Population, case definitions, and epidemiological investigation

Lansingerland is a municipality in the region Rotterdam-Rijnmond in the Netherlands, with 63,338 inhabitants (January 1^st 2021, www.cbs.nl). School X is located in Lansingerland and has two locations. It shares facilities with two primary schools, five after-school programs, and two day care centres for children under the age of 4 (hereafter ‘neighbouring schools’). Cases were individuals with a SARS-CoV-2 laboratory confirmed by RT-PCR or rapid antigen assay and were residents of Lansingerland (between November 16^th 2020 until February 22^th 2021), or had an epidemiological link to School X (November 16^th 2020 until January 1^st 2021), or the neighbouring schools (November 16^th 2020 until January 11^th 2021). Alpha and non-Alpha variant cases were cases confirmed by additional laboratory testing (variant PCR or whole genome sequencing). Symptomatic or post-exposure testing (routine testing) and population-based screening were performed in four phases based on the course of the outbreak (Table 1). Phase 1 targeted individuals with an epidemiological link to School X and their household members, and phase 2 targeted individuals with an epidemiological link to neighbouring schools of School X and their household members. Phases 3 (voluntary population-based screening) and 4 (post-outbreak monitoring) targeted all residents of Lansingerland. We distinguished cases identified through routine testing (A), and those who were found through voluntary population-based screening (B). Individuals with multiple test results were only included once in the analysis with priority given to the first positive test result. Ethical approval was not required for this study since outbreak investigations of notifiable diseases are a legal task of the Public Health Service as described under the national Public Health Act.

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Table 1. Number of cases stratified by study phase from November 16^th 2020 to February 22^nd 2021.

https://doi.org/10.1371/journal.pone.0276696.t001

National guidelines for routine SARS-CoV-2 testing and contact tracing

SARS-CoV-2 testing (RT-PCR or rapid antigen test) was provided free of charge by the regional Public Health Service (PHS) to individuals with COVID-19 like symptoms or after recent contact with a confirmed case. During the study period, national guidelines did not advise testing of children younger than 7 years (due to it being an invasive procedure). Children age 7–12 years were tested when symptomatic (fever, chest tightening), or after recent contact with a confirmed case. As a result, routine testing was biased towards children ≥7 years of age. Contact tracing was performed by phone interview to individuals with lab-confirmation of SARS-CoV-2 infection to assess symptom onset and to make an inventory of contacts.

Population-based screening and questionnaire

School children, staff, and their household members were invited by email for free of charge SARS-CoV-2 testing (phase 1B and 2B). For the population-based screening of the Lansingerland municipality, all residents received a personal letter and were informed through a town hall meeting chaired by the mayor which was broadcasted on regional and national media (phase 3B). Individuals in phase 1B and 2B were invited by email to fill in a questionnaire including name, address, date of birth, school, classroom, date of symptom onset, and COVID-19 symptoms.

SARS-CoV-2 diagnostics, S gene target failure, and whole genome sequencing

Nasopharyngeal/throat specimens were obtained by trained personnel at a SARS-CoV-2 test facility and specimens were sent to routine diagnostic laboratories for RT-PCR or tested by trained personnel using a rapid antigen test. Residual positive specimens (collected for either RT-PCR or rapid antigen test) obtained from individuals with an epidemiological link to School X, neighbouring schools, and residents of Lansingerland were requested from the diagnostic laboratories during the study period and tested using the TaqPath RTqPCR (ThermoFisher, Waltham, Massachusetts, United States) for S gene target failures (SGTF). Specimens with N and/or Orf1ab targets below cycle threshold (Ct) value 32, and negative for S were considered as SGTF. SGTF specimens below Ct value 32 were confirmed by whole genome sequencing (WGS) or N501Y PCR to determine the variant. WGS was performed using the Nanopore platform (Oxford Nanopore Technologies, Oxford, UK) or Illumina platform (Illumina, California, US) as described previously [9, 10].

SARS-CoV-2 detection in sewage

Wastewater specimen were collected at pumping station Bergschenhoek (a village with 18.250 inhabitants within the municipality Lansingerland), and Feijenoord (neighbourhood of the nearby city Rotterdam) as reference. Specimens were collected three days per week as 24h flow-dependent composite samples and processed as previously described [11]. The levels of SARS-CoV-2 were determined and normalized to the proportion of domestic sewage in the samples [12].

