Objectives To evaluate the performance of oral saliva swab (OSS) reverse transcription PCR (RT-PCR) compared with RT-PCR and antigen rapid diagnostic test (Ag-RDT) on nasopharyngeal swabs (NPS) for SARS-CoV-2 in children.
Design Cross-sectional multicentre diagnostic study.
Setting Study nested in a prospective, observational cohort (EPICO-AEP) performed between February and March 2021 including 10 hospitals in Spain.
Patients Children from 0 to 18 years with symptoms compatible with Covid-19 of ≤5 days of duration were included. Two NPS samples (Ag-RDT and RT-PCR) and one OSS sample for RT-PCR were collected.
Main outcome Performance of Ag-RDT and RT-PCR on NPS and RT-PCR on OSS sample for SARS-CoV-2.
Results 1174 children were included, aged 3.8 years (IQR 1.7–9.0); 73/1174 (6.2%) patients tested positive by at least one of the techniques. Sensitivity and specificity of OSS RT-PCR were 72.1% (95% CI 59.7 to 81.9) and 99.6% (95% CI 99 to 99.9), respectively, versus 61.8% (95% CI 49.1 to 73) and 99.9% (95% CI 99.4 to 100) for the Ag-RDT. Kappa index was 0.79 (95% CI 0.72 to 0.88) for OSS RT-PCR and 0.74 (95% CI 0.65 to 0.84) for Ag-RDT versus NPS RT-PCR.
Conclusions RT-PCR on the OSS sample is an accurate option for SARS-CoV-2 testing in children. A less intrusive technique for younger patients, who usually are tested frequently, might increase the number of patients tested.
- Molecular Biology
- Infectious Disease Medicine
Data availability statement
Data are available on reasonable request. Not applicable.
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What is already known on this topic
SARS-CoV-2 saliva tests have not been widely implemented in children.
The accuracy of reverse transcription PCR (RT-PCR) on saliva and nasopharyngeal swab is similar.
Studies in this population are scarce, particularly in preschoolers.
What this study adds
RT-PCR on oral saliva swab is an accurate option for SARS-CoV-2 testing in children.
RT-PCR on oral saliva swab shows higher sensitivity than the antigen rapid diagnostic test on nasopharyngeal swab.
How this study might affect research, practice or policy
Saliva swab may be more acceptable for younger patients than nasopharyngeal swab, possibly increasing the capacity of testing in this age group.
Saliva swab could be added to the SARS-CoV-2 diagnostic protocols as an alternative specimen to nasopharyngeal swab in children.
Nucleic acid amplification testing in nasopharyngeal swab (NPS) samples is considered as the gold standard technique for the diagnosis of SARS-CoV-2 infection1; however, this technique requires trained staff, is associated with an increased risk of complications (epistaxis, retained swabs, cerebrospinal fluid leak),2 is unpleasant and generates anxiety mainly in children. Indeed, patients with mild symptoms are often put off accessing the test.2 3
Saliva swab reverse transcription PCR (RT-PCR) is an alternative and minimally invasive test for the diagnosis of SARS-CoV-2 infection. Data from a recent systematic review showed that given a proper RT-PCR kit is chosen, the accuracy of RT-PCR on saliva and NPS is similar, particularly in ambulatory patients,4 yet saliva tests have not been widely implemented in children. Indeed, studies in this population are scarce, particularly in preschoolers.5–10
We performed this study with the hypothesis that oral saliva swab (OSS) RT-PCR is as sensitive as RT-PCR on NPS, which is considered the gold standard. Our objective was to analyse the performance of OSS RT-PCR in comparison to (1) the gold standard (RT-PCR on NPS) and (2) the Panbio SARS-CoV-2 antigen rapid test (Ag-RDT) on NPS, a technique widely implemented, in a symptomatic paediatric population evaluated at the emergency departments of 10 Spanish hospitals.
Cross-sectional multicentre diagnostic study nested in a prospective, observational cohort, the Epidemiological Study of Covid-19 in Children of the Spanish Society of Pediatrics (EPICO-AEP). Participants were children from 0 to 18 years old with symptoms compatible with SARS-CoV-2 infection of ≤5 days of duration, seen in the emergency departments of 10 secondary and tertiary hospitals, in Madrid and Almería, Spain.
