18 January 2021
Semaine: 2



Résultats cliniques de la sentinelle

La surveillance SENTINELLE des infections respiratoires aiguës est une collaboration entre le Laboratoire national de santé (LNS), la Direction de la Santé et un réseau de médecins-généralistes et de pédiatres répartis à travers tout le Luxembourg. Ce rapport hebdomadaire est publié par le départment de microbiologie du LNS dans le cadre de ses missions de surveillance, de vigilance et d’alerte dans les domaines de la santé publique, l'analyse des données rapportant l’activité clinique dans la Sentinelle et des données virologiques à l’appui.

Au cours de la semaine 2 (11/01/2021 - 17/01/2021), l'activité grippale reste nulle.

Le taux de consultations avec ILI1 est de 0% (seuil épidémiologique à 3%).

Le taux de consultations avec ARI2 est de 7%.

  1. ILI: - Influenza-Like Illness: Signes respiratoires aigus <10 jours, Fièvre 38C, Signes systémiques (myalgie, malaise, maux de tête, …)
  2. ARI: - Infection Respiratoire Aiguë: Signes respiratoires aigus (bronchite, pharyngite, rhinite, pneumonie…) avec ou sans fièvre

Number of Patients Percentage
ARI (acute respiratory infections) 15 6.9%
ILI (influenza-like illness) 0 0.0%
Consultations 217


Respiratory viruses circulating in Luxembourg

results
Cumulative number of tests performed 114
Cumulative number of positive samples 47
Positivity rate 41%

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). We noticed a high positivity rate of SARS-CoV-2 with 38 positive cases in 114 tests (33%). Moreover, there is a wave of Human Rhinovirus (HRV) circulating currently in Luxembourg.




SARS-CoV-2 Sequencing

Sequenced Samples

Since last week we are only reporting on samples which have been taken since the 01/09/2020.

As of today, the microbial genomics platform of the microbiology department has generated 2682 high quality consensus sequences with 90 % reference sequence3 coverage at a minimum sequencing depth of 20, wherein 335 sequences have been added this week. These 335 sequences cover a sampling time frame of 30th November until 11th of January. The relatively old sampling time is related to catch up sequencing to screen for possibly earlier introduced cases of B.1.1.7.

Referring to a sampling time frame of the last 14 days (28th December 2020 to 10th of January 2021) 203 high quality genomes have been generated. With 2324 new SARS-CoV-2 infections reported in Luxembourg in this time period, this correlates to 8.7% sequencing coverage.

  1. RefSeq ID NC_045512.2, Wuhan-1

Circulating Lineages

Lineages have been assigned based on Rambaut et al by means of pangolin (v2.1.6, pangoLEARN version 2021-01-06), allowing for a geographical classification of circulating viral genomes.

For the sampling time frame 4th-10th of January 6 circulating SARS-CoV-2 variants have been detected out of 69 positive samples been taken in this time period.

Confirming the observations made in previous weeks the three main circulating strains are lineages B.1.160 (35%), B.1.177 (25%) and B.1.221 (19%). A small fraction has been detected for B.1 (13%) and a singular case for B.1.258 (1.4%) both representing European clusters.

Lineage B.1.160 represents an EU/EEA and UK multi-country cluster which involves Belgium, France, Germany, Switzerland and the United Kingdom. It is characterized by a spike protein mutation S: S477N, occurring in combination with N: M234I, A376T, ORF1a: M3087I, ORF1b: A176S and most also with ORF1b: V767L K1141R, E1184D.

Lineage B.1.177, the so called “Spain to Europe lineage” is as well prevalent in many other European countries. It is characterized by the spike protein mutation A222V and A220V in the nucleoprotein.

Lineage B.1.221 is annotated as a Dutch and Belgian variant, but is also very common in Denmark and UK.

Internationally reported variants (B.1.1.7, B.1.351, P1)

Five new cases of the UK variant B.1.1.7 have been detected within 69 high quality sequences referring to week 1, 2021, representing 7.2 % of circulating strains (week 53/2020 3%). Three cases have been part of routine genomic surveillance, two have been detected by airport screening of returning travellers.

We herewith report another potential case of B.1.1.7. The respective sequence does not match the quality criteria applied for high quality genomes, but independent variant calling provides a high load of B.1.1.7 specific mutations; additional support is provided by epidemiological data.

Accordingly, by now, the number of detected B.1.1.7 cases is 12 (including the low quality sample from above). The earliest sampling date has been shifted to 19/12/2020 and the latest is 06/01/2021.

Lineage B.1.1.7 is characterized by several spike protein mutations, which seem to have a considerable epidemiological impact, as this variant has a higher transmissibility (more efficient and rapid).

Neither cases of the South African variant B.1.351 nor the Japanese/Brasilian variant P.1 or have been detected.

It is to note, that additional samples from this week are in preparation for sequencing, so that numbers might change with increasing representativeness of circulating strains and proportions.



