Ethics approval
Ethical approval for this study was obtained from Comitê de Ética para Pesquisa com Seres Humanos from the Federal University of Lavras (COEP/UFLA) with protocol number CAAE 43997221.6.0000.5148.
Area of study
The boarding school is located in the municipality of Lavras in the state of Minas Gerais. The establishment has a leisure and sports area, including courts, green areas and a religious temple, in addition to cafeterias and shared bathrooms. The educational area has classrooms and large auditoriums. The dormitories are mostly shared and separated into male and female wings.
Data collection
Clinical and epidemiological data were collected from all members at a private school located in the southeastern region of Brazil. A total of 291 people (Figure 1), including students, teachers, and staff, were tested.
The first 14 people with suspected COVID-19 underwent an antigen test at a municipal laboratory. In a second moment biological samples were collected from all members of the school community using nasopharyngeal swabs. The swabs were then transferred to tubes containing 3 mL of transport solutions for referral to the laboratory. Reverse transcription–polymerase chain reaction (RT–qPCR) tests for SARS-CoV-2 RNA were performed. After collection, 140 µL of the sample, jointly with 10 µL of exogenous internal control, was submitted to viral RNA extraction using the QIAamp Viral RNA Mini Kit (Qiagen, USA) following the manufacturer's instructions. One-step RT–qPCR was conducted using 8 µL of total RNA. Amplification for 4 different targets was evaluated using the Allplex™ SARS-CoV-2 Assay (Seegene, Brazil), following the manufacturer's instructions, with 3 viral targets for SARS-CoV-2: gene E; the RdRP gene; the N gene and RP-V internal control. The test was only considered valid with the amplification of the internal control. The samples were considered positive if at least one of the targets had a cycle threshold (Ct) below 40.
Patients positive for SARS-CoV-2 were isolated and the negative ones were retested between 3 to 5 days. This procedure was repeated until all tested patients had negative results. Sanitary measures were immediately implemented, like the adequate use of equipment of individual protection by all employees.
Environmental swab testing
For the collection of environmental samples, swabs with sterile phosphate-buffered saline were vigorously rubbed on the surfaces of areas with a high volume of people. The swabs were then transferred to tubes containing 3 mL of transport solutions.
Immediately after collection, 140 µL of the sample, jointly with 10 µL of exogenous internal control (Segeene, Belo Horizonte, Brazil), was submitted to viral RNA extraction using the QIAmp Viral RNA Mini Kit (QIAGEN, Maryland, USA). One-step qPCR was conducted by the Allplex 2019-nCoV Assay kit - RP10243X (Segeene, Brazil) using 8 µL of total RNA. Amplification for 4 different targets was evaluated, with 3 viral targets for SARS-CoV-2: gene E; the RdRP gene; the N gene [9]; and RP-V internal control. To be considered valid, the test must necessarily amplify the internal control. The samples were considered positive if at least one of the targets had a cycle threshold (Ct) below 40.
Whole-genome sequencing, assembly, and SARS-CoV-2 variant identification.
We sequenced the SARS-CoV-2 genomes of seven positive samples using the Ion AmpliSeq™ SARS-CoV-2 Research Panel (Thermo Fisher Scientific). This panel is a quick, accurate, cost-effective, and widely used tool to obtain complete SARS-CoV-2 genomes [10]. This panel consists of 2 pools containing 247 amplicons: 242 unique amplicons to detect 237 specific SARS-CoV-2 viral targets and five human gene expression controls to assess the presence of the virus in a variety of sample types (including nasal swabs), covering all potential serotypes. Briefly, viral RNA from the seven SARS-CoV-2-positive individuals was reverse transcribed using the Invitrogen™ SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufacturer's recommendations. Then, we used the Ion AmpliSeq SARS-CoV-2 Research Panel (Thermo Fisher Scientific) to amplify cDNA, and barcodes and adapters were ligated using Ion Xpress™ Barcode Adapters (Thermo Fisher Scientific). After ligation, all libraries were purified with MagSi-NGSPREP Plus (Magtivio) and quantified using a Qubit 4.0 fluorometer and dsDNA HS Assay kit (Invitrogen, Carlsbad, CA, USA). Barcoded libraries were pooled in equimolar concentrations and used for library preparation with the Ion OneTouch™ 2 System with the Ion PGM™ Hi-Q™ View OT2 Kit (Thermo Fisher Scientific). We performed sequencing using the Ion PGM™ Hi-Q™ View Sequencing Kit on the Ion PGM™ System in an Ion 318™ Chip v2 (Thermo Fisher Scientific).
Genomic data were collected and processed using the Ion PGM™ Torrent Server. Briefly, de novo assembly was performed using Trinity assembler v. 1.3.0.2, and then Torrent Variant Caller 5.12 was used for calling SNPs and/or INDELs. Finally, SnpEff was applied to annotate and predict the effects of genetic variants, and Pangolin 3.0 [10-11] was run to assign each genome to the most likely lineage according to the Pango dynamic lineage nomenclature scheme.
All genomes are available in GenBank (NCBI database - https://www.ncbi.nlm.nih.gov/) under the accession numbers MZ397170.1, MZ169917.1, MZ169913.1, MZ169912.1, MZ169914.1, MZ169915.1 and MZ169916.1.