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SARS-CoV-2 variants of concern are associated with lower RT-PCR amplification cycles between January and March 2021 in France

Open AccessPublished:September 30, 2021DOI:https://doi.org/10.1016/j.ijid.2021.09.076

      Highlights

      • SARS-CoV-2 variants replaced the wild type in France between January and March 2021
      • The Alpha variant is associated with a 40% increase in transmission on average
      • The mean transmission advantage of the Beta/Gamma variants is estimated to 28%
      • Infections by SARS-CoV-2 Alpha, Beta and Gamma variants exhibit lower Ct values
      • These results suggest these variants have a double viral load compared to wild type

      Abstract

      SARS-CoV-2 variants raise concern regarding the mortality caused by COVID-19 epidemics. We analyse 88,375 cycle amplification (Ct) values from variant-specific RT-PCR tests performed between January 26 and March 13, 2021. We estimate that on March 12, nearly 85% of the infections were caused by the Alpha variant and that its transmission advantage over wild type strains was between 38 and 44%. We also find that tests positive for Alpha and Beta/Gamma variants exhibit significantly lower cycle threshold (Ct) values.

      Context

      At least three SARS-CoV-2 variants are currently major sources of concern: Alpha from lineage B.1.1.7 [
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      ] and in the Appendix, we calculated the transmission advantage of each variant compared to the wild type strain after correction for several biases (region, sampling date, assay, and patient age). This inference was performed for individuals from 5 to 80 years old) and without the data from hospitals (to avoid sampling delay bias). For Alpha, the transmission advantage was 40% (95% confidence interval, CI: [38,42]%). For Beta-Gamma, the estimate was 28% (95%CI: [27,30]%).
      We then estimated the proportion of new infections caused by each type of strain on Mar 19, 2021. At a national level, the estimate was 87% for Alpha and 5,4% for Beta/Gamma, but with strong regional heterogeneity (Figure S1).

      Variants have lower Ct values

      We analysed tests and for which the strain has been determined from 1 to 89 year old patients with Ct values lower than 30, i.e. 76,745 tests (91% of all the tests with Ct values).
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      The virus strain effect was highly significant in the ANOVA (Figure 1). Samples from Alpha variants had a significantly smaller Ct than that from Beta/Gamma (21.8 vs. 22.1). Both had significantly smaller Ct values than wild type strains (22.8) and other variants.
      Fig. 1
      Fig. 1Cycles threshold (Ct) value for SARS-CoV-2 strains. Median estimates based on the linear model are shown in the box plot, and number of tests in each class are shown in the bottom of the graph. ‘other’ indicate tests with the Δ69-70 deletion and without the N501Y mutation. Start indicate the significance level (**** for a p-values strictly lower than 104 and *** for a p-value of 104, ns: not significant).
      The model also indicated a significant decrease of Ct with age, in line with existing data [
      • Alizon S.
      • Selinger C.
      • Sofonea M.T.
      • Boukobza S.
      • Giannoli J.M.
      • Ninove L.
      • Pillet S.
      • Vincent T.
      • Rougemont A.d.
      • Tumiotto C.
      • Solis M.
      • Stephan R.
      • Bressollette-Bodin C.
      • Salmona M.
      • L’Honneur A.S.
      • Behillil S.
      • Lefeuvre C.
      • Dina J.
      • Hantz S.
      • Hartard C.
      • Veyer D.
      • Delagreverie H.M.
      • Fourati S.
      • Visseaux B.
      • Henquell C.
      • Lina B.
      • Foulougne V.
      • Burrel S.
      Epidemiological and clinical insights from SARS-CoV-2 RT-PCR cycle amplification values.
      ]. Interestingly, this decrease was significantly (more than half) stronger for Alpha, while the inference cannot exclude equivalent slopes for the other variants (note that this trend holds even on the non-Alpha variant subset of Ct values). The sampling date, the sampling region and their interaction were also found to be significant. Samples from hospitals has a slightly higher Ct, likely due to the fact that testing in the general population occurs approximately one week after infection and one week before potenital hospitalization. Therefore, hospitalized patients data is likely to reflect an older state of the epidemic.

