Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 05/07/-11/07/2021)

Executive Summary

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 27/2021, the variant P.1 (Gamma) remains the dominant one with an overall frequency of 60% (CI 56% – 64%, p<0,05) in all sequenced specimens, followed by the B.1.617.2 (Delta) variant with 34,8% (CI 30,9% – 38,7%, p<0,05) and the B.1.1.7 (Alpha) variant with 4,1% (CI 2,5% – 5,7%, p<0,05).

The representative sample size was estimated, based on the number of positive cases in Luxembourg for week 27 (828). The minimum sample size required to detect prevalence of P.1 (71,5%) reported in week 26, with an error margin of 5%, was estimated to be 228 specimens. This number corresponds to a coverage of 27,5%, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 24 are not representative of the circulating variants in Luxembourg with an error margin of 5%.

The total number of sequences performed this week was 682, with 580 specimens having been collected in the time frame of week 27/2021. The sequencing coverage – based on sequenced samples collected in week 27 and corresponding to Luxembourg residents – was 67,3% of all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 293 consultations in week 27 (05/JULY/2021 – 11/JULY/2021). Four cases of ILI¹ have been reported, which is 1,4% of consultantions, as shown in Figure 1. The number of consultations for ARI² was 30, which represents 10,2%.

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

-Sequencing specimens from all hospitalized positive cases
-Sequencing specimens from all positive cases from Airport testing program
-Sequencing specimens from all outbreaks and identified clusters
-Systematic sequencing of specimens from reinfections and post-vaccination-infections
-Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 27 (828). The minimum sample size required to detect prevalence of P.1 (71,5%) reported in week 26 with an error margin of 5% was estimated to be 228 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 27,5%, which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 490. 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.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gisaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 682 specimens, with 580 collected in week 27/2021. The sequencing pool referring to Luxembourgish residents represents 67,3% of new infections reported in Luxembourg in week 27/2021. Among these 580 specimens, 68 specimens were reported to be part of a cluster or vaccine failure investigation, and 23 specimens were from non-residents (1 specimen overlapping). This leads to 490 specimens, collected in week 27, and being a representative population sequencing sample. In the population representative sample of residents, the frequency for P.1 was 61%, while the frequency for B.1.617.2 and B.1.1.7, was 33,7% and 4,3% respectively.

The population sequencing coverage in week 27/2021 was 59,2% (Figure 2). Based on statistical inference, the frequency of the reported variants in week 27/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v3.1.5, pangoLEARN version 2021-06-15).

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 27/2021, in the population representative sample, after removal of cluster samples, and excluding specimens collected from non-residents, there were 8 circulating SARS-CoV-2 variants, with the main three variants being the P.1 (61%, CI 56,7% – 65,3%), B.1.617.2 (33,7%, CI 29,5% – 37,9%) and B.1.1.7 (4,3%, CI 2,5% – 6,1%) as shown in Figure 3.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, B.1.525, B.1.617.2)

In week 27/2021, 348 new cases (60%) of the Gamma (P.1) variant have been detected (by comparison, the week 26/2021 pool had shown 71,5% of this variant). The total case count of sequenced Gamma variant was 887 by week 27/2021.

In week 27/2021, 202 additional cases (34,8%) of the Delta variant B.1.617.2 have been detected (by comparison, the week 26/2021 pool had shown a frequency of 26,4% of this variant). The case count by week 27 for B.1.617.2 rose to 627.

Among specimens collected within the week 27/2021, 24 cases of the Alpha (B.1.1.7) variant have been detected, representing 4,1% of the specimens in the week’s sequencing pool (by comparison, the week 26/2021 pool had shown a frequency of 1,1% of this variant). The total case count of sequenced variant Alpha was 6952 by week 27/2021.

In the collection period of week 27/2021, 1 case of the Beta (B.1.351) variant has been detected, representing 0,2% (by comparison, the week 26/2021 pool had shown 0,4% of this variant). The total case count of sequenced variant B.1.351 was 1196 by week 27/2021.

In week 27/2021, no new case of B.1.525 has been detected. The case count by week 27 for B.1.525 remains 50 (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 1^st  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 more transmissible than B.1.1.7 (increasing confidence). Neutralization studies show reductions in cross-neutralizing activity between B.1.1.7 and B.1.351.

WHO nomeculature to track VOC

The ReViLux will continue to use the (Pango) system to allow easier visualisation of links between any evolving variants and their ancestor (Figure 5).

Clinically relevant mutations

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 11/JULY/2021 (N=14977), as well as the frequencies in week 27/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 28/06/-04/07/2021)

Executive Summary

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 26/2021, the variant P.1 (Gamma) became the dominant one with an overall frequency of 72,4% (CI 68,6% – 76,2%, p<0,05) in all sequenced specimens, followed by the B.1.617.2 (Delta) variant with 25,4% (CI 21,7% – 29,1%, p<0,05) and the B.1.1.7 (Alpha) variant with 1,1% (CI 0,2% – 2%, p<0,05).

The representative sample size was estimated, based on the number of positive cases in Luxembourg for week 26 (787). The minimum sample size required to detect prevalence of B.1.617.2 (60,8%) reported in week 25, with an error margin of 5%, was estimated to be 251 specimens. This number corresponds to a coverage of 31,9%, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 24 are not representative of the circulating variants in Luxembourg with an error margin of 5%.

The total number of sequences performed this week was 637, with 540 specimens having been collected in the time frame of week 26/2021. The sequencing coverage – based on sequenced samples collected in week 26 and corresponding to Luxembourg residents – was 65,1% of all positive cases in Luxembourg.

 

Clinical Surveillance

The “Sentinel” surveillance network reported 290 consultations in week 26 (28/JUNE/2021 – 04/JULY/2021). No cases of ILI¹ have been reported as shown in Figure 1. The number of consultations for ARI² was 25, which represents 8,6%.

 

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 26 (787). The minimum sample size required to detect prevalence of B.1.617.2 (60,8%) reported in week 25 with an error margin of 5% was estimated to be 251 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 31,9%, which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 315. 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.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gisaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 637 specimens, with 540 collected in week 26/2021. The sequencing pool referring to Luxembourgish residents represents 65,1% of new infections reported in Luxembourg in week 26/2021. Among these 540 specimens, 200 specimens were reported to be part of a cluster or outbreak investigation, and 28 specimens were from non-residents (3 specimen overlapping). This leads to 315 specimens, collected in week 26, and being a representative population sequencing sample. In the population representative sample of residents, the frequency for P.1 was 74,3%, while the frequency for B.1.617.2 and B.1.1.7, was 24,4% and 0,95% respectively.

The population sequencing coverage in week 26/2021 was 40% (Figure 2). Based on statistical inference, the frequency of the reported variants in week 26/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

 

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v3.1.5, pangoLEARN version 2021-06-15).

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 26/2021, in the population representative sample, after removal of cluster samples, and excluding specimens collected from non-residents, there were 4 circulating SARS-CoV-2 variants, the P.1 (74,3%, CI 69,5% – 79,1%), B.1.617.2 (24,4%, CI 19,7% – 29,1%), B.1.1.7 (0,95%, CI 0% – 2%) and B.1.612 (0,3%, CI 0% – 0,9%) as shown in Figure 3.

 

 

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, B.1.525, B.1.617.2)

While an overall 8-fold increase of new infections has been detected, the Gamma (P.1) variant showed a 20-fold increase in case numbers, with 391 new cases (72,4%) in week 26/2021 (by comparison, the week 25/2021 pool had shown 24,1% of this variant). The total case count of sequenced Gamma variant was 537 by week 26/2021.

In week 26/2021, 137 additional cases of the Delta variant B.1.617.2 have been detected. The case count by week 26 for B.1.617.2 rose to 416.

Among specimens collected within the week 26/2021, 6 cases of the Alpha (B.1.1.7) variant have been detected, representing 1,1% of the specimens in the week’s sequencing pool (by comparison, the week 25/2021 pool had shown a frequency of 10,1% of this variant). The total case count of sequenced variant Alpha was 6904 by week 26/2021.

In the collection period of week 26/2021, 2 cases of the Beta (B.1.351) variant have been detected, representing 0,37% (by comparison, the week 25/2021 pool had shown 2,5% of this variant). The total case count of sequenced variant B.1.351 was 1192 by week 26/2021.

In week 26/2021, one new case of B.1.525 has been detected. The case count by week 26 for B.1.525 was 50 (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 1^st  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 more transmissible than B.1.1.7 (increasing confidence). Neutralization studies show reductions in cross-neutralizing activity between B.1.1.7 and B.1.351.

 

By week 26/2021, 99,4 % of the variants detected in the sequenced specimen pool are declared as Variants of Concern, including the P.1 (Gamma), the B.1.617.2 (Delta), the B.1.1.7 (Alpha) and B.1.351 (Beta) variants.

WHO nomeculature to track VOC

The ReViLux will continue to use the (Pango) system to allow easier visualisation of links between any evolving variants and their ancestor (Figure 5).

Clinically relevant mutations

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 04/JULY/2021 (N=14284), as well as the frequencies in week 26/2021.

 

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 21/06/-27/06/2021)

Executive Summary

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 25/2021, the variant B.1.617.2 (Delta) remained the dominant one in Luxembourg with an overall frequency of 60,1% (CI 49,3% – 70,9%, p<0,05) in all sequenced specimens, followed by the P.1 (Gamma) variant with 24,1% (CI 14,7% – 33,5%, p<0,05), the B.1.1.7 (Alpha) variant with 10,1% (CI 3.5% – 16,7%, p<0,05) and one case of the B.1.351 (Bêta) variant.

The representative sample size was estimated, based on the number of positive cases in Luxembourg for week 25 (107). The minimum sample size required to detect prevalence of B.1.617.2 (59%) reported in week 24, with an error margin of 5%, was estimated to be 84 specimens. This number corresponds to a coverage of 78,5 %, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 24 are not representative of the circulating variants in Luxembourg with an error margin of 5%.

The total number of sequences performed this week was 581, with 79 specimens having been collected in the time frame of week 25/2021. The sequencing coverage – based on sequenced samples collected in week 25 and corresponding to Luxembourg residents – was 69,2% of all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 268 consultations in week 25 (21/JUNE/2021 – 27/JUNE/2021) and accordingly there were 0 cases of ILI¹ and 31cases ARI² as shown in Figure 1. The number of consultations for ARI² was 31, which represents 11,6% of the consultations.

Virological Surveillance

Covid Consultation Centres have been closed since 16/May/2021. We are currently working on an alternative in partnership with private laboratories.

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 25 (107). The minimum sample size required to detect prevalence of B.1.617.2 (59%) reported in week 24 with an error margin of 5% was estimated to be 84 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 78,5%, which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 41. Therefore, our sequencing results this week are not 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.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gisaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 581 specimens, with 79 collected in week 25/2021. The sequencing pool referring to Luxembourgish residents represents 69,2% of new infections reported in Luxembourg in week 25/2021. Among these 79 specimens, 35 specimens were reported to be part of a cluster or outbreak investigation, and 5 specimens were from non-residents (2 specimen overlapping). This leads to 41 specimens, collected in week 25, and not being a representative population sequencing sample. In the population sample of residents, the frequency for B.1.617.2 was 61%, while the frequency for P.1 and B.1.1.7, was 19,5% and 14,6% respectively.

The population sequencing coverage in week 25/2021 was 38,3% (Figure 2). Based on statistical inference, the frequency of the reported variants in week 25/2021 is not representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v3.0.3, pangoLEARN version 2021-05-27).

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 25/2021, in the population sample, after removal of cluster samples, and excluding specimens collected from non-residents, there were 5 circulating SARS-CoV-2 variants, with the main three variants being B.1.617.2 (61%, CI 46,1% – 75,9%), P.1 (19,5%, CI 7,4% – 31,6%) and B.1.1.7 (14,6%, CI 3,8% – 25,4%), as shown in Figure 3.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, B.1.525, B.1.617.2)

Among specimens collected within the week 25/2021, 8 cases of the Alpha (B.1.1.7) variant have been detected, representing 10,1% of the specimens in the week’s sequencing pool (by comparison, the week 24/2021 pool had shown a frequency of 29,7% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant Alpha was 6882 by week 25/2021.

In the collection period of week 25/2021, 2 cases of the Beta (B.1.351) variant have been detected, representing 2,5% (by comparison, the week 24/2021 pool had shown 1,5% of this variant). The total case count of sequenced variant B.1.351 was 1177 by week 25/2021. A strong increase in case numbers has been detected for the Gamma (P.1) variant, with 19 new cases (24,1%) in week 25/2021. Since week 51/2020, the highest proportion the Gamma variant ever represented was in week 18 with 4,5%. The last case detected in the sequencing pool before week 25 is dated back to June 9^th. The total case count of sequenced Gamma variant was 145 by week 25/2021.

In week 25/2021, no new case of B.1.525 has been detected. The case count by week 25 for B.1.525 is 49 (including additional sequencing of previous weeks).

In week 25/2021, 48 additional cases of the Delta variant B.1.617.2 have been detected. The case count by week 25 for B.1.617.1 remained 9, and for B.1.617.2 it rose to 279. Since  May 6^th, 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 1^st  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 more transmissible as B.1.1.7 (increasing confidence). Neutralization studies show reductions in cross-neutralizing activity between B.1.1.7 and B.1.351.