Data and statistical analyses

The regional database with personal identifiers, SARS-CoV-2 test results, symptom onset, and symptoms was merged with the questionnaire and WGS databases. Data were analysed using SPSS Statistics for Windows, version 25 (IBM, Armonk, NY) and R version 4.1.0. Households with at least one individual tested in population-based phases were included in the household analysis (test results of all phases were included for these households). We assumed that all members of a household were infected by the same variant when not all household members’ specimens were available for variant testing. Households with infections with multiple variants were excluded from the analysis. Differences between groups were tested by t-test for continuous variables or chi-square for proportions. Confidence intervals were calculated using the Wilson method. Data obtained in this outbreak investigation could not be shared with a public repository due to the privacy and confidentiality of the individuals involved in this outbreak.

Sequence analysis

The raw sequence reads were demultiplexed using Porechop (https://github.com/rrwick/Porechop) and processed as previously described for Nanopore [9]. For Illumina sequencing adapter sequences and primer sequences were trimmed, sequence reads were mapped against the GISAID sequence EPI_ISL_412973, and consensus sequences were generated in Geneious v.9.1.8. All Alpha variant sequences from December 12^th 2020 until February 22^nd 2021 from the Netherlands and sequences from 21 September 2020 until 21 October 2020 from the UK were downloaded from the GISAID database [13]. Identical (or nearly identical) Dutch sequences (maximum 2 nucleotides difference) within the same collection month were removed using an in-house developed R script (https://github.com/dnieuw/alireduce). The remaining sequences were aligned with the outbreak sequences using MUSCLE [14]. The alignment was manually checked for discrepancies, and IQ-TREE was used to perform a maximum-likelihood phylogenetic analysis under the GTR + F + I + G4 model as the best predicted model using the ultrafast bootstrap option with 1,000 replicates [15]. The phylogenetic tree was visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). A cut off of maximal 2 nucleotides was used for sequence cluster detection based on the virus mutation rate and sequence diversity detected in previous outbreaks [16, 17]. The SARS-CoV-2 lineage was determined using the Pangolin tool [18].

Outbreak detection

On December 12^th 2020, a cluster of SARS-CoV-2 cases was detected at a school with two locations in Lansingerland (School X). The affected school was closed on the same day to prevent further transmission. On December 23^rd, routine random sequencing of samples as part of the genomic surveillance effort showed the presence of the Alpha virus variant in a patient tested through one of the test facilities operated by the regional PHS. The patient was indirectly linked to School X and this link suggested that the outbreak at School X was caused by the Alpha variant. Analysis of case notifications by postal code showed that the number of positive samples per 100,000 residents in Lansingerland in December 2020 were elevated compared to the rest of the Rotterdam-Rijnmond region (S1 Material). There were no other outbreaks reported to the PHS at the time.

Outbreak school X

Routine testing and population-based screening of children and staff of School X, and their household members (phases 1A and 1B) detected a total of 125 SARS-CoV-2 cases: 35 children, 17 staff members at School X and 73 household members (Table 1, Fig 1). Molecular typing showed that 77 out of 91 (85%) SARS-CoV-2 cases with a link to School X for which genotyping could be done were infected with the Alpha variant in phases 1A and 1B.

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Fig 1. Timeline of cases at School X by classroom and school location in Lansingerland.

Each line represents one case with symptom onset (black diagonal cross) and sampling date with variant (coloured box). School children and staff of School X were voluntarily tested regardless of symptoms between December 30^th 2020 and January 1^st 2021 (phase 1A and 1B).

https://doi.org/10.1371/journal.pone.0276696.g001

The symptom onset of the first two cases, a staff member and a child, was November 27^th 2021 at School X in classroom 1. The outbreak subsequently spread to a total of 11 classrooms at both locations (a and b) of School X with 1 to 16 cases per classroom (Fig 1). Out of 6 classrooms at location a, a child was the first symptomatic case in 4 classrooms, an adult was the first symptomatic case in 1 classroom, and in 1 classroom both a child and adult were symptomatic first. The first detected symptomatic case at school location b was an adult, which suggests that the staff of School X most likely transmitted the virus from location a to b. Out of 5 classrooms at location b, a child was the first symptomatic case in 1 classroom, an adult was the first symptomatic case in 3 classrooms, and in 1 classroom both a child and adult were symptomatic first. At least eight staff members worked at both locations, of whom four staff members tested positive. Of 18 households with children at both locations, at least four households had one or more positive cases. Of note, 5 symptomatic SARS-CoV-2 cases diagnosed as part of the outbreak in School X initially tested negative by routine testing (4 by rapid antigen test and 1 by RT-PCR). WGS analysis showed that 33 out of 35 (94%) Alpha variant sequences derived from specimens collected in phase 1A and 1B belonged to a cluster A (Fig 2), confirming the outbreak at School X and transmission to household members. The cases with a sequence indicated with b and c (household contact of a case of School X and child at School X, respectively) were considered unrelated.

