key: cord-0771301-xwwwkeqj
authors: Leli, Christian; Matteo, Luigi Di; Gotta, Franca; Cornaglia, Elisa; Vay, Daria; Megna, Iacopo; Pensato, Rosalia Emanuela; Boverio, Riccardo; Rocchetti, Andrea
title: Performance of a SARS CoV-2 antigen rapid immunoassay in patients admitted to the Emergency Department
date: 2021-07-22
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.07.043
sha: 0173682a8da2dc5088d3eae8ac8cb7f70b2095f3
doc_id: 771301
cord_uid: xwwwkeqj

OBJECTIVES: Given the ongoing emergency, there is a need to identify SARS CoV-2 infection in due course in various community settings. Rapid antigen testing is spreading worldwide, but diagnostic accuracy is extremely variable. We compared a microfluidic rapid antigen test with a reference molecular assay in patients admitted to the Emergency Department (ED) of a General Hospital from October 2020 to January 2021. METHODS: we included nasopharyngeal swabs collected in patients with suspected COVID-19 and in patients with no symptoms suggesting COVID-19, but requiring hospitalization. RESULTS: 792 patients of median age 71 years were included. With a prevalence of 21% we had: 68.7% [95% confidence interval (CI): 60.9-75.5] sensitivity; 95.2% (95% CI: 93.1-96.7) specificity; 79.2% (95% CI: 71.4-85.3) positive predictive value (PPV); 91.9% (95% CI: 89.5-93.9) negative predictive value; 3.8 (95% CI: 2.7-5.3) positive likelihood ratio (LR+); 0.09 (95% CI: 0.07-0.1) negative likelihood ratio. In the symptomatic subgroup sensitivity raised to 81% (95% CI: 70.3-88.6) and PPV to 96.9% (95% CI: 88.5-99.5) along with a LR+ of 32 (95% CI: 8.2-125.4). CONCLUSIONS: the new rapid antigen test showed an overall excellent diagnostic performance in a challenging condition such as that of an ED during the COVID-19 emergency.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a single-stranded positive-sense RNA virus and has four main structural proteins: spike, envelope, membrane protein and nucleocapsid protein (Naqvi et al., 2020) and is a member of the human coronavirus family (HCoV) of which, six were already known to cause disease (Yin and Wunderink, 2018) . Among these, four are known as human endemic coronaviruses: HCoV-229E, HCoV-NL63, HCoV-OC43

and HCoV-HKU1 causing acute self-limiting common cold symptoms (Yin and Wunderink, 2018) , while the other two, SARS-CoV and MERS-CoV, were cause of outbreaks of severe lower respiratory tract infection (Drosten et al., 2003; Zaki et al., 2012) .

The symptoms of COVID-19 patients are quite variable, ranging from asymptomatic cases to flu-like symptoms (fever, dry cough, dyspnea, fatigue) up to cases of acute respiratory distress syndrome caused by bilateral interstitial pneumonia requiring admission to an intensive care unit (Hassan et al., 2020; Sheleme et al., 2020) .

For these reasons, laboratory support is essential for a correct diagnosis of COVID-19 and the gold standard is RNA detection of SARS-CoV-2 by means of molecular methods (Cheng et al., 2020; Loeffelholz and Tang, 2020) . Nevertheless, molecular testing is expensive, time consuming, adequately skilled staff is needed and the time it takes for the result can be of hours. On the other hand, rapid antigen testing for detection of SARS-CoV-2 is spreading worldwide, allowing a quick diagnosis in many different community settings, but diagnostic accuracy is extremely variable, with sensitivity ranging from 0% to 94% (Dinnes et al., 2021) . The aim of this study was to compare the diagnostic accuracy of a new rapid antigen test based on microfluidic technology with a reference molecular assay in a population of patients admitted to the Emergency Department (ED) of a General Hospital.