By week 25/2021, 97,5 % of the variants detected in the sequenced specimen pool are declared as Variants of Concern, including the B.1.617.2 (Delta), the P.1 (Gamma), the B.1.1.7 (Alpha) and B.1.351 (Beta) variants.

WHO nomeculature to track VOC

The ReViLux will continue to use the (Pango) system to allow easier visualisation of links between any evolving variants and their ancestor (Figure 5).

Clinically relevant mutations

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 27/JUNE/2021 (N=13739), as well as the frequencies in week 25/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 14/06/-20/06/2021)

Executive Summary

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 24/2021, the variant B.1.617.2 became the dominant one in Luxembourg with an overall frequency of 59,4% (CI 47,4% – 71,4%, p<0,05), while the B.1.1.7 variant frequency dropped to 29,7% (CI 18,5% – 40,9%, p<0,05). One case of SARS-CoV-2 B.1.351 (1,5%, CI 0% – 4,5%, p<0,05) and no case of the P.1 variant were detected.

The representative sample size was estimated, based on the number of positive cases in Luxembourg for week 24 (90). The minimum sample size required to detect prevalence of B.1.617.2 (34%) reported in week 23, with an error margin of 5%, was estimated to be 72 specimens. This number corresponds to a coverage of 80 %, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 24 are not representative of the circulating variants in Luxembourg with an error margin of 5%.

The total number of sequences performed this week was 284, with 64 specimens having been collected in the time frame of week 24/2021. The sequencing coverage – based on sequenced samples collected in week 24 and corresponding to Luxembourg residents – was 66,7% of all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 374 consultations in week 24 (14/JUNE/2021 – 20/JUNE/2021). There were 3 cases of ILI¹, corresponding to 0,8% of the consultations, as shown in Figure 1. The number of consultations for ARI² was 37, which represents 9,9% of the consultations.

Virological Surveillance

Covid Consultation Centres have been closed since 16/May/2021. We are currently working on an alternative in partnership with private laboratories.

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 24 (90). The minimum sample size required to detect prevalence of B.1.617.2 (34%) reported in week 23 with an error margin of 5% was estimated to be 72 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 80%, which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 35. Therefore, our sequencing results this week are not 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.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gisaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 284 specimens, with 64 collected in week 24/2021. The sequencing pool referring to Luxembourgish residents represents 66,7% of new infections reported in Luxembourg in week 24/2021. Among these 68 specimens, 25 specimens were reported to be part of a cluster or outbreak investigation, and 6 specimens were from non-residents (2 specimen overlapping). This leads to 35 specimens, collected in week 24, and not being a representative population sequencing sample. In the population sample of residents, the frequency of B.1.617.2 was 54,3%, while the frequency for B.1.1.7 was 34,3%. One case has been detected for the B.1.351 variant, representing 2,9%.

The population sequencing coverage in week 24/2021 was 38,9% (Figure 2). Based on statistical inference, the frequency of the reported variants in week 24/2021 is not representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v3.0.3, pangoLEARN version 2021-05-27).

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 24/2021, in the population sample, after removal of cluster samples, and excluding specimens collected from non-residents, there were 5 circulating SARS-CoV-2 variants, with the main three variants being B.1.617.2 (54,3%, CI 37,8% – 70,8%), B.1.1.7 (34,3%, CI 18,6% – 50%) and B (5,7%, CI 0% – 13,4%), as shown in Figure 3.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, B.1.525, B.1.617.2)

Among specimens collected within the week 24/2021, 19 cases of the B.1.1.7 variant have been detected, representing 29.7% of the specimens in the week’s sequencing pool (by comparison, the week 23/2021 pool had shown a frequency of 51.6% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 (Alpha) was 6696 by week 24/2021.

In the collection period of week 24/2021, 1 case of the South African variant B.1.351(Bêta) has been detected, representing 1.6% (by comparison, the week 23/2021 pool had shown 0.8% of this variant). The total case count of sequenced variant B.1.351 was 1123 by week 24/2021.

No new case of the P.1 variant has been detected in week 24/2021.The total case count of sequenced variant P.1(Gamma) was 126 by week 24/2021 (including additional sequencing of previous weeks).

In week 24/2021, no new case of B.1.525 has been detected. The case count by week 24 for B.1.525 is 48 (including additional sequencing of previous weeks).

In week 24/2021, 38 additional cases of the Delta variant B.1.617.2 have been detected (latest sampling date 20/JUNE/2021). The case count by week 24 for B.1.617.1 increases by retrospective sequencing to 9, and for B.1.617.2 it raises to 231. Since  May 6^th, 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. Similarly to 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 1^st  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 more transmissible, as B.1.1.7 (increasing confidence). Neutralization studies show reductions in cross-neutralizing activity between B.1.1.7 and B.1.351.

By week 24/2021, 90.6 % of the variants detected in the sequenced specimen pool are declared as Variants of Concern, including the B.1.617.2, B.1.1.7 and B.1.351 variants.

WHO nomeculature to track VOC

The ReViLux will continue to use the (Pango) system to allow easier visualisation of links between any evolving variants and their ancestor (Figure 5).

Clinically relevant mutations

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 20/JUNE/2021 (N=13149), as well as the frequencies in week 24/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 07/06/-13/06/2021)

Executive Summary

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 23/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all sequenced specimens slightly increased to 50,5% (CI 40,8% – 60,2%, p<0,05). One case of SARS-CoV-2 B.1.351 (1%, CI 0% – 2,9%, p<0,05) and 4 cases of P.1 variant (3,9%, CI 0% – 7,6%, p<0,05) were detected, while the frequency of the variant B.1.617.2 increased slightly to 36% (CI 26,7% – 45,3%, p<0,05).

The representative sample size was estimated, based on the number of positive cases in Luxembourg for week 23 (189). The minimum sample size required to detect prevalence of B.1.1.7 (47%) reported in week 22, with an error margin of 5%, was estimated to be 127 specimens. This number corresponds to a coverage of 67,2 %, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 23 are not representative of the circulating variants in Luxembourg with an error margin of 5%.

The total number of sequences performed this week was 283, with 103 specimens having been collected in the time frame of week 23/2021. The sequencing coverage – based on sequenced samples collected in week 23 and corresponding to Luxembourg residents and to non-targeted collections – was 36% of all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 375 consultations in week 23 (07/JUNE/2021 – 13/JUNE/2021). There were 4 cases of ILI¹, corresponding to 1,1% of the consultations, as shown in Figure 1. The number of consultations for ARI² was 35, which represents 9,3% of the consultations.

Virological Surveillance

Covid Consultation Centres have been closed since 16/May/2021. We are currently working on an alternative in partnership with private laboratories.

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 23 (189). The minimum sample size required to detect prevalence of B.1.1.7 (47%) reported in week 22 with an error margin of 5% was estimated to be 127 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 67,2% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 68. Therefore, our sequencing results this week are not 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.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gisaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 283 specimens, with 103 collected in week 23/2021. The sequencing pool referring to Luxembourgish residents represents 52,4% of new infections reported in Luxembourg in week 23/2021. Among these 103 specimens, 34 specimens were reported to be part of a cluster or outbreak investigation, and 5 specimens were from non-residents (4 specimen overlapping). This leads to 68 specimens, collected in week 23, and not being a representative population sequencing sample. In the population sample of residents, the frequency of B.1.1.7 was 58,8%, while the frequency for B.1.617.2 was 30,9%. Two cases have been detected for the P1 and 1 case for the B.1.351 variant, representing 2,9% and 1,5% respectively.

The population sequencing coverage in week 23/2021 was 36% (Figure 2). Based on statistical inference, the frequency of the reported variants in week 23/2021 is not representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v3.0.3, pangoLEARN version 2021-05-27).

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 23/2021, in the population sample, after removal of cluster samples, and excluding specimens collected from non-residents, there were 7 circulating SARS-CoV-2 variants, with the main three variants being B.1.1.7 (58,8%, CI 47,1% – 70,5%), B.1.617.2 (30,9%, CI 19,9% – 41,9%) and P.1 (2,9%, CI 0% – 6,9%), as shown in Figure 3.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, B.1.525, B.1.617.2)

Among specimens collected within the week 23/2021, 52 cases of the B.1.1.7 variant have been detected, representing 50,5% of the specimens in the week’s sequencing pool (by comparison, the week 22/2021 pool had shown a frequency of 47,4% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 6460 by week 23/2021.

In the collection period of week 23/2021, 1 case of the South African variant B.1.351 has been detected, representing 1% (by comparison, the week 22/2021 pool had shown 0% of this variant). The total case count of sequenced variant B.1.351 was 1079 by week 23/2021.

Four new cases of the P.1 variant have been detected in week 23/2021, corresponding to 3,9%, while the previous week no case had been detected. The total case count of sequenced variant P.1 was 122 by week 23/2021.

In week 23/2021, no new case of B.1.525 has been detected. The case count by week 23 for B.1.525 is 46.

In week 23/2021, 36 additional cases of the Delta variant B.1.617.2 have been detected (latest sampling date 11/JUNE/2021). The case count by week 23 for B.1.617.1 increases by retrospective sequencing to 8, and for B.1.617.2 it raises to 186. Since  May 6^th 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 1^st  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 more transmissible as B.1.1.7 (increasing confidence). Neutralization studies show reductions in cross-neutralizing activity between B.1.1.7 and B.1.351.

By week 23/2021, 89,3 % of the variants detected in the sequenced specimen pool are declared as either Variants of Concern, including the B.1.1.7, the B.1.617.2 (increasing trend) and P.1 variant.

WHO nomeculature to track VOC

The ReViLux will continue to use the (Pango) system to allow easier visualisation of links between any evolving variants and their ancestor (Figure 5).

Clinically relevant mutations

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 13/JUNE/2021 (N=12723), as well as the frequencies in week 23/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 31/05/-06/06/2021)

Executive Summary

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 22/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all sequenced specimens decreased to 47,4% (CI 40,4% – 54,4%, p<0,05). No case was detected for the SARS-CoV-2 B.1.351 and P.1 variant, while the frequency the variant B.1.617.2 doubled to 30,9% (CI 24,4% – 37,4%, p<0,05).

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 22 (324). The minimum sample size required to detect prevalence of B.1.1.7 (66%) reported in week 21, with an error margin of 5%, was estimated to be 168 specimens. This number corresponds to a coverage of 51,8 %, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 22 are not representative of the circulating variants in Luxembourg with a margin of error of 5%.

The total number of sequences performed this week was 371, with 194 specimens having been collected in the time frame of week 22/2021. The sequencing coverage – based on sequenced samples collected in week 22 and corresponding to Luxembourg residents and to non-targeted collections – was 45,1% of all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 222 consultations in week 22 (31/MAY/2021 – 06/June/2021). There was 1 case of ILI¹, corresponding to 0,5% of the consultations, as shown in Figure 1. The number of consultations for ARI² was 18, which represents 8,1% of the consultations.

Virological Surveillance

Covid Consultation Centres have been closed by 16/May/2021. We are currently working on an alternative in partnership with private laboratories.

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 22 (324). The minimum sample size required to detect prevalence of B.1.1.7 (66%) reported in week 20 with an error margin of 5% was estimated to be 168 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 51,8% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 146. Therefore, our sequencing results this week are not 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.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 371 specimens, with 194 collected in week 22/2021. The sequencing pool referring to Luxembourgish residents represents 52,8% of new infections reported in Luxembourg in week 22/2021. Among these 194 specimens, 40 specimens were reported to be part of a cluster or outbreak investigation, and 23 specimens were from non-residents (15 specimen overlapping). This leads to 146 specimens, collected in week 22, and being the representative population sequencing sample. In the population representative sample of residents, the frequencies of B.1.1.7 and  B.1.617.2 were 46,6% and 30,8% respectively. No cases have been detected for the B.1.351 and P.1 variants.

The population sequencing coverage in week 22/2021 was 45,1% (Figure 2). Based on statistical inference, the frequency of the reported variants in week 22/2021 is not representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v3.0.3, pangoLEARN version 2021-05-27).

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 the week 22/2021, in the population representative sample, after removal of cluster specimens, and excluding specimens collected from non-residents, there were 13 circulating SARS-CoV-2 variants, with the main three variants being  B.1.1.7 (46,6%, CI 38,5% – 54,7%), B.1.617.2 (30,8%, CI 23,3% – 38,3%) and B.1.1 (5,5%, CI 1,8% – 9,2%), as shown in Figure 3.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, B.1.525, B.1.617.2)

Among specimens collected within the week 22/2021, 92 cases of the B.1.1.7 variant have been detected, representing 47,4% of the specimens in the week’s sequencing pool (by comparison, the week 21/2021 pool had shown a frequency of 65,2% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 6230 by week 22/2021.

In the collection period of week 22/2021, no case of the South African variant B.1.351 has been detected (by comparison, the week 20/2021 pool had shown 3,1% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 1048 by week 22/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 29/MAY/2021.

In week 22/2021, no new case of B.1.525 and P.1 has been detected. The case count by week 22 for B.1.525 remains 45 (latest sampling date 17/MAY/2021) and for P.1 the case count increases due to retrospective sequencing to 112 (latest sampling date 30/MAY/2021).