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Fig 2. Maximum likelihood analysis of 60 Alpha variant SARS-CoV-2 whole genome sequences obtained from individuals living in the municipality Lansingerland, the Netherlands, or with an epidemiological link to School X or neighbouring schools and child care centres from December 30^th 2020 to January 22^nd 2021, in a background of 140 Alpha variant sequences obtained from the Netherlands from November 29^th 2020 until February 22^th 2021 and 20 Alpha variant sequences from the United Kingdom from September 21^th until October 21^th 2020 (black).

Colours indicate the study phase in which specimens were collected. Scale bar represents the number of nucleotide substitutions per site. The text next to each sequence indicates the GISAID accession id and sample collection date.

https://doi.org/10.1371/journal.pone.0276696.g002

Outbreak phase 2 neighbouring schools

After three teachers at a neighbouring primary school tested positive for the Alpha variant on December 31^st 2020, retrospective case finding of routinely tested individuals and population-based screening were extended to children and staff of 2 neighbouring primary schools, 5 afterschool programs, and 2 child day care centres neighbouring School X (hereafter ‘neighbouring schools’), and their household members (phase 2A and 2B). A total of 44 SARS-CoV-2 cases were detected with a link to the neighbouring schools. 25 samples were successfully sequenced or typed by variant PCR, of which 16 (64%) were confirmed Alpha variant cases (Table 1), suggesting the Alpha variant spread from School X to the neighbouring schools. These 16 cases infected with Alpha variant were four staff members, four children, and eight household members. WGS analysis showed that four out of six available full genome sequences belonged to the same sequence cluster as School X (cluster A), supporting the hypothesis that cases with an epidemiological link to School X transmitted the virus to cases with a link to the neighbouring schools (Fig 2). Two out of six Alpha variant sequences obtained in phase 2A (cluster D) had three identical mutations when compared to cluster A, potentially reflecting a separate introduction.

Lansingerland municipality

The rapid spread of the Alpha variant among individuals of both schools and their household members raised the question to which extent the Alpha variant was circulating in the community of Lansingerland. Therefore, samples from SARS-CoV-2 cases confirmed by routine testing were retrieved for variant typing from November 16^th 2020 until January 22^nd 2021 (phase 3A). The Alpha variant was retrospectively detected in 34 out of 311 (11%) positive specimens for which genotyping could be done and which had no (known) epidemiological link to the School X or neighbouring schools (Table 1). Alpha variant sequences obtained through this approach (n = 10) were more diverse, grouping in clusters A and J, and unrelated sequence h (Fig 2). All remaining residents of Lansingerland were subsequently invited for testing from January 11^th until 22^nd 2021 (Phase 3B), and 36,534 of 63,338 (58%) residents were tested. 196 out of 36,534 (0,5%) residents tested positive. Thirteen out of 106 (12%) cases for which genotyping could be done were infected by the Alpha variant (Table 1). Out of 9 available Alpha variant Phase B sequences, only 1 sequence clustered with sequences from School X; other sequences were found in clusters F, I, and J, or as unrelated sequences e and g (Fig 2). The sequence results of phases 3A and 3B combined indicate that the Alpha variant had been introduced multiple times in the municipality of Lansingerland until January 22^nd 2021.

After the population-based screening campaign, enhanced surveillance was done to continue monitoring the circulation of the Alpha virus variant. SARS-CoV-2 positive specimens from routinely tested residents of Lansingerland were requested from diagnostic laboratories and tested by variant PCR to monitor the circulation of the Alpha variant in Lansingerland from January 23^rd 2021 until February 22^nd 2021 (phase 4). Of 265 of these SARS-CoV-2 cases, 176 specimens (66%) could be retrieved for variant testing. The Alpha variant was detected at low levels (5 out of 24 [21%] of cases that had sufficient loads for variant typing) in week 4 (January 25^th to 31^st 2021), but increased to 45 out of 53 (85%) cases with sufficient loads for genotyping in week 7 (February 15^th to 21^st 2021) (Fig 3 bottom). Variant replacement in Lansingerland was 1 to 2 weeks ahead compared to national surveillance data (Fig 3 bottom). Positive specimens in phase 4 were tested by variant PCR and were not included in the sequence analysis.

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Fig 3.

