key: cord-0809442-xgajeibp authors: Flinck, Heini; Kerimov, Dominik; Luukinen, Bruno; Seiskari, Tapio; Aittoniemi, Janne title: Evaluation of the Roche-SD Biosensor rapid antigen test: antigen is not reliable in detecting SARS-CoV-2 at the early stage of infection with respiratory symptoms date: 2021-12-25 journal: Diagn Microbiol Infect Dis DOI: 10.1016/j.diagmicrobio.2021.115628 sha: f596e154b4b11a7128a20510210add5070878cea doc_id: 809442 cord_uid: xgajeibp We evaluated a rapid antigen test against SARS-CoV-2 virus (Roche-SD Biosensor; RSDB-RAT) in children and adults with respiratory symptoms compared to those with non-respiratory symptoms or asymptomatic. Also the performance of RSDB-RAT with respect to the duration of respiratory symptoms was assessed. A viral cross-reactivity panel was included. RSDB-RAT was reliable in detecting SARS-CoV-2 in children and adults if the respiratory symptoms had endured 1-7 days. If the respiratory symptoms had lasted less than 1 day, the sensitivity was significantly lower. No cross-reactivity with other respiratory viruses was observed. The laboratory diagnostics of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rests on nucleic acid amplification tests (NAATs) based mainly on reverse-transcriptase polymerase chain reaction (RT-PCR), which is the most sensitive and highly specific golden standard method. However, its limitations in the point-of-care (POC) testing are a relatively long turnaround-time, high test cost per sample, and the need for specialized equipment. Rapid antigen tests (RATs), in turn, are less sensitive, but usually faster and cheaper than NAATs. [1, 2] According to the guidelines, the use of RATs is acceptable in certain conditions, if the NAAT test is not reasonably feasible [3] [4] [5] . The European Centre for Disease Prevention and Control (ECDC) guideline allows the use of RATs even in pre-symptomatic and early symptomatic phase up to five days from the onset of symptoms, the guideline being exceptionally more front-loaded in the testing than the others [3] . According to the World Health Organization (WHO), the testing should be conducted within the first 5-7 days after the onset of symptoms [4] . The Infectious Diseases Society of America (IDSA) guideline concluded that RATs should be used within seven days after symptom onset [5] . They also concluded that the RATs perform equally in adults and children, but in children the data is still very limited [6] [7] [8] . The guidelines also allow the use of RATs in screening of asymptomatic cases in certain conditions like in high prevalence of COVID-19 or in identifying highly infectious cases [3] [4] [5] . We evaluated the performance of Roche-SD Biosensor SARS-CoV-2 (RSDB) RAT both in children and adults with respiratory symptoms compared to other cases with non-respiratory symptoms or asymptomatic, and to the duration of the respiratory symptoms. A cross-reactivity panel containing positive samples regarding other respiratory viruses was included in the material. The test was reliable in detecting SARS-CoV-2, if the respiratory symptoms had endured 1-7 days. However, if the respiratory symptoms had lasted less than 1 day, the sensitivity was significantly lower. No cross-reactivity with other respiratory viruses was observed. The sensitivities and specificities of the RSDB-RAT in different case groups are shown in Table 1 . The overall specificity was 99.3%, and the only false positive result was detected in an adult male. The sensitivity of the test was significantly higher among all COVID-19 cases including children whose respiratory symptoms had endured 1-7 days (94.6%) before sampling compared to those with the duration of less than 1 day [64.3%, confidence interval (CI) 35.1-87.2%; P=0.007], or to those with non-respiratory symptoms or whom were asymptomatic (77.4%; P=0.031). The majority of the false negative RAT results occurred in the cases where the respiratory symptoms had continued less than 1 day before sampling (Figure 1.) groups, no statistically significant difference between children and adults, nor between respiratory patients and the others (asymptomatic or non-respiratory symptoms) was detected. The study population contained 84 children under the age of 15 years (median age 10 years, quartiles 5-13 years; 36 boys). Among the 18 NAAT positive children, 6 had had respiratory symptoms 1-7 days before sampling, of whom the RAT was positive in all (100.0%). Of the other 12 NAAT positive children, 2 had had respiratory symptoms less than 1 day before sampling, of whom the RAT was positive in none (0%), and 10 had had only non-respiratory symptoms or they had been asymptomatic, of whom the RAT was positive in 7 (70.0%). The results of the cross-reactivity testing against respiratory samples containing other viruses than SARS-CoV-2 are shown in Table 2 . No cross-reactivity against other viruses was detected. The RSDB-RAT has been included in the common list of COVID-19 RATs that are considered mutually appropriate for use in context of the situations by the European Commission's (EC) Health Security Committee, the minimum performance requirements of being ≥90% sensitivity and ≥97% specificity compared to NAAT [9] . EC has also purchased RSDB RATs to the European Union member states via Emergency Support Instrument [10] . According to our study, the RSDB-RAT was reliable with >94% sensitivity and >99% specificity in detecting SARS-CoV-2 in both children and adults, if the respiratory symptoms had endured 1-7 days. In recent Cochrane report, RSDB-RAT's specificity was observed high (>98%) in every case group [11] . In the same report, the overall sensitivity in symptomatic patients was 80%, but if restricted to instructions for use (IFU) compliant studies the sensitivity was 88%. Our sample handling deviated from IFU regarding the sampling tube and the time delay of making the test, but