In week 22/2021, 60 additional cases of the Delta variant B.1.617.2 have been detected (latest sampling date 06/June/2021). The case count by week 22 for B.1.617.1 remains 6, and for B.1.617.2 it raises to 149. Since  May 6^th 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 1^st  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 more transmissible as B.1.1.7 (increasing confidence). Neutralization studies show reductions in cross-neutralizing activity between B.1.1.7 and B.1.351.

By week 22/2021, 78,3 % of the variants detected in the sequenced specimen pool are declared as either Variants of Concern. These include B.1.1.7, which is continuously decreasing, apparently being displaced by B.1.617.2.

WHO nomeculature to track VOC

The ReViLux will continue to use the “Pango” nomenclature system to allow easier links to any evolving variants with their ancestor (Figure 5).

Clinically relevant mutations

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 30/MAY/2021 (N=12080), as well as the frequencies in week 22/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

 

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 24/05/-30/05/2021)

Executive Summary

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 21/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all sequenced specimens decreased to 66% (CI 59,6% – 72,4%, p<0,05). For the SARS-CoV-2 B.1.351 and P.1 variant, we found for each an overall frequency of 2,8% (CI 0,6% – 5%, p<0,05) within the sequenced sample. The frequency detected for the variant B.1.617.2 doubled to 14.6% (CI 9,8% – 19,3%, p<0,05).

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 21 (354). The minimum sample size required to detect prevalence of B.1.1.7 (76%) reported in week 20, with an error margin of 5%, was estimated to be 157 specimens. This number corresponds to a coverage of 44.3 %, 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 293, with 212 specimens having been collected in the time frame of week 21/2021. The sequencing coverage this week was 55,4% from all positive cases in Luxembourg (based on sequenced samples collected in the time frame of week 21 and corresponding to Luxembourg residents).

Clinical Surveillance

The “Sentinel” surveillance network reported 187 consultations in week 21 (24/MAY/2021 – 30/MAY/2021). There were 2 cases of ILI¹, corresponding to 1,1% of the consultations, as shown in Figure 1. The number of consultations for ARI² was 34, which represents 18.2% of the consultations.

Virological Surveillance

Covid Consultation Centres have been closed by 16/May/2021. We are currently working on an alternative in partnership with private laboratories.

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 21 (354). The minimum sample size required to detect prevalence of B.1.1.7 (76%) reported in week 20 with an error margin of 5% was estimated to be 157 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 44,3% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 196. 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 will introduced the PCR screening for VOC on 14/06/2021, using the Allplex™ SARS-CoV-2 Variants I Assay which will allow simultaneous detection and differentiation of SARS-CoV-2 and three mutation sites in S gene for identification of SARS-CoV-2 variants to detect the Variants of Concern (VOCs. This will enable a faster investigation time of any outbreak and allow us to identify variants of concerns that do not pass the quality criteria for sequencing.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gisaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 293 specimens, with 212 collected in week 21/2021. The sequencing pool referring to Luxembourgish residents represents 55,4% of new infections reported in Luxembourg in week 21/2021. Among these 212 specimens, 12 specimens were reported to be part of a cluster or outbreak investigation, and 15 specimens were from non-residents (5 specimen overlapping). This leads to 190 specimens, collected in week 21, 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 65,3%, 16,3%, 3,2% and 2,1% respectively.

The population sequencing coverage in week 21/2021 was 55,4% (Figure 2). Based on statistical inference, the frequency of the reported variants in week 21/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v3.0.3, pangoLEARN version 2021-05-27).

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 21/2021, in the population representative sample, after removal of cluster specimens, and excluding specimens collected from non-residents, there were 13 variants, with the main three variants being B.1.1.7 (65,3%, CI 58,5% – 72,1%), followed by B.1.617.2 (16,3%, CI 11% – 21,5%) and P.1.1 / B.1.1 (3,7% each , CI 1% – 6,4%), as shown in Figure 3.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, B.1.525, B.1.617.2)

Among specimens collected within the week 21/2021, 140 cases of the B.1.1.7 variant have been detected, representing 66% of the specimens in the week’s sequencing pool (by comparison, the week 20/2021 pool had shown a frequency of 76,1% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 6053 by week 21/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 30/05/2021.

In the collection period of week 21/2021, 6 cases of the South African variant B.1.351 have been detected, representing 2,8% of the specimens in the week’s sequencing pool (by comparison, the week 20/2021 pool had shown 2,5% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 1031 by week 21/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 28/MAY/2021.

In week 21/2021, no new case of B.1.525 has been detected, and 6 new cases of the Brazilian variant P.1. The case count by week 21 for B.1.525 remains 45 (latest sampling date 17/MAY/2021) and for P.1 the case count increases to 111 (latest sampling date 30/MAY/2021)

In week 21/2021, 31 additional cases of the Delta variant B.1.617.2 have been detected (latest sampling date 30/MAY/2021). The case count by week 21 for B.1.617.1 remains 6, and for B.1.617.2 it raises to 85. Since  May 6^th, 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 1^st  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 (increasing confidence). Neutralization studies show reductions in cross-neutralizing activity between B.1.1.7 and B.1.351.

By week 21/2021, 86,3 % 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).

New WHO nomeculature to track VOC

The World Health Organization (WHO) has recently announced a new nomeculature system for tracking SARS-CoV-2 variants of interest (VOI) and variants of concern (VOC). The system is based on the Greek alphabet.

According to the new system, B.1.1.7 was the first VOC designated by WHO and it is now called Alpha. The B.1.351, which originated in Brazil, is now called Beta. The two other VOCs are the P.1, the variant first identified in Brazil and now referred to as Gamma, and the B.1.617.2 that originated in India, now called Delta (Figure 5).

The ReViLux will continue to use the Rambaut et al. (“Pango”) nomenclature system to enable linking between any evolving variants and their ancestor.

Clinically relevant mutations

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 30/MAY/2021 (N=12080), as well as the frequencies in week 21/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 17/05/-23/05/2021)

Executive Summary

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).

Clinical Surveillance

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.

Virological Surveillance

Covid Consultation Centres have been closed by 16/May/2021. We are currently working on an alternative surveillance solution in partnership with private laboratories.

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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.

Sequenced specimens

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%.

Circulating lineage detection

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.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, B.1.525, B.1.617.2)

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 6^th 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 1^st  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).

Clinically relevant mutations

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.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

 

 

 

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 10/05/-16/05/2021)

Executive Summary

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 19/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all sequenced specimens remained rather stable on 79% (CI 75,6% – 82,4%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 5,7% (CI 3,7% – 7,6%, p<0,05) within the sequenced sample, while for P.1, the frequency decreased to 3,7% (CI 2,1% – 5,3%, p<0,05). The frequency of the “indian variant” B.1.617.2 increased to 3,9% (CI 2,3% – 5,5%, p<0,05).

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 19 (759). The minimum sample size required to detect prevalence of B.1.1.7 (82%) reported in week 18, with an error margin of 5%, was estimated to be 175 specimens. This number corresponds to a coverage of 23,1 %, 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 630, with 543 specimens having been collected in the time frame of week 19/2021. The sequencing coverage this week was 67,5% from all positive cases in Luxembourg (based on sequenced samples collected in the time frame of week 19 and corresponding to Luxembourg residents).

Clinical Surveillance

The “Sentinel” surveillance network reported 299 consultations in week 19 (10/MAY/2021 – 16/MAY/2021). There were 3 cases of ILI¹, corresponding to 1% of the consultations, as shown in Figure 1. The number of consultations for ARI² was 37, which represents 12% of the consultations.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SARS-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 36 positive cases (33%). The wave of Human Rhinovirus (HRV) continued in week 19/2021, with 9 positive cases in 108 tests (8,3%). Four cases of MPV (3,7%) have been detected and no cases of other respiratory viruses like ADV, PIV or RSV.

No case  of Influenza A/B was detected. (c.f. Figure 2).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 19 (759). The minimum sample size required to detect prevalence of B.1.1.7 (82%) reported in week 18 with an error margin of 5% was estimated to be 175 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 23,1% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 449. 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.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 630 specimens, with 543 collected in week 19/2021. The sequencing pool referring to Luxembourgish residence represents 67,5% of new infections reported in Luxembourg in week 19/2021. Among these 543 specimens, 65 specimens were reported to be part of a cluster or outbreak investigation, and 31 specimens were from non-residents (2 specimen overlapping). This leads to 449 specimens, collected in week 19, and being the representative population sequencing sample. In the population representative sample of residents, the frequency of B.1.1.7 and B.1.351 was 81,7% and 6% respectively, while the frequency of P.1 and B.1.617.2 was 3,6% and 2,2% respectively.

The population sequencing coverage in week 19/2021 was 67,5% (Figure 3). Based on statistical inference, the frequency of the reported variants in week 19/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

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 19/2021, in the population representative sample, after removal of cluster specimens, and excluding specimens collected from non-residents, there were 14 circulating SARS-CoV-2 variants, with the main three variants being B.1.1.7 (81,7%, CI 78,1% – 85,3%), B.1.351 (6,0%,CI 3,8% – 8,2%), followed by P.1 (3,6%, CI 1,9% – 5,3%), as shown in Figure 4.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, A.23.1, B.1.525, B.1.617.2)

Among specimens collected within the week 19/2021, 429 cases of the B.1.1.7 variant have been detected, representing 79% of the specimens in the week’s sequencing pool (by comparison, the week 18/2021 pool had shown a frequency of 81,6% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 5610 by week 19/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 16/05/2021.

In the collection period of week 19/2021, 31 cases of the South African variant B.1.351 have been detected, representing 5,7% of the specimens in the week’s sequencing pool (by comparison, the week 18/2021 pool had shown 7,0% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 1007 by week 19/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 15/MAY/2021.

In week 19/2021, 13 additional cases of B.1.525 has been detected, and 20 new cases of the Brazilian variant P.1. Thus, the case count by week 19 for B.1.525 is 39 (latest sampling date 14/MAY/2021) and for P.1 the case count is 93 (latest sampling date 16/MAY/2021).

In week 19/2021, 21 additional cases of the B.1.617 subtype B.1.617.2 have been detected (latest sampling date 16/MAY/2021). The case count by week 19 for B.1.617.1 remains 6, and for B.1.617.2 it raises to 26. Since  May 6^th B.1.617.2 has been escalated by Public Health England from a variant under investigation to a variant of concern.

By now, no case of A.23.1 has been detected in Luxembourg (Figure 5).

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 1^st  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 moderate confidence).

Figure 5  Evolution of variants in Sequencing pool of all specimens including targeted sequencing (cluster/contact tracing/ non residents) since first detection of B.1.1.7 in Luxembourg

By week 19/2021, 95,6 % 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).

Clinically relevant mutations

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 16/MAY/2021 (N=11447), as well as the frequencies in week 19/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

 

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 03/05/-09/05/2021)

Executive Summary

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 18/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all sequenced specimens remained stable on 81,1% (CI 77,6% – 84,6%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 6,8% (CI 4,5% – 9%, p<0,05) within the sequenced sample, while for P.1, the frequency increased to 4,6% (CI 2,7% – 6,5%, p<0,05).

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 18 (936). The minimum sample size required to detect prevalence of B.1.1.7 (81%) reported in week 17, with an error margin of 5%, was estimated to be 189 specimens. This number corresponds to a coverage of 20,2 %, 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 500, with 482 specimens (deduplicated) having been collected in the time frame of week 18/2021. The sequencing coverage this week was 47,2% from all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 346 consultations in week 18 (03/MAY/2021 – 09/MAY/2021). There were 2 cases of ILI¹, corresponding to 0,6% of the consultations, as shown in Figure 1. The number of consultations for ARI² was 47, which represents 13,6% of the consultations.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SARS-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 33 positive cases (28,7%). The wave of Human Rhinovirus (HRV) continued in week 18/2021, with 10 positive cases in 115 tests (8,7%). Sporadic cases of other respiratory viruses continued to appear, such as 2 cases of PIV (1,7%) and MPV (each 1,7%) and 1 case of RSV (0,9%). No case  of Influenza A/B was detected. (c.f. Figure 2).

In Luxembourg, we have tested 115 samples from the Sentinel surveillance network, as compared to 555 specimens tested in Europe, in the week 18/2021. None of these 555 specimens tested positive for Influenza type A/B virus. Overall, surveillance was very efficient over the course of the 2020-2021 season and although there was a small decrease in the number of samples tested as compared with previous seasons, there was a remarkable decrease (>99% decrease) in the number of influenza infections detected, with numbers detected on a weekly basis being similar to those reported during interseasonal periods. (Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 18 (936). The minimum sample size required to detect prevalence of B.1.1.7 (81%) reported in week 17 with an error margin of 5% was estimated to be 189 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 20,2% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 397. 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.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 500 specimens, with 482 collected in week 18/2021. The sequencing pool represents 47,2% of new infections reported in Luxembourg in week 18/2021. Among these 482 specimens, 50 specimens were reported to be part of a cluster or outbreak investigation, and 40 specimens were from non-residents (5 specimen overlapping). This leads to 397 specimens, collected in week 18, and being the representative population sequencing sample. In the population representative sample of residents, the frequency of B.1.1.7 and B.1.351 was 83,4% and 5,8% respectively, while the frequency of P.1 was 3,8%.