The study was performed between February and March 2021. At that time, the peak incidence of Covid-19 in Spain was 559/100 000 individuals.11 Standards for Reporting Diagnostic Accuracy (STARD) 2015 guidelines for reporting diagnostic accuracy studies were followed throughout.12
Trained nurses collected three samples consecutively from each patient following a standardised procedure: two NPS samples (for Ag-RDT and RT-PCR) and one OSS sample for RT-PCR. The Panbio SARS-CoV-2 Ag-RDT was purchased from Abbott Rapid Diagnostics (Jena, Germany). OSS samples were collected using the identical brand of the swab for the NPS samples. The procedure included smearing the swab under the tongue, between the gums and lips and in the inner part of both cheeks, but not the pharynx. Older patients were invited to cough with their mouths closed before sampling. A minimum amount of saliva was not necessary for the oral smear. Clinicians recommended patients not eat or drink at least 30 min before sampling. OSS and NPS to be used for RT-PCR were placed in standard viral transport media (Delta Labs, Barcelona, Spain).
The nurses in charge of collecting samples performed Ag-RDT on-site and results were interpreted following the manufacturer’s instructions, whereas OSS and NPS for RT-PCR were immediately transported to each local microbiology laboratory of each participating centre after specimen collection.
RT-PCR testing in OSS and NPS was performed immediately after specimen collection, targeting at least two viral genomic fragments following the manufacturer’s recommendations in each laboratory. RNA was extracted from samples by using the automated systems in place in each of the participating centres. OSS were processed using the same protocol as NPS in each centre. It was considered indeterminate those samples in which only one of the genes was amplified with a high cycle threshold (Ct). Turnaround time was less than 12 hours at all the centres. Nurses who interpreted the Ag-RDT did not have access to the RT-PCR result. Lab technicians had access to the Ag-RDT result. Clinicians attending each patient had the information of the three tests once available. Clinical information was available for all of them.
Patients with discordant diagnostic test results from any of the three different tests were invited to a second visit and a blood sample was drawn for SARS-CoV-2 IgG detection. Samples were performed in less than 24 hours after the researchers were aware of the results and less than 48 hours from the initial clinical visit. IgG antibodies directed against the SARS-CoV-2 surface S1 domain of the spike protein or the internal nucleocapsid protein were measured in the serum samples using commercial enzyme-linked immunosorbent assay (ELISA) or chemiluminescent immunoassay kits, depending on availability in each centre following manufacturer’s instructions. Besides, in these patients, a study of viral viability was performed whenever possible. All the information related to this additional analysis is included in the online supplemental material.
Clinical and laboratory data were collected using RedCap electronic data capture tools13 14 hosted at the 12 de Octubre Hospital.
The intended sample size was 1500 patients to estimate a 70% sensitivity for OSS and 90% of sensitivity for Ag-RDT to reach a statistical power of 80% with acceptable precision.
The study population was described using counts and percentages for categorical variables and medians and IQRs for continuous variables. Categorical variables were compared with χ² or Fisher’s test, and continuous variables with Mann-Whitney U test. The performance of the test was evaluated estimating the sensitivity, specificity, negative and positive predictive values and the kappa index. Additionally, we provided the sensitivity and specificity of the different diagnostic techniques stratified by age (≤3 and >3 years old). The performance of the test was evaluated by estimating the sensitivity, specificity, negative and positive predictive values and the kappa index taking RT-PCR on NPS as the gold standard. A confusion matrix was assessed to calculate the diagnostic accuracy. CIs for sensitivity and specificity are exact Clopper-Pearson CIs. Besides, the performance of the three tests was also estimated by Bayesian Latent Class Models (BLCA) using extensions of the three tests in one population model implemented in a simplified interface application.15 The BLCA16 was used to approximate the prevalence and the sensitivities and specificities of all tests. This model does not assume that any test is perfect but considers that each test could be imperfect in diagnosing the true disease status. The true disease status of the patient population was then defined based on overall prevalence (the probability that a patient with suspected SARS-CoV-2 infection is truly infected with SARS-CoV-2). BLCA estimates the prevalence and accuracy of each test based on the observed frequency of the possible combinations of test results.
Overall, 1186 patients were included in the study. Eight children did not provide the three samples, and four children presented indeterminate RT-PCR results (two for OSS and two for NPS) and were excluded. Thus, 1174 children were included in the final analysis (figure 1). Median age was 3.8 years (IQR 1.7–9.0), and 516/1174 (44.0%) were ≤3 years old. A total of 647/1174 (55.1%) children were males. The median duration of symptoms before emergency department admission was 1.0 days (IQR 1.0–2.0) (table 1).
