This is a study of the prevalence of SARS-CoV-2 positivity and a time series of weekly rates of absenteeism due to COVID-19 among HCWs in a hospital during the first months of the epidemic, March to August 2020. The impact of implementing a universal serial RT-PCR testing program for asymptomatic HCW, tracking of contacts, and absenteeism were evaluated. Through the phylogenetic reconstruction of some of the positive samples, potential transmission clusters were investigated and correlated with information on function and place of work.
Study location and period
The hospital has 325 beds, of which 196 are dedicated to public health, placing it as a regional clinical reference center (3 million inhabitants), and in the city of Campinas (1.2 million inhabitants), 100 km from the capital São Paulo. The hospital sectors enrolled in the research were the intensive care units (ICUs), COVID-19 wards, clinical and surgical wards, the adult emergency room, administrative, and cleaning and support sectors, with a total of 170 beds and 473 professionals.
Protocols for testing/tracking/absence from work on leave for symptomatic patients and personal protective equipment (PPE) were already implemented in the hospital according to the recommendations of the National Health Surveillance Agency (ANVISA)  and Centers for Disease Control and Prevention (CDC) .
The hospital and the COVID-19 pandemic
The first imported case in Campinas was registered on March 12, 2020; the first patient with COVID-19 admitted to the PUC-Campinas Hospital was on March 15, and the first positive case in an HCW was on March 18. The increase in the incidence curves for COVID-19 in the city followed the transmission growth noted within the state of São Paulo, with a peak in epidemiological weeks 23–27 (June 2020), with a total of 8523 cases accumulated up to week 27 .
The intervention: universal testing and placing on leave of asymptomatic HCW
On May 11 (epidemiological week 20), the intervention began by testing all asymptomatic health professionals who agreed to participate in the research, from all the hospital sectors enrolled in the study.
After signing the informed consent form, professionals were invited to answer a questionnaire and then a biological sample was collected using a nasopharynx swab to perform the RT-PCR Fleury test (Charité  and CDC  protocols) and/or Gene-X-pert (Cepheid-USA) for faster results. If positive, HCW were place on leave. Three serial collections were performed on each professional who had been negative in previous tests, with an interval of 20 days between them. Symptomatic workers were not included in the study; that is, those who presented at least one of the following symptoms: headache, fever, cough, sore throat, anosmia, ageusia, myalgia, chills, cough, or runny nose.
Study variables and data source
Using a structured questionnaire, data on demographics (gender and age), workplace, and role performed in the hospital, as well as symptoms (in the last 14 days) were obtained.
RT-PCR test positivity rates were calculated according to the variables of interest (gender, age, professional category, work place in the hospital). The number of daily leaves and the total days of leave for any reason and for COVID-19 were obtained from the Department of Human Resources and Occupational Medicine of the hospital from January 1 to September 12, 2019, and 2020 (epidemiological weeks 1–37).
The impact of the testing intervention in asymptomatic individuals was evaluated based on the temporal trend of weekly leave rates by COVID-19 (number of leaves per week/total number of employees scheduled for the week) from March 8 to September 12, 2020 (epidemiological weeks 11–37).
Data on the COVID-19 epidemic (confirmed cases of COVDI-19 severe acute respiratory syndrome) of the population of Campinas and HCW were obtained from publications of the Health Surveillance Department of the Municipal Health Department of Campinas, allowing for comparison of the incidence curves in the study institution and in the community (https://covid-19.campinas.sp.gov.br/).
Health care-associated COVID-19 was considered when the onset of symptoms occurred at least 7 days after admission .
Viral genetic sequencing and phylogenetic analysis
Biological samples from professionals with positive RT-PCR results collected in different sectors of the hospital were sequenced to assess clusters and the lineage of SARS-CoV-2.
After extraction of viral RNA, cDNA synthesis was performed using the Protoscript II First Strand transcription kit (New England Biolabs, UK) and random hexamers (Thermo Fisher Scientific, Waltham, MA, USA). Amplification of the total genome was carried out using the multiplex PCR reaction with specific primers for SARS-CoV-2 (https://artic.network/ncov-2019), together with the Q5 High-Fidelity DNA Polymerase Kit (New England Biolabs, UK). The PCR conditions have been described previously (https://artic.network/ncov-2019). Amplicons were purified using magnetic beads (1× AMPure XP, Beckman Coulter, UK). After purification, the product was quantified using fluorometry techniques with Qubit dsDNA high sensitivity reagents, and reading was performed using the Qubit 3.0 instrument (Life Technologies, Carlsbad, CA, USA).
Complete genome sequencing of SARS-CoV-2
To achieve good coverage of the genome, only samples with more than 4 ng/µL of DNA were used to prepare the library for sequencing. The amplicons of each sample were normalized to be equimolar. After this process, the normalized amplicons were processed to continue preparing the library for sequencing according to the previously published protocol  using the EXP-NBD104 (1–12) and EXPNBD114 (13–24) kits (Oxford Nanopore Technologies, Oxford, UK). The libraries were loaded into a flow cell and sequenced by MinION for 8–24 h using the SQK-LSK109 kit (Oxford Nanopore Technologies). To monitor the sequencing in real time and estimate the depth of coverage (200 times target), ARTIC Network RAMPART software (https://artic.network/ncov-2019) was used. Minimap2 v2.28.0 software was used to obtain the consensus sequence and structure the fast five files against the reference genome of the SARS-CoV-2 Wuhan-Hu 1 1 isolate (GenBank accession number MN908947).
Consensus sequences were initially submitted to a quality control check using Nextclade. To retain the maximum amount of information possible, sequences with coverage above 80% were used for further analysis. Our final dataset consisted of 49 original SARS-CoV-2 complete genomes and nine sequences collected in Campinas downloaded from GISAID as of July 31, 2020.
Fast multiple sequence alignment was applied in our dataset with MAFFT v. 7.450. A maximum likelihood tree was reconstructed using IQ-TREE version 1.6.12  under the TIM + F + I nucleotide substitution model (ModelFinder). Statistical support was evaluated using 1000 bootstrap replicates. Internal nodes with > 90% statistical support were assigned on the tree. To infer the existence of potential transmission clusters, we used Phydelity v. 2.1. SARS-CoV-2 lineages were identified using Pangolin software.
The prevalence of positivity and the respective 95% confidence intervals were calculated by the ratio of positive tests to the population tested at the three testing moments, according to the variables of interest. Proportions were compared using the chi-squared test with Yates’ correction, with a significance level of 5% (P < 0.05).
After verifying the assumption of homoscedasticity, absence of autocorrelation, and normality of residuals, a segmented regression of weekly absenteeism rates was adjusted. The percentage and mean variations in the rates and their statistical significance were obtained using the Joint Point Regression Program version 126.96.36.199.
The median and quartiles (1st and 3rd) of daily leaves for all causes of health care workers from January 1 to September 12, 2019, and 2020 were also evaluated. The daily averages of HCW sick leave from 2019 and 2020 were compared using analysis of variance analysis (ANOVA) and the F test, considering a significance level of 5% (P < 0.05).
All methods were carried out in accordance with relevant guidelines and regulations and this study was approved by Pontifical Catholic University of Campinas Medical Ethics Committee of Pontifical Catholic University of Campinas, under protocol no. 31042120.4.0000.5481.