ORIGINAL RESEARCH published: 07 January 2022 doi: 10.3389/fpubh.2021.788581 Analytical Performance of Quantitative DiaSorin Liaison SARS-COV-2 Antigen Test for the Asymptomatic Population Gema Fernández-Rivas 1,2*, Jaume Barallat 3 , Victoria Gonzalez 1,4,5 , Silvia Martinez 3 , Antoni E. Bordoy 6 , Laura Jimenez 3 , Cristina Casañ 1 and Ignacio Blanco 6 1 Microbiology Department, Clinical Laboratory North Metropolitan Area, Germans Trias i Pujol University Hospital, Badalona, Spain, 2 Department of Genetics and Microbiology, Autonomous University of Barcelona, Badalona, Spain, 3 Biochemistry Department, Germans Trias i Pujol University Hospital, Badalona, Spain, 4 Center for Epidemiological Studies on Human Immunodeficiency Virus Infection and Acquired Immunodeficiency Syndrome (HIV/AIDS) and Sexually Transmitted Infections (STI) of Catalonia (CEEISCAT), Generalitat de Catalunya, Badalona, Spain, 5 Centro de Investigación Biomédica en Red (CIBER) in Epidemiology and Public Health (CIBERESP), Madrid, Spain, 6 Metropolitana Nord Laboratory, Germans Trias i Pujol University Hospital, Badalona, Spain Edited by: Zisis Kozlakidis, Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigen International Agency for Research on Cancer (IARC), France (Ag) tests have been widely employed to identify patients for a rapid diagnosis and Reviewed by: pandemic control. Rapid lateral-flow techniques are currently the most used, but Luigi De Maria, automated technologies have emerged as another viable alternative to molecular University of Bari Aldo Moro, Italy methods. We aimed to evaluate the analytical performance of the DiaSorin Liaison Gregorio Paolo Milani, University of Milan, Italy SARS-CoV-2 Ag test in asymptomatic population and close contacts, for its use as a *Correspondence: tool in pandemic control efforts. Gema Fernández-Rivas gfernandezr.germanstrias@gencat.cat Material and Methods: A retrospective study was conducted. A total of 861 samples were included, 291 (34%) were positive for SARS-CoV-2 with cycle threshold (Ct) <40, Specialty section: and 570 (66%) were negative. This article was submitted to Infectious Diseases - Surveillance, Results: A strong correlation was observed between reverse transcriptase-PCR Prevention and Treatment, (RT-PCR) Ct and Ag 50% Tissue Culture Infectious Dose per milliliter (TCID50 /ml; a section of the journal Frontiers in Public Health r = 0.6486; p < 0.0001) and all RT-PCR negative samples tested negative for the Received: 02 October 2021 200 TCID50 /ml SARS-Cov-2 Ag cutoff, i.e., a specificity of 100% was reached (95% Accepted: 22 November 2021 CI: 99.4–100.0%). Samples with <25 Ct and/or >106 extrapolated copies/ml were Published: 07 January 2022 reached a sensitivity of 100% (95% IC 97.0–100.0%). For intermediate viral loads (>105 Citation: extrapolated copies/ml or <30 Ct), the sensitivity value still exceeded 80%. As with Fernández-Rivas G, Barallat J, Gonzalez V, Martinez S, Bordoy AE, other Ag methods, samples between 30 and 40 Ct could not be detected with a Jimenez L, Casañ C and Blanco I reliable sensitivity. (2022) Analytical Performance of Quantitative DiaSorin Liaison Conclusions: The LIAISON® SARS-CoV-2 Ag assay displays an acceptable sensitivity SARS-COV-2 Antigen Test for the and a very high specificity that is useful for detecting the presence of SARS-CoV-2 Asymptomatic Population. Front. Public Health 9:788581. in nasal swabs (NPS) of asymptomatic population or to regular monitoring of risk doi: 10.3389/fpubh.2021.788581 groups in controlled settings. Additionally, the flexibility in processing different samples Frontiers in Public Health | www.frontiersin.org 1 January 2022 | Volume 9 | Article 788581 Fernández-Rivas et al. Quantitative Antigen Detection for SARS-CoV-2 and in the sampling preparation process makes this test an option for its use in high throughput laboratories. Automated tests may facilitate result reporting and yield consistent data, while avoiding some of the pitfalls