key: cord-0852461-ypetwg1g authors: Yokota, I.; Shane, P. Y.; Teshima, T. title: Logistic advantage of two-step screening strategy for SARS-CoV-2 at airport quarantine date: 2021-01-26 journal: nan DOI: 10.1101/2021.01.25.21250509 sha: abd18ff2efb5cfcadca66d59cdd2c716c233c6a4 doc_id: 852461 cord_uid: ypetwg1g Background: Airport quarantine is required to reduce the risk of entry of travelers infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, it is challenging for both high accuracy and rapid turn-around time to coexist in testing; polymerase chain reaction (PCR) is time-consuming with high accuracy, while antigen testing is rapid with less accuracy. Methods: 88,924 (93.2%) of 95,457 arrivals at three international airports in Japan were tested for SARS-CoV-2 using self-collected saliva by a screening strategy with initial chemiluminescent enzyme immunoassay (CLEIA) followed by confirmatory nucleic acid amplification tests (NAAT) only for intermediate range antigen concentrations. Results: 254 (0.27%) persons were found to be SARS-CoV-2 antigen positive ([≥] 4.0 pg/mL) by CLEIA. NAAT was required for confirmatory testing in 513 (0.54%) persons with intermediate antigen concentrations (0.67-4.0 pg/mL) whereby the virus was detected in 34 (6.6%) persons. This two-step strategy dramatically reduced the utilization of NAAT to approximately one out of every 200 test subjects. Estimated performance of this strategy did not show significant increase in false negatives as compared to performing NAAT in all subjects. Further reduction in imported cases may be achieved by post-screening quarantine. Conclusions: Point of care testing by quantitative CLEIA using self-collected saliva is less labor-intensive and yields results rapidly, thus suitable as an initial screening test. Reserving NAAT for CLEIA indeterminate cases may prevent compromising accuracy while significantly improving the logistics of administering mass-screening at large venues. Keywords: SARS-CoV-2, COVID-19, PCR, CLEIA, quarantine The coronavirus disease-19 pandemic has forced many countries to introduce border closures to prevent the entry of infected travelers from regions where COVID-19 is rife 1-3 . However, this decision has brought on heavy social and economic repercussions. Now several countries have been increasingly accepting international flights, albeit with quarantine for any traveler suspected of infection by various tests 4-7 . Testing measures include PCR before departure, temperature and symptom check at airports, and PCR testing at arrival. Due to the high volume of international air travel, performing the virus tests with high accuracy in rapid turnaround time has been challenging. Currently, the "gold standard" of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is nucleic acid amplification tests (NAAT), such as the quantitative reverse transcriptase polymerase chain reaction (PCR) using nasopharyngeal swab (NPS) samples 8, 9 . Recently, self-collected saliva has attracted attention as an alternative sample with significant logistic advantages over NPS 10 . Specifically, self-collection eliminates the requirement of specialized medical personnel and allows simultaneous parallel sample collection thus more suitable for mass screening 9, 11 . We and others have shown that the accuracy of self-collected saliva and NPS samples are equivalent in large scale direct comparative studies 12-14 . However, although PCR is highly accurate and reliable, results may take 24-48 hours to return. Such delays may lead to further transmission of disease 15 , especially in the confines of airport transit. Recently, we and others have shown the utility of a novel quantitative antigen test using chemiluminescent enzyme immunoassay (CLEIA) and reverse transcriptase loop-mediated isothermal All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.25.21250509 doi: medRxiv preprint amplification (LAMP) to detect SARS-CoV-2 proteins and nucleic acids, respectively, within 30 minutes 14, 16 . Accordingly, a novel two-step strategy using these tests have been implemented for mass screening of SARS-CoV-2 at airport quarantines in Japan 17 . We herein evaluated utility of this strategy in 88,924 persons and estimated its performance in several clinical scenarios. Testing for SARS-CoV-2 using either self-collected saliva or NPS samples obtained by medical officers was mandatory for all international arrivals between July 29 and September 30, 2020. Due to logistical advantages, vast majority of tests were performed using self-collected saliva. Samples were collected in a sterilized 15mL polystyrene sputum collection tube (Toyo Kizai, Warabi, Japan) and all specimens were analysed at the airport quarantine laboratories. This study was approved by the Institutional Ethics Number: 020-0116) and anonymously processed data were provided by the quarantine stations. Initially, all specimens were tested by CLEIA with positive and negative thresholds of 4.0pg/mL and 0.67pg/mL, respectively, as previously reported 17 . Concentrations in between the thresholds were considered indeterminate, and only these specimens underwent confirmatory testing by NAAT. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Lumipulse SARS-CoV-2 Ag kit (Fujirebio, Tokyo, Japan), a sandwich CLEIA using monoclonal antibodies that recognize SARS-CoV-2 N-Ag on LUMIPULSE G1200 automated machine (Fujirebio), was used as previously described 17 . The detection range is between 0.01 pg/mL and 5000 pg/mL. Saliva was diluted 4-fold with phosphate buffered saline and centrifuged at 20,000 × g for 5 min to remove cells and debris. RNA was extracted from 200 µL of the supernatant using QIAsymphony DSP Virus/Pathogen kit and QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Then, nucleic acids of SARS-CoV-2 were detected by either PCR or LAMP. PCR tests were performed as previsouly described 14 . The cycle threshold (Ct)-values were obtained using N2 primers (NIID_2019-nCOV_N_F2, NIID_2019-nCOV_N_R2) and a probe (NIID_2019-nCOV_N_P2). LAMP was carried out to detect Reagent Kit (Eiken Chemical, Tokyo, Japan) and the Loopamp Real-time Turbidimeter (Eiken Chemical) as previously described 14 . We compared the estimated effectiveness of three mass-screening strategies at border quarantine: the two-step strategy, NAAT only for all entrants (without CLEIA), and test-free entry, expressed as the rate of false negatives per 100,000 persons and the number of NAATs performed. The rate of false negatives by NAAT was estimated by p × FN/Pos where p is the assumed proportion of the test population with positive NAAT, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 26, 2021. (which was not certified by peer review) 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.25.21250509 doi: medRxiv preprint respectively. In specimens negative by NAAT, the frequency of high antigen concentrations monotonically approached zero, while NAAT positives did not follow any trend (Fig. 2) . Comparing the effectiveness of the three strategies, the number of false negatives was greater in the two-step strategy compared to NAAT only in all scenarios of NAAT positivity, although both tests reduced false negative rates by more than half compared to test-free entry (Table 1) . Conversely, the two-step strategy allowed the reduction of NAAT by approximately 95% compared to when NAAT was used in all persons. For example, in the scenario when p=0.1%, the NAAT only strategy detected 40 false negatives by 100,000 NAATs, whereas the two-step strategy resulted in 64 false negatives but only required 549 NAATs to be performed. Furthermore, as the majority of CLEIA indeterminates were NAAT negative, the number of NAATs needed did not significantly increase with varying scenarios of p. When Table 2) . Regardless of FN/Pos, post-screening 14-day quarantine substantially reduced imported false negatives, although the efficacy of quarantine was highly dependent on the degree of adherence (Table 2 ). Although PCR is a standard of care for the detection of SARS-CoV-2, mandatory mass-screening should ideally avoid time-consuming and labor-intensive All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.25.21250509 doi: medRxiv preprint procedures. In this regard, quantitative antigen test by CLEIA is rapid albeit with slightly less accuracy than PCR 17 . Therefore, our two-step strategy combined the utility of initial CLEIA with the accuracy of NAAT only for diagnosis of indeterminate cases. This two-step strategy has been adopted in quarantine stations at the international airports in Japan, especially for the prevention of long waiting times spent in closed spaces in crowds and close-contact situations. Here, we showed the benefits of this strategy using 88,924 samples, providing numerical estimates of undetected infectees under various circumstances. The quantitative antigen testing allows for setting appropriate positive and negative thresholds to freely define the indeterminate range with a trade-off; a wider range improves test performance but at the expense of increasing the requirement of confirmatory NAAT. These thresholds may be altered to suit different clinical