This is a retrospective observational study including all nasopharyngeal swabs collected for diagnostic purposes from patients aged ≥18 years admitted to the ED of the SS. Antonio e Biagio e Cesare Arrigo Hospital of Alessandria, Italy, from October 2020 to January 2021. All patients included in the study were evaluated by the ED Physician that took medical history and performed physical examination. The symptoms considered as associated with possible COVID-19 were those already described by other Authors (Siordia, 2020) . Nasopharyngeal swab sampling was performed in patients with suspected COVID-19 and in patients without symptoms suggesting COVID-19, but requiring hospitalization. This because up to 45% of individuals infected with SARS-CoV-2 can be asymptomatic (Oran and Topol, 2020 ) and IDSA's COVID-19 diagnostic guidelines (Hanson et al., 2020) suggest direct SARS-CoV-2 RNA testing in asymptomatic individuals with no known contact with COVID-19 who are being hospitalized in areas with a high prevalence of COVID-19 in the community. From all patients were collected two swabs concurrently, one nasopharyngeal swab for SARS-CoV-2 RNA detection and one nasal swab for rapid antigen-testing. SARS-CoV-2 RNA detection Nasopharyngeal swabs were collected by means of Universal Transport Medium for Viruses, Chlamydia, Mycoplasma and Ureaplasma (Copan UTM ® system; Copan, Italy). severe acute respiratory syndrome coronavirus 2 detection was performed by means of The Alinity m SARS-CoV-2 AMP Kit (ALINITY m SARS-COV-2 ASSAY, 2021) run on the Abbott Alinity m system (Abbott Molecular Inc., Des Plaines, IL, USA). Both kit and instrumentation were employed according to the manufacturer's instructions for both the handling and the interpretation of the results. The assay is a real-time reverse transcriptase polymerase chain reaction (RT-PCR) test for qualitative detection of nucleic acid from SARS-CoV-2 in nasal, nasopharyngeal or oropharyngeal swabs and in bronchoalveolar lavage specimens. The system employs magnetic microparticle technology to capture, wash, and elute the nucleic acid. An internal control is introduced into each specimen at the beginning of the sample preparation and both a positive control and a negative control are processed concurrently. After disruption of SARS-CoV-2 virions by guanidine isothiocyanate, nucleic acids are captured on the magnetic microparticles and inhibitors and unbound sample components are removed by subsequent washing steps. The purified RNA is then combined with liquid unit-dose Alinity m SARS-CoV-2 activation reagent and liquid unit-dose Alinity m SARS-CoV-2. During the amplification step, the target RNA is converted to cDNA by the reverse transcriptase. The target sequences for the assay are in the SARS-CoV-2 RdRp and N genes of the SARS-CoV-2 genome, highly conserved and specific. Amplification of the three targets (SARS-CoV-2 RdRp, SARS-CoV-2 N and internal control) takes place simultaneously in the same reaction amplification/detection reagents. If in the sample are present the target sequences, the hybridization to complementary sequences separates the fluorophore and the quencher and allows fluorescent emission and detection. The lowest concentration level with observed positive rates ≥ 95% is 100 virus copies/ml and the maximum number of amplification cycles is 45.

The nucleocapsid protein antigen of SARS-CoV-2 was detected by means of The LumiraDx SARS-CoV-2 Ag Test (Technical Validation for LumiraDx SARS-CoV-2 Ag test, 2021), a rapid microfluidic immunofluorescence assay for use with the LumiraDx Platform for the qualitative detection of the nucleocapsid protein antigen to SARS-CoV-2 directly from nasal swab samples.

Each sample was collected by means of a standard dry swab eluted into a vial containing extraction buffer. By a vial dropper cap a single drop of the sample in the extraction buffer was added to the test strip containing dried reagents. The test result was determined from the amount of fluorescence detected by the device within 12 minutes.

Continuous variables were expressed as median and interquartile range (IQR), categorical variables as absolute numbers and percentages. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), positive likelihood ratio (LR+) and negative likelihood ratio (LR-), with 95% confidence intervals (95% CI), agreement by Cohen's kappa between the antigen test results and the RT-PCR results were calculated as described by Eusebi et al. (2013) .

