#Stayathome If You Have a Cold: High SARS-CoV-2 Salivary Viral Loads in Pediatric Patients with Nasopharyngeal Symptoms

The choice of the best SARS-CoV-2 detection approach is crucial to predict which children with SARS-CoV-2 are at high risk of spreading the virus in order to manage public health measures and policies. In this prospective observational study of 35 children admitted to the Pediatric Emergency Departments of two tertiary hospitals in Northern Italy who tested positive for SARS-CoV-2 by standard RT-PCR in nasopharyngeal swab (NPS), we evaluated their presenting symptoms according to their salivary viral load (SVL) determined by droplet digital PCR (ddPCR). Despite an overall low concordance between SARS-CoV-2 detected by salivary ddPCR and NPS RT-PCR (54.3%), when only patients with nasopharyngeal symptoms were analyzed, the sensitivity of ddPCR in saliva specimens increased to 71.4%, and over half of these patients had high SVL (>105 copies/mL), which was significantly more frequent than in children without nasopharyngeal symptoms (57.1% vs. 14.3%, OR = 8, CI 95% 1.28–50.03, p = 0.03). All asymptomatic children had low SVL values. Our findings support the hypothesis that children with nasopharyngeal symptoms are at higher risk of spreading SARS-CoV-2 due to their high SVL and, conversely, asymptomatic children are unlikely to spread the virus due to their low SVL, regardless of their NPS positivity.


Introduction
Predicting which children with SARS-CoV-2 are at high risk of spreading the virus would be useful for managing public health, reaching a proper balance between COVID-19 containment and the limitation of common life activities, but this relies on choosing the best SARS-CoV-2 detection approach. The current gold standard for SARS-CoV-2 detection is RT-PCR amplification of viral genes from nasopharyngeal swab samples (NPS), although saliva has also been explored as an alternative [1,2]. In pediatric patients, the reported concordance rates between upper respiratory tract swabs and saliva have been highly variable (52.9-89.5%) [1,[3][4][5], with better performances in older children and after a shorter duration of symptoms [1,5]. However, the association between COVID-19 symptoms and SARS-CoV-2 salivary viral load (SVL) in children is poorly defined. We therefore examined the clinical features of a cohort of children who tested positive for SARS-CoV-2 by standard RT-PCR in NPS samples according to their SVL determined by droplet digital PCR (ddPCR).

Materials and Methods
This was a prospective observational study of patients admitted to the Pediatric Emergency Departments (ED) of two tertiary hospitals in Novara and Alessandria, Pied-mont, Italy between November 2020 and May 2021, when the Alpha variant was the main circulating SARS-CoV-2 variant in Italy (https://www.epicentro.iss.it/coronavirus/ pdf/sars-cov-2-monitoraggio-varianti-rapporti-periodici-19-maggio-2021.pdf, accessed on 24 December 2022). A ddPCR test was performed on saliva specimens from non-vaccinated pediatric patients (0-15 years) with a SARS-CoV2-positive NPS. SVL assessed by ddPCR was correlated with clinical symptoms at admission and the NPS RT-PCR cycle threshold (Ct) values. Parents or guardians provided written informed consent, and the local ethics committee (Comitato Etico Interaziendale Novara, CE 8/21, Novara, Italy) approved the study protocol.

Demographic and Clinical Data
General information including age, gender, and ethnicity were collected for each patient. The presenting symptoms at ED admission were collected and categorized according to fever, dyspnea, cough, diarrhea/abdominal pain, vomiting, asthenia, headache, rhinitis/pharyngodynia, ageusia/anosmia, exanthema, and conjunctivitis. COVID-19 related symptoms were always asked for at the ED admission, even when they were not the reason for the ED referral. When no symptoms were reported, patients who had been admitted for injuries or foreign body ingestion but tested positive on NPS RT-PCR performed at ED admission were also recorded. Prescribed therapy at discharge or need for hospitalization were also documented. Data were recorded by the investigators in an electronic case report form (REDCap v10.3.3, Vanderbilt University, Nashville, TN, USA).

Study Procedures
NPS and saliva samples were collected on the same day, with no clinical interventions occurring between sample collection. NPS were collected by trained healthcare workers in tubes containing 2 mL of 1x Hanks' balanced salt solution without phenol red (Thermo Fisher Scientific, Waltham, MA, USA). Samples were taken at any hour of the day and were sent the same day or the next morning to the molecular diagnostics laboratory for RT-PCR analyses. As soon as the NPS tested positive, saliva was collected by a pipette from the sublingual region (at least 1 mL of specimen) and then transferred in a 2 mL sterile tube and sent to the Molecular Virology Laboratory (University of Piemonte Orientale, Novara, Italy) for ddPCR analysis.

SARS-CoV-2 RNA Detection and Quantification in Saliva
Commercially available kits were used to detect SARS-CoV-2 virus in COVID-19 patients [Allplex 2019-nCoV Assay (Seegene, Seoul, Republic of Korea): Xpert Xpress CoV-2 plus (GeneXpert, Cepheid, Sunnyvale, CA, USA); Simplexa COVID-19 Direct (DiaSorin Molecular, Cypress, CA, USA); Viasure SARS-CoV-2 BD MAX System (CERTest Biotec, Zaragoza, Spain)]. Nasopharyngeal swabs were processed according to the manufacturers' instructions. A specimen was considered positive if the gene target had a Ct < 40 [6]. Only the qualitative result of positivity was initially provided by the molecular diagnostics laboratory and the Ct value was recorded in REDCap at a later time.
For ddPCR, total RNA was extracted from 200 µL of saliva using the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. SARS-CoV-2 genomic RNA was quantified using the QX200 Droplet Digital PCR System (ddPCR, Bio-Rad, Hercules, CA, USA) using the SARS-CoV-2 Droplet Digital PCR Kit (Bio-Rad, Hercules, CA, USA). SARS-CoV-2 quantification was expressed as copy number/mL of saliva.