      Discussion

      We show that variant of concern Alpha is now vastly dominant in France compared to wild type strains (87% vs. less than 10%). Beta or Gamma remain limited (approximately 5.4% of the new infections). These results are consistent with earlier reports of a marked transmission-advantage of the Alpha variant [
      • Davies N.G.
      • Abbott S.
      • Barnard R.C.
      • Jarvis C.I.
      • Kucharski A.J.
      • Munday J.D.
      • Pearson C.A.B.
      • Russell T.W.
      • Tully D.C.
      • Washburne A.D.
      • Wenseleers T.
      • Gimma A.
      • Waites W.
      • Wong K.L.M.
      • Zandvoort K.v.
      • Silverman J.D.
      • Group1 C.C.W.
      • C.U. Consortium C.G.U.K.
      • Diaz-Ordaz K.
      • Keogh R.
      • Eggo R.M.
      • Funk S.
      • Jit M.
      • Atkins K.E.
      • Edmunds W.J.
      Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England.
      ,
      • Gaymard A.
      • Bosetti P.
      • Feri A.
      • Destras G.
      • Enouf V.
      • Andronico A.
      • Burrel S.
      • Behillil S.
      • Sauvage C.
      • Bal A.
      • Morfin F.
      • Werf S.V.D.
      • Josset L.
      • Covid-19 A.M.A.
      • Group F.v.C.
      • Blanquart F.
      • Coignard B.
      • Cauchemez S.
      • Lina B.
      Early assessment of diffusion and possible expansion of SARS-CoV-2 Lineage 20I/501Y.V1 (B.1.1.7, variant of concern 202012/01) in France, January to March 2021.
      ,
      • Haim-Boukobza S.
      • Roquebert B.
      • Trombert-Paolantoni S.
      • Lecorche E.
      • Verdurme L.
      • Foulongne V.
      • Selinger C.
      • Michalakis Y.
      • Sofonea M.T.
      • Alizon S.
      Detecting rapid spread of SARS-cov-2 variants, france, january 26–february 16, 2021.
      ,
      • Volz E.
      • Mishra S.
      • Chand M.
      • Barrett J.C.
      • Johnson R.
      • Geidelberg L.
      • Hinsley W.R.
      • Laydon D.J.
      • Dabrera G.
      • O’Toole A.
      • et al.
      Assessing transmissibility of SARS-cov-2 lineage b. 1.1. 7 in england.
      ].
      By investigating the RT-PCR Ct values, which can inform us on clinical features of the infection [
      • Alizon S.
      • Selinger C.
      • Sofonea M.T.
      • Boukobza S.
      • Giannoli J.M.
      • Ninove L.
      • Pillet S.
      • Vincent T.
      • Rougemont A.d.
      • Tumiotto C.
      • Solis M.
      • Stephan R.
      • Bressollette-Bodin C.
      • Salmona M.
      • L’Honneur A.S.
      • Behillil S.
      • Lefeuvre C.
      • Dina J.
      • Hantz S.
      • Hartard C.
      • Veyer D.
      • Delagreverie H.M.
      • Fourati S.
      • Visseaux B.
      • Henquell C.
      • Lina B.
      • Foulougne V.
      • Burrel S.
      Epidemiological and clinical insights from SARS-CoV-2 RT-PCR cycle amplification values.
      ,
      • Néant N.
      • Lingas G.
      • Hingrat Q.L.
      • Ghosn J.
      • Engelmann I.
      • Lepiller Q.
      • Gaymard A.
      • Ferré V.
      • Hartard C.
      • Plantier J.C.
      • Thibault V.
      • Marlet J.
      • Montes B.
      • Bouiller K.
      • Lescure F.X.
      • Timsit J.F.
      • Faure E.
      • Poissy J.
      • Chidiac C.
      • Raffi F.
      • Kimmoun A.
      • Etienne M.
      • Richard J.C.
      • Tattevin P.
      • Garot D.
      • Moing V.L.
      • Bachelet D.
      • Tardivon C.
      • Duval X.
      • Yazdanpanah Y.
      • Mentré F.
      • Laouénan C.
      • Visseaux B.
      • Guedj J.
      • C.I. Groups f.t.F.C.
      • Study F.C.
      Modeling SARS-CoV-2 viral kinetics and association with mortality in hospitalized patients from the French COVID cohort.
      ], we show that infections caused by variants significantly differ from that caused by wild type strains. That variants are associated with lower Ct values could be an indication of higher viral load, although care must be taken because of the biology of SARS-CoV-2 [
      • Michalakis Y.
      • Sofonea M.T.
      • Alizon S.
      • Bravo I.G.
      Sars-cov-2 viral rna levels are not ǣviral loadǥ.
      ] and of the variability inherent to such values [
      • Alizon S.
      • Selinger C.
      • Sofonea M.T.
      • Boukobza S.
      • Giannoli J.M.
      • Ninove L.
      • Pillet S.
      • Vincent T.
      • Rougemont A.d.
      • Tumiotto C.
      • Solis M.
      • Stephan R.
      • Bressollette-Bodin C.
      • Salmona M.
      • L’Honneur A.S.
      • Behillil S.
      • Lefeuvre C.
      • Dina J.
      • Hantz S.
      • Hartard C.