The median values of threshold cycles (CTs) were also calculated and compared by the Wilcoxon rank-sum test. SPSS statistical package, release 17.0 (SPSS Inc, Chicago, IL, USA) was used for all statistical analyses. The significance level was set at p≤0.05.

A total of 792 patients were included in the study. The median age was 71 years (IQR: 53-82.7) and 50.9% (403/792) were males. The 52.4% (415/792) had one or more symptoms associated with possible COVID-19 and the clinical characteristics are described in Table 1 

The comparison between the two tests in the whole population is described in Table 2 

Repeating the same analysis considering only the subgroup of patients who had at least one of the three main symptoms potentially associated with COVID-19 (fever, cough and dyspnea) at the time of the evaluation at the ED, therefore patients with fever and/or cough and/or dyspnea, 207/792 (26.1%) patients were considered. The results of the comparison are described in 

The same analysis performed only on the subgroup of patients without any of the symptoms potentially associated with COVID-19 described in Table 1 at the time of the evaluation in the ED, is described in Table 2 ; 377 patients were considered. In this subgroup, the prevalence of disease 

Looking at the median age of the patients corresponding to nasopharyngeal swabs positive by the molecular method we can see how the population sampled in this study was quite older compared to the median age described by other Authors in other countries. Indeed, in a retrospective review of SARS-CoV-2 molecular testing results from over 1000 hospitals across the United States of America, median age of positive people was 40.8 years in the period March-April 2020, decreasing to 35.8 years in June-July 2020 (Greene et al., 2020) . Likewise, in the epidemiological reports by the Center for Disease Control, overall median age was 48 years as of June 2020 (Stokes et al., 2020) declining from 46 years in May to 37 years in July and 38 in August 2020 (Boehmer et al., 2020) . Conversely, the median age of the patients included in this study is in line with a report about the case-fatality rate in Italy of March 2020 (Onder et al., 2020) , in which individuals aged 70 years or older were the 37.6% of cases.

The overall sensitivity found in this study for the rapid antigen test was better than that described in most of other reports for immunochromatographic tests, that reported values from 0% to 75.5% (Fenollar et al., 2021; Lambert-Niclot et al., 2020; Mertens et al., 2020; Pérez-García et al., 2021; Scohy et al., 2020; Weitzel et al., 2021) , whereas the specificity was substantially comparable. On the other hand, in a prospective controlled observational study that evaluated 907 patients (Bianco et al., 2021) , higher values of sensitivity were found; nevertheless in that study, both adults and pediatric patients were included (mean age 47.9 years) and the prevalence of disease was higher (298/907; 32.9%). In this study, from the likelihood ratios adjusted for the prevalence of the study population, it can be seen how in the presence of a positive antigen test it is 3.8 times more likely that the molecular test will also be positive rather than negative. Conversely, in the presence of a negative antigen test, the molecular test is 0.08 times more likely to be positive. The median of RT-PCR cycles required to reach the threshold of positivity was significantly higher in molecular swabs corresponding to negative antigen swabs.

For the comparative analysis in the group of symptomatic, the subgroup of patients with fever and/or cough and/or dyspnea was chosen because these three main symptoms are the most frequent in patients suffering from severe disease (Borges do Nascimento et al., 2020) . Therefore, the prevalence of disease in the subgroup of patients with symptoms raised to 38.2% and consequently, the diagnostic accuracy of the rapid antigen test improved. This finding has been described in all the studies above reported (Fenollar et al., 2021; Lambert-Niclot et al., 2020; Mertens et al., 2020; Pérez-García et al., 2021; Scohy et al., 2020; Weitzel et al., 2021) and also in a systematic review with meta-analysis (Dinnes et al., 2021) that evaluated 78 study cohorts for a total of 24,087 samples, estimates of sensitivity varied between symptomatic and asymptomatic (72% vs 58.1%, respectively). Also in this subgroup of symptomatic patients the median of the RT-PCR cycles required to reach the threshold was significantly higher in the molecular swabs corresponding to negative antigen swabs.