Phylogenetic Analysis

Phylogenetic analysis has been done by means of nextstrain4 and is based on genetic distance including a timeline on the x-axis. The tree is rooted versus the first sequence obtained in Luxembourg (29/02/2020). Sequences obtained before 01/11/2020 are in grey and those obtained within the last 2 weeks (28th December – 10th January) are represented in colour and by greater diameter. Due to the increasing number of sequences represented, we performed a random subsampling of 300 sequences over the complete sequencing time frame since 01/09/2020, adding on top the sequences related to B.1.1.7.

The tree shows four well separated clusters. As in previous weeks, the biggest clusters are formed by the lineages B.1.160, B.1.177 and B.1.221. The fourth cluster is more diverse, including the B.1.1.7 lineage.

The B.1.1.7 lineage is genetical distance wise well separated from the remaining sequences, mirroring the relatively high amount of accumulated mutations in this lineage. It can be found at the bottom of the tree represented by a mini cluster of overlapping light brownish dots.


  1. Nextstrain applied an additional filter on genome length > 27000, 310 sequences shown

Clinically relevant mutations

Currently genomic surveillance is observing the occurrence of 13 different known SARS-Cov-2 mutations, which are assumed to have a clinical or epidemiological relevance. The pool of observed mutations is updated continuously, depending on the development of SARS-CoV-2 variants and their prevalence.

Due to the increasing incidence of B.1.1.7 variant in European countries like the UK, Denmark and Ireland, the characterizing mutations N501Y, H69/V70del, Y144del and P681H have been integrated in weekly reporting. Additionally, the mutation E848K has been added, which is in combination with N501Y characteristic for the South African and Japanese/Brazilian variant.

The following table provides the overall frequency of these mutations between 01/09/2020 and the last sequenced sampling date 11th January 2021 (N=2683), as well as a close-up of the last 14 days.



Mutation Gene Genomic position in reference Frequency overall Frequency recent 14days Characteristics Reference
D614G S gene 23402 100.00% 100.00% Higher infectivity, higher case fatality rate, higher transmission; replaced original Wuhan strain, became globally dominant form of the virus Eaaswarkhanth, al Madhoun, and Al-Mulla 2020, Becerra-Flores and Cardozo 2020, Hu et al. 2020, Plante et al. 2020
E848K S gene 23012 0.00% 0.00% 501Y.V2 / possible impact on antibody neutralization activity (escape mutation), improved ACE2 binding affinity Greaney et al, 2020
G204R N gene 28883 7.00% 7.00% Fitness advantage for the virus Leary et al. 2020
H69/V70del S gene 21765-21770 2.00% 5.00% possible impact on antibody neutralization activity and reinfection; included in "mink" mutation Kemp et al 2020, Kemp et al 2020
K417N S gene 22813 0.00% 0.00% 501Y.V2 / possible impact on antibody binding affinity (escape mutation) Kemp et al 2020
L37F Nsp6 11081 8.00% 8.00% Favored viral infection, higher severity Aiewsakun et al. 2020
N439K S gene 26143 2.00% 0.00% Improved ACE2 binding affinity Zhou et al, 2020
N501Y S gene 23063 0.00% 4.00% 501Y.V1/V2; Improved ACE2 binding affinity/higher transmissibility Filip Fratev 2020 COVID-19 Genomics Consortium UK, 2020
P323L ORF1ab (RdRP gene, nsp12) 14407 99.00% 98.00% Higher severity Biswas and Mudi 2020
P681H S gene 23604 4.00% 12.00% immediately adjacent to the furin cleavage site, a known location of biological significance COVID-19 Genomics Consortium UK, 2020
Q57H ORF3a 25561 44.00% 44.00% Higher severity Biswas and Mudi 2020
R203K N gene 28880 7.00% 7.00% Fitness advantage for the virus Leary et al. 2020
Y144del S gene 21991-21993 1.00% 5.00% possible impact on antibody binding affinity Dawood et al 2020

Clinically relevant mutations frequencies over time

The D614G and P323L mutations have been and are continuously present in a vast majority of sequences. They mainly occur together.

In comparison to the previous week, the frequency within the last 14 days increased for S:N501Y from 2% to 4%, for S:H69/V70 deletion from 2% to 5%, S:P681H from 11% to 12% and S:Y144del from 4% to 5%. However, as mentioned above additional samples are in preparation, so that these numbers might change.

The mutation E848K (South African and Brazilian/Japanese variant) has by now not been detected in Luxembourg.



Additional Information

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

For more information on protein structures and a global phylogeny visit: https://genome.lux-covid19.lu/

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

References

Rambaut, A., Holmes, E.C., O’Toole, Á. et al. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat Microbiol 5, 1403–1407 (2020). https://doi.org/10.1038/s41564-020-0770-5

Pangolin (pangolin assigns lineages to query sequences as described in Rambaut et al 2020); Áine O’Toole, JT McCrone, Emily Scher; github.com/cov-lineages/pangolin

Hadfield et al., Nextstrain: real-time tracking of pathogen evolution, Bioinformatics (2018)

Guangchuang Yu, David Smith, Huachen Zhu, Yi Guan, Tommy Tsan-Yuk Lam. ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution 8(1) 28-36 (2017). https://doi.org/10.1111/2041-210X.12628