key: cord-0715713-n3s53zja authors: Labriola, Laura; Ruelle, Jean; Scohy, Anaïs; Seghers, François; Perlot, Quentin; De Greef, Julien; Desmet, Christine; Romain, Cécile; Yombi, Jean Cyr; Rodriguez‐Villalobos, Hector; Kabamba, Benoît; Jadoul, Michel title: Dynamics of spreading of SARS‐CoV‐2 in a Belgian hemodialysis facility: The importance of the analysis of viral strains date: 2021-11-30 journal: J Med Virol DOI: 10.1002/jmv.27471 sha: 18490eb89917d2c14150ecd2721e75efc3ea54e6 doc_id: 715713 cord_uid: n3s53zja In‐center maintenance hemodialysis (HD) patients are at high risk of acquiring coronavirus disease 2019 (COVID‐19) by cross‐contamination inside the unit. The aim of this study was to assess retrospectively the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) transmission during the very first pandemic phase (March–July 2020) in a cohort of in‐center maintenance HD patients and in nurses the same HD facility, using a phylogenetic approach. All SARS‐CoV‐2 quantitative reverse‐transcription polymerase chain reaction positive patients and nurses from our HD unit‐respectively 10 out of 98, and 8 out of 58‐ and two other positive patients dialyzed in our self‐care unit were included. Whole‐genome viral sequencing and phylogenetic analysis supported the cluster investigation. Five positive patients were usually dialyzed in the same room and same shift before their COVID‐19 diagnosis was made. Viral sequencing performed on 4/5 patients' swabs showed no phylogenetic link between their viruses. The fifth patient (whose virus could not be sequenced) was dialyzed at the end of the dialysis room and was treated by a different nurse than the one in charge of the other patients. Three nurses shared the same virus detected in both self‐care patients (one of them had been transferred to our in‐center facility). The epidemiologically strongly suspected intra‐unit cluster could be ruled out by viral genome sequencing. The infection control policy did not allow inter‐patient contamination within the HD facility, in contrast to evidence of moderate dissemination within the nursing staff and in the satellite unit. Epidemiologic data without phylogenetic confirmation might mislead the interpretation of the dynamics of viral spreading within congregate settings. In-center maintenance hemodialysis (HD) patients are at potentially high risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (coronavirus disease 2019 ) by crosscontamination during HD sessions and travels to and from HD units. Furthermore, dialysis patients are particularly vulnerable due to their high burden of comorbidities, uremia-associated immune dysfunction, and older age. 1, 2 Indeed, in the early pandemic period, the incidence of COVID-19 in maintenance HD patients from 65 centers in Wuhan (n = 7154), China, was reported to be higher compared with the local general population (2% vs. 0.5%, respectively). 3 Although various preventative policies to avoid the dissemination of SARS-CoV-2 have now become standard of care in hospital settings, clusters can occur despite robust infection control measures. 4 We recently reported the evolution (over 3 months) of anti-SARS-CoV-2 antibodies in a cohort of adult, in-center chronic HD patients. 5 We now assess the dynamics of SARS-CoV-2 transmission during the same early pandemic period in this carefully studied cohort and in staff members of the same HD facility. We used a phylogenetic approach, based on viral whole-genome sequences. Our in-center HD facility is located at the Cliniques Universitaires Saint-Luc (the main teaching hospital of UCLouvain, 983 beds), Brussels, Belgium. The unit has two shifts per day (up to 31 patients per shift), 6 days a week. At the time of this study, 98 patients were on maintenance HD in our facility (aged 68. 8 [±14] years, 58% males, 47% diabetics). The COVID-19 preventative protocol implemented in two phases is described in Table S1 . On March 9, 2020, we became aware through personal contacts of the first cases of COVID-19 in Belgian HD units, soon followed by an outbreak in one of these units. We thus implemented in our unit on March 13 a protocol aiming at the early diagnosis and immediate isolation of HD patients infected with SARS-CoV-2. Our initial protocol (Table S1A ) was implemented almost a month before but was largely consistent with the recommendations of the Centers for the Disease Control and Prevention (CDC), issued on April 12. 