The population sequencing coverage in week 18/2021 was 47,2% (Figure 3). Based on statistical inference, the frequency of the reported variants in week 18/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

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 18/2021, in the population representative sample, after removal of cluster specimens, and excluding specimens collected from non-residents, there were 13 circulating SARS-CoV-2 variants, with the main three variants being B.1.1.7 (83,4%, CI 79,7% – 87,1%), B.1.351 (5,8%, CI 3,5% – 8,1%), followed by P.1 (3,8%, CI 1,9% – 5,7%), as shown in Figure 4.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, A.23.1, B.1.525, B.1.617.2)

Among specimens collected within the week 18/2021, 391 cases of the B.1.1.7 variant have been detected, representing 81,1% of the specimens in the week’s sequencing pool (by comparison, the week 17/2021 pool had shown a frequency of 80,6% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 4098 by week 18/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 09/05/2021.

In the collection period of week 18/2021, 33 cases of the South African variant B.1.351 have been detected, representing 6,8% of the specimens in the week’s sequencing pool (by comparison, the week 17/2021 pool had shown 10,8% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 967 by week 18/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 09/MAY/2021.

In week 18/2021, 1 additional case of B.1.525 has been detected, and 22 new cases of the Brazilian variant P.1. Thus, the case count by week 18 for B.1.525 is 26 (latest sampling date 07/MAY/2021) and for P.1 the case count is 72 (latest sampling date 08/MAY/2021).

In week 18/2021, 4 additional case of the B.1.617 subtype B.1.617.2 have been detected (latest sampling date 07/MA/2021). The case count by week 18 for B.1.617.1 remains 6, and for B.1.617.2 it raises to 5. Since  May 6^th B.1.617.2 has been escalated by Public Health Engand from a variant under investigation to a variant of concern.

By now, no case of A.23.1 has been detected in Luxembourg (Figure 5).

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 1^st  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 moderate confidence).

By week 18/2021, 96,5 % 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).

Clinically relevant mutations

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 09/MAY/2021 (N=10831), as well as the frequencies in week 18/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 26/04/-02/05/2021)

Executive Summary

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 17/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all sequenced specimens decreased to 80,8% (CI 77,6% – 84%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 10,7% (CI 8,2% – 13,2%, p<0,05) within the sequenced sample, while for P.1, the frequency was 2,6% (CI 1,3% – 3,9%, p<0,05).

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 17 (1117). The minimum sample size required to detect prevalence of B.1.1.7 (85%) reported in week 16, with an error margin of 5%, was estimated to be 167 specimens. This number corresponds to a coverage of 14,9 %, 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 678, with 588 specimens (deduplicated) having been collected in the time frame of week 17/2021. The sequencing coverage this week was 52,6% from all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 324 consultations in week 17 (26/APR/2021 – 02/MAY/2021). There were 4 cases of ILI¹, corresponding to 1,2% of the consultations, as shown in Figure 1. The number of consultations for ARI² was 23, which represents 7,1% of the consultations.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SARS-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 37 positive cases (28,9%). The wave of Human Rhinovirus (HRV) continued in week 17/2021, with 9 positive cases in 128 tests (7%). Sporadic cases of other respiratory viruses continued to appear, such as 2 cases of PIV and RSV (each 1,5%) and 1 case of ADV. No case  of Influenza A/B was detected. (c.f. Figure 2).

In Luxembourg, we have tested 128 samples from the Sentinel surveillance network, as compared to 822 specimens tested in Europe, in the week 17/2021. None of these 822 specimens tested positive for Influenza type A/B virus (Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 17 (1117). The minimum sample size required to detect prevalence of B.1.1.7 (85%) reported in week 16 with an error margin of 5% was estimated to be 167 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 14,9% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 498. 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.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 678 specimens, with 588 collected in week 17/2021. This represents 52,6% of the new infections reported in Luxembourg in week 17/2021. Among these 588 specimens, 60 specimens were reported to be part of a cluster or outbreak investigation, and 40 specimens were from non-residents (10 specimen overlapping). This leads to 498 specimens, collected in week 17, and being the representative population sequencing sample. In the population representative sample of residents, the frequency of B.1.1.7 and B.1.351 was 82,1% and 10,2% respectively, while the frequency of P.1 was 2,2%.

The population sequencing coverage in week 17/2021 was 52,6% (Figure 3). Based on statistical inference, the frequency of the reported variants in week 17/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

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 2021-04-28).

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 17/2021, in the population representative sample, after removal of cluster specimens, and excluding specimens collected from non-residents, there were 13 circulating SARS-CoV-2 variants, with the main three variants being B.1.1.7 (82,1%, CI 78,7% – 85,5%), B.1.351 (10,2%, CI 7,5% – 12,9%), followed by P.1 (2,2%, CI 0,9% – 3,5%), as shown in Figure 4.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, A.23.1, B.1.525, B.1.617.2)

Among specimens collected within the week 17/2021, 475 cases of the B.1.1.7 variant have been detected, representing 80,8% of the specimens in the week’s sequencing pool (by comparison, the week 16/2021 pool had shown a frequency of 85,4% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 4704 by week 17/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 02/05/2021.

In the collection period of week 17/2021, 63 cases of the South African variant B.1.351 have been detected, representing 10,7% of the specimens in the week’s sequencing pool (by comparison, the week 16/2021 pool had shown 9,9% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 933 by week 17/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 02/MAY/2021.

In week 17/2021, 10 additional cases of B.1.525 have been detected, and 15 new cases of the Brazilian variant P.1. Thus, the case count by week 17 for B.1.525 is 25 (latest sampling date 30/APR/2021) and for P.1 the case count is 49 (latest sampling date 30/APR/2021).

In week 17/2021, 1 additional case of the B.1.617 subtype B.1.617.1 has been detected (latest sampling date 26/APR/2021). The case count by week 17 for B.1.617.1 is six, and for B.1.617.2 one. Since  May 6^th 2021, B.1.617.2 has been escalated by Public Health Engand from a variant under investigation to a variant of concern.

By now, no case of A.23.1 has been detected in Luxembourg (Figure 5).

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 1^st  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 moderate confidence).

By week 17/2021, 97,5 % of detected variants in Luxembourg 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).

Clinically relevant mutations

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 02/MAY/2021 (N=10333), as well as the frequencies in week 17/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 19/04/-25/04/2021)

Executive Summary

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 16/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all sequenced specimens remained stable at 85,2% (CI 82,6% – 87,8%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 9,9% (CI 7,7% – 12,1%, p<0,05) within the sequenced sample, while for P.1, the frequency was 1,4% (CI 0,5% – 2,3%, p<0,05).

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 16 (1226). The minimum sample size required to detect prevalence of B.1.1.7 (85%) reported in week 14, with an error margin of 5%, was estimated to be 170 specimens. This number corresponds to a coverage of 13,8 %, 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 714, with 695 specimens having been collected in the time frame of week 16/2021. The sequencing coverage this week was 56,7% from all positive cases in Luxembourg, hence representative.

Clinical Surveillance

The “Sentinel” surveillance network reported 140 consultations in week 16 (19/APR/2021 – 25/APR/2021). There was no case of ILI¹, as shown in Figure 1. The number of consultations for ARI² was 11, which represents 7,9% of the consultations.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SARS-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 80 positive cases (37,9%). The wave of Human Rhinovirus (HRV) continued in week 16/2021, with 17 positive cases in 211 tests (8%). Sporadic cases of other respiratory viruses continued to appear, such as 2 cases of PIV (0,9%) and 3 cases of ADV. No case  of Influenza A/B was detected. (c.f. Figure 2).

In Luxembourg, we have tested 211 samples from the Sentinel surveillance network, as compared to 996 specimens tested in Europe, in the week 16/2021. None  of these 996 specimens tested positive for Influenza type A/B virus. Influenza activity remained at interseasonal levels (Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 16 (1226). The minimum sample size required to detect prevalence of B.1.1.7 (85%) with an error margin of 5% was estimated to be 170 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 13,8% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 605. 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 are evaluating 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 monthly basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 714 specimens, with 695 collected in week 16/2021. This represents 56,7% of the new infections reported in Luxembourg in week 16/2021. Among these 650 specimens, 52 specimens were reported to be part of a cluster or outbreak investigation, and 44 specimens were from non-residents (6 specimen overlapping). This leads to 605 specimens, collected in week 16, and being the representative population sequencing sample. In the population representative sample of residents, the frequency of B.1.1.7 and B.1.351 was 85,3% and 10,4% respectively, while the frequency of P.1 was 1,2%.

The population sequencing coverage in week 16/2021 was 56,7% (Figure 3). Based on statistical inference, the frequency of the reported variants in week 16/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v2.3.8, pangoLEARN version 2021-04-21).

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 16/2021, in the population representative sample, after removal of cluster specimens, and excluding specimens collected from non-residents, there were 14 circulating SARS-CoV-2 variants, with the main three variants being B.1.1.7 (85,3%, CI 82,5% – 88,1%), B.1.351 (10,4%, CI 8% – 12,8%), followed by P.1 (1,2%, CI 0,3% – 2,1%), as shown in Figure 4.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 16/2021, 592 cases of the B.1.1.7 variant have been detected, representing 85,2% of the specimens in the week’s sequencing pool (by comparison, the week 15/2021 pool had shown a frequency of 85,8% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 4229 by week 16/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 15/APR/2021.

In the collection period of week 16/2021, 69 cases of the South African variant B.1.351 have been detected, representing 9,9% of the specimens in the week’s sequencing pool (by comparison, the week 15/2021 pool had shown 8,7% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 870 by week 16/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 25/APR/2021.

In week 16/2021, 7 additional cases of B.1.525 have been detected, and 10 new cases of the Brazilian variant P.1. Thus, the case count by week 16 for B.1.525 is 15 (latest sampling date 23/APR/2021) and for P.1 the case count is 34 (latest sampling date 23/APR/2021).

In week 16/2021, 3 cases of the so called “double mutant” or “indian variant” B.1.617, subtype B.1.617.1, have been detected in Luxembourg. By now, no case of A.23.1 has been detected in Luxembourg (Figure 5).

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 1 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.

By week 16/2021, 98,5 % of detected variants in Luxembourg are declared as either Variants of Concern (VOC – B.1.1.7, B.1.351, P.1) or Variants Under Investigation (VUI – B.1.525, B.1.1.318, B.1.617).

Clinically relevant mutations

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 25/APR/2021 (N=9653), as well as the frequencies in week 16/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 12/04/-18/04/2021)

Executive Summary

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 15/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all sequenced specimens increased to 85,8% (CI 83,1% – 88,5%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 8,8% (CI 6,6% – 11%, p<0,05) within the sequenced sample, while for P.1, the frequency was 0,77% (CI 0,1% – 1,5%, p<0,05).

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 15 (1261). The minimum sample size required to detect prevalence of B.1.1.7 (81%) reported in week 14, with an error margin of 5%, was estimated to be 200 specimens. This number corresponds to a coverage of 15,8 %, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 15 are representative of the circulating variants in Luxembourg with a margin of error of 5%.

The total number of sequences performed this week was 687, with 650 specimens having been collected in the time frame of week 15/2021. The sequencing coverage this week was 51,6% from all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 146 consultations in week 15 (12/APR/2021 – 18/APR/2021). There was no case of ILI¹, as shown in Figure 1. The number of consultations for ARI² was 14, which represents 9,6% of consultations.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SARS-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 66 positive cases (29,7%). The wave of Human Rhinovirus (HRV) continued in week 15/2021, with 14 positive cases in 222 tests (6,3%). Sporadic cases of other respiratory viruses continued to appear, such as 5 cases of PIV (2,3%), 2 cases of MPV and 1 case of ADV. No case  of Influenza A/B was detected. (c.f. Figure 2).

In Luxembourg, we have tested 222 samples from the Sentinel surveillance network, as compared to 1271 specimens tested in Europe, in the week 15/2021. Four of these 1271 specimens tested positive for Influenza type A virus. Influenza activity remained at interseasonal levels.(Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 15 (1261). The minimum sample size required to detect prevalence of B.1.1.7 (81%) with an error margin of 5% was estimated to be 200 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 15,8% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens from Luxembourgish residents sequenced this week was 561. 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 are evaluating 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.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 687 specimens, with 650 collected in week 15/2021. This represents 51,6% of the new infections reported in Luxembourg in week 15/2021. Among these 650 specimens, 46 specimens were reported to be part of a cluster or outbreak investigation, and 43 specimens were from non-residents (10 specimen overlapping). This leads to 561 specimens, collected in week 15, and being the representative population sequencing sample. In the population representative sample of residents, the frequency of B.1.1.7 and B.1.135 was 86,3% and 8,9% respectively, while the frequency pf P.1 was 0,5%.

The population sequencing coverage in week 15/2021 was 51,6% (Figure 3). Based on statistical inference, the frequency of the reported variants in week 15/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v2.3.8, pangoLEARN version 2021-04-14).

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 15/2021, in the population representative sample, after removal of cluster samples, 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 (86,3%, CI 83,4% – 89,1%), B.1.351 (8,9%, CI 6,5% – 11,3%), followed by B.1 (0,5%, CI 0% – 1,1%), as shown in Figure 4.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 15/2021, 558 cases of the B.1.1.7 variant have been detected, representing 85,8% of the specimens in the week’s sequencing pool (by comparison, the week 14/2021 pool had shown a frequency of 81,5% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 3626 by week 15/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 18/APR/2021.

