key: cord-1038387-1ql89m5j
authors: HALFON, P.; PENARANDA, G.; KHIRI, H.; GARCIA, V.; DROUET, H.; PHILIBERT, P.; PSOMAS, C.; DELORD, M.; ALLEMAND-SOURRIEU, J.; RETORNAZ, F.; CHARPIN, C.; Gonzales, T.; PEGLIASCO, H.; ALLARDET-SERVENT, J.
title: An optimized stepwise algorithm combining rapid antigen and RT-qPCR for screening of COVID-19 patients
date: 2021-01-15
journal: nan
DOI: 10.1101/2021.01.13.21249254
sha: 2588a790f31eb5769ad1da9ce7737b21afdf0bca
doc_id: 1038387
cord_uid: 1ql89m5j

Background: Diagnosing SARS CoV-2 infection with certainty is essential for appropriate case management. We investigated the combination of rapid antigen detection (RAD) and RT-qPCR assays in a stepwise procedure to optimize the detection of COVID-19. Methods: From August 2020 to November 2020, 43,399 patients were screened in our laboratory for COVID-19 diagnostic by RTqPCR using nasopharyngeal swab. Overall, 4,691 of the 43,399 were found to be positive, and 200 were retrieved for RAD testing allowing comparison of diagnostic accuracy between RAD and RT-qPCR. Cycle threshold (Ct) and time from symptoms onset (TSO) were included as covariates. Results: The overall sensitivity, specificity, PPV, NPV, LR-, and LR+ of RAD compared with RT-qPCR were 72% (95%CI 62%;81%), 99% (95% CI95%;100%), 99% (95%CI 93%;100%), and 78% (95%CI 70%;85%), 0.28 (95%CI 0.21;0.39), and 72 (95%CI 10;208) respectively. Sensitivity was higher for patients with Ct [≤] 25 regardless of TSO: TSO [≤] 4 days 92% (95%CI 75%;99%), TSO > 4 days 100% (95%CI 54%;100%), and asymptomatic 100% (95%CI 78%;100%). Overall, combining RAD and RT-qPCR would allow reducing from only 4% the number of RT-qPCR needed. Conclusions: This study highlights the risk of misdiagnosing COVID-19 in 28% of patients if RAD is used alone. Thus, negative results from RAD needs to be confirmed by RT-qPCR prior to making treatment decisions. A stepwise analysis that combines RAD and RT-qPCR would be an efficient screening procedure for COVID-19 detection and may facilitate the control of the outbreak.

Since the beginning of the COVID-19 outbreak, one of the most important challenges for the scientific community is to quickly establish the diagnosis of SARS-CoV-2 infection. To establish an effective COVID-19 filter that will stop this pandemic, tests that can enable both mass screening and reliable detection of infected people are needed 1 . Consequently, the use of surveillance testing to identify infectious individuals represents one possible method for breaking enough transmission chains in order to suppress the ongoing pandemic and reopen societies with or without a vaccine. The reliance on testing underscores the importance of analytical sensitivity of virus assays with gold-standard being the real-time quantitative polymerase chain reaction (RT-qPCR) from nasopharynx swab 2 .

These assays have analytical limits of detection that are usually within 10 3 viral RNA copies per ml (cp/ml) 3, 4 . However, RT-qPCR remains expensive and often has sample-to-result times of 24-48 h as a laboratory-based assay. New developments in SARS-CoV-2 diagnostics have the potential to reduce cost significantly, thus allowing for expanded testing or greater frequency of testing and reducing turnaround time to minutes [5] [6] [7] [8] [9] [10] [11] [12] [13] . Several diagnostic strategies are available for identifying or ruling out current infection, identifying people in need of care escalation, or testing for past infection and immune response. Point-of-care (POC) antigen and molecular tests for the detection of current SARS-CoV-2 infections could allow for the earlier detection and isolation of confirmed cases than laboratory-based diagnostic methods, with the aim of reducing household and community transmission. These tests exist today in the form of rapid molecular assays or rapid antigen detection (RAD) tests. The latter can quickly detect fragments of proteins found on or within the virus by testing samples collected from the nasal cavity via swabs. Additionally, RAD tests, which are widely used to detect viral infections other than COVID-19, are not only rapid (15-30 minutes) but also simple to use, require short training, and can be performed outside the laboratory for mass screening when RT-qPCR assays are not or insufficiently available. RAD tests are cheap (<5 USD), can be produced in tens of millions or more per week; these advantages can theoretically lead towards effective COVID-19 filter regimens. However, these assays do not meet the gold standard for analytical sensitivity, thus hindering their widespread use 14 . Among factors that potentially alter the sensibility of RAD tests are the viral load, which has been found to be highly variable in COVID-19 patients and depends on factors such as time from symptoms . CC-BY 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.13.21249254 doi: medRxiv preprint onset (TSO), sample collection (i.e., type and quality), disease severity, and patient age [14] [15] [16] .