      • Veyer D.
      • Delagreverie H.M.
      • Fourati S.
      • Visseaux B.
      • Henquell C.
      • Lina B.
      • Foulougne V.
      • Burrel S.
      Epidemiological and clinical insights from SARS-CoV-2 RT-PCR cycle amplification values.
      ].
      This result contrasts with earlier findings. One study did not find a significant result when comparing Ct values for tests with or without the S-gene target failure [
      • Walker P.G.T.
      • Whittaker C.
      • Watson O.J.
      • Baguelin M.
      • Winskill P.
      • Hamlet A.
      • Djafaara B.A.
      • Cucunubá Z.
      • Mesa D.O.
      • Green W.
      • Thompson H.
      • Nayagam S.
      • Ainslie K.E.C.
      • Bhatia S.
      • Bhatt S.
      • Boonyasiri A.
      • Boyd O.
      • Brazeau N.F.
      • Cattarino L.
      • Cuomo-Dannenburg G.
      • Dighe A.
      • Donnelly C.A.
      • Dorigatti I.
      • Elsland S.L.v.
      • FitzJohn R.
      • Fu H.
      • Gaythorpe K.A.M.
      • Geidelberg L.
      • Grassly N.
      • Haw D.
      • Hayes S.
      • Hinsley W.
      • Imai N.
      • Jorgensen D.
      • Knock E.
      • Laydon D.
      • Mishra S.
      • Nedjati-Gilani G.
      • Okell L.C.
      • Unwin H.J.
      • Verity R.
      • Vollmer M.
      • Walters C.E.
      • Wang H.
      • Wang Y.
      • Xi X.
      • Lalloo D.G.
      • Ferguson N.M.
      • Ghani A.C.
      • et al.
      The impact of COVID-19 and strategies for mitigation and suppression in low- and middle-income countries.
      ]. However, our results are based on a variant-specific PCR. Another study on the Gamma variant [
      • Faria N.R.
      • Mellan T.A.
      • Whittaker C.
      • Claro I.M.
      • Candido D.d.S.
      • Mishra S.
      • Crispim M.A.E.
      • Sales F.C.S.
      • Hawryluk I.
      • McCrone J.T.
      • Hulswit R.J.G.
      • Franco L.A.M.
      • Ramundo M.S.
      • Jesus J.G.d.
      • Andrade P.S.
      • Coletti T.M.
      • Ferreira G.M.
      • Silva C.A.M.
      • Manuli E.R.
      • Pereira R.H.M.
      • Peixoto P.S.
      • Kraemer M.U.G.
      • Gaburo N.
      • Camilo C.d.C.
      • Hoeltgebaum H.
      • Souza W.M.
      • Rocha E.C.
      • Souza L.M.d.
      • Pinho M.C.d.
      • Araujo L.J.T.
      • Malta F.S.V.
      • Lima A.B.d.
      • Silva J.d.P.
      • Zauli D.A.G.
      • de F.A.C.
      • Schnekenberg S.R.P.
      • Laydon D.J.
      • Walker P.G.T.
      • Schlüter H.M.
      • Santos A.L.P.d.
      • Vidal M.S.
      • Caro V.S.D.
      • Filho R.M.F.
      • Santos H.M.d.
      • Aguiar R.S.
      • Proença Modena J.L.
      • Nelson B.
      • Hay J.A.
      • Monod M.
      • Miscouridou X.
      • Coupland H.
      • Sonabend R.
      • Vollmer M.
      • Gandy A.
      • Prete C.A.
      • Nascimento V.H.
      • Suchard M.A.
      • Bowden T.A.
      • Pond S.L.K.
      • Wu C.H.
      • Ratmann O.
      • Ferguson N.M.
      • Dye C.
      • Loman N.J.
      • Lemey P.
      • Rambaut A.
      • Fraiji N.A.
      • S. Carvalho M.d.P.S.
      • Pybus O.G.
      • Flaxman S.
      • Bhatt S.
      • Sabino E.C.
      • et al.
      Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil.
      ] did not find a significant difference after accounting for the symptom onset to sampling delay. However, their study was performed on a limited number of samples (n=147). Finally, our estimates, based on a large number of interpretable samples with Ct lower than 30, suggest that differences in within-host replication and transcription between Alpha and non-Alpha variants might be lower than previous quantified [
      • Jones T.C.
      • Biele G.
      • Mühlemann B.
      • Veith T.
      • Schneider J.
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      • Bleicker T.
      • Tesch J.
      • Schmidt M.L.
      • Sander L.E.
      • et al.
      Estimating infectiousness throughout SARS-cov-2 infection course.
      ].

      Data availability

      The data and scripts used for the analysis will be shared upon publication.

      Funding Source

      This work received no specific funding.

      Ethical Approval

      This study was approved by the Internal Review Board of the CHU of Montpellier (ClinicalTrial.gov identier NCT04738331).

      Acknowledgements

      We thank the ETE modelling team for discussion, as well as the CNRS, the IRD, the ANR, and the Région Occitanie for funding (PHYEPI grant).

      Appendix A. Supplementary materials

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