6 As soon as a first nasopharyngeal swab tested positive by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) for SARS-CoV-2 (Patient 1, March 20), the preventative policy was intensified, with the aim to avoid viral spread from any potentially infected individual (Table S1B ). This intensified protocol particularly stressed the use of surgical facemasks by all staff members and all HD patients as long as they are in the facility. As soon as they were diagnosed SARS-CoV-2 positive, patients were moved for their dialysis sessions to the dedicated COVID-19 isolation room (two shifts on the same day, three times a week). This room is usually reserved for either carriers of HBsAg or patients with protective anti-HBs. At the end of each day with sessions of COVID-19 patients, this room (including chairs and all other surfaces) was disinfected by vaporization of a 6% hydrogen peroxide solution (Nocolyse Oxy'Pharm), 6 allowing its safe use for COVID-19 positive (none of which was HBV+) and COVID-19 negative patients on alternate days. The three HBsAg-carrier patients, all COVID-19 negative, were transferred to another isolation room. The on-board iSeq sequence analysis software generated the Fastq files, where reads were trimmed for primers and indexes sequences. Fastq files were uploaded on the cloud-based ASP-IDNS ® −5 analysis software (SmartGene). Analysis was made using the "beta-Coronavirus pipeline" version 2.2.0_COV_v0.2. Briefly, paired-end reads were generated and automatically filtered for low-quality sections. The resulting reads were mapped against the SARS-CoV-2 profiles and mutations were detected in a quantitative manner (% reads aligned). A consensus genome was generated using a 40% cut-off for base determination and a minimal number of 30 reads per position. Nextclade Beta version 0.8.1 was used as a first sequence aligner, allowing comparison to other documented SARS-CoV-2 strains and clade assignment (https://clades.nextstrain.org). The 22 sequences were aligned with CLUSTAL O (1. 5ʹ-and 3ʹ-ends not included in the consensus alignment were trimmed: sequences used for the tree shared the same length of 29770 nt. Fasta files were then submitted to the NGPhylogeny web interface. 8 The workflow included: sequence alignment using the MAFFT software, 9 curation of the sequences with the block mapping and gathering with entropy (BMGE) software, 10 tree generation using the fast distance-based phylogeny inference program FastME 2.0, 11 and tree output formatted with the Newick display. 12 Felsenstein's bootstrap analysis (not shown) was not informative given the very low diversity in our data set with some identical sequences' clusters. Whole-genome sequences analyzed here were submitted to the GISAID platform and are accessible through the following identifiers: EPI_ISL_949244 to API_ISL_949250 and from EPI_ISL_1029958 to EPI_ISL_1029972. The aim of our study was to identify the viral strains circulating in our unit, to understand the modalities of the spread of SARS-CoV-2 among patients and staff members within the facility (including potential clusters). The study was performed in compliance with relevant laws and in- HD 1-5 ). Ten sequences obtained from patients hospitalized at the same institution but not on HD served as the control group. Table S2 details the clade and the mutation associated with each sequence, and Figure 3 shows the phylogenetic relationships. The samples belonged to various lineages, frequently found in Belgium during the first wave of the pandemic, such as B.1.6, B.1.83, or B38, as detailed in Table S2 . We found no direct phylogenetic link between the viral strains of Patients 2, 5, 6, and 10, that is, four out of the five positive patients belonging to an epidemiological cluster. For Patient 5, the genomic sequence was incomplete, however, the coverage was sufficient to classify his strain in clade 20C/B.1 which is a different strain from those carried by Patients 2, 6, and 10. RT-qPCR sample from Patient 4 was not available; however, his chair was at the end of the dialysis room ( Figure 2) To the best of our knowledge, this is the first study using the gold standard viral genome sequencing to assess the modalities of the Admittedly, as the viral load in nasopharyngeal samples was too low in some patients (1, 3, 4, 7, and 9) , a within-unit spread of SARS-CoV-2 from patient to patient cannot be completely ruled out ( Figure 2 ). Yet, in that case, it was at most minor, as compared with many other reports. Furthermore, these patients whose virus could not be sequenced were dialyzed in different rooms or different shifts, making this hypothesis very unlikely. Interestingly in a study comparing aggregated daily counts of University hospital (7.8%), 19 but less than the 35% reported by week 13 after the first diagnosed COVID-19 patient in a pediatric HD unit from Indianapolis. 20 Although these differences might be due to differences in the earlier or later antibody testing in the evolution of the epidemic, they emphasize the high-risk associated with HD, that is, care provided to patients coming repeatedly from outside the fa- Our study has some limitations. First, it was performed in a single-center, and the number of patients included was relatively low, thanks to the preventative protocol. Second, the phylogenetic analysis showed a definite link between two patients from our self-care unit (one of them transferred to our in-center facility) and three members of the nursing staff of the in-center facility, but some links could be missed as some positive samples could not be sequenced due to low viral load. Indeed, a staff-to-patient transmission caused by an infected, asymptomatic HCW cannot be completely ruled out. 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