Statistical Analysis
Quantitative data are presented as medians and interquartile ranges (IQR). Categorical variables are summarized as counts and percentages. Differences in median were evaluated using the Mann-Whitney test. Associations between categorical variables were tested using Pearson's chi-squared test or Fisher's exact test, as appropriate. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated. Pearson's correlation coefficient was applied to measure the relationship between two quantitative variables. A two-sided p-value < 0.05 was considered statistically significant. Analyses were performed using STATA software, v17 (Stata-Corp. 2021. Statistical Software: Release 17.0. College Station, TX, USA: Stata Corporation).

Results
Of 78 eligible subjects testing positive for SARS-CoV-2 by RT-PCR of NPS samples, 12 were excluded due to insufficient saliva samples and 31 were excluded because consent was not obtained (Figure 1). Thus, SARS-CoV-2 SVL was determined by ddPCR in 35 children (Table 1).

Statistical Analysis
Quantitative data are presented as medians and interquartile ranges (IQR). Categorical variables are summarized as counts and percentages. Differences in median were evaluated using the Mann-Whitney test. Associations between categorical variables were tested using Pearson's chi-squared test or Fisher's exact test, as appropriate. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated. Pearson's correlation coefficient was applied to measure the relationship between two quantitative variables. A two-sided p-value < 0.05 was considered statistically significant. Analyses were performed using STATA software, v17 (Stata-Corp. 2021. Statistical Software: Release 17.0. College Station, TX, USA: Stata Corporation).

Discussion
Here, we report high SARS-CoV-2 SVL in children with rhinitis/pharyngodynia. Despite an overall low concordance between SARS-CoV-2 detected by salivary ddPCR and NPS RT-PCR (54.3%), when only patients with nasopharyngeal symptoms were analyzed, the sensitivity of ddPCR in saliva specimens increased to 71.4%, and over half of these patients had high SVL (>10 5 copies/mL), which was significantly more frequent than in children without nasopharyngeal symptoms. The overall low concordance between the two techniques is consistent with previous reports, showing high variability between 53% and 100% both in children and in adults [1,[3][4][5]7,8]. It could be, at least in part, explained by the lower number of copies per ml in saliva than in swabs and by the variable interval between the onset of symptoms and sample taking [1,4], as shown by Kam KQ et al., who reported that peak SARS-CoV-2 viral loads occurred around day two of illness in infected children [9]. Moreover, four different RT-PCR assays were interchangeably used by our molecular diagnostics laboratory for a diagnostic purpose, and it was not possible to find out which one was used for every test. Indeed, this could represent another possible source of low concordance between saliva specimens and NPS. In our cohort, all asymptomatic children had low SVL values. Taken together, our findings support the hypothesis that children with nasopharyngeal symptoms are at higher risk of spreading SARS-CoV-2 due to their high SVL, and asymptomatic children are unlikely to spread the virus due to their low SVL, regardless their NPS positivity. Our data suggest that salivary ddPCR cannot replace standard NPS RT-PCR for SARS-CoV-2 infection, but it may be useful for stratifying patients with a low or high risk of viral spreading.
In conclusion, children with nasopharyngeal symptoms are likely to spread the virus, as confirmed by their high SVL values and recent data from the delta and omicron variant waves [10][11][12].

Discussion
Here, we report high SARS-CoV-2 SVL in children with rhinitis/pharyngodynia. Despite an overall low concordance between SARS-CoV-2 detected by salivary ddPCR and NPS RT-PCR (54.3%), when only patients with nasopharyngeal symptoms were analyzed, the sensitivity of ddPCR in saliva specimens increased to 71.4%, and over half of these patients had high SVL (>10 5 copies/mL), which was significantly more frequent than in children without nasopharyngeal symptoms. The overall low concordance between the two techniques is consistent with previous reports, showing high variability between 53% and 100% both in children and in adults [1,[3][4][5]7,8]. It could be, at least in part, explained by the lower number of copies per ml in saliva than in swabs and by the variable interval between the onset of symptoms and sample taking [1,4], as shown by Kam KQ et al., who reported that peak SARS-CoV-2 viral loads occurred around day two of illness in infected children [9]. Moreover, four different RT-PCR assays were interchangeably used by our molecular diagnostics laboratory for a diagnostic purpose, and it was not possible to find out which one was used for every test. Indeed, this could represent another possible source of low concordance between saliva specimens and NPS. In our cohort, all asymptomatic children had low SVL values. Taken together, our findings support the hypothesis that children with nasopharyngeal symptoms are at higher risk of spreading SARS-CoV-2 due to their high SVL, and asymptomatic children are unlikely to spread the virus due to their low SVL, regardless their NPS positivity. Our data suggest that salivary ddPCR cannot replace standard NPS RT-PCR for SARS-CoV-2 infection, but it may be useful for stratifying patients with a low or high risk of viral spreading.
In conclusion, children with nasopharyngeal symptoms are likely to spread the virus, as confirmed by their high SVL values and recent data from the delta and omicron variant waves [10][11][12].
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v15010081/s1. Table S1: Clinical data, Ct value from RT-PCR of NPS, and SVL by ddPCR of the enrolled subjects.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee (Comitato Etico Interaziendale Novara, CE 8/21, Novara, Italy).

Informed Consent Statement:
Informed consent was obtained from parents or guardians of all children involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.