In the collection period of week 15/2021, 57 cases of the South African variant B.1.351 have been detected, representing 8,8% of the specimens in the week’s sequencing pool (by comparison, the week 14/2021 pool had shown 14% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 799 by week 15/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 18/APR/2021.

In week 15/2021, three additional cases of B.1.525 have been detected, and 5 new cases of the Brazilian variant P.1. Thus, the case count by week 15 for B.1.525 is 8 (latest sampling date 13/APR/2021) and for P.1 the case count is 24 (latest sampling date 17/APR/2021).

By now, no case of A.23.1 has been detected in Luxembourg.

In week 15/2021, 3 cases of the so called “double mutant” or “indian variant” B.1.617 have been detected in Luxembourg. All detected cases are linked to travelling (Figure 5).

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 1 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.

By week 15/2021, 96,6 % of detected variants in Luxembourg are declared as either Variants of Concern (VOC – B.1.1.7, B.1.351, P.1) or Variants Under Investigation (VUI – B.1.525, B.1.1.318, B.1.617).

Clinically relevant mutations

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 18/APR/2021 (N=8938), as well as the frequencies in week 15/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 05/04/-11/04/2021)

Executive Summary

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 14/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all samples sequenced increased to 81,4% (CI 78,4% – 84,4%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 14,2% (CI 11,5% – 16,9%, p<0,05) within the sequenced samples and for P.1, 1,25% (CI 0,4% – 2,1%, p<0,05).

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 14 (1265). The minimum sample size required to detect prevalence of B.1.1.7 (75%) reported in week 13, with an error margin of 5%, was estimated to be 235 specimens. This number corresponds to a coverage of 18,6 %, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 14 are representative of the circulating variants in Luxembourg with a margin of error of 5%.

The total number of sequences performed this week was 661, with 641 specimens having been collected in the time frame of week 14/2021. The sequencing coverage this week was 50,7% from all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 102 consultations in week 14 (05/APR/2021 – 11/APR/2021). There was no case of ILI¹, as shown in Figure 1. The number of consultations for ARI² was 15, which represents 15% of consultations.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SARS-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 92 positive cases (32%). The wave of Human Rhinovirus (HRV) continued in week 14/2021, with 13 positive cases in 289 tests (4,4%). Sporadic cases of other respiratory viruses continued to appear, such as 8 cases of PIV (2,8%), 4 cases of MPV and 1 case of RSV. No case  of Influenza A/B was detected. (c.f. Figure 2).

In Luxembourg, we have tested 289 samples from the Sentinel surveillance network, as compared to 948 specimens tested in Europe, in the week 14/2021. Two of these 948 specimens tested positive for Influenza type A virus. Influenza activity remained at interseasonal levels.(Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 for week 14 (1265). The minimum sample size required to detect prevalence of B.1.1.7 (75%) with an error margin of 5% was estimated to be 235 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 18,6% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens of Luxembourgish residents sequenced this week was 565. 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.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gisaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 661 specimens, with 641 collected in week 14/2021. This represents 50,7% of the new infections reported in Luxembourg in week 14/2021. Among the 641 specimens, 28 specimens were reported to be part of a cluster or outbreak investigation, and 48 specimens were from non-residents. This leads to 565 specimens, collected in week 14, and being the representative population sequencing sample. In the population representative sample of residents, the frequency of B.1.1.7 and B.1.135 was 81,4% and 14,2% respectively.

The population sequencing coverage in week 14/2021 was 50,7% (Figure 3). Based on statistical inference, the frequency of the reported variants in week 14/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v2.3.6, pangoLEARN version 2021-03-29).

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 14/2021, in the population representative sample, after removal of cluster samples, and excluding specimens collected from non-residents, there were 13 circulating SARS-CoV-2 variants, with the main three variants being B.1.1.7 (81,4%, CI 78,4% – 84,4%) followed by B.1.351 (14,2%, CI 11,5% – 16,9%) and P.1 (1,25% , CI 0,4% – 2,1%), as shown in Figure 4.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 14/2021, 522 cases of the B.1.1.7 variant have been detected, representing 81,4% of the specimens in the week’s sequencing pool (by comparison, the week 13/2021 pool had shown a frequency of 75,5% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 3028 by week 14/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 11/APR/2021.

In the collection period of week 14/2021, 91 cases of the South African variant B.1.351 have been detected, representing 14,2% of the specimens in the week’s sequencing pool (by comparison, the week 13/2021 pool had shown 17,7% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 734 by week 14/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 11/APR/2021.

In week 14/2021 two additional cases of B.1.525 and 8 new cases of the Brazilian variant P.1 have been detected. Thus, the case count by week 14 for B.1.525 is 5 (latest sampling date 07/APR/2021) and for P.1, 17 cases (latest sampling date 10/APR/2021).

By now, no case of A.23.1 has been detected in Luxembourg (Figure 5).

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.

Clinically relevant mutations

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 11/APR/2021 (N=8251), as well as the frequencies in week 14/2021.

References

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

https://github.com/cov-lineages/pangolin

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

 

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 29/03/-04/04/2021)

Executive Summary

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 13/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all samples sequenced increased to 75,5% (CI 71,6% – 79,4%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 17,9% (CI 14,4% – 21,3%, p<0,05) within the sequenced samples.

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 13 (1544). The minimum sample size required to detect prevalence of B.1.1.7 (74%) reported in week 12, with an error margin of 5%, was estimated to be 249 specimens. This number corresponds to a coverage of 16,1 %, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 13 are representative of the circulating variants in Luxembourg with a margin of error of 5%.

The total number of sequences performed this week was 529, with 474 specimens having been collected in the time frame of week 13/2021. The sequencing coverage this week was 30,7% from all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 178 consultations in week 13 (29/MAR/2021 – 04/APR/2021). There was no case of ILI¹, as shown in Figure 1. The number of consultations for ARI² was 35, which represents 20% of consultations.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SAR-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 46 positive cases (22%). The wave of Human Rhinovirus (HRV) continued in week 13/2021, with 21 positive cases in 208 tests (10,1%). Sporadic cases of other respiratory viruses continued to appear, such as 6 cases of PIV in 208 tests (2,8%), 2 cases of MPV and 1 case of ADV. One case  of Influenza B was detected and no case of Influenza A (Figure 2).

In Luxembourg, we have tested 208 samples from the Sentinel surveillance network, as compared to 1019 specimens tested in Europe, in the week 13/2021. One of these 1189 specimens tested positive for Influenza type B virus. The influenza epidemic in the European Region has usually reached its peak and starts to decline by this time of the year but, despite widespread and regular testing for influenza, reported influenza activity still remains at a very low level, likely due to the impact of the various public health and social measures, implemented to reduce transmission of SARS-CoV-2 (Source: FluNews Europe).

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SAR-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 46 positive cases (22%). The wave of Human Rhinovirus (HRV) continued in week 13/2021, with 21 positive cases in 208 tests (10,1%). Sporadic cases of other respiratory viruses continued to appear, such as 6 cases of PIV in 208 tests (2,8%), 2 cases of MPV and 1 case of ADV. One case  of Influenza B was detected and no case of Influenza A (Figure 2).

In Luxembourg, we have tested 208 samples from the Sentinel surveillance network, as compared to 1019 specimens tested in Europe, in the week 13/2021. One of these 1189 specimens tested positive for Influenza type B virus. The influenza epidemic in the European Region has usually reached its peak and starts to decline by this time of the year but, despite widespread and regular testing for influenza, reported influenza activity still remains at a very low level, likely due to the impact of the various public health and social measures, implemented to reduce transmission of SARS-CoV-2 (Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 for week 13 (1544). The minimum sample size required to detect prevalence of B.1.1.7 (74%) with an error margin of 5% was estimated to be 249 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 16,1% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens of Luxembourgish residents sequenced this week was 382. 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.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gisaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 529 specimens, with 474 collected in week 13/2021. This represents 30,7% of the new infections reported in Luxembourg in week 13/2021. Among the 474 specimens, 58 specimens were reported to be part of a cluster or outbreak investigation, and 37 specimens were from non-residents (3 specimens belong to both groups). This leads to 382 specimens, collected in week 13, and being the representative population sequencing sample. In the population representative sample of residents, the frequency of B.1.1.7 and B.1.135 was 74,1% and 19,4% respectively.

The population sequencing coverage in week 13/2021 was 30,7% (Figure 3). Based on statistical inference, the frequency of the reported variants in week 13/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v2.3.6, pangoLEARN version 2021-03-29).

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 13/2021, in the population representative sample, after removal of cluster samples, and excluding specimens collected from non-residents, there were 13 variants, with the main three variants being B.1.1.7 (74,1%, CI 69,7% – 78,5%), B.1.351 (19,4%, CI 15,4% – 23,4%), followed by P.1 / B.1 (1% each, CI 0% – 2%) as shown in Figure 4.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 13/2021, 358 cases of the B.1.1.7 variant have been detected, representing 75,5% of the specimens in the week’s sequencing pool (by comparison, the week 12/2021 pool had shown a frequency of 74,1% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 2496 by week 13/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 04/APR/2021.

In the collection period of week 13/2021, 85 cases of the South African variant B.1.351 have been detected, representing 17,9% of the specimens in the week’s sequencing pool (by comparison, the week 12/2021 pool had shown 18,2% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 643 by week 13/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 04/APR/2021.

In week 13/2021 one additional case of B.1.525 has been detected and 6 new cases of the Brazilian variant P.1. Thus, the case count by week 13 for B.1.525 is 3 (latest sampling date 31/MAR/2021) and for P.1 nine (latest sampling date 03/APR/2021).

By now, no case of A.23.1 has been detected in Luxembourg (Figure 5).

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.

Clinically relevant mutations

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 04/APR/2021 (N=7583), as well as the frequencies in week 13/2021.

References

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

https://github.com/cov-lineages/pangolin

Y Guangchuang et al. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 2017;8:28-36

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 22-28/03/2021)

Executive Summary

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 12/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all samples sequenced increased to 73,5% (CI 69,8% – 77,2%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 18,4% (CI 15,2% – 21,6%, p<0,05) within the sequenced samples.

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 12 (1690). The minimum sample size required to detect prevalence of B.1.1.7 (68%) reported in week 11, with an error margin of 5%, was estimated to be 280 specimens. This number corresponds to a coverage of 16,5 %, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 12 are representative of the circulating variants in Luxembourg with a margin of error of 5%.

The total number of sequences performed this week was 689, with 548 specimens having been collected in the time frame of week 12/2021. The sequencing coverage this week was 32,4% from all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 178 consultations in week 12 (22/MAR/2021 – 28/MAR/2021). There was no case of ILI¹, as shown in Figure 1. The percentage of consultations for ARI² was 19.6%.

1. ILI: – Influenza-Like Illness: Acute respiratory symptoms <10 days, Fever 38^∘C, systematic symptoms (myalgia, malaise, …)
2. ARI: – Acute Respiratory Infection: Acute respiratory symptoms (bronchitis, pharyngitis, rhinitis, pneumonia…) with or without fever.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SAR-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 69 positive cases (28%). The wave of Human Rhinovirus (HRV) continued in week 12/2021, with 37 positive cases in 243 tests (15,2%). Sporadic cases of other respiratory viruses continued to appear, such as 6 cases of PIV in 243 tests (2,4%). No cases of Influenza A/B were detected, indicating absence of circulation of Influenza viruses in Luxembourg, as shown in Figure 2.

In Luxembourg, we have tested 243 samples from the Sentinel surveillance network, as compared to 1189 specimens tested in Europe, in the week 12/2021. None of these 1189 specimens tested positive for type A Influenza virus. The influenza epidemic in the European Region has usually reached its peak by this point of the year but, despite widespread and regular testing for influenza, reported influenza activity still remains at a very low level, likely due to the impact of the various public health and social measures, implemented to reduce transmission of SARS-CoV-2 (Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 for week 12 (1690). The minimum sample size required to detect prevalence of B.1.1.7 (68%) with an error margin of 5% was estimated to be 280 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 16,5% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens of Luxembourgish residents sequenced this week was 498. 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.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gisaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 548 specimens having been collected in week 12/2021. This represents 32,4% of the new infections reported in Luxembourg in week 12/2021. Among the 548 specimens, 29 specimens were reported to be part of a cluster or outbreak investigation, and 21 specimens were from non-residents. This leads to 498 specimens, collected in week 12, and being the representative population sequencing sample. In the population representative sample of residents, the frequency of B.1.1.7 and B.1.135 was 74,1% and 18,1% respectively.

The population sequencing coverage in week 12/2021 was 32,4% (Figure 3). Based on statistical inference, the frequency of the reported variants in week 12/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v2.3.6, pangoLEARN version 2021-03-29).

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 12/2021, in the population representative sample, after removal of cluster samples, and excluding specimens collected from non-residents, there were 13 variants, with the main three variants being B.1.1.7 (74,1%, CI 70,2% – 77,9%), B.1.351 (18,1%, CI 14,7% – 21,5%), followed by B.1 (1,6%, CI 0,5% – 2,7%), as shown in Figure 4.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 12/2021, 403 cases of the B.1.1.7 variant have been detected, representing 73,5% of the specimens in the week’s sequencing pool (by comparison, the week 11/2021 pool had shown a frequency of 67,8% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.1.7 was 2115 by week 12/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 28/MAR/2021.