In total, 73/1174 (6.2%) patients tested positive by at least one of the diagnostic techniques. Out of those 73 patients, 68 tested positive by NPS RT-PCR (93.2%; 5.8% of the total), 53 by OSS RT-PCR (72.6%; 4.5% of the total) and 43 were positive by NPS Ag-RDT (58.9%; 3.7% of the total) (figure 1).
The comparison of OSS RT-PCR and the Ag-RDT in NPS samples versus NPS RT-PCR (gold standard) is shown in table 2. The overall sensitivity was 72.1% (95% CI 59.7% to 81.9%) for OSS RT-PCR and 61.8% (95% CI 49.1% to 73.0%) for the Ag-RDT. The specificity was 99.6% (95% CI 99.0% to 99.9%) for OSS RT-PCR and 99.9% (95% CI 99.4% to 100%) for the Ag-RDT. The kappa index for the OSS RT-PCR and NPS RT-PCR was 0.79 (95% CI 0.72 to 0.88) versus 0.74 (95% CI 0.65 to 0.84) for the Ag-RDT. OSS RT-PCR showed similar performance in children stratified by age. By contrast, the Ag-RDT showed lower sensitivity in children younger than 3 years (table 3).
The estimated medians with 95% credible intervals (Crs) in the whole cohort and in the BLCA model are shown in table 2. Sensitivity for OSS RT-PCR was 84.8% (95% Cr 71.5%–93.6%) and 72.5% (95% Cr 58.8%–83.6%) for the Ag-RDT. Specificity for OSS RT-PCR was 99.7% (95% Cr 99.2%–99.9%) and 99.9% (95% Cr 99.6%–100%) for the Ag-RDT.
The median Ct values for those patients with positive RT-PCR results were 21.1 (IQR 17.8–30.0) on NPS samples and 32.9 (IQR 28.8–35.6) on OSS samples. The Cts were higher in OSS than in NPS samples (Ct (NPS)=0.5×(Ct saliva)+4.5), p=0.027).
In total, 36/1174 patients (3.1%) tested positive with the three diagnostic tests, 13 cases (1.1%) tested positive only by NPS RT-PCR, 4 cases (0.3%) only by OSS RT-PCR and 1 case (0.1%) only by Ag-RDT (table 4). Ct values for patients with discordant results are shown in online supplemental table S1.
A total of 37 patients had a discordant result in at least one of the three tests (table 3 and online supplemental material). All were invited to provide a blood sample for SARS-CoV-2 serology, and it was available for 22/37 (59.5%) patients.
A total of 19/68 (27.9%) patients were positive for NPS RT-PCR and negative for OSS RT-PCR. Of these 19 patients, 4 were serology positive and could be considered not contagious.20 Four patients had OSS RT-PCR as the unique positive test, but three of them with serology available and positive, suggesting old infections.
This study evaluates the performance of three SARS-CoV-2 diagnostic tests in a large paediatric population with Covid-19 symptoms showing that the RT-PCR on OSS is a valid diagnostic option in the paediatric population, even better than the widely used Ag-RDT on NPS. If the turnaround time is sufficiently rapid, OSS RT-PCR would seem to be the better choice for children. The performance of the test improved when NPS RT-PCR was considered as an imperfect gold standard.21 22
NPS is the most widely used sample for SARS-CoV-2 testing, although it is not always well accepted by patients.1–3 23 Several studies have shown that different sample types of saliva (dribble pots, suck swabs, oral and oropharynx swabs) are a valid alternative for SARS-CoV-2 testing, but most of the studies included only adults.4 To our knowledge, this study is the largest paediatric study comparing OSS with the established gold standard. The results are in concordance with the few studies performed on children.5 7 9 10 24 A recent study performed in Dubai with 476 children demonstrated a sensitivity even higher (87.7%) than found here; however, the authors used a saliva sample rather than saliva swabs, and the children were older (mean 10.8 years old).10 Obtaining direct saliva samples in children younger than 3 years of age and children with special needs, who could be at a higher risk of severe Covid-19 (ie, neurological diseases) is challenging. However, unlike direct saliva, oral swabs can be obtained with practically no collaboration from patients. For this reason, we used oral swabs, which is a sample easily feasible and painless for all ages including those children with special needs. We had a high proportion of children younger than 3 years old, which we believe fills the gap of knowledge on the performance of saliva samples at this age. Moreover, analysis by age showed that the accuracy is similar for both groups (≤3 years vs >3 years), unlike the Ag-RDT, which performed poorly in children under 3 years. Children with special needs were not specifically included in this study so the results cannot be directly extrapolated to them.