of rapid lateral-flow techniques, such as observer variability. Keywords: antigen detection, chemiluminescence, COVID-19 infection, asymptomatic patients, monitoring population INTRODUCTION Several authors have evaluated Ag-detection tests as a screening method for a symptomatic population obtaining very Since the Coronavirus Disease-2019 (COVID-19) pandemic good results in terms of sensitivity and specificity (5). Also, has emerged, efforts in its control have been focused on when the risk of contracting COVID-19 is lower, such as for the development of high-sensitivity diagnostic tools and rapid asymptomatic individuals in low prevalence settings, the high systems for more afford able strategies. NPVs of the Ag-detection rapid diagnostic tests (Ag-RDTs) could The European Center for Disease Control (ECDC) in be useful to rule out infection. In any case, it is important to know its document of November 19, 2020 “Options for the use the limitations of these tests when the option is to scale up for of rapid antigen (Ag) tests for COVID-19 in the EU/EEA community and healthcare policies (6). and the UK” indicates the need to implement policies and The option to test asymptomatic individuals has been rapid systems of detection in certain settings where there considered to ensure a safe environment in certain settings, such is a high risk of transmission, such as social healthcare as hospitals or schools (6), due to their low incidence rates. This centers or hospital settings. The use of rapid Ag tests is strategy could also be boosted with the weekly performance of appropriate in high-prevalence settings when a positive these tests. result is likely to indicate true infection and in low- Recently, the DiaSorin company has launched a SARS-CoV- prevalence settings to quickly identify highly infectious 2 Ag sandwich-type direct chemiluminescence immunoassay cases (1). (CLIA) for a quantitative determination of SARS-CoV-2 Given the different rapid Ag-detection tests on the market, nucleocapsid protein Ag in nasal (NS) or nasopharyngeal swabs only those that meet the WHO criteria of sensitivity (S) (NPS). The final reaction consists of the emission of a light ≥80% and specificity (E) ≥97% and that have undergone signal, giving rise to relative light units (RLU) that are directly independent validation studies (2) should be used for diagnostic proportional to the concentration of SARS-CoV-2 viral Ag purposes. Likewise, this document refers to the possibility present in the samples. Automation allows greater performance of using these tests to monitor trends in the incidence of and control of the entire analytical and post-analytical process of diseases in communities and particularly among healthcare the sample. This technology could be applied in those settings workers (HCWs) in case of outbreaks or in areas of high with low incidence even in an asymptomatic population or to community transmission where the positive and negative trace COVID-19 contacts for a faster result than using PCR. predictive values (NPVs) of Ag detection are enough to allow We aimed to evaluate the capacity of the LIAISON R SARS- effective control policies. Rapid Ag tests were mostly developed CoV-2 Ag assay in comparison with the reference technique used in lateral flow devices to obtain quick results, having some for the diagnosis of SARS-CoV-2 infection (AllplexTM SARS- limitations, such as inter-observer variability between readers CoV-2 Assay, Seegene, Seoul, Korea). or false-positive results (3). Other technologies have been developed by high throughput laboratories, minimizing reader bias and claiming to provide more consistent results than lateral MATERIALS AND METHODS flow techniques. Antigen tests identify the presence of the nucleocapsid Severe Study Design Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Ag, This retrospective study was approved by the Ethics Committee which is usually detectable in samples from the upper respiratory of the Germans Trias i