Asymptomatic infections with SARS-CoV-2 are associated with viral transmission and have a key role in the propagation of the pandemic¹. Understanding viral shedding during asymp tomatic infections is critical. Unfortunately, data on asymptomatic SARS-CoV-2 infection in chil dren is extremely limited².

In adult patients with SARS-CoV-2 infection RT-PCR in respiratory samples can be positive 3 days up to several weeks from symptoms onset. Furthermore, some patients can become posi tive again after a period of negative testing³. In children, the mean time of positive RT-PCR is 11.1 days in symptomatic and 9.4 days in asymptomatic subjects⁴. However, a positive RT-RCR test does not necessarily reflect shedding of viable virus.

Infectiousness of SARS-CoV-2 can begin 2-3 days prior to symptoms onset and declines 7 days from symptoms onset. Viable SARS-CoV-2 detected in cell culture virus is isolated in as ymptomatic adults mostly within 7 days after the initial positive RT-PCR⁵. In symptomatic chil dren, viable virus was detected in cell culture up to 5 days after onset of symptoms⁶. Longer viral shedding has been described in very few pediatric cases, either from critical patients or from children with oncohematologic diseases. Specifically, viable SARS-CoV-2 was isolated up to day 54 in a critical pediatric patient and up to 139 days in an immunocompromised child with severe COVID-19². To our knowledge, there is a lack of data on isolation of SARS-CoV-2 in asymptomatic and otherwise healthy children.

The objective of this study was to determine the presence of viable virus by cell culture in sa liva samples from two asymptomatic and other wise healthy children infected with SARS-CoV-2.

A prospective study in a family group in fected with SARS-CoV-2 was conducted. Family members were prospectively followed for up to 28 days (during April, 2021). Demographic and clinical data were collected. This study was ap proved by the Ethics Committee of CEMIC (Pro tocol: 1298/20).

Sequential saliva and fecal samples were ob tained every 3 days. Nucleic acid was extracted from 100μl and eluted in 15μl using manual col umns (Quick-RNA TM Viral Kit, Zymo Research CORP.), following manufacturer’s recommenda tion.

Detection of SARS-CoV-2 was performed with an in‐house one‐step real time RT‐PCR multi plex assay targeting the E gene of SARS‐CoV‐2 and the human RNAsa P gene as an internal control, in a CFX 96 Deep Well™ Real Time Sys tem (BioRad). A positive result was considered when the human RNAse gene or the internal amplification control were positive and the cycle threshold (Ct) value was less than 40⁷.

To analyze the signature amino acid mu tations on the Spike protein of the variants of SARS-CoV-2, Sanger sequencing of segment 29 of the CDC amplification protocol that includes amino acids 428 to 750 was performed⁸.

SARS-CoV-2 isolation was performed in a BSL3 facility at Instituto de Investigaciones Bio médicas en Retrovirus y SIDA (INBIRS), Universi dad de Buenos Aires. Vero cell monolayers were inoculated with 300 μl of pre-filtered infec tious saliva sample diluted in 300ul of DMEM (Sigma) supplemented with Fetal Bovine Serum (4%), streptomycin (50 μg/ml), penicillin (50 U/ ml) and amphotericin B (125 ng/ml). Cells were monitored for virus-associated cytopathic ef fect (CPE) for 96 hours. Positive supernatants were confirmed by RT-qPCR.

SARS-CoV-2 serology was evaluated in both children using COVIDAR IgG assay, which uses a combination of a trimer stabilized spike pro tein and the receptor binding domain (RBD) in a single enzyme-linked immunosorbent assay (ELISA) plate (Fundación Instituto Leloir- CONICET-Laboratorio Lemos, Argentina)⁹.

A breakthrough male case with COVID-19 was identified on April 6, 2021 (Patient#1). His fam ily included his wife (43 years old, Patient#2), a 9 years old boy (Asymptomatic#1) and a 12 years old girl (Asymptomatic#2). Both parents were healthcare workers who had completed the Sputnik V vaccine scheme (2 doses) in Feb ruary 2021. All subjects were previously healthy. The family returned from a short holiday trip on April 4, 2021. On the same day, Asymptomat ic#1 developed pharyngitis. A rapid pharyngeal test obtained the following day was positive for Streptococcus pyogenes, and he received antibiot ics. His nasopharyngeal swab (NPS) for SARS-CoV-2 was negative.

