of detection was 1.49 × 10−2 copies/µL (95% confidence interval [CI]: 1.46×10−2−1.51 × 10−2 copies/µL) for Cobas;1.43 × 10−1 copies/µL (95% CI: 8.01×10−3−2.78 × 10−1 copies/µL) for Xpert® Xpress SARS-CoV-2 test (Xpert);2.00 × 10−1 copies/µL (95% CI: 1.95×10−1-2.05 × 10−1 copies/µL) for FilmArray Respiratory Panel v2.1 (FilmArray);and 3.33 × 10 copies/µL (95% CI: 1.93 × 10–4.72×10 copies/µL) for Smart Gene® SARS-CoV-2 (Smart gene). Cobas also had a high sensitivity (100%) compared with Shimadzu assay. The sensitivities of Xpert, FilmArray, and Smart Gene were 97.2%, 97.2%, and 75.0%, respectively. The specificity of all tests was 100%.In conclusion, the four rapid SARS-CoV-2 molecular test kits have high specificity and sensitivity for detecting SARS-CoV-2. As they are easy to use, they could be a useful method for detecting SARS-CoV-2.

Viral load may impact the diagnostic performance of nasal swabs in nucleic acid amplification test and quantitative antigen test for SARS-CoV-2 detection.

INTRODUCTION: Compared to nasopharyngeal swabs (NPS), there has been insufficient evaluation of the diagnostic performance of nasal swabs (NS) for the detection of severe acute respiratory coronavirus 2 (SARS-CoV-2) in the nucleic acid amplification test (NAAT) and quantitative SARS-CoV-2 antigen test (QAT). METHODS: We prospectively compared healthcare worker-collected and flocked NS within nine days after symptom onset to paired NPS to detect SARS-CoV-2 in NAAT and QAT on the fully automated Lumipulse system. The agreement between sample types was evaluated, and cycle threshold (Ct) values and antigen levels were used as surrogate viral load measures. RESULTS: Sixty sets of NPS and NS samples were collected from 40 patients with COVID-19. The overall agreements between NAAT and QAT samples were 76.7% and 65.0%, respectively. In NAAT, the Ct value of NS was significantly higher, 5.9, than that of NPS. Thirty-nine (95.1%) NS tested positive in 41 positive-paired NPS with Ct ≤ 30. The negative correlation was observed between antigen levels of NS in QAT and Ct values of NS in NAAT (r = -0.88). In QAT, the antigen level of NS was significantly lower than that of NPS. Thirty-six (90.0%) NS tested positive in 40 positive-paired NPS with antigen levels >100 pg/mL, which were collected significantly earlier than those with antigen levels ≤100 pg/mL. CONCLUSIONS: In NAAT and QAT, NS had limited performance in detecting SARS-CoV-2 compared to NPS. However, NS may be helpful for patients with COVID-19 with high viral loads or those in the early stages of the illness.

Does the timing of saliva collection affect the diagnosis of SARS-CoV-2 infection?

We evaluated the optimal timing of saliva sample collection to diagnose the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We obtained 150 saliva samples at four specific time points from 13 patients with confirmed SARS-CoV-2 infection. The time points were (1) early morning (immediately after waking), (2) immediately after breakfast before tooth brushing, (3) 2 h after breakfast, and (4) before lunch. On the 2nd hospital day, patients collected saliva at the four time points by themselves. We collected samples at two time points, (1) and (3), from the 3rd hospital day to day 9 following symptom onset. In 52 samples collected at the four time points, there was no significant difference. Meanwhile, there was no significant difference in the positive proportion or the viral load between the two time points in both analyses by the day from symptom onset and by all samples. In this study, there was no difference in the positive proportions in saliva collected at various time points within 9 days after symptom onset. The timing of saliva collection was not affected by the diagnosis of SARS-CoV-2 infection.

Evaluation of false positives in the SARS-CoV-2 quantitative antigen test.

INTRODUCTION: Highly sensitive reagents for detecting SARS-CoV-2 antigens have been developed for accurate and rapid diagnosis till date. In this study, we aim to clarify the frequency of false-positive reactions and reveal their details in SARS-CoV-2 quantitative antigen test using an automated laboratory device. METHODS: Nasopharyngeal swab samples (n = 4992) and saliva samples (n = 5430) were collected. We measured their SARS-CoV-2 antigen using Lumipulse® Presto SARS-CoV-2 Ag and performed a nucleic acid amplification test (NAAT) using the Ampdirect™ 2019 Novel Coronavirus Detection Kit as needed. The results obtained from each detection test were compared accordingly. RESULTS: There were 304 nasopharyngeal samples and 114 saliva samples were positive in the Lumipulse® Presto SARS-CoV-2 Ag test. All positive nasopharyngeal samples in the antigen test were also positive for NAAT. In contrast, only three (2.6%) of all the positive saliva samples in the antigen test were negative for NAAT. One showed no linearity with a dilute solution in the dilution test. Additionally, the quantitative antigen levels of all the three samples did not decrease after reaction with the anti-SARS-CoV-2 antibody. CONCLUSIONS: The judgment difference between the quantitative antigen test and NAAT seemed to be caused by non-specific reactions in the antigen test. Although the high positive and negative predictive value of this quantitative antigen test could be confirmed, we should consider the possibility of false-positives caused by non-specific reactions and understand the characteristics of antigen testing. We recommend that repeating centrifugation before measurement, especially in saliva samples, should be performed appropriately.