We found that OSS RT-PCR could detect some cases that were missed by the NPS RT-PCR; four cases were positive for OSS RT-PCR but negative for NPS RT-PCR, as has been described previously.7–9 Also, three of those four cases had positive serology but were negative for the Ag-RDT. These data suggest that the positivity on saliva RT-PCR could remain even longer than in NPS RT-PCR, but a SARS-CoV-2 reinfection cannot be fully discarded.
A similar finding was observed in the majority of the 19 cases with a negative result on the OSS but positive on the NPS. Of these 19, 4 had positive serology. An observational study showed that the concordance between paired samples (NPS and saliva) decreased with time, with saliva false negatives increasing in older infections.25 We observed a lower sensitivity of the Ag-RDT in children younger than 3 years old. Moreover, among the four cases with negative Ag-RDT and positive OSS, three were younger than 3 years. Thus, younger children might benefit from the OSS RT-PCR in testing guidelines possibly before Ag-RDT.
At the time of performing the study, the cost of SARS-CoV-2 Ag-RDT was US$2–US$5, which was around 10 times lower than the average price of NPS RT-PCR including the swab and reagents. OSS RT-PCR uses the same diagnostic approach as NPS RT-PCR, but in a different sample, so the cost in our study was the same. Additionally, RT-PCR tests require the prior acquisition of expensive devices to perform the analysis and specialised staff. Considering only the cost, Ag-RDT is a better choice than RT-PCR tests. Furthermore, the turnaround is lower (15 min vs 2–12 hours). However, compared with OSS RT-PCR, it has some disadvantages, such as a lower sensitivity and NPS being a more uncomfortable sample than OSS. So, OSS RT-PCR could be a good alternative to NPS RT-PCR in case of aiming a high sensitivity test and comfortable for patients. Besides, a new approach for SARS-CoV-2 RT-PCR (SalivaDirect) that simplifies RNA extraction has been published.26 In this study, the standard RNA extraction and the SalivaDirect procedure were performed followed by the same RT-PCR with similar results. The potential implementation of the simplified procedure could greatly reduce the cost and the time of the OSS RT-PCR testing, making this method even more attractive.
No systematic or standardised information of the acceptance of the patients, families and operators were collected in this study, but several publications support that OSS is the preferred technique by patients.27 28 The acceptability of participating in the study (>99%, data not shown) shows how families acknowledge that finding a more accepted technique to test children is needed. Besides, the collection and processing of samples showed scarce difficulties as only eight children did not provide the three samples, and four children presented indeterminate RT-PCR results.
This study has several limitations. RT-PCR tests and serology were not centralised, which may have introduced some heterogeneity in the results and difficulties in reproducibility. It accounted for only one wave, including the alpha variant as dominant (>70% of isolations), and it may not be fully extrapolated to Omicron or future variants. The possibility of reinfections was not addressed, and not all discordant patients had serology available. Finally, this study included only symptomatic patients (≤5 days of duration), so the results cannot be extrapolated to asymptomatic patients and new studies will be necessary to evaluate this test in SARS-CoV-2 contacts and as a screening test in the general paediatric population. However, as a strength, we evaluate a novel, comfortable and easy-to-collect sample in children, which has not been previously thoroughly evaluated in the paediatric population, mainly in the younger children.
In conclusion, OSS RT-PCR seems a more suitable and friendly technique for younger patients who must be tested very frequently and might help to maximise the number of patients tested, playing an important role in the control of the disease.
Data availability statement
Data are available on reasonable request. Not applicable.
Patient consent for publication
This study involves human participants and was approved by Ethics Committee of the “Fundación de Investigación Biomédica del Hospital 12 de Octubre” (code 20/101) and by the Ethics Committees of the other participating hospitals. Participants gave informed consent to participate in the study before taking part.
We thank all the patients and families for their participation in the study, and the laboratory staff and clinical staff members who cared for them and the EPICO working group. We also thank Kenneth McCreath for the meaningful comments and suggestions on English writing. K.M. is a medical English consultant funded by Universidad Europea de Madrid.
