Epidemiological, virological and serological investigation of a SARS-CoV-2 outbreak (Alpha variant) in a primary school: A prospective longitudinal study

Objectives To report a prospective epidemiological, virological and serological investigation of a SARS-CoV-2 outbreak in a primary school. Methods As part of a longitudinal, prospective, school-based surveillance study, this investigation involved repeated testing of 73 pupils, 9 teachers, 13 non-teaching staff and 26 household members of participants who tested positive, with rapid antigen tests and/or RT-PCR (Day 0–2 and Day 5–7), serologies on dried capillary blood samples (Day 0–2 and Day 30), contact tracing interviews and SARS-CoV-2 whole genome sequencing. Results We identified 20 children (aged 4 to 6 years from 4 school classes), 2 teachers and a total of 4 household members who were infected by the Alpha variant during this outbreak. Infection attack rates were between 11.8 and 62.0% among pupils from the 4 school classes, 22.2% among teachers and 0% among non-teaching staff. Secondary attack rate among household members was 15.4%. Symptoms were reported by 63% of infected children, 100% of teachers and 50% of household members. All analysed sequences but one showed 100% identity. Serological tests detected 8 seroconversions unidentified by SARS-CoV-2 virological tests. Conclusions This study confirmed child-to-child and child-to-adult SARS-CoV-2 transmission and introduction into households. Effective measures to limit transmission in schools have the potential to reduce the overall community circulation.


Introduction
within primary schools and early childhood education centres in a sample of five institutions in Geneva, and the risk of introduction of SARS-CoV-2 into the children's households. The study started in March 2021. Participants (pupils and their teachers) had a baseline assessment which included SARS-CoV-2 serology from a capillary blood test, an antigen rapid diagnostic test (RDT) from an oropharyngeal swab sample, and the completion of an online questionnaire. Then, a surveillance phase started, with weekly questionnaires and self-declarations (anytime outside of the weekly questionnaires) allowing participants to report COVID-19-like symptoms, contact with a positive case or the diagnosis of a SARS-CoV-2 infection. An outbreak investigation was triggered when a positive case was diagnosed from a positive real-time reverse transcription polymerase chain reaction (RT-PCR), which was the case on April 26, 2021. The date of diagnosis of the first positive case in a school class was referred to as Day 0 for that school class. The investigation involved repeated virological and serological testing of the participants: RDT and/or RT-PCR were performed at Day 0-2 and Day 5-7, serologies were performed at Day 0-2 and Day 30.

Study population
In the investigated school, all 9 school classes with children aged 3 to 6-7 years old participated in the SEROCoV-Schools study. The present study population consisted of children and school staff in school classes with a positive case as well as household members of the confirmed cases. School staff included teachers (including assistants) and non-teaching staff (administrative, cleaners, catering). Two children in Class 2 were absent from school for the 2 weeks preceding this outbreak, therefore they were considered as non-exposed and not included in the analysis.

Context and public health measures
During the period of this outbreak investigation, non-pharmaceutical public health measures in Geneva were gradually relaxed, with restaurants and bars opening their outdoor spaces and entertainment venues opening their indoor spaces.
Mitigation of the COVID-19 pandemic in Switzerland included school closures during the first wave from March to May 2020. Thereafter, priority was given to keeping schools open with several types of preventive measures in place which varied widely across institutions. In the investigated school, measures in place during the outbreak included checking children's temperature every morning, sending children home if they had fever or sickness beyond very mild symptoms, restricted access for parents and requiring all adults (but not young children) to wear facemasks.

Epidemiological investigation
Case definition. Cases were defined according to laboratory results: confirmed SARS-CoV-2-infected cases were those with positive SARS-CoV-2 RDT and/or RT-PCR results, and/or a seroconversion between Day 0-2 and Day 30 (from seronegative to seropositive according to the test-specific cut-off, unrelated to vaccination). Confirmed cases could be further classified as symptomatic or asymptomatic.
Outcomes. Only descriptive analyses are presented here. Infection attack rates (IAR) were defined as the number of children and school staff with a positive RDT and/or RT-PCR detected within a 21-day period of the index case identification, and/or a seroconversion, divided by the total number of children and school staff participating in the study. Household secondary attack rates (SAR) were defined as the number of non-index household members who tested positive by RDT and/or RT-PCR within a 21-day period of the first infection detected in each household or who seroconverted, divided by the total number of non-index members participating in the study. Adults were defined as individuals �18 years, whereas children were defined as individuals <18 years of age. Plausible directions of transmission were determined, when possible, on the basis of symptom onset and testing dates.
Contact tracing. Positive cases (or their parents in the case of children) were interviewed using a structured questionnaire investigating symptoms, contact with a positive person, school attendance, extracurricular activities, play dates/birthday parties, family/friend gatherings and best friends, in the 14 days prior to diagnosis.