As conceivable, in the subgroup of asymptomatic patients, lower values of PPV were observed.

Nevertheless, this means that even in patients completely asymptomatic, near half of the positive ones by molecular test can be identified also by the rapid antigen test. This is in line with other reports, indeed, a study comparing an antigen fluorescent immunoassay with a reference molecular test, the sensitivity found in the asymptomatic patients was of 41.2%, with a PPV of 33.3% (Pray et al., 2021) , likewise, in another study evaluating a rapid immunochromatographic test, in the group of 22 PCR positive swabs from the 159 asymptomatic subjects the test showed a sensitivity of 45.4% (Fenollar et al., 2021) . Even in the subgroup of asymptomatic patients, the median of the cycles of RT-PCR required to reach the threshold was significantly higher in the molecular swabs corresponding to negative antigen swabs.

The finding of a significantly higher median number of CTs of positive molecular tests corresponding to negative rapid antigen tests in all analyses is most probably due to the inverse correlation between viral load and CTs already described by several studies (Rao et al., 2020) .

Similarly, in a study that evaluated 100 nasopharyngeal swab samples collected from individuals living in a shared facility (Kohmer et al., 2021) , sensitivity varied from 50% in the whole population to 100% in the subgroup of potentially infectious samples (≥6 log 10 RNA copies/mL).

The choice of considering as low viral load samples the nasopharyngeal swabs with CTs ≥35 in the final comparative analysis was driven by the study of La Scola et al. (2020) that among 183 nasopharyngeal samples inoculated for virus isolation, none of the samples with CTs ≥34 led to virus isolation and the study of Bullard et al. (2020) that on a total of 90 samples found that for every 1-unit increase in CT, the odds ratio for infectivity decreased by 32%. Even considering positive molecular swabs with CTs ≥35 as low viral load samples, the median of the CTs required to reach the threshold was significantly higher for the molecular swabs corresponding to negative antigen swabs. In a prospective cohort study that evaluated the same assay of the present study, but in a population of 512 among children and adults (Drain et al., 2021) , a 100% sensitivity was found among the subjects with CT ≤33. The same analysis in our series showed an increase of 16,1% for sensitivity and of 9,4% for PPV (data not shown).

With regard to the similar sensitivity values found in the subgroups with main symptoms (fever and/or cough and/or dyspnea) and high viral load (CTs ≥35 as low viral load samples), this finding is conceivable, since it has been found that the viral load at the time of initial sample collection was significantly higher in symptomatic than in asymptomatic patients (Kawasuji et al., 2020) .

Concerning the use of this new rapid antigen test outside hospital, in the light of the findings of this study and the cost of the test, some Authors suggest to use it only by people who have no COVID-19 symptoms and want to exclude infection before travelling or meeting people vulnerable to an infection (Cairns, 2020) .

This study has limitations. This is a single-center study, therefore the results are not completely applicable to other settings, we could not test the viability of the viruses in the swabs corresponding to negative antigen tests and the sampling methods for the two tests were different. Information regarding days after clinical onset for symptomatic COVID-19 patients were not available, analysis could not be performed.

Compared with a molecular test considered as reference, the new rapid antigen test showed an overall excellent diagnostic performance in a population of nearly 800 patients in a challenging condition such as that of the ED during the Covid-19 emergency. The diagnostic performance was better in symptomatic patients, but even in asymptomatic patients it anyway allowed the identification of near half of the patients positive by the molecular test, improving the patient tracking and speeding up possible isolation. In each condition evaluated, the negative antigen tests corresponding to positive molecular tests, had a significantly higher number of CTs needed for the positivity threshold. This shows how the combination of the two tests can be exploited to better figure out if the patient has a very low viral load and therefore a lower risk of infectivity. 

The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This research did not receive any grant from funding agencies in the public, commercial or not-forprofit sectors.

The work described has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. Ethical approval was not needed because this is a retrospective analysis of data from samples collected as part of standard care and those included in the database were deidentified before access. No personal information was stored in the study database. No patient intervention occurred with the obtained results

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