Zou et al. 14 reported cycle threshold (Ct) values in the range of 19-40 in the upper respiratory specimens of infected patients. Antigen tests are less sensitive than RT-qPCR and could be less reliable in the clinical diagnosis of COVID-19 patients with low viral load. Caraguel et al. 17 reported that epidemiologic strategies use criteria based on either the probability or the cost of a false test result associated with a specified cutoff. Recent studies on four different commercial RAD tests demonstrated a wide range of sensitivities from 16.7% to 85% (with 100% specificity) in COVID-19 clinical samples 12, 18, 19 . World Health Organization recommends RAD tests that meet the minimum performance requirements of ≥80% sensitivity and ≥97% specificity, while the European Centre for Disease Prevention and Control suggests to use tests with a performance closer to those of RT-PCR, i.e. ≥90% sensitivity and ≥97% specificity 20, 21 . The objective of this study was to assess the combination of RAD and RT-qPCR assays in a stepwise procedure to optimize the detection of COVID-19 in a large cohort of patients. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint According to French regulations, the study was submitted to French ethics committee (CPP Sud-Méditerranée II) and registered as a reference methodology (MR-004) on the Health Data Hub French registering website platform (registration number: F20201028125903, https://www.health-data-hub.fr). Patients were informed that their biological results could be used for research purposes and that they were free to refuse participation in the study. 

Among the 200 patients tested by RAD, there were 104 females (52%) and 96 males (48%); mean age was 48 years old (standard deviation, 21). The overall sensitivity, specificity, PPV, NPV, LR-, and LR+ of RAD compared with RT-qPCR were 72% (95% CI 62%-81%), 99% (95% CI 95%-100%), 99% (95% CI 93%-100%), 78% (95% CI 70%-85%), 0.28 (95% CI 0.21-0.39), and 72 (95% CI 10-208) respectively. The sensitivity of RAD according to Ct values was significantly higher in Ct ≤ 25 than in Ct > 25: 96% (95% CI 87%-100%) vs. 44% (95% CI 29%-59%), respectively (p < .0001). Sensitivity was even lower in CT > 30: 20% (95% CI 3%-56%). The sensitivity of RAD according to TSO was 83% if TSO ≤ 4 days from RT-qPCR (95% CI 66%-93%), 69% if TSO > 4 days from RT-qPCR (95% CI 41%-89%), and 57% in asymptomatic patients (95% CI 39%-74%) (p = 0.0661). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.13.21249254 doi: medRxiv preprint

The interactions between Ct values and TSO showed a predictably higher sensitivity for Ct ≤ 25 regardless of TSO: TSO ≤ 4 days 92% (95% CI 75%-99%), TSO > 4 days 100% (95% CI 54%-100%), and asymptomatic 100% (95% CI 78-100%). For patients with Ct > 25, sensitivity was higher when TSO ≤ 4 days than when TSO > 4 days or even in asymptomatic patients but was still not significant: 56% (95% CI 21%-86%), 50% (95% CI 19%-81%), and 25% (95% CI 9%-49%) respectively (p = 0.2099) (Figure 2 ). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.13.21249254 doi: medRxiv preprint current surveillance approach can lead to the identification of infected people when the test is positive. However, this approach is slow, thus leading to the potential spread of infection when the test is negative. This limits the impact of isolation and contact tracing. If RAD has a sensitivity of at least 80%, it might be used only in in patients with TSO ≤ 4 days and need to be confirmed by an RT-qPCR assay when negative.