Laboratory investigation
Virological testing. SARS-CoV-2 testing was performed on oropharyngeal swabs taken by nurses or medical doctors of the study team. This swabbing method is less invasive than nasopharyngeal swabs and more acceptable by young children. Children who were not attending school on the testing days were tested by their paediatrician or at a testing centre and communicated the results to our team. Depending on the visit, we performed RT-PCR or RDT tests. We used the Panbio COVID-19 Ag rapid test (Abbott) which has been validated in adults for use with oropharyngeal instead of nasopharyngeal swabs [31]. All oropharyngeal swab samples used for the RDT were tested a second time from the same swab by RT-PCR to confirm the result (in-house SARS-CoV-2 RT-qPCR or Cobas1 SARS-CoV-2 Test, Cobas 6800, Roche, Switzerland). SARS-CoV-2 whole genome sequencing was performed for positive samples at the Health 2030 Genome Center (Geneva) using the Illumina COVIDSeq library preparation reagents following the protocol provided by the supplier. All results were communicated to the participants as soon as they became available.
Serological testing. We collected capillary blood on a Neoteryx Mitra1 collection device at Day 0-2 and Day 30, and tested for anti-Spike-SARS-CoV-2 IgG on a microfluidic nanoimmunoassay as described previously [32]. This sampling method is minimally invasive, easily implementable in schools and more acceptable than traditional venipuncture by children. Serologies were communicated to the families approximately 2 months after the visit.

Ethics
This study was approved by the ethics committee of the Canton of Geneva (Project ID 2020-02957). All parents and teachers were informed about the study (orally and through a flyer and a detailed information note) and gave written consent. We also provided a short letter written in a child-friendly style, and a schema illustrating concepts of infection and antibodies, which parents could use to inform their child. Before each test, a team of trained nurses discussed with each child, explained the tests to be taken and answered any questions. Children gave verbal assent to participate.

Outbreak description, epidemiological and serological investigation
The first COVID-19 case (index case: Teacher 1 [T1], Class 1) was diagnosed by RT-PCR performed in a laboratory on April 26, 2021 (Fig 1), 3 days after the onset of symptoms (April 23). This participant reported the result to our team, which triggered the investigation. The second teacher in Class 1 (T2) had symptoms onset on April 24 with an initially negative RT-PCR on April 26 followed by a positive RT-PCR on April 28 (Fig 2). Both attended school until April 23 included. Children in another school class from the same grade (Class 2) were identified as having close contacts with children of Class 1 during breaks and specific activities. Class 2 was therefore tested on April 29, 2021. Of the 19 children aged 4 years old, 17 were tested by RT-PCR, of whom 2 (K6, K7) tested positive (IAR 11.8%), and 2 did not participate in the outbreak investigation (they both had only a negative RT-PCR performed externally on May 6). No additional case was identified at subsequent visits. We also repeatedly tested 4 teachers and 2 non-teaching staff in contact with Classes 1 and 2, none of whom was diagnosed with a SARS-CoV-2 infection (Table 1).
Of note, all children from these two school classes were placed in quarantine by local health authorities for 10 days from April 30, 2021.
The sibling of a positive case from Class 1, attending Class 3 in the same school, tested positive on May 2 (K9), triggering the testing protocol in Class 3. All 17 6-year old tested in Class 3 had a negative RT-PCR on May 4, and 16/16 had a negative RDT on May 11 (subsequently confirmed with a RT-PCR). Serological tests at Day 30 revealed 2 additional seroconversions (K17, K20, of whom one was not tested at Day 2 of the outbreak but seroconverted since baseline at the end of March 2021) (IAR 16.7%). In the other school class from the same grade, Class 4, tested because of close and regular contacts with Class 3, 15/15 had a negative RT-PCR on May 4, 13/13 had a negative RDT on May 11, and 2 additional cases of seroconversion (K18, K19) were identified (IAR 13.3%); 2 children in the school class did not take part in the study (Table 1, Fig 2). Among 3 teachers and 11 non-teaching staff, none tested positive.
Transmission occurred in 3 out of 10 investigated households of participants with a positive SARS-CoV-2 test, leading to a secondary attack rate among household members of 4/26 (15.4%). Three members of the same household refused to participate in this investigation, two of whom were vaccinated. Secondary attack rates were 1/2 (50.0%) with an adult index case, and 3/24 (12.5%) with a child index case. A teacher spent a few days at his/her parents' place while having symptoms, his/her parent then developed symptoms and tested positive (adult household member [HA17]). The parent (HA5) of a positive child from Class 1 tested negative twice (RDT on May 1, RT-PCR on May 7), though he/she seroconverted (Figs 2 and  3). He/she reported no contacts outside his/her household, strictly followed all recommendations, and was not vaccinated between the two blood draws. Finally, in the family of two children from Classes 1 and 3 who tested positive one after the other, a parent (HA1) who initially tested negative by RT-PCR, tested positive after quarantine with his/her children.
Among the 15 cases with a positive SARS-CoV-2 test, 3/3 (100%) adults and 9/12 (69%) children reported symptoms either before or after the positive test, and 3 children were asymptomatic. Among the 8 children who seroconverted without a positive RDT or RT-PCR test, 3 were symptomatic, 4 were asymptomatic, and one did not provide symptom information. Overall, 12/19 (63%) infected children reported symptoms. No severe form of COVID-19 (requiring hospitalization) was reported, and all cases recovered well.