Pujol Hospital, Badalona, Spain: tract during the acute phase of infection. These tests do not PI-21-096. The study enrolled 861 asymptomatic individuals have an amplification step, so their analytical detection limits are from primary healthcare centers of the Metro Nord health higher than PCR. Nonetheless, SARS-CoV-2 has an exponential administrative region of Catalonia during the month of January growth in infected patients, so even with initial low viral loads; 2021. NS specimens from these 861 individuals were collected it is possible that in just a few hours, the viral levels reach the in 3 ml Universal Transport Media (UTM) or Viral Transport detection thresholds of Ag tests. This pattern of viral load kinetics Media (VTM). could explain why repeated population screenings with Ag tests The aim of this study was to assess the analytical performance may lead to effective detection policies. Taking into account of the SARS-CoV-2 Ag-detection test in asymptomatic these criteria, some authors state that Ag-detection tests used individuals who were contacts of positive patients and in frequently can still have a high sensitivity to detect carriers or healthcare workers undergoing repeated screenings. infected patients without the need to meet the analytical limit of Asymptomatic population was defined as patients with a detection of the PCR as the reference test (4). definitive molecular diagnosis of COVID-19 positive without Frontiers in Public Health | www.frontiersin.org 2 January 2022 | Volume 9 | Article 788581 Fernández-Rivas et al. Quantitative Antigen Detection for SARS-CoV-2 presenting symptoms, such as fever, cough, myalgia, or other TABLE 1 | Sensitivity, specificity, negative predictive value, positive predictive symptoms related to COVID-19. value, and positive likelihood ratio for LIAISON® SARS-CoV-2 antigen assay and (A) RT-PCR (Ct < 30) and (B) RT-PCR (Ct < 40) for the different cutoffs (200 Close contacts were defined as a person who has spent more TCID50 /ml; 100 TCID50 /ml). than 15 consecutive minutes with the positive case at a distance closer than 2 m, within 48 h of the person getting symptoms or Ct = 30 Ag cut off 200 Ag cut off 100 testing positive for SARS-CoV-2. TCID50 /mL TCID50 /mL (A) Laboratory Methods TP 132 148 Nasal specimens were processed by reverse transcription-PCR FN 570 566 (RT-PCR; AllplexTM SARS-CoV-2 Assay, Seegene, Seoul, Korea) FP 0 4 according to the instructions of the manufacturer. A total of 1,000 µl of these samples were pipetted into a tube containing FN 30 14 1 ml of Liaison SARS-CoV-2 sample inactivation buffer for virus Positive likelihood ratio Not applicable 130.19 (48.97–346.08) inactivation. The tubes were kept at room temperature for Negative likelihood ratio 0.19 (0.13–0.26) 0.09 (0.05–0.14) 120 min before testing. This procedure was performed on a type VPP 100 56.8 (33.10–77.76) II biological safety cabinet. The quantification of SARS-CoV- VPN 99.81 (99.74–99.86) 99.91 (99.86–99.95) 2 Ag was determined using the Liaison SARS-CoV-2 Ag assay Ct = 40 Ag cut off 200 Ag cut off 100 on the Liaison XL platform according to the instructions of TCID50 /mL TCID50 /mL the manufacturer. SARS-CoV-2 concentrations are expressed as 50% tissue culture infectious doses per milliliter (TCID50 /ml). (B) The Liaison XL instrument directly calculates SARS-CoV-2 viral TP 134 158 concentrations from 22 up to 105 TCID50 /ml. The specimens FN 570 566 with ≥200 TCID50 /ml values are considered positive, results FP 0 4 <200 TCID50 /ml are considered negative. In a previous iteration FN 157 133 of the test, results between ≥100 and <200 TCID50 /ml were Positive likelihood ratio Not applicable 77.37 (28.97–206.61) classified as equivocal. To facilitate comparisons with other Negative likelihood ratio 0.54 (0.49–0.60) 0.46 (0.41–0.52) authors, results have been presented with both cutoffs. VPP 100 43.87 (22.64–67.61) For absolute quantification based on RT-PCR cycle threshold VPN 99.46 (99.40–99.51) 99.54 (99.48–99.59) (Ct) values, a standard curve was built using 1/2 serial dilutions of a SARS-CoV-2 RNA (Amplirun R Coronavirus RNA Control, Ct, cycle threshold; TCID, tissue culture infectious dose per milliliter; TP, true positive; TN, true negative; FP, false positive; FN, false negative. catalog ref. MBC090, Vircell Microbiologists, Granada, Spain) in a range of concentrations from 1,400,000 copies/ml to 684 copies/ml. The measurements were made in different CFX positive for SARS-CoV-2, with Ct < 40 (49 samples with a Ct instruments, by different technicians, and on the same day < 20, 113 in a range of 20–30 Ct, and 129 in a range of 30–40 to account for the inherent variability of PCRs. The resulting Ct; Figure 1). RT-PCR result distribution and Ag results are standard curve was used to extrapolate the viral load of each summarized in Table 2. sample (in copies/ml) from their respective Ct. A strong correlation was observed between RT-PCR and Ct and Ag TCID50 /ml (r = 0.6486; p < 0.0001; Figure 2). Significant Statistical Analysis differences were observed between Ct in Ag positive and Ag Concordance between results obtained for Liaison SARS-CoV- negative samples with median values of 21 [interquartile range, 2 Ag assay and RT-PCR was established using the Spearman IQR: 18–24] and 34 [IQR: 32–36], respectively. correlation index. In addition, NPV, positive predictive value All RT-PCR negative samples were tested negative for the 200 (PPV), and positive and negative likelihood ratio (LR+, LR−) TCID50 /ml cutoff SARS-CoV-2 Ag with a specificity of 100% were calculated. These data were obtained with an estimated (95% CI: 99.4–100.0%). If we consider the gray zone cutoff of incidence of 1% according to Government Data supplied in 100–199 TCID50 /ml previously proposed by the manufacturer, January 2021 with RT-PCR results (Ct < 30 and Ct < 40; the specificity was 99.3% (95% CI: 98.2–99.8%). Classification Tables 1A,B). The RT-PCR was used as a gold standard for the differences between both cutoffs were observed only in the 25–35 assessment of sensitivity and specificity. Statistical analyses were Ct range. Both thresholds detected all samples with Ct < 25 performed using SPSS v20 and MedCalc V19.0.6. and were not able to detect any sample with Ct > 35. Adjusted sensitivities for different Ct and extrapolated copies/ml are listed in Table 3. Ct-Copies extrapolation was performed using the RESULTS following equation: Copies/ml = 1014 ·e(−0.709 Ct) . A total of 861 samples were analyzed, being 616 (72%) from female individuals, with an age distribution ranging from 1 to 94 DISCUSSION years old (median: 44 years old). There were a total of 87 samples from patients younger than 15 years old that were included in One of the goals of clinical microbiology is to develop new tools the analysis. Two hundred and ninety-one samples (34%) were for accurate diagnostic. In this sense, the COVID pandemic Frontiers in Public Health | www.frontiersin.org 3 January 2022 | Volume 9 | Article 788581 Fernández-Rivas et al. Quantitative Antigen Detection for SARS-CoV-2 FIGURE 1 | STARD diagram. STARD, standards for the reporting of diagnostic accuracy studies. TABLE 2 | Sample distribution according to Ct and Ag results by using two amplification tests (NAATs) (7) but could be sufficient on high different cutoffs >200 TCID50 /ml and >100 TCID50 /ml previously classified as viral load specimens (defined as samples with real-time RT-PCR undetermined cutoffs as described by the manufacturer (100–199 TCID50 /ml). Ct values <25). Ct values do not lineally correlate with viral Ct n Ag cut-off: 200 TCID50 /mL Ag cut-off: 100 TCID50 /mL load and are dependent on the technique and equipment used. Moreover, it is not possible to predict viral transmissibility based Positive Negative Positive Negative only on Ct values at the individual level and results could <20 49 49 0 49 0 overlap between symptomatic and asymptomatic non-spreader 20–24.99 62 62 0 62 0 groups (8). 