Number of confirmed SARS-CoV-2 cases per week in Lansingerland (including individuals with an epidemiological link to School X or neighbouring schools) by variant (bottom). Line shows the percentage of Alpha variant among national surveillance specimens per week on the right y-axis (data obtained via https://www.rivm.nl/coronavirus-covid-19/virus/varianten) (bottom). Number of individuals tested for SARS-CoV-2 in Lansingerland (including individuals with an epidemiological link to School X or neighbouring schools) (middle). Levels of normalized SARS-CoV-2 copies per mL of domestic wastewater in Bergschenhoek (village in Lansingerland) and Feijenoord (neighbourhood in Rotterdam) (top). Domestic wastewater data were only available from January 11^th 2021 onwards. Vertical dashed lines indicate: (+) First confirmed case with Alpha variant at School X, (x) School X closed, (1B) Period of population-based screening at School X (phase 1B), (2B) Period of population-based screening neighbouring schools (phase 2B), (3B) Period of population-based screening residents Lansingerland (phase 3B). *Data incomplete for this week.

https://doi.org/10.1371/journal.pone.0276696.g003

Levels of SARS-CoV-2 in waste water were quantified to study SARS-CoV-2 circulation independent of testing strategy in Lansingerland, and compared to a different community within the city of Rotterdam (Feijenoord) during and after phase 3B. The normalized SARS-CoV-2 RNA copies per mL in Lansingerland followed a similar trend compared to Feijenoord at lower levels, suggesting less virus circulation in Lansingerland compared to Feijenoord from January 11^th 2021 onwards (Fig 3 top).

Clinical symptoms and secondary attack rate

The population-based testing allowed us to make comparisons of the clinical symptoms and secondary attack rate between Alpha and non-Alpha infected individuals. Of 478 cases with available data on symptoms and virus variant, 97 out of 127 (76%) Alpha variant infected cases and 256 of 351 (73%) non-Alpha infected cases reported to be symptomatic (no significant difference, p = 0.449). The mean number of reported symptoms was 2.1 and 2.3 for respectively Alpha and non-Alpha infected symptomatic cases (p = 0.442). Stratification of the analysis by age group did not show significant differences in reported clinical symptoms among age groups (S2 Material).

Households with prospectively tested cases and for whom variant typing could be done were selected to determine the household secondary attack rate (Table 2). The mean age among household members with infection(s) with the Alpha variant was significantly younger compared to household members with non-Alpha variant infection(s) (p = 0.019). There were significantly more children in the households infected with the Alpha variant (p = 0.003) and significantly more adults in non-Alpha infected households (p<0.001). Of 100 individuals at risk in Alpha variant infected households, 41 (41%, 95% confidence interval (CI) 32%-51%) individuals became infected, and 95 of 346 (27%, 95% CI 23%-32%) individuals at risk in non-Alpha variant infected households became infected; a 52% higher transmission among contacts in Alpha variant infected households (p = 0.010).

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Table 2. Characteristics of households and household members infected by Alpha versus non-Alpha variants.

https://doi.org/10.1371/journal.pone.0276696.t002

Two households were excluded since both variants were detected. Among 37 Alpha variant infected households, 21 households (57%, 95% CI 41%-71%) showed transmission to at least one contact, which was 24% higher than for non-Alpha variant households (63/136; 46%, 95% CI 38%-55%). This difference was not significant (p = 0.260). The household size was not significantly different between Alpha and non-Alpha variant infected households (p = 0.410).

Members of the outbreak investigation team

Paul Bijkerk¹, Timo Boelsums*¹, Marjan Boter², Rachida Bouali¹, Danny Colijn¹, Adnan Elkaka¹, Diana Filipov¹, Bibianne Geerts¹, Marijke Langenberg¹, Bas Lohman¹, Marloes Mattijssen¹, Corien Middelbeek¹, David F. Nieuwenhuijse², Hanna Poell¹, Vivian Pouw¹, Annemieke de Raad¹, Mustafa Sener¹, Reina Sikkema², Madeleine ter Kuile¹, Anne van der Linden², Jans Velzing², Jolanda Voermans², Atze Vonk¹, Babette Weller², and Pinar Yilmaz^1,3

*Lead outbreak investigation team, tl.boelsums@rotterdam.nl

Members of the national genomic surveillance team

Dirk Eggink⁴, Chantal Reusken^§4, and Harry Vennema⁴

^§Lead national genomic surveillance team, chantal.reusken@rivm.nl

Affiliations

¹ Department of Infectious Disease Control, Public Health Service Rotterdam-Rijnmond, Rotterdam, the Netherlands

² Department of Viroscience, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands

³ Departments of Epidemiology and Radiology and Nuclear Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands

⁴ National Institute for Public Health and the Environment, Bilthoven, the Netherlands