In the collection period of week 12/2021, 101 cases of the South African variant B.1.351 have been detected, representing 18,4% of the specimens in the week’s sequencing pool (by comparison, the week 11/2021 pool had shown 22,9% of this variant, including additional specimens having been sequenced from previous weeks). The total case count of sequenced variant B.1.351 was 554 by week 12/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 28/MAR/2021.

In week 12/2021 one additional case has been detected for B.1.525, but not any of the other variants of concern (P.1, A.23.1) (Figure 5).

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.

Clinically relevant mutations

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 22/MAR/2021 (N=7040), as well as the frequencies in week 12/2021.

References

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

https://github.com/cov-lineages/pangolin

Y Guangchuang et al. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 2017;8:28-36

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 15-21/03/2021)

Executive Summary

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 11/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all samples sequenced increased 68,1% (CI 64,2% – 72%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 23% (CI 19.4% - 26,5%, p<0,05) within the sequenced samples. The representative sample was estimated, based on the number of positive cases in Luxembourg for week 11 (1570). The minimum sample size required to detect prevalence of B.1.1.7 (68%) reported in week 11, with an error margin of 5%, was estimated to be 276 specimens. This number corresponds to a coverage of 17,6%, which exceeds the minimum coverage recommended by ECDC (10%). The sequencing results of week 11 are representative of the circulating variants in Luxembourg with a margin of error of 5%. The total number of sequences performed this week was 540, with all specimens having been collected in the time frame of week 11/2021. The sequencing coverage this week was 34,4% from all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 263 consultations in week 11 (15/MAR/2021 – 21/MAR/2021). There was 5 cases of ILI1, as shown in Figure 1, corresponding to 1,9% of consultations. The percentage of consultations for ARI2 was 17,9%.

1. ILI: – Influenza-Like Illness: Acute respiratory symptoms <10 days, Fever 38^∘C, systematic symptoms (myalgia, malaise, …)
2. ARI: – Acute Respiratory Infection: Acute respiratory symptoms (bronchitis, pharyngitis, rhinitis, pneumonia…) with or without fever.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SAR-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 64 positive cases (22%). The wave of Human Rhinovirus (HRV) continued in week 11/2021, with 34 positive cases in 291 tests (11,7%). Sporadic cases of other respiratory viruses continued to appear as one case of PIV was identified in week 11 (0,3%) and two cases of Adenovirus (ADV), corresponding to 0,7% of all tests. No cases of Influenza A/B were detected, indicating absence of circulation of Influenza viruses in Luxembourg, as shown in Figure 2.

In Luxembourg, we have tested 291samples from the Sentinel surveillance network, as compared to 1256 specimens tested in Europe, in the week 11/2021. One of these 1256 specimens tested positive for type A Influenza virus. The influenza epidemic in the European Region has usually reached its peak by this point of the year but, despite widespread and regular testing for influenza, reported influenza activity still remains at a very low level, likely due to the impact of the various public health and social measures, implemented to reduce transmission of SARS-CoV-2 (Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

1. Sequencing specimens from all hospitalized positive cases
2. Sequencing specimens from all positive cases from Airport testing program
3. Sequencing specimens from all outbreaks and identified clusters
4. Systematic sequencing of specimens from reinfections and post-vaccination-infections
5. Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 for week 11 (1570). The minimum sample size required to detect prevalence of B.1.1.7 (68%) with an error margin of 5% was estimated to be 276 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 17,6% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens sequenced this week was 515. Therefore, our sequencing results this week are representative of the circulating variants in Luxembourg with a margin of error of 5%.

The starting material used for sequencing is respiratory specimens (nasopharyngeal or oropharyngeal swabs) that have already been tested positive by RT PCR.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gsaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 540 specimens. All of these were collected in week 11/2021. This represents 32,8% of the new infections reported in Luxembourg in week 11/2021. Among the 540 specimens, 25 specimens were reported to be part of a cluster or outbreak investigation, and 41 specimens were from non-residents. This leads to 474 specimens, collected in week 11, and being the representative population sequencing sample. In the population representative sample of residents, the frequency of B.1.1.7 and B.1.135 was 68.3% and 22.5% respectively.

The population sequencing coverage in week 11/2021 was 34,4% (Figure 3). Based on statistical inference, the frequency of the reported variants in week 11/2021 is representative of the circulating variants in Luxembourg with a margin of error of 5%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software (v2.3, pangoLEARN version 2021-02-21).

In week 11/2021, in the population representative sample, after removal of the 25 cluster samples, and excluding specimens collected from non-residents, there were 13 variants, with the main three variants being B.1.1.7 (68,3%, CI 64,1% – 72,5%), B.1.351 (22.5%, CI 18,7% – 26,3%), followed by B.1.1.29 (2.1%, CI 0,8% – 3,4%), as shown in Figure 4.

Variants of concern tracker (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 11/2021, 368 cases of the B.1.1.7 variant have been detected, representing 68,1% of the specimens in the week’s sequencing pool (by comparison, the week 10/2021 pool had shown a frequency of 63,5% of this variant). The total case count of sequenced variant B.1.1.7 was 1623 by week 11/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 21/MAR/2021.

In the collection period of week 11/2021, 124 cases of the South African variant B.1.351 have been detected, representing 23% of the specimens in the week’s sequencing pool (by comparison, the week 10/2021 pool had shown 20,4% of this variant). The total case count of sequenced variant B.1.351 was 423 by week 10/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 21/MAR/2021.

In week 11/2021 no additional cases have been dected for any of the other variants of concern (P.1, B.1.525 or A.23.1) (Figure 5).

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.

Of note, additional specimens are in the pipeline for sequencing, so that numbers may change with increasing representativeness of circulating variants and proportions.

Clinically relevant mutations

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 21/MAR/2021 (N=6345), as well as the frequencies in week 11/2021.

References

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
https://github.com/cov-lineages/pangolin

Y Guangchuang et al. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 2017;8:28-36

For more information on lineages visit: https://cov-lineages.org

For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 08-14/03/2021)

Executive Summary

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 10/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all samples sequenced remained stable at 63,5% (CI 57,6% – 69,3%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 20,4% (CI 15.5% – 25,3%, p<0,05) within the sequenced samples.

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 10 (1249). The minimum sample size required to detect prevalence of B.1.1.7 (63%) reported in week 9, with an error margin of 5%, was estimated to be 279 specimens. This number corresponds to a coverage of 22,34%, which exceeds the minimum coverage recommended by ECDC (10%). Our sequencing results this week are representative of the circulating variants in Luxembourg with a margin of error of 6%.
The total number of sequences performed this week was 260, with all specimens having been collected in the time frame of week 10/2021. The sequencing coverage this week was 20,8% from all positive cases in Luxembourg.

Clinical Surveillance

The “Sentinel” surveillance network reported 146 consultations in week 10 (08/MAR/2021 – 14/MAR/2021). There was no case of ILI1, as shown in Figure 1. The percentage of consultations for ARI2 was 16,4%.

1. ILI: – Influenza-Like Illness: Acute respiratory symptoms <10 days, Fever 38^∘C, systematic symptoms (myalgia, malaise, …)
2. ARI: – Acute Respiratory Infection: Acute respiratory symptoms (bronchitis, pharyngitis, rhinitis, pneumonia…) with or without fever.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SAR-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 62 positive cases (24,9%). The wave of Human Rhinovirus (HRV) continued in week 10/2021, with 19 positive cases in 249 tests (7,6%). Three cases of PIV were identified in week 10 (1,2%). No cases of Influenza A/B were detected, indicating absence of circulation of Influenza viruses in Luxembourg, as shown in Figure 2.

In Luxembourg, we have tested 249 samples from the Sentinel surveillance network, as compared to 1110 samples tested in Europe, in the week 10/2021. None of these 1110 specimens tested for Influenza viruses were positive. The influenza epidemic in the European Region has usually reached its peak by this point of the year but, despite widespread and regular testing for influenza, reported influenza activity still remains at a very low level, likely due to the impact of the various public health and social measures, implemented to reduce transmission of SARS-CoV-2 (Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

1. Sequencing specimens from all hospitalized positive cases
2. Sequencing specimens from all positive cases from Airport testing program
3. Sequencing specimens from all outbreaks and identified clusters
4. Systematic sequencing of specimens from reinfections and post-vaccination-infections
5. Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 for week 10 (1249). The minimum sample size required to detect prevalence of B.1.1.7 (63%) with an error margin of 5% was estimated to be 279 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 22.3% which exceeds the minimum coverage recommended by ECDC (10%). The number of non-targeted specimens sequenced this week was 232, which is slightly lower that the above estimated sample size. Therefore, our sequencing results this week are representative of the circulating variants in Luxembourg with a margin of error of 6%.

For the purposes of the weekly genomic surveillance, and for this week’s representative population sample, we removed all cluster samples (28) from our sequencing pool. The remaining sample was considered as this week’s representative population sample (N=232, corresponding to 18,7% of the new infections).

The starting material used for sequencing is respiratory specimens (nasopharyngeal or oropharyngeal swabs) that have already been tested positive by RT PCR.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gsaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 260 specimens. All of these were collected in week 10/2021. This represents 20,8% of the new infections reported in Luxembourg in week 10/2021. Among the 260 specimens, 28 specimens were reported to be part of a cluster or outbreak investigation, and 23 specimens were from non-residents.
The population sequencing coverage in week 10/2021 was 20,34% (Figure 3). Based on statistical inference, the frequency of the reported variants in Week 10/2021 is representative of the circulating variants in Luxembourg with a margin of error of 6%.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software (v2.3, pangoLEARN version 2021-02-21).
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 the sampling period of week 10/2021, in the population representative sample, after removal of the 28 cluster samples, and excluding specimens collected from non-residents, there were 8 variants, with the main three variants being B.1.1.7 (66,2%, CI 59,8% – 72,6%), B.1.351 (20%, CI 14,6% – 25,4%), followed by B.1.1.29 (6%, CI 2,8% – 9,2%), as shown in Figure 4.

Variants of concern tracker (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 10/2021, 165 cases of the B.1.1.7 variant have been detected, representing 63,5% of the specimens in the week’s sequencing pool (by comparison, the week 9/2021 pool had shown the same frequency of 63,5% of this variant). The total case count of sequenced variant B.1.1.7 was 1255 by week 10/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 14/MAR/2021.
In the collection period of week 10/2021, 53 cases of the South African variant B.1.351 have been detected, representing 20,4% of the specimens in the week’s sequencing pool (by comparison, the week 9/2021 pool had shown 18,7% of this variant). The total case count of sequenced variant B.1.351 was 299 by week 10/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 14/MAR/2021.
In week 10/2021 no additional cases have been dected for any of the other variants of concern (P.1, B.1.525 or A.23.1) (Figure 5).
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.

Of note, additional specimens are in the pipeline for sequencing, so that numbers may change with increasing representativeness of circulating variants and proportions.

Clinically relevant mutations

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 14/MAR/2021 (N=5802), as well as the frequencies in week 10/2021.

References

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
https://github.com/cov-lineages/pangolin
Y Guangchuang et al. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 2017;8:28-36
For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 01-07/03/2021)

Executive Summary

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 9/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all specimens sequenced remained stable at 62,8% (CI 58,1% – 67,5%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 18,5% (CI 14,7% – 22,2%, p<0,05) within the sequenced specimens.

The representative sample was estimated, based on the number of positive cases in Luxembourg for week 9 (1191). The minimum sample size required to detect prevalence of B.1.1.7 (63%) reported in week 8 with an error margin of 5% was estimated to be 276 specimens. This number corresponds to a coverage of 23.1% which exceeds the minimum coverage recommended by ECDC (10%). Hence our sequencing results this week are representative of the circulating variants in Luxembourg.

The total number of sequences performed this week was 475, with 411 specimens collected in the time frame of week 9/2021. The sequencing coverage this week was 34% of all positive cases in Luxembourg.

Scope

At present, the scope of the ReViLux report is to provide (i) surveillance and descriptive epidemiology data, including data on molecular phylogenies (identification of importation events, changes in outbreak size over time), and (ii) phylogenetic interpretation of regional virus spread and circulation in Luxembourg, and cluster investigation. The scope of the ReViLux report is not to attempt phylogeographical reconstructions in Luxembourg, as a whole.

Clinical Surveillance

The “Sentinel” surveillance network reported 294 consultations in week 9 (01/MAR/2021 – 07/MAR/2021). There was no case of ILI¹, as shown in Figure 1. The percentage of consultations for AR^I2 was 7,8%.

1. ILI: – Influenza-Like Illness: Acute respiratory symptoms <10 days, Fever 38^∘C, systematic symptoms (myalgia, malaise, …)
2. ARI: – Acute Respiratory Infection: Acute respiratory symptoms (bronchitis, pharyngitis, rhinitis, pneumonia…) with or without fever.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2).