situations 19 , most importantly the local prevalence of disease. Initial CLEIA has excellent specificity with the upper cutoff value at 4·00 pg/mL, as increasing antigen concentrations of NAAT negative samples consistently approach zero (Fig. 2b) . Assuming that the frequency continues to decrease by one for every 0·5 pg/mL, 36 NAAT-negative samples would be included between 4 pg/mL and 8 pg/mL, giving specificity as high as 99·96% (=88,636/(88,636+36)). To further reduce the false positive rates, the upper cutoff may be set at higher values, but at the expense of increased requirement for confirmatory NAAT. For example, raising the upper cut-off from 4 pg/mL to 8 pg/mL would increase the number of indeterminate results and hence the number of NAAT from 513 to 603 (Fig. 2a) . The main objective of screening for SARS-CoV-2 is to detect all transmissible persons, and this has been a great challenge with presymptomatic false negative PCR at 67% one day prior to and 38% on the day of symptom onset 20 . Therefore, in order to accurately identify presymptomatic infectees, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. pre-departure testing should be conducted several days before departure. Assuming that all passengers were asymptomatic with negative results before departure, infectees will most likely be in the latent phase at the time of screening. Given the median incubation period of 5 days [21] [22] [23] , testing five days prior to departure should reveal half the infections Regardless of testing strategy, post-screening quarantine performed very well at limiting import cases of false negatives in any scenario. However, perfect adherence to two weeks of compulsory isolation by all travelers is unrealistic, with detrimental psychological, social, and economic impact for those who do comply [24] [25] [26] Furthermore, screening is useful in monitoring the dynamics of test positivity, which may influence various immigration and health policies as well as suggest the possibility of viral mutations. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.25.21250509 doi: medRxiv preprint The main limitation of our study was the lack of clinical information after screening to assess the rates of observed false positivity. Post-screening investigation of more than 80,000 persons was simply out of the scope of this study. An additional limitation was that the probability of CLEIA-positive given NAAT-positive could not be validated, as NAAT was not performed for CLEIA-negative samples (antigen levels below 0·67pg/mL). Finally, although not truly a limitation of our study, we alluded to NAAT positivity as infectiousness, whereas this may not be true in cases of high Ct values 28, 29 . In summary, we examined the data from mass screening of more than 88,000 persons at airport quarantines and showed the effectiveness of the two-step strategy. We believe the logistic advantage of reducing the burden of NAAT by approximately 95% far outweigh the cost of slightly higher imported cases of false negatives. Two-step testing by CLEIA followed by NAAT is effective in real-world situations, especially when combined with appropriately timed pre-departure testing and/or with quarantine optimized with repeat testing. IY, PYS and TT provided statistical analysis and drafted the manuscript and all authors reviewed critically and approved the final manuscript. This study was supported by Health, Labour and Welfare Policy Research Grants 20HA2002. We thank the international quarantine stations for their cooperation. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 26, 2021. We declare no competing interests. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. FN/Pos*=0.4) . The two-step strategy reduced the number of NAATs performed by approximately 95% compared to NAAT only, with an increase in false negatives by only 24 per 100,000 persons. Both NAAT only and two-step performed significantly better than free entry at limiting the number of false negatives. (which was not certified by peer review) 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.25.21250509 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 26, 2021. NAAT was only performed for CLEIA results with antigen concentrations between 0.67 and 4.0pg/mL. The frequency of NAAT negative samples consistently approach zero with increasing antigen concentrations, while NAAT positives did not show any trend. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.25.21250509 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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