25–27.49 28 17 11 28 0 Our data suggest that for samples with a Ct value lower 27.5–29.99 23 4 19 9 14 than 25 or >106 extrapolated copies per ml, the Liaison Ag test displays a 100% of sensitivity and specificity with the currently 30–32.49 31 2 29 8 23 recommended cutoff (200 TCID50 /ml). Even with lower viral 32.50–34.99 40 0 40 2 38 loads (>105 extrapolated copies or <30 Ct), the sensitivity 35–40 58 0 58 0 58 value is still >80% (84.08%). If the previously recommended Negative 570 0 570 4 566 undetermined cutoff of 100–200 TCID50 /ml is used, a slightly Ct, cycle threshold; Ag, antigen; TCID, Tissue Culture Infectious Dose per milliliter. better sensitivity is obtained (93.63%) for samples >105 copies or <30 (91.36%) Ct, with a still high specificity of 99.3%. These data are similar to other groups who also study the performance was a cornerstone for the introduction of molecular and new of this technology (9, 10) but with a higher number of samples approaches for the diagnosis of infectious diseases in most analyzed, strengthening our results. clinical microbiology laboratories. The rapidity in results, such Despite lower sensitivity compared to molecular testing, it has as the one offered by rapid Ag devices, is mandatory to manage been postulated that Ag tests may serve as a better indicator of the isolation of patients and to create a safe environment viral infectivity (11) and that even the increase in Ag quantitation for healthcare workers. However, a shorter diagnosis time is related to lower Ct. In this sense, the LIAISON R SARS- must not significantly reduce the ability to perform a CoV-2 Ag assay could serve as an indicator to the evolution correct diagnosis. to the infection due to it being a quantitative technology, Rapid Ag devices, such as lateral flow tests, have many but more clinical and analytical studies are required to prove advantages but sometimes fail to detect viral Ags in specimens this hypothesis. from which the virus was isolated and therefore pose a risk of Likelihood ratio is used to assess how good a diagnostic test misdiagnosing infected people. The limit of detection of Ag- is and to help in selecting an appropriate diagnostic test. It is RDTs could be 10,000-fold lower than those of nucleic acid better than S (Sensitivity) or E (Especificity) because it is less Frontiers in Public Health | www.frontiersin.org 4 January 2022 | Volume 9 | Article 788581 Fernández-Rivas et al. Quantitative Antigen Detection for SARS-CoV-2 FIGURE 2 | (A) Ag TCID50 /ml represented against RT-PCR Ct and (B) RT-PCR Ct represented against antigen negative (<200 TCID50 /ml) and positive (>200 TCID50 /ml) results. Ct, cycle threshold; Ag, antigen; TCID, Tissue Culture Infectious Dose per milliliter. TABLE 3 | Antigen determination sensitivity according to both TCID50 /ml cutoff the Ct. Therefore, it seems clear that 200 cutoff is better for adjusted for (a) different Ct and (b) extrapolated copies/ml. the accuracy of the test and is now the cutoff established by Ct Ag Cut off 100 TCID50 /mL Ag Cut off 200 TCID50 /mL DiaSorin as the reference cutoff. The 100 cutoff still could be useful in populations with high suspicion of COVID-19 or high Sensitivity IC 95% Sensitivity IC 95% incidence rates, maintaining an indeterminate zone as a marker of follow-up. (a) Based on our results, values between 100 and 199 TCID50 /ml, <25 100 96.7–100.0 100 96.7–100.0 which could be considered equivocal, must be carefully <27.5 100 96.7–100.0 92.09 86.3–96.0 regarded depending on clinical symptoms and the timing of <30 91.36 85.9–95.2 81.48 74.6–87.1 possible exposure. It would be recommended that patients with <32.5 80.83 74.6–86.1 69.43 62.4–75.8 equivocal results underwent several follow-up determinations 67.81 61.4–73.8 57.51 50.9–63.9 <35 to definitively rule out SARS-CoV-2 infection. As NS, 54.3 48.4–60.1 46.05 40.2–52.0 <40 oropharyngeal swabs, or even saliva samples (10) are useful Copies/mL Ag Cut off 100 TCID50 /mL Ag Cut off 200 TCID50/mL for