Collaborators EPICO working group: Francisco José Sanz MD, María Isabel Iglesias-Bouzas MD, Jose Antonio Alonso Cadenas MD, Blanca Herrero (Hospital Infantil Universitario Niño Jesús, Madrid, Spain), Teresa del Rosal MD, Ana Méndez-Echevarría MD, Talía Sainz MD, Clara Udaondo MD, Fernando Baquero MD, Cristina Calvo MD, Carlos Grasa MD, Paula R Molino MD, María José Mellado MD, María Ceano MD, Victor Galán MD, Paula Garcia Sanchez MD, Sonsoles San Roman (Hospital Universitario La Paz Madrid, Spain), Alicia Hernanz Lobo MD, Mar Santos MD, Marisa Navarro MD, Elena Rincón MD, David Aguilera-Alonso MD, Begoña Santiago MD, Jesús Saavedra, Jorge Huerta MD, Eduardo Bardón MD, Jorge Lorente MD, Pilar Catalán MD (Hospital Universitario Gregorio Marañón, Spain), Pablo Rojo MD, Daniel Blázquez MD, Luis Prieto MD, Elisa Fernández-Cooke MD, David Torres-Fernández MD, Ángela Manzanares MD, Jaime Carrasco MD, Cristina Epalza MD, Jesús Contreras MD, Sara Domínguez MD, Sara Villanueva MD, Arantxa Gonzalez (Hospital Universitario 12 de Octubre, Madrid, Spain), Cinta Moraleda MD, Alfredo Tagarro MD, Elena Cobos MD, Álvaro Ballesteros MD, Sara Domínguez-Rodriguez (Instituto de Investigación 12 de Octubre, Madrid, Spain), Gemma Pons MD, Silvia Simó MD, Miguel Lanaspa MD, Victoria Fumadó MD, Rosa María Pino MD, María Melé (Hospital Sant Joan de Déu, Barcelon, Spain), María Espiau MD, Jacques G. Rivière MD, Pere Soler-Palacín MD, Antonio Soriano Arandes MD, Natalia Mendoza (Hospital Universitari Vall d’Hebron, Barcelona, Spain), Mercedes Herranz MD, María Urretavizcaya Martínez (Complejo Hospitalario de Navarra, Navarra, Spain), Fernando Cabañas MD, Fátima Ara MD, Marta Baragaño (Hospital Universitario Quirón salud Madrid, Spain), Rut del Valle MD, Ana González-de-Zárate MD, Mónica Pacheco MD, María Luisa Herreros MD, Julia Yebra MD, Beatriz Pérez-Seoane MD, María Fernández MD, Teresa Raga MD, María de la Serna MD, Ane Plazaola MD, Juan Miguel Mesa MD, Rosa Batista MD, Ana Barrios MD, Ignacio Navarro MD, Jana Rizo MD, Teresa Reinoso MD, Alfonso Cañete (Hospital Universitario Infanta Sofía, Madrid, Spain), María Dolores Martín MD, Elena Sáez MD, Olga Nerea Coya MD, Fernando Cava (BR Salud, Madrid, Spain), Enrique Otheo MD, Juan Carlos Galán MD, José Luis Vázquez MD, Carmen Vázquez MD, Victor Quintero (Hospital Universitario Ramón y Cajal, Madrid, Spain), Lola Falcón MD, Olaf Neth MD, Peter Olbrich MD, Walter Goicoechea MD, Cati Márquez MD, Marisol Camacho MD, Inés Marín Cruz (Hospital Universitario Virgen del Rocío, Sevilla, Spain), Laura Martín (Hospital Universitario Regional de Málaga, Málaga, Spain), Lucía Figueroa (Hospital de Villalba, Madrid, Spain), María Llorente (Hospital Universitario del Sureste, Madrid, Spain), María Penin MD, Claudia García MD, María García MD, Teresa Alvaredo (Hospital Universitario Príncipe de Asturias, Madrid, Spain), Mª Inmaculada Olmedo MD, Agustín López MD, María Jose Pérez (Hospital Universitario Puerta de Hierro, Madrid, Spain), Elvira Cobo (Hospital Fundación Alcorcón, Madrid, Spain), Mariann Tovizi (Hospital del Tajo, Madrid, Spain), Pilar Galán (Hospital Universitario de Fuenlabrada, Madrid Spain), Beatriz Soto MD, Sara Guillén (Hospital de Getafe, Madrid, Spain), Adriana Navas (Hospital Universitario Infanta Leonor, Madrid, Spain) M. Luz García (Hospital de Leganés, Madrid, Spain), Sara Pérez (Hospital de Torrejó, Madrid, Spain), Amanda Bermejo MD, Pablo Mendoza (Hospital de Móstoles, Spain), Gema Sabrido (Hospital Rey Juan Carlos, Madrid, Spain), María José Hernández (Hospital Central de la Defensa, Madrid, Spain), Ana