The aim of the “Sentinel” national surveillance program is to monitor the circulating respiratory viruses, including SARS-CoV-2 variants, and hence underpin public health actions.
In week 20/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all sequenced specimens decreased to 76,5% (CI 72,1% – 80,9%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 2,5% (CI 0,9% – 4,1%, p<0,05) within the sequenced sample, while for P.1, the frequency decreased to 2,2% (CI 0,7% – 3,7%, p<0,05). The frequency for the “indian variant” B.1.617.2 increased to 7,5% (CI 4,8% – 10,2%, p<0,05).
The representative sample was estimated, based on the number of positive cases in Luxembourg for week 20 (495). The minimum sample size required to detect prevalence of B.1.1.7 (79%) reported in week 19, with an error margin of 5%, was estimated to be 169 specimens. This number corresponds to a coverage of 34,1 %, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 16 are representative of the circulating variants in Luxembourg with a margin of error of 5%.
The total number of sequences performed this week was 395, with 358 specimens having been collected in the time frame of week 20/2021. The sequencing coverage this week was 66,3% from all positive cases in Luxembourg (based on sequenced samples collected in the time frame of week 20 and corresponding to Luxembourg residents).
The “Sentinel” surveillance network reported 291 consultations in week 20 (17/MAY/2021 – 23/MAY/2021). There were 3 cases of ILI1, corresponding to 1,0% of the consultations, as shown in Figure 1. The number of consultations for ARI2 was 34, which represents 11,7% of the consultations. This exceeds the epidemiological threshold for the third consecutive week for ILI in Luxembourg, which is 2 cases per week.
Covid Consultation Centres have been closed by 16/May/2021. We are currently working on an alternative surveillance solution in partnership with private laboratories.
The National Reference Laboratory for Acute Respiratory Infections at LNS continues to improve the representativeness of the pool of sequenced specimens to reach real-time epidemiology, by implementing the following weekly sequencing activities:
The representative sequencing sample was based on the minimum number of specimens required to extrapolate prevalence of VOC variants with error rate of 5%. The representative sample was estimated based on the number of positive cases in Luxembourg in week 20 (495). The minimum sample size required to detect prevalence of B.1.1.7 (79%) reported in week 19 with an error margin of 5% was estimated to be 169 specimens. The calculation was based on a sample size calculation tool that uses the expected prevalence of the variant in the total population. (Population Proportion – Sample Size – Select Statistical Consultants (select-statistics.co.uk). This number represented a coverage of 34,1% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 294. Therefore, our sequencing results this week are representative of the circulating variants in Luxembourg.
The starting material used for sequencing is respiratory specimens (nasopharyngeal or oropharyngeal swabs) that have already been tested positive by RT PCR.
In parallel, we continue to evaluate commercial PCR screening, using the same starting material, to detect the Variants of Concern (VOCs), with specific PCR. This will enable a faster investigation time of any outbreak and allow us to screen 100% of positive cases referred to LNS, including those that do not pass the quality criteria for sequencing. Specimens that are not eligible for sequencing can thus be used in a PCR to detect the presence of variants of concern.
The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gisaid.org ) on a periodic basis.
Last week the microbial genomics platform at the LNS sequenced 395 specimens, with 358 collected in week 20/2021. The sequencing pool referring to Luxembourgish residence represents 66,3% of new infections reported in Luxembourg in week 20/2021. Among these 358 specimens, 36 specimens were reported to be part of a cluster or outbreak investigation, and 30 specimens were from non-residents (7 specimens overlapping). This leads to 294 specimens, collected in week 20, and being the representative population sequencing sample. In the population representative sample of residents, the frequencies of B.1.1.7, B.1.617.2, B.1.351 and P.1 were 75,5%, 8,2%, 2,7% and 2,4% respectively.
The population sequencing coverage in week 20/2021 was 66,3% (Figure 2). Based on statistical inference, the frequency of the reported variants in week 20/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.
Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software (v2.4.2, pangoLEARN version 2021-05-12).
The lineage nomenclature system that we use is the one proposed by Rambaut et al. that focuses on actively circulating virus lineages (https://cov-lineages.org ).
