Sequencing analysis of SARS-CoV-2 cases in Slovenian long-term care facilities to support outbreak control

Abstract

Introduction: Residents of long-term care facilities (LTCFs) are at high risk of morbidity and mortality due to COVID-19, especially when new variants of concern (VOC) emerge. To provide intradisciplinary data in order to tailor public health interventions during future epidemics, available epidemiologic and genomic data from Slovenian LTCFs during the initial phases of the COVID-19 pandemic was analyzed.

Methods: The first part of the study included SARS-CoV-2 reverse-transcription Real-Time PCR (rtRT-PCR) positive LTCF residents, from 21 facilities with COVID-19 outbreaks occurring in October 2020. The second part of the study included SARS-CoV-2 rtRT-PCR positive LTCF residents and staff between January and April 2021, when VOC Alpha emerged in Slovenia. Next-generation sequencing (NGS) was used to acquire SARS-CoV-2 genomes, and lineage determination. In-depth phylogenetic and mutational profile analysis were performed and coupled with available field epidemiological data to assess the dynamics of SARS-CoV-2 introduction and transmission.

Results: 370/498 SARS-CoV-2 positive residents as well as 558/699 SARS-CoV-2 positive residents and 301/358 staff were successfully sequenced in the first and second part of the study, respectively. In October 2020, COVID-19 outbreaks in the 21 LTCFs were caused by intra-facility transmission as well as multiple independent SARS-CoV-2 introductions. The Alpha variant was confirmed in the first LTCF resident approximately 1.5 months after the first Alpha case was identified in Slovenia. The data also showed a slower replacement of existing variants by Alpha in residents compared to staff and the general population.

Discussion: Multiple SARS CoV-2 introductions as well as intra-facility spreading impacted disease transmission in Slovenian LTCFs. Timely implementation of control measures aimed at limiting new introductions while controlling in-facility transmission are of paramount importance, especially as new VOCs emerge. Sequencing, in conjunction with epidemiological data, can facilitate the determination of the need for future improvements in control measures to protect LTCF residents from COVID-19 or other respiratory infections.

Keywords: SARS-CoV-2; long-term care facility; next-generation sequencing; outbreak control; phylogeny.

Figure 1

Study design flowchart.

Figure 2

Number of sequenced SARS-CoV-2 cases in a specific LTCF per day in October 2020.

Figure 3

Phylogenetic tree of all SARS-CoV-2 positive residents with successfully obtained complete viral genome sequence within a month (110 samples) from the same LTCF in October 2020. Colours represent the week of detection.

Figure 4

Enlargement of the B.1.1.70 part of the complete SARS-CoV-2 genome analysis phylogenetic tree from one LTCF. The numbers represent delay times in days when the PCR test was positive after the first detected case on October 9th, 2020 (0).

Figure 5

The distinct mutation profile of the B.1.1.70 subgroup in one specific LTCF. The y-axis represents the classification beyond Pangolin, with a sequential number denoting a potential independent introduction event and a letter if the mutation profile represents an expected change within a certain timeframe (possible intra-LTCF transmission). Green represents substitutions that are fixed, grey represents substitutions that differ between samples, and black represents the presence of substitutions in a mutation profile. The last column represents the time delay after the first detection of SARS-CoV-2.

Figure 6

Estimated number of SARS-CoV-2 mutations accumulated in a closed system based on 1.8 × 10⁻³ mutations acquisition per base per year (calculated for the SARS-CoV-2 reference genome NC_045512.2). The purple dots represent the time points observed in our study and the expected number of mutations if only intra LTCF transmission were to occur.

Figure 7

Temporal distribution of detected SARS-CoV-2 lineages in (A) LTCF residents and (B) LTCF staff per week of detection.

Figure 8

(A) Number of SARS-CoV-2 infections in LTCF residents (red) and LTCF staff (blue) and, the total number of PCR positive tests (grey) in Slovenia (all PCR testing sites) per week from January 2021 to April 2021. (B) Proportion of VOC Alpha in LTCF residents, LTCF staff, and the general population per day from January 2021 to April 2021. (C) Number of different lineages among LTCF residents, LTCF staff, and the general population per day from January 2021 to April 2021. The colored lines represent the median value in the respective populations, and the grey areas represent the 95% confidence interval.