Contact tracing analysis
The two infected teachers live alone and reported no contact with a positive or symptomatic case in their private life or activities, during the 14 days prior to infection. Their presumed source of infection was school, as several children from Class 1 were coughing during the week of April 19. The two infected parents were likely infected by their children, as they reported no contact outside the household.
Three social activities outside the school setting occurred in the two weeks before the triggering case was diagnosed: the first one (April 18, outdoors) brought together 6 children from Class 1 of whom 5 subsequently tested positive and 3 kids from another school (not included in the study); the second one (April 21, both indoors and outdoors) gathered 4 children from Class 1 and Class 2 of whom 3 tested positive (the other one had antibodies at baseline and at Visit 1); the last one (April 24, outdoors) was attended by 2 kids from Class 1 and 2 kids from another school (not included in the study), 1 tested positive at the 2nd visit.

Virological investigation
We conducted whole genome sequencing on samples from the 15 participants who tested positive for SARS-CoV-2. Coverage of three isolate genomes (HA1, K9 and K10) were insufficient for any comparisons due to low viral load in the specimen (32%, 20% and 16% coverage only, respectively). The other sequenced genomes belonged to the Alpha variant. The virus sequence of K12 differed from the others in one region covered. Here, in position 15824-15827 a deletion and one addition that restores the reading frame was observed, resulting in a total of 4 nucleotides difference compared to the other sequences. All 9 sequences with >99% coverage shared 100% identity between genomes in comparison to the reference sequence (Fig 4). Virus specimens that could only be partially sequenced retrieved the same sequence without additional mutations in the regions covered.

