key: cord-0910669-jotx7s6v authors: Kurihara, Y.; Kiyasu, Y.; Akashi, Y.; Takeuchi, Y.; Narahara, K.; Mori, S.; Takeshige, T.; Notake, S.; Ueda, A.; Nakamura, K.; Ishikawa, H.; Suzuki, H. title: The evaluation of a novel digital immunochromatographic assay with silver amplification to detect SARS-CoV-2 date: 2021-05-13 journal: nan DOI: 10.1101/2021.05.06.21256738 sha: f7fcfdb90f5979df20ca8078221c000732a5480b doc_id: 910669 cord_uid: jotx7s6v Introduction Rapid antigen tests are convenient for diagnosing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, they have lower sensitivities than nucleic acid amplification tests. In this study, we evaluated the diagnostic performance of Quick Chaser Auto SARS-CoV-2, a novel digital immunochromatographic assay that is expected to have higher sensitivity than conventional antigen tests. Methods A prospective observational study was conducted between February 8 and March 24, 2021. We simultaneously obtained two nasopharyngeal samples, one for evaluation with the QuickChaser Auto SARS-CoV-2 antigen test and the other for assessment with reverse transcription PCR (RT-PCR), considered the gold-standard reference test. The limit of detection (LOD) of the new antigen test was compared with those of four other commercially available rapid antigen tests. Results A total of 1401 samples were analyzed. SARS-CoV-2 was detected by reference RT-PCR in 83 (5.9%) samples, of which 36 (43.4%) were collected from symptomatic patients. The sensitivity, specificity, positive predictive value, and negative predictive value were 74.7% (95% confidence interval (CI): 64.0-83.6%), 99.8% (95% CI: 99.5-100%), 96.9% (95% CI: 89.2-99.6%), and 98.4% (95% CI: 97.6-99.0%), respectively. When limited to samples with a cycle threshold (Ct) <30 or those from symptomatic patients, the sensitivity increased to 98.3% and 88.9%, respectively. The QuickChaser Auto SARS-CoV-2 detected 34-120 copies/test, which indicated greater sensitivity than the other rapid antigen tests. Conclusions QuickChaser Auto SARS-CoV-2 showed sufficient sensitivity and specificity in clinical samples of symptomatic patients. The sensitivity was comparable to RT-PCR in samples with Ct<30. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic and continues to place an immense burden on healthcare systems [1] despite the introduction of effective vaccines [2] . Since rapid and accurate testing is a critical element in containing viral transmission [3] , the development of reliable point-of-care testing is necessary. However, several limitations have reduced their test capacity and clinical utility, including long processing times and the need for expensive equipment and skilled staff [3] . By contrast, antigen tests are convenient and have moderate sensitivities and high specificities [4] . These tests have made it possible to diagnose COVID-19 in low-resource settings [5] , despite the possibility of missing a certain proportion of infected patients [6] . Therefore, increasing sensitivity should enhance the clinical utility of antigen tests. Quick Chaser ® Auto SARS-CoV-2 (Mizuho Medy, Saga, Japan) is a new antigen test based on the silver amplification method. This test uses the same reagent as FUJI DRY-CHEM IMMUNO AG Cartridge COVID-19 Ag (Fujifilm, Tokyo, Japan), and is tailored for digital immuno-chromatographic assays. The test provides results in 15 minutes when used with the QuickChaser Immuno Reader II dedicated reader (Mizuho Medy). Both the silver amplification method and digital immuno-chromatographic assays were reported to increase the sensitivity of antigen tests for the influenza virus . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 13, 2021. ; https://doi.org/10.1101/2021.05.06.21256738 doi: medRxiv preprint [7] . Although Quick Chaser ® Auto SARS-CoV-2 is expected to have higher sensitivity than conventional antigen tests, its diagnostic performance for detecting SARS-CoV-2 has not been evaluated in clinical samples. In this study, we evaluated the diagnostic performance of Quick Chaser ® Auto SARS-CoV-2 and QuickChaser Immuno Reader II with nasopharyngeal specimens, and performed comparisons with the reverse transcription PCR (RT-PCR) method. This study was carried out as an extension of our previous research [8] and followed a similar protocol. The investigation was performed between February 8 and March 24, 2021, at Tsukuba Medical Center Hospital (TMCH), a tertiary hospital in Ibaraki Prefecture, Japan. Nasopharyngeal samples and clinical information were gathered from individuals who had possibly contracted SARS-CoV-2. The enrolled patients were referred from 67 nearby clinics and a local public health center, and by healthcare workers at TMCH. All patients provided informed consent to participate in the study, which was approved by the ethics committee of TMCH (approval number: 2020-071). Two nasopharyngeal samples were obtained from each patient for further testing: one with a sponge swab™ (NIPRO, Osaka, Japan) for antigen testing, and the . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) After sample collection, antigen testing was performed immediately using the QuickChaser ® Auto SARS-CoV-2 and QuickChaser Immuno Reader II. FLOQSwab samples were diluted in 3 mL of Universal Transport Medium™ (UTM™) (Copan Italia) for in-house RT-PCR and reference RT-PCR. For in-house RT-PCR, magLEAD 6gC (Precision System Science, Chiba, Japan) was used to extract and purify RNA from UTM™ samples. The samples were then The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of QuickChaser ® Auto SARS-CoV-2 were calculated using the Clopper and Pearson method, with 95% confidence intervals (CIs). All calculations were conducted using the R 4.0.3 software program (www.r-project.org). . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 13, 2021. ; https://doi.org/10.1101/2021.05.06.21256738 doi: medRxiv preprint During the study period, 1,416 nasopharyngeal samples were initially included. Samples with missing clinical data (n = 14) or measurement errors (n = 1) were excluded. A total of 1,401 samples were eventually analyzed. Reference real-time RT-PCR detected SARS-CoV-2 in 83 (5.9%) of the 1401 samples. The results of reference and in-house RT-PCR were consistent in all but one sample, which was negative by in-house RT-PCR and positive by reference RT-PCR. This sample was re-evaluated by in-house RT-PCR and GeneXpert ® using preserved UTM. Both tests showed positive results, and the sample was finally considered to be positive for SARS-CoV-2. Of the 83 samples, 36 (43.4%) were collected from symptomatic patients, and 47 (56.6%) were obtained from asymptomatic participants. The relationship between the interval from symptom onset and the sensitivity of QuickChaser ® Auto SARS-CoV-2 is shown in Figure 1 . The results of LOD tests using clinical specimens are summarized in Table 1 Sensitivity, specificity, PPV, and NPV of QuickChaser ® Auto SARS-CoV-2 The clinical performance of QuickChaser ® Auto SARS-CoV-2 is summarized in Tables 2 and 3 . Sixty-two of the 83 samples that were positive by reference RT-PCR were also positive by the antigen test. The sensitivity, specificity, PPV, and NPV were 74.7% (95% CI: 64.0-83.6%), 99.8% (95% CI: 99.5-100%), 96.9% (95% CI: 89.2-99.6%), and 98.4% (95% CI: 97.6-99.0%), respectively ( Table 2 ). In samples from symptomatic patients, 32 of 36 reference RT-PCR-positive samples were also positive by antigen testing (Table 3a) . The sensitivity, specificity, PPV, and NPV were 88.9% (95% CI: 73.9-96.9%), 100% (95% CI: 99.3-100%), 100% (95% CI: 84.2-100%), and 99.5% (95% CI: 98.8-99.9%), respectively. In samples from asymptomatic individuals, 30 of 47 reference RT-PCR-positive samples were positive by antigen testing (Table 3b ). The sensitivity, specificity, PPV, and NPV were 63.8% (95% CI: 48.5-77.3%), 99.6% (95% CI: 98.5-100%), 93.8% (95% CI: 79.2-99.2%), and 96.7% (95% CI: 94.7-98.0%), respectively. The sensitivities of the antigen test stratified by Ct value are shown in Table 4 . clinical samples [11] . Therefore, direct comparison using clinical samples should be conducted to evaluate the real-life performance of each test. Viral load influences overall sensitivity, as shown by the fact that the sensitivity of antigen tests generally plummets in samples with Ct >30 [8, 12] . Samples with Ct >30 comprised 30.1% (25/83) of our study population, which may have decreased the overall sensitivity of Quick Chaser ® Auto SARS-CoV-2. Another factor that may influence sensitivity is the swab type used. Quick Chaser ® Auto SARS-CoV-2 includes . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 13, 2021. ; https://doi.org/10.1101/2021.05.06.21256738 doi: medRxiv preprint sponge swabs, although flocked-type swabs can collect samples more efficiently [13] . Despite the aforementioned challenges, this antigen test successfully detected SARS-CoV-2 in all but one of the samples with Ct <30. The remaining case was a false negative, and considering that re-examination with the UTM™ sample showed a positive result, the original finding may have been caused by a low viral concentration due to a flawed sample collection technique. The good performance of this test indicates that it can accurately identify contagious patients, given that those with Ct values <30 are considered to be highly infectious [14] . We observed false positives in only two samples, and the specificity of Quick Chaser ® Auto SARS-CoV-2 was over 99%. False positives should be avoided as they lead to unnecessary further testing or quarantine measures [15] ; thus, the specificity is recommended to be over 97% [5] . Positive results should be cautiously interpreted, especially when the prevalence of SARS-CoV-2 or possibility of infection is low. To maximize its sensitivity, Quick Chaser ® Auto SARS-CoV-2 uses two methods: silver amplification and digital interpretation of the results. Similar to many antigen tests [5], Quick Chaser ® Auto SARS-CoV-2 implements sandwich methods using labeled antibodies and capture antibodies. Antibodies labeled with gold colloid attach to specific antigens in a sample. The labeled antigens are then sandwiched by capture antibodies, which indicate the positive bands. The silver amplification method generates large silver particles using the gold colloid as a catalyst, and thus enhances the visibility of the labeled antibody complex [7] . A previous study showed that among antigen tests for the influenza virus, those that used this method had higher sensitivity . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 13, 2021 There are several limitations regarding this study. First, reference real-time RT-PCR used frozen samples. While samples were stored at −80 °C, their viral load may have decreased during storage process. Second, we did not investigate whether mutations in SARS-COV-2 affected the diagnostic performance. Third, we did not evaluate saliva or anterior nasal cavity samples. Saliva collection and anterior nasal swabs cause less pain and coughing than nasopharyngeal swabs [18] . Future studies should compare the diagnostic performance of samples obtained using each of these methods. In conclusion, Quick Chaser ® Auto SARS-CoV-2 showed satisfactory diagnostic performance of symptomatic patients. The sensitivity was especially high in samples of Ct <30, indicating that the test can accurately detect highly infectious patients. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 13, 2021. ; https://doi.org/10.1101/2021.05.06.21256738 doi: medRxiv preprint Center Hospital for their intensive support of this study. We thank all of the medical institutions for providing their patients' clinical information. Mizuho Medy provided fees for research expenses and provided the Quick . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 13, 2021. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 13, 2021 RT-PCR, reverse transcription polymerase chain reaction Data in parentheses are 95% confidence intervals. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 13, 2021. ; https://doi.org/10.1101/2021.05.06.21256738 doi: medRxiv preprint Ct, cycle threshold . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 1 World Health Organisation. Coronavirus disease (COVID-19) Weekly Epidemiological Update and Weekly Operational Update SARS-CoV-2 Vaccines Evaluation of a rapid antigen test (Panbio TM COVID-19 Ag rapid test device) for SARS-CoV-2 detection in asymptomatic close contacts of COVID-19 patients Real-life validation of the Panbio TM COVID-19 antigen rapid test (Abbott) in community-dwelling subjects with symptoms of potential SARS-CoV-2 infection Evaluation of the panbio COVID-19 rapid antigen detection test device for the screening of patients with COVID-19 Comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients Predicting Infectious Severe Acute Respiratory Syndrome Coronavirus 2 From Diagnostic Samples International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 13, 2021. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted May 13, 2021. ; https://doi.org/10.1101/2021.05.06.21256738 doi: medRxiv preprint