In week 20/2021, in the population representative sample, after removal of cluster specimens, and excluding specimens collected from non-residents, there were 15 circulating SARS-CoV-2 variants, with the main three variants being B.1.1.7 (75,5%, CI% 70,6% – 80,4%) followed by B.1.617.2 (8,2%, CI 5,1% – 11,3%) and R.1 (2,7%, CI 0,8% – 4,5%), as shown in Figure 3.
Among specimens collected within the week 20/2021, 274 cases of the B.1.1.7 variant have been detected, representing 76,5% of the specimens in the week’s sequencing pool (by comparison, the week 19/2021 pool had shown a frequency of 78,9% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 5904 by week 20/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 23/05/2021.
In the collection period of week 20/2021, 9 cases of the South African variant B.1.351 have been detected, representing 2,5% of the specimens in the week’s sequencing pool (by comparison, the week 19/2021 pool had shown 5,9% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 1025 by week 20/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 22/MAY/2021. In week 20/2021, 6 additional cases of B.1.525 has been detected, and 8 new cases of the Brazilian variant P.1. Thus, the case count by week 20 for B.1.525 is 45 (latest sampling date 17/MAY/2021) and for P.1 the case count is 103 (latest sampling date 22/MAY/2021). In week 20/2021, 27 additional cases of the B.1.617 subtype B.1.617.2 have been detected (latest sampling date 22/MAY/2021). The case count by week 20 for B.1.617.1 remains 6, and for B.1.617.2 it raises to 53. Since May 6th B.1.617.2 has been escalated by Public Health England from a variant under investigation to a variant of concern (Figure 4).
Lineage B.1.1.7 is characterized by several spike protein mutations, including N501Y, H69/V70del and P861H. The variant seems to have a considerable epidemiological impact, as it has a higher transmissibility rate.
Lineage B.1.351 holds numerous spike protein mutations, of which three are located in the receptor binding domain (K417N, E484K and N501Y), and are therefore relevant for antibody binding. As for B.1.1.7, a higher transmissibility rate and viral loads seem to be associated with this variant. Due to the K417N and E484K mutations, an impact on vaccination efficacy and possibility of reinfection is subject to scientific investigation.
Lineage P.1 (descendent of B.1.1.28), initially found in the Amazon region, has a similar mutation profile as the South African variant, including E484K and N501Y. Concerns are, as for the South African variant, higher transmissibility and a decreased protection by neutralizing antibodies.
Lineage B.1.525 carries several mutations of biological significance, including E484K, Q677H and F888L. It does not carry N501Y, but a set of deletions similar to the B.1.1.7 variant.
Lineage B.1.617 is a variant first detected in India and was designated “Under Investigation” on 1st April 2021 by Public Health England. It contains a number of spike mutations associated with antigenic escape or found in other variants of concern, including L452R, E484Q and P681R. Subtype B.1.617.2 does not carry S:E484Q and seems to be at least as transmissible as B.1.1.7 (with increasing confidence). Neutralization studies show reduction in cross-neutralizing activity between B.1.1.7 and B.1.351.
By week 20/2021, 92,5 % of the variants detected in the sequenced specimen pool are declared as either Variants of Concern (VOC – B.1.1.7, B.1.351, P.1, B.1.617.2) or Variants Under Investigation (VUI – B.1.525, B.1.1.318, B.1.617.1, B.1.1).
Currently the LNS genomic surveillance program – independently from lineage calling – notes the occurrence of 13 different known SARS-CoV-2 mutations, assumed to have clinical and epidemiological relevance. The list of observed mutations is being updated continually, based on the appearance and prevalence of SARS-CoV-2 variants.
The following table provides the overall frequencies of these mutations, detected in the lineage-assignable genome sequences, analyzed between 01/SEP/2020 and 23/MAY/2021 (N=11849), as well as the frequencies in week 20/2021.
Genomic sequencing of SARS-CoV-2. A guide to implementation for maximum impact on public health. WHO, 8 January 2021.
COVID-19 data portal. 2020 (https://www.covid19dataportal.org/sequences )
J Hadfield et al. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics 2018;34:4121-4123
A Rambaut et al. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat Microbiol 2020;5:1403-1407