these did not seem to have effect on the results. In contrast, the strength of our study was that the RAT and NAAT had been made from the same sample. Less information is available of the RAT performance in children. L'Huillier et al. concluded that the sensitivity of RAT in symptomatic children was 73% and peaked at the day 2 after the onset of symptoms [6] . Eleftheriou and colleagues observed that the overall sensitivity of RAT in children was 82%, and the sensitivity in symptomatic cases was even >95% [7] . In one cohort of symptomatic children, the overall sensitivity of RAT was 88% compared to NAAT [8] . Thus, our findings in children are in line with current literature, with RATs revealed to be reliable also in children with respiratory symptoms endured 1-7 days. The overall sensitivity of the RSDB-RAT was significantly lower if the respiratory symptoms had lasted less than one day (64%), or the patients had had only non-respiratory symptoms or they had been asymptomatic (77%). According to Cochrane report, the overall sensitivity of the RSDB-RAT in asymptomatic patients was 61%, and in IFU compliant studies 69% [11] . Thus, the decreased sensitivity of the RATs in asymptomatic COVID-19 patients is well known. However, the decreased sensitivity of the RSDB-RAT was more striking in the cases with the duration of respiratory symptoms less than 1 day, since all guidelines consider RATs reliable already at the very early stage of the respiratory symptoms or even before the symptom onset [3] [4] [5] . L'Huillier et al. has observed in children that RAT sensitivity peaks at the day 2 after the onset of symptoms [6] . Berger et al. has observed in community-based setting that the sensitivity of RAT is over 95% 1-5 days after the onset of symptoms, but only 88% at the day of onset [12] . Thus we conclude, that if the respiratory symptoms have lasted less than 1 day, RAT is not reliable in detecting SARS-CoV-2, and the RAT should be repeated next day. Most of the false negative RAT samples gave NAAT Ct values above 30. This finding is in concordance with other literature [12] . According to WHO, infectiousness is associated with high viral loads resulting in NAAT Ct values below 25-30. [4] Based on our findings, the identification of potentially infectious cases by RSDB-RAT is possible in those with respiratory symptoms with timely sampling, but RSDB-RAT is not suitable for detecting cases with low viral loads. In our study, only one sample was considered as a false positive in RAT, since the more sensitive NAAT result from the same sample had remained negative. The cause of this false positive result is unknown. Corman and colleagues observed that some other factor than the tested cross-reacting pathogens were likely to have caused the false positive signals [13] . Chaimayo et al. observed that thick and highly viscous mucous may cause false positive results [14] . Recent study with another RAT showed that the changes in the test buffer pH due to the added sample may cause non-specific interactions between SARS-CoV-2 specific conjugated antibodies and capture antibodies and cause a false positive result [15] . Cross-reactivities of the RATs with the other respiratory viruses has not been profoundly investigated. SARS-CoV-2 belongs to Betacoronaviruses like HCoV-OC43 and HCoV-HKU1. Thus cross-reactions might be expected especially with these [16] . Corman et al. tested 100 samples containing other respiratory viruses than SARS-CoV-2, and only one sample with human parainfluenzavirus 3 cross-reacted with RSDB-RAT. However, they tested only one sample containing Betacoronavirus (HCov-OC43) [13] . In our cross-reactivity testingincluding also 18 HCoV-OC43 samples -no cross-reactivity against any of the viruses was detected. The SARS-CoV-2 testing strategy in developed countries is changingmainly due to the intensive respectively) were calculated with different assumed COVID-19 prevalence (2 and 5%) in the population to demonstrate the performance of the test in different epidemic situations. References 1. European Centre for Disease Prevention and Control. COVID-19 testing strategies and objectives Considerations for diagnostic COVID-19 tests European Centre for Disease Prevention and Control. Options for the use of rapid antigen tests for COVID-19 in the EU/EEA and the UK the Diagnosis of SARS-CoV-2 Infection Using Rapid Immunoassays. Interim Guidance The Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: Antigen Testing Diagnostic Accuracy of SARS-CoV-2 Rapid Antigen Detection Testing in Symptomatic and Asymptomatic Children in the Clinical Setting Real-life evaluation of a COVID-19 rapid antigen detection test in hospitalized children Diagnostic Accuracy of SARS-CoV-2 Antigen Detection Test in Children: A Real-Life Study A Common List of COVID-19 Rapid Antigen Tests and a Common Standardised Set of Data to Be Included in COVID-19 Test Result Certificates Coronavirus: Commission puts forward rules on rapid antigen tests and secures 20 million tests for Member States. Brussels Rapid, point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 infection Diagnostic accuracy of two commercial SARS-CoV-2 antigen-detecting rapid tests at the point of care in community-based testing centers Comparison of seven commercial SARS-CoV-2 rapid point-of-care antigen tests: a single-centre laboratory evaluation study Rapid SARS-CoV-2 antigen detection assay in comparison with real-time RT-PCR assay for laboratory diagnosis of COVID-19 in Thailand Generation of False-Positive SARS-CoV-2 Antigen Results with Testing Conditions outside Manufacturer Recommendations: A Scientific Approach to Pandemic Misinformation. Microbiol Spectr A systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease. medRxiv We thank all who have enabled this study and taken part in developing COVID-19 diagnostics in Fimlab Laboratories operation region.