The SAR-COV-2 was the most prevalent respiratory virus detected in the “Sentinel” network, with 33 positive cases (19%). The wave of Human Rhinovirus (HRV) continued in week 9/2021, with 16 positive cases in 174 tests (9%). Sporadic cases of other respiratory viruses were identified in week 9, including 2 cases of PIV (1%), 2 cases of ADV (1%) and 1 case of MPV (0,5%). No cases of Influenza A/B were detected, indicating absence of circulation of Influenza viruses in Luxembourg, as shown in Figure 2.

In Luxembourg, we have tested 174 specimens from the Sentinel surveillance network, as compared to 1026 specimens tested in Europe, in the week 09/2021. Three of these 1026 specimens, tested for influenza viruses, were positive. The influenza epidemic in the European Region has usually reached its peak by this point of the year but, despite widespread and regular testing for influenza, reported influenza activity still remains at a very low level, likely due to the impact of the various public health and social measures, implemented to reduce transmission of SARS-CoV-2 (Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

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 for week 9 (1191). The minimum sample size required to detect prevalence of B.1.1.7 (63%) with an error margin of 5% was estimated to be 276 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 23.1% which exceeds the minimum coverage recommended by ECDC (10%). Hence our sequencing results this week are representative of the circulating variants in Luxembourg.

This week, the Health Inspection provided a set of 300 randomly selected cases. Between this list and our sequencing pool, there was an overlap of 91 specimens. This set of 91 specimens represents an alternative representative sample for the purposes of the weekly genomic surveillance. We will call this sample “randomized sample”.

The starting material used for sequencing is respiratory specimens (nasopharyngeal or oropharyngeal swabs) that have already been tested positive by RT PCR.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GISAID EpiCov database (www.gsaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 475 specimens. Out of these, 411 specimens were collected in week 9/2021. This represents 34% of the new infections reported in Luxembourg in week 9/2021. Among the 475 specimens, 70 specimens were reported to be part of a cluster or outbreak investigation.

The population sequencing coverage in week 9/2021 was 34% (Figure 3). Based on statistical inference, the frequency of the reported variants in Week 9/2021 is representative of the circulating variants in Luxembourg.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v2.3, pangoLEARN version 2021-02-21).

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 the sampling period of week 9/2021, 20 circulating SARS-CoV-2 variants were detected within our sequencing pool, as shown in Figure 4a. The most prevalent lineage was B.1.1.7 (62,8%, CI 58,1% – 67,5%), followed by B.1.351 (18.5%, CI 14,7% – 22,2%) and B.1.1.29 (9%, CI 6,2% – 11,8%).

The “randomized sample” of 91 specimens included 10 variants, again with the main three variants being B.1.1.7 (58,2%, CI 48,1% – 68,3%), B.1.351 (19,8%, CI 11,6% – 28%), followed by B.1.1.29 (12,1, CI 5,9% – 19,5%), as shown in Figure 4b.

Variants of concern tracker (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 9/2021, 258 cases of the B.1.1.7 variant have been detected, representing 62,7% of the specimens in the week’s sequencing pool (by comparison, the week 8/2021 pool, which also included specimens from previous weeks’ collections, had shown 63,7% of this variant). The total case count of sequenced variant B.1.1.7 was 1090 by week 9/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 07/MAR/2021.

In the collection period of week 9/2021, 76 cases of the South African variant B.1.351 have been detected, representing 18,5% of the specimens in the week’s sequencing pool (by comparison, the week 8/2021 pool, which also included specimens from previous weeks’ collections, had shown 17,2% of this variant). The total case count of sequenced variant B.1.351 was 246 by week 9/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 07/MAR/2021.

In week 9/2021, an additional case of the Brazilian variant P.1 was detected, therefore the total case count of variant P.1 was 3 by week 9/2021 (latest collection date 05/MAR/2021).

No additional cases have been detected for lineage B.1.525, and by now no specimen in Luxembourg corresponded to the A.23.1 variant (Figure 5).

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.

Of note, additional specimens are in the pipeline for sequencing, so that numbers may change with increasing representativeness of circulating variants and proportions.

Clinically relevant mutations

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 07/MAR/2021 (N=5542), as well as the frequencies in week 9/2021.

Conclusion

The ReViLux data are communicated as support to the understanding of respiratory virus transmission dynamics, including introduction of new variants, to the evaluation of the impact of response measures, to the contact tracing and the investigation of clusters.

References

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

https://github.com/cov-lineages/pangolin

Y Guangchuang et al. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 2017;8:28-36

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 22-28/02/2021)

Executive Summary

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 8/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant, in all sequenced samples, increased to 64.4% (CI 59,4% – 69,4%, p<0,05). For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 16.4% (CI 12,5% – 20,3%, p<0,05).

The representative sample size was estimated, based on the number of positive cases in Luxembourg for week 8 (1315). The minimum sample size required to detect prevalence of B.1.1.7 (56%) with an error margin of 5% was estimated to be 295 specimens. This number corresponds to a coverage of 22.4%, which exceeds the minimum coverage recommended by ECDC (10%). Hence our sequencing results this week are representative of the circulating variants in Luxembourg.

The total number of sequences performed this week was 570, with 354 samples collected in the time frame of week 8/2021. The sequencing coverage this week was 27% of all positive cases in Luxembourg.

Scope

At present, the scope of the ReViLux report is to provide (i) surveillance and descriptive epidemiology data, including data on molecular phylogenies (identification of importation events, changes in outbreak size over time), and (ii) phylogenetic interpretation of regional virus spread and circulation in Luxembourg, and cluster investigation. The scope of the ReViLux report is not to attempt phylogeographical reconstructions in Luxembourg, as a whole.

Clinical Surveillance

The “Sentinel” surveillance network reported 210 consultations  in week 8 (22/FEB/2021 – 28/FEB/2021). There was one case of ILI¹, representing 0.47% of the consultations (epidemiological threshold is 2 new cases per week), as shown in Figure 1. The percentage of consultations for ARI² was 7,6%.

1. ILI: – Influenza-Like Illness: Acute respiratory symptoms <10 days, Fever 38^∘C, systematic symptoms (myalgia, malaise, …)
2. ARI: – Acute Respiratory Infection: Acute respiratory symptoms (bronchitis, pharyngitis, rhinitis, pneumonia…) with or without fever.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2). The SAR-COV-2 was the most prevalent respiratopry virus detected in the “Sentinel” network, with 39 positive cases (25%). The wave of Human Rhinovirus (HRV) continued in week 8/2021, with 19 positive cases in 156 tests (12.2%). Three cases of PIV were identified in week 8. No cases of Influenza A/B were detected, indicating absence of circulation of influenza viruses in Luxembourg, as shown in Figure 2.

In Luxembourg, we have tested 156 samples from the “Sentinel” surveillance network, as compared to 1045 samples tested in Europe, in the week 08/2021. Three of these 1045 specimens tested for influenza viruses were positive. The influenza epidemic in the European Region has usually reached its peak by this point of the year but, despite widespread and regular testing for influenza, reported influenza activity still remains at a very low level, likely due to the impact of the various public health and social measures, implemented to reduce transmission of SARS-CoV-2 (Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population representative sequencing of specimens to follow the evolution of the different variants in the Luxembourg population.

The representative sequencing sample size calculation was based on the minimum number of specimens required to extrapolate prevalence of VOC variants with error rate of 5%. The representative sample size was estimated based on the number of positive cases in Luxembourg for week 8 (1315). The minimum sample size required to detect prevalence of B.1.1.7 (56%) with an error margin of 5% was estimated to be 295 specimens. The calculation was based on a sample size calculation tool that uses the expected prevalence of the variant in the population of interest. (Population Proportion – Sample Size – Select Statistical Consultants (select-statistics.co.uk). This number represented a coverage of 22.4% which exceeds the minimum coverage recommended by ECDC (10%). Hence 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.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GSAID EpiCov database (www.gsaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 570 specimens. Out of these, 354 specimens were collected in week 8/2021. This represents 27% of the new infections reported in Luxembourg in week 8/2021. Among the 354 specimens, no specimens were reported to be part of a cluster investigation.

The population sequencing coverage in week 8/2021 was 27%. Based on statistical inference, the frequency of the reported variants in Week 8/2021 is representative of the circulating variants in Luxembourg.

In the weekly population sequencing pool from representative regions, 17 specimens were collected from non-residents. This leads to 337 specimens, collected in Week 8, and being the representative population sequencing sample.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v2.3.3, pangoLEARN version 2021-02-21).

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 the sampling period of week 8/2021, 17 circulating SARS-CoV-2 variants were detected within our representative sequencing pool, as shown in Figure 3. The most prevalent lineage was B.1.1.7 (65.5%, CI 60,4% – 70,6%), followed by B.1.351 (16%, CI 12,1% – 19,9%) and B.1.1.29 (6%, CI 3,4% – 8,4%).

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 8/2021, 221 cases of the B.1.1.7 variant have been detected, representing 65.5% of the specimens in the week’s representative sample (by comparison, the week 7/2021 had shown 53.7% of this variant). The total case count of sequenced variant B.1.1.7 was 813 by week 8/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 28/FEB/2021.

In the collection period of week 8/2021, 54 cases of the South African variant B.1.351 have been detected, representing 16% of the specimens in the week’s sequencing pool (by comparison, the week 7/2021 had shown 14.8% of this variant). The total case count of sequenced variant B.1.351 was 159 by week 8/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 28/FEB/2021.

In week 8/2021, one case of the Brazilian variant P.1 was detected, therefore the total case count of variant P.1 was 2 by week 8/2021 (collection date 02/FEB/2021 and 26/FEB/2021).

The pangolin group, which provides a global report on novel coronavirus haplotypes, published two additional variants of concern, assigned as A.23.1 and B.1.525. Both variants have already been reported by neighboring countries (Belgium, France). Retrospectively, one case of the B.1.525 variant has been detected for the week 7/2021 (sampling date 20/FEB/2021).

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.

Of note, additional specimens are in the pipeline for sequencing, so that numbers may change with increasing representativeness of circulating variants and proportions.

Clinically relevant mutations

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 28/FEB/2021 (N=5098), as well as the mutation frequencies in week 8/2021.

Conclusion

The ReViLux data are communicated as support to the understanding of respiratory virus transmission dynamics, including introduction of new variants, to the evaluation of the impact of response measures, to the contact tracing and the investigation of clusters.

References

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

https://github.com/cov-lineages/pangolin

Y Guangchuang et al. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 2017;8:28-36

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 15-22/02/2021)

Executive Summary

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 7/2021, the overall frequency of the SARS-CoV-2 B.1.1.7 variant in all samples sequenced remained stable at 53%. After excluding specimens obtained via target sequencing, the prevalence of the B.1.1.7 variant, in the non-targeted sequencing sample, was 56% and comparable to last week’s prevalence. For the SARS-CoV-2 B.1.351 variant, we found an overall frequency of 22.7% within the sequenced samples. However, as 8 cases were from non-residents, the corrected frequency in all sequenced samples was 15.9%.

The total number of sequences performed this week was 272, with only 97 samples sequenced for the time frame of week 7/2021. The sequencing coverage dropped this week to 7,77% from all positive cases in Luxembourg, while the newly sequenced specimens had improved the sequencing coverage in week 5/2021 (39.6%) and week 6/2021 (22.2%). Therefore, this week’s sequencing coverage cannot be considered as representative of the general population.

Scope

At present, the scope of the ReViLux report is to provide (i) surveillance and descriptive epidemiology data, including data on molecular phylogenies (identification of importation events, changes in outbreak size over time), and (ii) phylogenetic interpretation of regional virus spread and circulation in Luxembourg, and cluster investigation. The scope of the ReViLux report is not to attempt phylogeographical reconstructions in Luxembourg, as a whole.

Clinical Surveillance

The “Sentinel” surveillance network is composed of 37 general practicioners and paediatricians around Luxembourg (General Practice and Paediatrics). The “Sentinel” surveillance network reported 133 consultations in week 7 (15/2/2021 – 22/2/2021). There were no cases of ILI1, representing 0% of consultations (epidemiological threshold is 2 new cases per week), as shown in Figure 1. The percentage of consultations for ARI2 was 12%.

Figure 1 Percentage of patients with ILI over the epidemiological surveillance weeks

1. ILI: – Influenza-Like Illness: Acute respiratory symptoms <10 days, Fever 38^∘C, systematic symptoms (myalgia, malaise, …)
2. ARI: – Acute Respiratory Infection: Acute respiratory symptoms (bronchitis, pharyngitis, rhinitis, pneumonia…) with or without fever.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2). The SAR-COV-2 was the most prevalent respiratopry virus detected in the “Sentinel” network, with 48 positive cases (25.3%). The wave of Human Rhinovirus (HRV), circulating currently in Luxembourg, continued in week 7/2021, with 30 positive cases in 183 tests (15.8%). No cases of Influenza A/B were detected, indicating absence of circulation of Influenza viruses in Luxembourg, as shown in Figure 2.

In Luxembourg, we have tested 190 samples from the Sentinel surveillance network, as compared to 996 samples tested in Europe, in the week 07/2021. Two of these 996 specimens tested for influenza viruses were positive. The influenza epidemic in the European Region has usually reached its peak by this point of the year but, despite widespread and regular testing for influenza, reported influenza activity still remains at a very low level, likely due to the impact of the various public health and social measures, implemented to reduce transmission of SARS-CoV-2 (Source: FluNews Europe).