Belén Jiménez (Fundación Jiménez Díaz, Madrid, Spain), Arantxa Berzosa MD, José Tomás Ramos MD, Marta Illán (Hospital Clínico San Carlos, Madrid, Spain), Ana López MD, Nerea Gallego (Hospital Universitari Son Espases, Mallorca, Spain), Beatriz Ruiz (Hospital Universitario Reina Sofía, Granada, Spain), Santiago Alfayate MD, Ana Menasalvas MD, Eloísa Cervantes (Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain), María Méndez (Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Barcelona, Spain), Ángela Hurtado (Instituto Hispalense de Pediatría, Sevilla, Spain), Cristina García MD, Inés Amich MD, Yolanda Ruiz (Hospital San Pedro, Logroño, Spain), Manuel Oltra MD, Álvaro Villarroya-Villalba MD, Adela Cañete MD, Bienvenida Argiles (Hospital Universitari i Politècnic La Fe, Valencia, Spain), Angustias Ocaña (Hospital La Moraleja, Madrid, Spain), Isabel Romero MD, María Fernanda Guzmán (Hospitales Madrid, Madrid, Spain), M.J. Pascual (Hospital Nisa, Madrid, Spain), Francisco Llorente de Gracia, Jovita Fernández-Pinero, Miguel Ángel Jiménez-Clavero, Pilar Aguilera-Sepúlveda, Cristina Cano-Gómez (Centro de Investigación en Sanidad Animal INIA-CSIC, Valdeolmos, Madrid, Spain), María Sánchez-Códez (Hospital Universitario Puerta del Mar, Cádiz, Spain), Elena Montesinos (Consorci Hospital General Universitari de València, Valencia, Spain), Julia Jensen MD, María Rodríguez (Hospital Universitario Infanta Cristina, Madrid, Spain), Gloria Caro (Hospital Universitario Infanta Elena, Madrid, Spain), Neus Rius MD, Alba Gómez (Hospital Universitari Sant Joan de Reus, Reus, Spain), Rafael Bretón (Hospital Clínico Universitario de Valencia, Valencia, Spain), Margarita Rodríguez MD, Julio Romero MD, Juan Francisco Pascual Gazquez (Hospital Universitario Virgen de las Nieves, Granada, Spain), Ana Campos (Hospital Universitario Sanitas La Zarzuela, Madrid, Spain), Mercedes García (Hospital de Mérida, Mérida, Spain), Rosa María Velasco (Complejo Hospitalario de Toledo, Toledo, Spain), Zulema Lobato Althaia MD, (Xarxa Assistencial Universitària de Manresa, Manresa, Spain), Fernando Centeno MD, Elena Pérez MD, Alfredo Cano (Hospital Universitario Río Hortega, Valladolid, Spain), Paula Vidal (Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain), Corsino Rey MD, Ana Vivanco MD, Maruchi Alonso (Hospital Universitario Central de Asturias, Oviedo, Spain), Pedro Alcalá MD, Javier González de Dios MD, Laura Ureña Horno (Hospital General Universitario de Alicante, Alicante, Spain), Eduard Solé MD, Laura Minguell (Hospital Universitari Arnau de Vilanova, Catalunya, Spain), Itziar Astigarraga MD, Olatz Villate MD, Susana García Obregón (Hospital Universitario de Cruces, Bilbao, Spain), Mª Ángeles Vázquez MD, Miguel Sánchez MD, Leticia Martínez Campos (Hospital Universitario Torrecárdenas, Almería, Spain), Elena Díaz (Hospital Virgen de la Luz, Spain), Eduardo Consuegra MD, Susana Riesco MD, Almudena González MD, Maika Mendoza (Hospital Universitario de Salamanca, Salamanca, Spain), María Cabanillas (Complejo Asistencial Universitario de Palencia, Palencia, Spain), Yeray Novoa-Medina MD, Elena Colino-Gil MD, Ana Reyes-Domínguez MD, Luis Peña-Quintana (Hospital Universitario Materno Infantil de las Palmas, Las Palmas, Spain), Elisa Garrote MD, Maite Goicoechea (Hospital Universitario de Basurto, Basurto, Spain), Irene Centelles (Hospital General Universitari de Castelló, Castelló, Spain), Santiago Lapeña MD, Sara Gutiérrez MD, Soraya Gutiérrez (Complejo Asistencial Universitario de León, León, Spain), Amparo Cavalle (PIUS Hospital de Valls, Valls, Spain), José