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Influenza-Überwachung

La surveillance sentinelle de la grippe est une collaboration entre le département de microbiologie du Laboratoire national de santé, la Direction de la Santé et un réseau de médecins-généralistes et de pédiatres sentinelles répartis dans tous le pays. Entre octobre et avril, chaque médecin/pédiatre communique une fois par semaine les données cliniques concernant un jour de la semaine. Ces données cliniques comportent le nombre de patients soignés ayant des symptômes similaires à la grippe, le nombre de patients ayant des infections respiratoires aiguës et le nombre de patients consultés au total.

En parallèle, les médecins font des prélèvements de nez/gorge chez les patients ayant des symptômes similaires à la grippe. Ces prélèvements sont analysés au LNS pour détecter la présence du virus influenza et de charactériser le type du virus.

Pour recevoir des informations supplémentaires à ce sujet, veuillez nous contacter via email. 

Vous trouverez l’évolution de la grippe lors de la saison 2017/2018 dans la rubrique téléchargement.


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  • Semaine 13

    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

  • Semaine 12

    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 38C, 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

  • Semaine 11

    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 38C, 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

  • Semaine 10

    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 38C, 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

  • Semaine 9

    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 ARI2 was 7,8%.

    1. ILI: – Influenza-Like Illness: Acute respiratory symptoms <10 days, Fever 38C, 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

  • Semaine 8

    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 38C, 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

  • Semaine 7

    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 38C, 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

  • Semaine 6

    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 38C, 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

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