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

The National Reference Laboratory for Acute Respiratory Infections at LNS strives to achieve representativeness of the pool of sequenced specimens to reach real-time epidemiology, by implementing the following weekly sequencing activities:

• Sequencing specimens from all hospitalized positive cases
• Sequencing specimens from all positive cases from Airport testing program
• Sequencing specimens from all outbreaks and identified clusters
• Systematic sequencing of specimens from reinfections and post-vaccination-infections
• Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

A representative sequencing sample calculation was based on the minimum number of specimens required to extrapolate prevalence of VOC variants with error rate of 5%. Based on the total number of positive specimens during the period 01-21/FEB/2021 (3443) , and their geographic distribution across Luxembourg, we concluded on the minimum number of specimens per each canton, as shown in Figure 3. An average sequencage coverage of 11% is the minimum coverage to allow extrapolation of frequencies of variants with confidence.

The starting material used for sequencing is respiratory specimens (nasopharyngeal or oropharyngeal swabs) that have already been tested positive by RT PCR.

The LNS sequencing data sharing strategy includes sharing of the sequencing data with GSAID EpiCov database (www.gsaid.org ) on a periodic basis.

Sequenced specimens

Last week the microbial genomics platform at the LNS sequenced 272 specimens. Out of these, 97 specimens were collected in week 7/2021. This represents 7.77% of new infections reported in Luxembourg in week 7/2021. Among the 97 specimens, 16% (15 samples) corresponded to targeted specimens (contact tracing with 14 specimens and cluster investigation with 1 specimen).

The population sequencing coverage in week 7/2021 was 6,57%. This is below the estimated coverage of 11% being the minimum requirement for a representative population sequencing sample. Based on statistical inference, the frequency of the reported variants in Week 7/2021 is not representative of the circulating variants in Luxembourg.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v2.3, pangoLEARN version 2021-02-18).

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 the sampling period of week 7/2021, 11 circulating SARS-CoV-2 variants were detected within our sequencing pool, as shown in Figure 3. The most prevalent lineage was B.1.1.7 (52.6%), followed by B.1.351 (22.7%) and B.1.160 (12.4%). However, the number of local cases of B.1.351 variant was corrected to be 14 only, with 6 cases originating from France and 2 cases from Belgium.

Variants of Concern tracker (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 7/2021, 51 cases of the B.1.1.7 variant have been detected, representing 52,6% of the specimens in the week’s sequencing pool (by comparison, the week 6/2021 pool, which also included specimens from previous weeks’ collections, had shown 54,1% of this variant). The total case count of sequenced variant B.1.1.7 was 543 by week 7/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 19/FEB/2021.

In the collection period of week 7/2021, 22 cases of the South African variant B.1.351 have been detected, representing 22,7% of the specimens in the week’s sequencing pool (by comparison, the week 6/2021 pool, which also included specimens from previous weeks’ collections, had shown 10,7% of this variant). The total case count of sequenced variant B.1.351 was 83 by week 7/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 19/FEB/2021.

In week 7/2021, no cases of the Brazilian variant P.1 were detected, therefore the total case count of variant P.1 was 1 by week 7/2021 (collection date 02/FEB/2021). Last week, the pangolin group, which provides a global report on novel coronavirus haplotypes, published two additional variants of concern, assigned as A.23.1 and B.1.525. Both variants have already been reported by neighboring countries (Belgium, France). However, none of these variants has been detected in Luxembourg.

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.

Of note, additional specimens are in the pipeline for sequencing, so that numbers may change with increasing representativeness of circulating variants and proportions.

Phylogenetic analysis

Phylogenetic analysis is done by means of Nextstrain (https://nextstrain.org/sars-cov-2/) and is based on genetic distances including a timeline on the x-axis. The phylogenetic tree is rooted against the first sequence obtained in Luxembourg (29/FEB/2020), which is considered as our “outgroup”. Sequences obtained before 01/NOV/2020 are shown as grey dots. Sequences obtained within the last 2 weeks, are represented in colour and by greater dot diameter. In the context of increasing numbers of sequences, a random subsampling of 300 sequences over the whole sequencing period, starting 01/SEP/2020, was performed.

The phylogenetic tree shows four visually well separated clusters. These clusters correspond to the lineages B.1.160, B.1.177, B.1.1.7 and B.1.221. Due to random selection of sequences and the intentional insertion of variants of concern, the cluster representation is mostly qualitative and does not necessarily correlate with relative prevalence of the respective variants (Figure 5).

Lineage B.1.1.7 (represented by light brownish dots) forms a well distinguishable cluster, due to the high number of accumulated mutations. B.1.1.7 subtrees might indicate different subgroups, possibly related to different routes of infection.

Clinically relevant mutations

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 19/FEB/2021 (N=4665), as well as the frequencies in week 7/2021.

Conclusion

The ReViLux data are communicated as support to the understanding of respiratory virus transmission dynamics, including introduction of new variants, to the evaluation of the impact of response measures, to the contact tracing and the investigation of clusters.

 

References

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

https://github.com/cov-lineages/pangolin

Y Guangchuang et al. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 2017;8:28-36

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html

Respiratory Viruses in Luxembourg (ReViLux)

Weekly report (Period 08-14/02/2021)

Executive Summary

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. ECDC and WHO have recently recommended that national sequencing programs should cover a minimum of 10% of all SARS-CoV-2 positive specimens, or 500 specimens, whichever is less.

In week 5/2021, the SARS-CoV-2 sequencing coverage had reached 27% of all identified positive cases, yielding a representative population sequencing sample. Luxembourg reported the second highest sequencing coverage in EU, surpassed only by Denmark. In week 6/2021, this coverage was 19.5%.

In week 6/2021, the overall prevalence of the SARS-CoV-2 B.1.1.7 variant in the sequencing sample was 53%. After correction for the bias linked to sequencing of targeted, instead of random specimens, the prevalence of the B.1.1.7 variant, in the representative population sequencing sample, was 57%. A circumscript outbreak of 17 cases of the B.1.351 variant, appeared in the hospital environment.

 

 

Scope

At present, the scope of the ReViLux report is to provide (i) surveillance and descriptive epidemiology data, including data on molecular phylogenies (identification of importation events, changes in outbreak size over time), and (ii) phylogenetic interpretation of regional virus spread and circulation in Luxembourg, and cluster investigation. The scope of the ReViLux report is not to attempt phylogeographical reconstructions in Luxembourg, as a whole.

Clinical Surveillance

The “Sentinel” surveillance network is composed of 37 general practicioners and paediatricians around Luxembourg (General Practice and Paediatrics). This week we had 145 consultations reported by 9 of these general paractitioners and pediatricians (24% of the network size).

During week 6 (8/2/2021 – 14/2/2021), there were 2 cases of ILI1 representing 1.37% of consultations (epidemiological threshold is 2 new cases per week), as shown in Figure 1. The percentage of consultations for ARI2 was 14%.

Figure 1 Percentage of patients with ILI over the epidemiological weeks

1. ILI: – Influenza-Like Illness: Acute respiratory symptoms <10 days, Fever 38^∘C, systematic symptoms (myalgia, malaise, …)
2. ARI: – Acute Respiratory Infection: Acute respiratory symptoms (bronchitis, pharyngitis, rhinitis, pneumonia…) with or without fever.

Virological Surveillance

Patients presenting with ILI and ARI at the Covid Consultation Centre (CCC) in Luxembourg were tested using a respiratory virus panel (ADV = Adenovirus, FLU A = Influenza A, FLU B = Influenza B, HRV = Human Rhinovirus, MPV = Human metapneumovirus, PIV = Parainfluenza virus, RSV = Respiratory Syncytial Virus, SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2). The SAR-COV-2 was the most prevalent respiratopry virus detected in the “Sentinel” network, with 38 positive cases (20.8%). The wave of Human Rhinovirus (HRV), circulating currently in Luxembourg, continued in week 6/2021, with 36 positive cases in 183 tests (19.7%). Only three positive cases of Parainfluenza viruses were detected. No cases of Influenza A/B were detected, indicating absence of circulation of Influenza viruses in Luxembourg as shown in Figure 2.

In Luxembourg, we have tested 183 samples from the Sentinel surveillance network, as compared to 1268 samples tested in Europe, in the week 06/2021. None of these 1268 specimens tested for influenza viruses were positive. The influenza epidemic in the European Region has usually reached its peak by this point of the year but, despite widespread and regular testing for influenza, reported influenza activity still remains at a very low level, likely due to the impact of the various public health and social measures, implemented to reduce transmission of SARS-CoV-2 (Source: FluNews Europe).

 

 

SARS-CoV-2 Genomic Surveillance

The current sequencing strategy

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:

1. Sequencing specimens from all hospitalized positive cases
2. Sequencing specimens from all positive cases from Airport testing program
3. Sequencing specimens from all outbreaks and identified clusters
4. Systematic sequencing of specimens from reinfections and post-vaccination-infections
5. Population sequencing of specimens from representative regions and age groups, to follow the evolution of the different variants in the Luxembourg population.

The starting material used for sequencing is respiratory specimens (nasopharyngeal or oropharyngeal swabs) that have already been tested positive by RT PCR.

Sequenced specimens

Last week the microbial genomics platform at the LNS has sequenced 390 specimens. Out of these, 214 specimens were collected in week 6/2021. This represents 19.5% of new infections reported in Luxembourg in week 6/2021. Among the 214 specimens, 38% (82 samples) corresponded to targeted specimens (contact tracing with 46 specimens and cluster investigation with 36 specimens).

After filtering out specimens referred for sequencing in a targeted way, the population sequencing coverage in week 6/2021 was 12%. This exceeds the ECDC recommendation of 10% being the minimum requirement for a representative population sequencing sample.

Circulating lineage detection

Lineages (variants) have been assigned based on Rambaut et al by means of Phylogenetic Assignment of Named Global Outbeak LINeages (pangolin) software  (v2.3, pangoLEARN version 2021-02-18).

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 the sampling period of week 6/2021, in the population representative sample (132 specimens), 13 variants were detected, with the main three variants being B.1.1.7 (57.6%), B.1.160 (13.6%), followed by B.1.221 (7.6%), as shown in Figure 3.

 

 

Variants of Concern (B.1.1.7, B.1.351, P.1, A.23.1 and B.1.525)

Among specimens collected within the week 6/2021, 114 cases of the B.1.1.7 variant have been detected, representing 53,5% of the specimens in the week’s sequencing pool (by comparison, the week 5/2021 pool, which also included specimens from previous weeks’ collections, had shown 54,2% of this variant). The total case count of sequenced variant B.1.1.7 was 403 by week 6/2021. The earliest collection date for this variant remains 19/DEC/2020 and the latest is 14/FEB/2021.

In the collection period of week 6/2021, 24 cases of the South African variant B.1.351 have been detected. The total case count of sequenced variant B.1.351 was 52 by week 6/2021. The earliest collection date for this variant remains 11/JAN/2021 and the latest is 14/FEB/2021.

In week 6/2021, no additional cases of the Brazilian variant P.1 were detected, therefore the total case count of variant P.1 was 1 by week 6/2021 (collection date 02/FEB/2021). Last week, the pangolin group, which provides a global report on novel coronavirus haplotypes, published two additional variants of concern, assigned as A.23.1 and B.1.525. Both variants have already been reported by neighboring countries (Belgium, France). However, none of these variants has been detected in Luxembourg (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.

Of note, additional specimens are in the pipeline for sequencing, so that numbers may change with increasing representativeness of circulating variants and proportions.

 

Phylogenetic analysis

Phylogenetic analysis is done by means of Nextstrain (https://nextstrain.org/sars-cov-2/) and is based on genetic distances including a timeline on the x-axis. The phylogenetic tree is rooted against the first sequence obtained in Luxembourg (29/FEB/2020), which is considered as our “outgroup”. Sequences obtained before 01/NOV/2020 are shown as grey dots. Sequences obtained within the last 2 weeks, are represented in colour and by greater dot diameter. In the context of increasing numbers of sequences, a random subsampling of 300 sequences over the whole sequencing period, starting 01/SEP/2020, was performed.

The phylogenetic tree shows four visually well separated clusters. These clusters correspond to the lineages B.1.160, B.1.177, B.1.1.7 and B.1.221. Due to random selection of sequences and the intentional insertion of variants of concern, the cluster representation is mostly qualitative and does not necessarily correlate with relative prevalence of the respective variants (Figure 5).

Lineage B.1.1.7 (represented by light brownish dots) forms a well distinguishable cluster, due to the high number of accumulated mutations. B.1.1.7 subtrees might indicate different subgroups, possibly related to different routes of infection.

Clinically relevant mutations

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 14/FEB/2021 (N=4392), as well as the frequencies in week 6/2021.

 

Conclusion

The ReViLux data are communicated as support to the understanding of respiratory virus transmission dynamics, including introduction of new variants, to the evaluation of the impact of response measures, to the contact tracing and the investigation of clusters.

The SARS-CoV-2 sequencing program at the LNS continues to expand, both with higher coverage and with retrospective epidemiological metadata annotations.

References

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

https://github.com/cov-lineages/pangolin

Y Guangchuang et al. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 2017;8:28-36

For more information on lineages visit: https://cov-lineages.org
For more information and statistics on Covid-19 infections in Luxembourg visit: https://covid19.public.lu/en.html