Discussion
This is one of the first investigation of a SARS-CoV-2 outbreak caused by the Alpha variant in a primary school. It involved 20 children from 4 school classes, 2 teachers and 4 household members (of whom one triggered the investigation in another school class). The primary case could not be formally identified, but it is likely that this outbreak started during the week of April 19 and was only identified on April 26 when a teacher tested positive and triggered the investigation. Of note, children in this age group were not routinely tested by the official testing recommendations in Switzerland when symptomatic [12]. This prospective school classbased study provides evidence of SARS-CoV-2 circulation among young children, school teachers, and introduction into households. Since at least 9 positive cases of this outbreak shared viruses with identical sequence, we conclude that they are part of the same cluster. This could have been either by simultaneous infection through the same source, or transmission chains between affected individuals. One divergent virus sequence was found in one of the positive children, which could be either a de novo mutation occurring during the outbreak, or constitute independent introductions into the school, with one leading to this cluster. It cannot be ruled out that, by coincidence, several infection events with viruses sharing the same sequence were introduced from the community independently into the school. However, this hypothesis seems less likely given the number of cases involved, the epidemiological link and time frame, and the limited period of potentially other exposures before the quarantine decision. Overall community circulation at the cantonal level was also relatively low at the time (weekly incidence: about 200 cases/100,000 inhabitants). Between April 15 and May 15, the Alpha variant was causing 92% of new SARS-CoV-2 infections in Geneva [33].  Other main insights are as follows. First, viral circulation of the Alpha variant in young children aged 4-6 years old was high, with a majority of unspecific and mild symptomatic infections, which might explain relatively high secondary attack rates [13]. The observed cluster probably started in one class and spread to two other classes, most likely through direct childto-child contacts and interactions as per our epidemiological investigation, although there could be non-sampled child or adult intermediaries. We identified two seroconversions in a fourth class, but with no identification of SARS-CoV-2 virus, thereby limiting the conclusions on transmission linked to the other school classes. Similar patterns were found in a prospective study of three B.1.1.7 variant outbreaks which occurred in three childcare centers in Germany in January-February 2021: 11/12 groups had secondary cases, and secondary attack rates ranged from 7 to 64% among groups of children aged 1-6 years [34].
Second, child-to-parent transmission occurred in two different households, and child-toteacher transmission is probable, which supports previous findings [18,[34][35][36]. Of note, vaccination was opened to all persons aged 45 years or older from April 12, and to all people in the 16-44 age group from May 19. Therefore, only a minority of adults (1 household member and 1 non-teaching staff) were vaccinated at the beginning of the outbreak. Child-to-adult transmission seems to depend on the duration of contacts, as no infection was identified among non-teaching staff who spent only limited periods of time (meals) with children. This is contrary to previous findings [37], and may be explained by the young age of our participants and their behaviour involving physical proximity with their teachers [38].
Third, social activities outside the school could have contributed to the spread of the infection, as previously reported [39,40]. However, they could also reflect the bonding between children and a closer contact at school, thus facilitating transmission.
Fourth, we evidenced low sensitivity of RDTs with an oropharyngeal specimen for identifying both symptomatic and asymptomatic infected children, which confirms previous results showing that only children with high viral load are identified by such tests [23,41]. We conclude that RDTs with an oropharyngeal specimen are not the most appropriate for surveillance and/or outbreak investigation purposes. The analysis of joint RT-PCR/serological data shows a substantial under-detection of infections in young children, even with RT-PCR testing, although the optimal time point for viral testing might have been missed in the fixed testing scheme of this study. This finding is consistent with prospective research based on comprehensive testing protocols which identified a higher incidence of COVID-19 among children and higher rates of transmission than previously reported, including in households [14,35,36]. Under-detection of acutely infected children may be an explanation for the discrepancy between official numbers of infected children [42] and studies on virus prevalence and seroprevalence in school settings [24,28,[43][44][45][46][47].
As a take-home message, we suggest that routinely reported infections are likely to be just the tip of the iceberg, highlighting the importance of testing contacts, regardless of symptoms, to identify SARS-CoV-2 infections and stop transmission by rapidly isolating positive cases.

Strengths and limitations
Few large SARS-CoV-2 outbreaks in young children in school settings have been documented so far [48,49] even fewer involved an investigation of variants of concern. As part of an ongoing prospective study, this investigation started less than 24 hours after the first case was laboratory-confirmed and involved the use of three complementary approaches. We followed up and repeatedly tested all contacts within four school classes regardless of symptoms. Repeated serological tests proved useful to retrieve seroconversions following asymptomatic or undiagnosed infections. However, we relied on a limited number of cases, even though, by definition, the sample size of a school class outbreak can only be as large as the number of pupils in each school class. Not all children and adults were tested, which could lead to underestimating IAR and SAR. We might have missed infections among adults who were vaccinated between Day 0-2 and Day 30, as we could not distinguish between antibodies due to vaccination and those due to infection. Also, we could not test the household members of cases only detected by seroconversion at Day 30 with no positive RT-PCR/RDT, leading to a potential underestimation of secondary attack rates. The study was performed before the circulation of the more infectious Delta and Omicron variants; estimates are therefore likely to be underestimated [50][51][52][53].

Conclusion
This prospective school-based study provides evidence of SARS-CoV-2 transmission among young children and school teachers and introduction into households. Epidemiological investigation confirmed child-to-child and child-to-adult direction of transmission of the infection. Children may be a significant source of extra-household infections and have the potential to play a role in community transmission, potentially even more so with more contagious variants. With most of the adult and adolescent population vaccinated, young children could act as disease reservoirs. Effective strategies are needed to limit transmission in school settings and vaccination of school staff and children should be encouraged.
of Diagnostics, Geneva University Hospitals, Geneva, Switzerland; Department of Pediatrics,