One of the main issues that the scientific community have to deal with when using RAD as a mass screening process is its low sensitivity (30%-75%) depending on the assays and the type of population analyzed; this issue has been described by several teams 2 . In a recent Cochrane study, antigen test sensitivity varied considerably across studies (from 0%-94%): the average sensitivity and average specificity were 56.2% (95% CI 29.5%-79.8%) and 99.5% (95% CI 98.1%-99.9%), respectively (based on 8 evaluations in 5 studies on 943 samples) 24 . Data for individual antigen tests were limited, with no more than two studies for any test 24 . Rapid molecular assay sensitivity showed less variation than antigen tests (from 68% to 100%), with an average sensitivity of 95.2% (95% CI 86.7%-98.3%) and average specificity of 98.9% (95% CI 97.3%-99.5%) (based on 13 evaluations in 11 studies of 2,255 samples).

A high sensitivity for SARS-CoV-2 detection in nasopharynx swab samples was observed only for samples with Ct < 25 (corresponding to viral loads higher than 10 6 copies/mL, which has been proposed as the threshold of transmissibility) 17 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.13.21249254 doi: medRxiv preprint self-collected COVID-19 positive saliva samples when using antigen tests 19 . Given the increasing global need for COVID-19 tests, rapid and inexpensive assays are required to supplement current nucleic acid amplification-based assays, and the wide variation in the sensitivities of RAD needs to be evaluated and understood.

Regardless of whether 72% of the overall sensitivity of the RAD (PANBIO COVID-19) is in agreement with those found by Fenollar et al. 25 , the scientific community has to pay attention to the panel of nucleic acid amplification testing positive controls used for testing the sensitivity of RAD. We showed that sensitivity varies from 25% to 100% according to TSO and Cts (≤25, >25).

Based on our experience and others, we would consider using RAD only for patients with TSO ≤ 4 days (sensitivity: 83% [95% CI 66%-93%]) if the test is positive, when RT-qPCR is unreliable, and when RT-qPCR cannot be reported in a short time (<24 h). Otherwise, any negative RAD should be confirmed by an RT-qPCR assay. This timing of TSO ≤ 4 days is in agreement with the fact that the RT-qPCR false-negative rate is the lowest three days after the onset of symptoms or approximately eight days after exposure 26 . Clinicians should consider waiting one to three days after the onset of symptoms to minimize the probability of a false-negative result. Notably, we found that age (>65 or <65 years) had no effect on the decision to use RAD first or RT-qPCR first (results not shown). If the availability of POC or self-administered surveillance tests leads to a faster turnaround time or more frequent testing, our results suggest that they would have a high epidemiological value. We showed that using RAD may allow reducing from only 4% the number of RT-qPCR performed. One of the major advantages of RAD in the effectiveness of surveillance of the outbreak beside shorter turnaround time and lowest cost is its speed of reporting more than its sensitivity 27 Our study has some limitations. First, we do not assess RAD in the whole population of 43,399 patients but RAD was performed on a sample representative for TSO and Cts distribution among all RT-qPCR samples. Another limit is that calculations were performed on samples among whom 11% were positive for COVID-19; thus, the usefulness of RAD must be reassessed according to the prevalence of COVID-19 by RT-qPCR. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint In summary, surveillance should prioritize sensibility, accessibility, frequency, and sample-toanswer time. However, based on the current understanding of sensitivity challenges, our study may alert the scientific community to the fact that extensive use and misinterpretation of RAD can lead to the misdiagnoses of COVID-19 patients due to its low predictive negative value. Negative results from an antigen test should be considered in context of the clinical observations, patient history, and epidemiological information and may need to be confirmed with a molecular test prior to making treatment decisions. A stepwise analysis that combines RAD and RT-qPCR would be an efficient screening procedure for COVID-19 detection and may facilitate the control of the outbreak.

Data Sharing Statement: Data will be available immediately after publication with no end date to researchers who provide a methodologically sound proposal. Requests should be addressed by email to g.penaranda@alphabio.fr. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.13.21249254 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.13.21249254 doi: medRxiv preprint

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