María Olmos (Hospital Mare de Déu dels Lliris, Catalunya, Spain), Alejandro Cobo MD, Sara Díaz MD, Cristina Martinez Faci MD, Macarena Gonzalez Cruz MD, Beatriz Castro (Hospital Universitario de Canarias, Tenerife, Spain), Beatriz Jiménez MD, Cristina Alvarez Alvarez (Hospital Universitario Marqués de Valdecilla, Santander, Spain), Raúl González (Hospital Sant Joan d’Alacant, Alicante, Spain), Miguel Lafuente MD, Matilde Bustillo (Hospital Infantil de Zaragoza, Zaragoza, Spain), Natividad Pons MD, Julia Morata (Hospital Lluís Alcanyis, Catalunya, Spain), Elsa Segura (Hospital Universitario Son Llatzer de Palma de Mallorca, Spain), María Bernardino (Universidad Europea de Madrid, Spain), Marta Pareja León (Complejo Hospitalario Universitario de Albacete, Albacete, Spain), Ana Domingo Ruiz (Hospital de Manacor, Mallorca, Spain), Eider Oñate MD, Nagore García de Andoin Baran (Hospital Universitario Donostia, Donostia, Spain), Nerea Domínguez-Pinilla (Hospital Virgen de la Salud, Spain), María Teresa Coll Sibina (Hospital General de Granollers, Granollers, Spain), María Jesús García García (Hospital Universitario de Cáceres, Cáceres, Spain), Marta Osuna (Hospital HM Montepríncipe, Madrid, Spain), Raquel Portugal (Hospital Universitario de Burgos, Burgos, Spain), Leonor García Maset (Hospital de Sagunto, Valencia, Spain), Belén Sevilla (Hospital Universitario San Cecilio, Granada, Spain), Noelia Berciano Jiménez (Hospital Virgen Macarena, Sevilla, Spain).
Contributors AT, CM, EC-C and JMM designed the study, contributed to the collection and interpretation of the data, drafted the initial manuscript and reviewed the final version. SD-R and AB designed the study, made the statistical analysis and drafted the initial manuscript, and reviewed the final version. FL, JF-P, EP-R and II carried out the viral cultures, and JCG and JL-F carried out the sgRNA analysis. PGS, MdlS, JAA-C, AB, GS, LM-C, AFG-P, MI-R, DA-A, ABJ, PC, ED, IN, BF-G, PM, CP-J, TC-F, DB-G, GR, PG-D, PM, SM, and JL contributed to the collection and interpretation of the data, the creation of the manuscript and reviewed the final version. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work. AT and CM accepts full responsibility for the work and/or the conduct of the study, had access to the data, and controlled the decision to publish.
Funding The study was funded by: Project PI20/00095, from the Instituto de Salud Carlos III (Ministry of Economy, Industry and Competitiveness) and cofounded by the European Regional Development Fund, by Infanta Sofia University Hospital and Henares University Hospital Foundation for Biomedical Research and Innovation (FIIB HUIS HHEN), and by SERMAS-Fundación para la Investigación Biomédica del Hospital 12 de Octubre. EC-C is supported by the Spanish Society of Paediatrics (Asociación Española de Pediatría); Grant COVID-19 EPICO-AEP 2020. JMM is funded by SERMAS-Fundación para la Investigación Biomédica del Hospital Infanta Sofía y del Henares and by Universidad Europea de Madrid, Spain. MdlS is funded by Grant Cantera de Investigación Santander, Fundación Universidad Europea de Madrid, Spain. ED is funded by the Juan de la Cierva–Incorporación granted by the Spanish Ministry of Science and Innovation. DB-G is funded by the Spanish Ministry of Science and Innovation—Instituto de Salud Carlos III and Fondos FEDER by 'Contratos para la intensificación de la actividad investigadora en el Sistema Nacional de Salud, 2020 (INT20/00086)'.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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