Chemiluminescence assays (CLIAs); this is a strength for an easy way for sampling and therefore, patient’s adherence. Sensitivity IC 95% Sensitivity IC 95% Chemiluminescence assays are commonly used in highly automated instruments and are known for their high sensitivity (b) and practicability compared to other immunological assays (12). >106 100 97.0–100.0 100 97.0–100.0 So, CLIA should be considered for diagnostics and control >105 93.63 88.6–96.9 84.08 77.4–89.4 efforts in the COVID-19 pandemic. More advantages of the >104 80.83 74.6–86.1 69.43 62.4–75.8 automated CLIA technology are the multiple types of specimens >103 65.29 58.9–71.3 55.37 48.9–61.7 (13) and the sampling procedure. The option to deliver the >102 54.3 48.4–60.1 46.05 40.2–52.0 sample preparation tube in the sampling settings allows saving TCID, Tissue Culture Infectious Dose per milliliter. time because while the specimens are being sent to central laboratories, the inactivation process is already taking place. This fact is not only useful for reducing time to results but likely to change with the prevalence of the disorder. With a 200 also to reduce a hand-on time for the laboratory technicians, TCID50/ml cutoff, the LR+ is not calculable because it would to avoid relabeling tubes, to allow a more automated process have to be divided by zero, since we have not had false positives. compared to other molecular systems, and to reduce the In any case, it can be seen that the LR+ is very good in all cases biohazard risk in laboratories. Additionally, notification of the and excellent at the 200 cutoff. LR– at Ct <40 is poor with both new COVID-19 diagnostics to Public Health Authorities could cutoffs. On the other hand, if we take 30 Ct as a cutoff point, also be automatic and, hence more efficient than with the the LR– becomes good or very good at 200 and 100 (0.54 and lateral flow devices. Pandemic control efforts will be safer 0.46, respectively). with these samples traceability. According to other authors (3), The PPV with a 200 cutoff is 100%, but on the other hand, this technology could be useful in settings where controlled the PPV with a cutoff of 100 is 43% or 56% depending on groups are identified and where there is a low incidence Frontiers in Public Health | www.frontiersin.org 5 January 2022 | Volume 9 | Article 788581 Fernández-Rivas et al. Quantitative Antigen Detection for SARS-CoV-2 of COVID-19 infections. In these cases, a repeated sampling DATA AVAILABILITY STATEMENT weekly is a cheaper and faster alternative for monitoring than molecular tests. The original contributions presented in the study are included This study has several limitations. Firstly, our study in the article/supplementary material, further inquiries can be populations are asymptomatic patients in whom the evolution of directed to the corresponding author. the infection is unknown, and it might be a problem because we do not work with homogeneous specimens. Additionally, some ETHICS STATEMENT subjects develop symptoms a few days after the test, and we were not able to record this information, so some information about The studies involving human participants were reviewed symptoms is not well-documented. In addition, we used frozen and approved by Comitè d’Etica de la Investigació amb samples and freeze-thaw cycles may affect the integrity (14) of medicaments Hospital Universitari Germans Trias i Pujol. the N protein and hence, modify some results because small ice Written informed consent for participation was not required for crystals could modify their structure, and additionally, ice could this study in accordance with the national legislation and the alter the concentration of proteins. institutional requirements. In addition, the pediatric population was included in the analysis despite it was suggested that children could be facilitators AUTHOR CONTRIBUTIONS in the spread of SARS-CoV2 infection because many affected children might be asymptomatic (15) but several studies