On April 6, (day 1), Patient#1 (the index case) developed fever and myalgia and his NPS was SARS-CoV-2 positive. On the same day, Pa tient#2, who was asymptomatic, also tested positive for SARS-CoV-2. The following day, both asymptomatic children were RT-PCR negative for SARS-CoV-2 in saliva and NPS samples. On day 7, Patient#2 developed COVID-19 symptoms including fever, myalgia, arthralgia and head ache. On day 8, both asymptomatic children became SARS-CoV-2 positive. Both adults de veloped mild COVID-19 and remained RT-PCR positive for 21 and 25 days. Children remained asymptomatic throughout the study period and they had RT-PCR positive in saliva for 25 and 28 days. Viable viruses were detected in children by cell culture on days 8 and 17 (Fig. 1). Positive cell culture samples correlated with RT-PCR Ct val ues ranging from 22.3 to 33.4. In addition, stool samples were SARS-CoV-2 positive in both chil dren for up to 21 and 28 days.

Figure 1 SARS-CoV-2 isolation and detection by RT-PCR in saliva and NPS samples Sequential samples of Patient #1, #2, Asymptomatic #1 and #2. Filled circle: positive culture. Empty circle: negative culture. Dotted vertical line: Theoretical de-isolation date. Ct value ≥ 40 are RT-PCR negative results 

Viral sequencing in Patient#2 and both chil dren showed four mutations corresponding to E484K, N501Y, D614G and H655Y, consistent with B.1.1.28.1 lineage (Variant Gamma or Variant P.1). Both asymptomatic children seroconverted and showed detectable SARS-CoV-2 anti-Spike IgG levels (65 and 227 UI/ml).

SARS-CoV-2 pandemic affects mostly adult patients and shedding time of viable SARS-CoV-2 has been well established. However, data on viable shedding in asymptomatic healthy children is lacking. In this study, we describe the presence of viable SARS-CoV-2 from saliva samples in two asymptomatic healthy children.

Given the low rate of infections in pediat rics, asymptomatic children, even those with close contacts to positive cases, are usually not screened for SARS-CoV-2. In this study, two asymptomatic children living with their infect ed parents, who were prospectively followed, showed RT-PCR positivity and viable virus. This observation underscores the potential role of asymptomatic children in the spread of the vi rus, especially considering that most children remain asymptomatic¹⁰.

Interestingly, the asymptomatic child with viable viral shedding for at least 17 days from index case’s symptoms onset, would have been potentially contagious beyond the isola tion period that was suggested, in this mo ment, by the Ministry of Health in the region. The isolation period for asymptomatic close contacts of a positive case had been deter mined for 10 days from the case’s symptoms onset. Other works have shown that most of the children with COVID-19 have silent dis ease, but SARS-CoV-2 RNA can still be de tected in the respiratory tract for a prolonged period¹¹.

Successful cell culture isolation was associ ated with Ct values lower than 2312. In our study, isolation was successful even on samples with higher Ct values. This finding suggests that at least in children Ct value >23 cannot rule out the presence of viable virus. Murata et al. found similar results, from nasopharyngeal swab sam ples from an older adult who became infected with SARS-CoV-2 on a cruise ship⁵. In this study, sequence analysis demonstrated the presence of Gamma variant (lineage P1), which was cir culating in Argentina in 2021, but was later dis placed by Omicron variant¹³. Whether this vari ant remains contagious for longer periods or has a different kinetic in children is still to be determined.

RT-PCR in saliva samples was shown to be con venient and successful in detecting SARS-CoV-2 in symptomatic adult patients⁷. In our study, sa liva samples were also useful in detecting SARS-CoV-2 in asymptomatic children. Furthermore, these samples were also useful for successful viral isolation in cell culture. As nasopharyngeal swabs can be painful and bothersome, particu larly in children, saliva samples represent a more convenient, non-invasive and painless option^{14, 15}. Confirming the presence of true infections, both children were found to have anti-S IgG for SARS-CoV-2 in subsequent serum samples.

The main limitation of this study is that only two children were evaluated. Despite this limi tation, our observation showed that the pres ence of viable virus in saliva samples from as ymptomatic children can last for at least 10 days from the initial PCR positivity and can represent a source for spreading.

In summary, our observation underscores the importance of testing asymptomatic children since they can also shed viable virus for several days. Given the difficulties for obtaining nasal swabs in children, saliva samples can provide a reasonable alternative for detection of SARS-CoV-2.