GF-R, JB, VG, and SM: conceptualization and writing—original demonstrated that the frequency of asymptomatic SARS-CoV-2 draft. GF-R, JB, VG, SM, and LJ: data curation. JB, VG, carriers was similar among children and adults (16). SM, and LJ: formal analysis. GF-R, JB, VG, SM, LJ, AB, and Lastly, the study was performed in January, so we do not CC: methodology. GF-R and IB: resources. GF-R, JB, and know the accuracy of this test with new variants of concern; IB: supervision. GF-R, JB, VG, SM, LJ, AB, CC, and IB: despite data demonstrate that the SARS-CoV-2 proteome is writing—review and editing. All authors contributed to the slowly accumulating mutations, fortunately (17). article and approved the submitted version. In conclusion, LIAISON R SARS-CoV-2 Ag is a good alternative for diagnostic purposes for symptomatic individuals FUNDING but also in close contact and asymptomatic patients. The good agreement with RT-PCR and its high specificity makes it an ideal Diasorin Iberia supports this project by granting part of choice as a diagnostic tool in controlled low-incidence settings. the funds for reagents. However, this commercial sponsor The easiness in sample management and processing is a strength had no involvement in study design, collection, analysis, or for choosing LIAISON R SARS-CoV-2 Ag in the COVID-19 interpretation of data, writing the manuscript, and the decision diagnostic algorithms. to submit the manuscript for publication. REFERENCES 7. Safiabadi Tali SH, LeBlanc JJ, Sadiq Z, Oyewunmi OD, Camargo C, Nikpour B, et al. Tools and techniques for severe acute respiratory syndrome coronavirus 1. European Centre for Disease Prevention and Control. 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Quantitative Antigen Detection for SARS-CoV-2 13. Infantino M, Grossi V, Lari B, Bambi R, Perri A, Manneschi M, et al. Conflict of Interest: The authors declare that the research was conducted in the Diagnostic accuracy of an automated chemiluminescent immunoassay for absence of any commercial or financial relationships that could be construed as a anti-SARS-CoV-2 IgM and IgG antibodies: an Italian experience. J Med Virol. potential conflict of interest. (2020) 92:1671–5. doi: 10.1002/jmv.25932 14. Cao E, Chen Y, Cui Z, Foster PR. Effect of freezing and thawing rates Publisher’s Note: All claims expressed in this article are solely those of the authors on denaturation of proteins in aqueous solutions. Biotechnol Bioeng. (2003) and do not necessarily represent those of their affiliated organizations, or those of 82:684–90. doi: 10.1002/bit.10612 the publisher, the editors and the reviewers. Any product that may be evaluated in 15. Milani GP, Bottino I, Rocchi A, et al. Frequency of children vs adults this article, or claim that may be made by its manufacturer, is not guaranteed or carrying severe acute respiratory syndrome coronavirus 2 asymptomatically. endorsed by the publisher. JAMA Pediatr. (2021) 175:193–4. doi: 10.1001/jamapediatrics.202 0.3595 16. Milani GP, Marchisio P, Rocchi A, Bertolozzi G, Furlan L, La Vecchia Copyright © 2022 Fernández-Rivas, Barallat, Gonzalez, Martinez, Bordoy, Jimenez, A, et al. Frequency of asymptomatic carriers of SARS-CoV-2 among Casañ and Blanco. This is an open-access article distributed under the terms children and adults after school reopening. Ital J Pediatr. (2021) 47:65. of the Creative Commons Attribution License (CC BY). The use, distribution or doi: 10.1186/s13052-021-01016-5 reproduction in other forums is permitted, provided the original author(s) and the 17. Vilar S, Isom DG. One year of SARS-CoV-2: how much has copyright owner(s) are credited and that the original publication in this journal the virus changed? Biology. (2021) 10:91. doi: 10.3390/biology100 is cited, in accordance with accepted academic practice. No use, distribution or 20091 reproduction is permitted which does not comply with these terms. Frontiers in Public Health | www.frontiersin.org 7 January 2022 | Volume 9 | Article 788581