Ct values from SARS-CoV-2 diagnostic PCR assays should not be used as direct estimates of viral load

  • Elias Dahdouh
    Affiliations
    Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ, Paseo de La Castellana 261, Madrid 28046, Spain
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  • Fernando Lázaro-Perona
    Affiliations
    Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ, Paseo de La Castellana 261, Madrid 28046, Spain
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  • María Pilar Romero-Gómez
    Affiliations
    Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ, Paseo de La Castellana 261, Madrid 28046, Spain
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  • Jesús Mingorance
    Correspondence
    Corresponding author.
    Affiliations
    Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ, Paseo de La Castellana 261, Madrid 28046, Spain
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  • Author Footnotes
    # SARS-CoV-2 Working Group: M. Dolores Montero, C. Toro-Rueda, S. García-Bujalance, G. Ruiz-Carrascoso, E. Cendejas-Bueno, I. Falces-Romero, M. Ruiz-Bastián, A. Gutiérrez-Arroyo, P. Girón De Velasco-Sada, B. Gómez-Arroyo, C. García-Sánchez, V. Guedez-López, I. Bloise, M. Alguacil-Guillén, M. Gracia Liras-Hernández, M. Sánchez-Castellano, P. García-Clemente, P. González- Donapetry, S. San José-Villar, M. de Pablos, R. Gómez-Gil, M. Corcuera, A. Rico-Nieto, B. Loeches
    Julio García-Rodriguez
    Footnotes
    # SARS-CoV-2 Working Group: M. Dolores Montero, C. Toro-Rueda, S. García-Bujalance, G. Ruiz-Carrascoso, E. Cendejas-Bueno, I. Falces-Romero, M. Ruiz-Bastián, A. Gutiérrez-Arroyo, P. Girón De Velasco-Sada, B. Gómez-Arroyo, C. García-Sánchez, V. Guedez-López, I. Bloise, M. Alguacil-Guillén, M. Gracia Liras-Hernández, M. Sánchez-Castellano, P. García-Clemente, P. González- Donapetry, S. San José-Villar, M. de Pablos, R. Gómez-Gil, M. Corcuera, A. Rico-Nieto, B. Loeches
    Affiliations
    Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ, Paseo de La Castellana 261, Madrid 28046, Spain
    Search for articles by this author
  • Author Footnotes
    # SARS-CoV-2 Working Group: M. Dolores Montero, C. Toro-Rueda, S. García-Bujalance, G. Ruiz-Carrascoso, E. Cendejas-Bueno, I. Falces-Romero, M. Ruiz-Bastián, A. Gutiérrez-Arroyo, P. Girón De Velasco-Sada, B. Gómez-Arroyo, C. García-Sánchez, V. Guedez-López, I. Bloise, M. Alguacil-Guillén, M. Gracia Liras-Hernández, M. Sánchez-Castellano, P. García-Clemente, P. González- Donapetry, S. San José-Villar, M. de Pablos, R. Gómez-Gil, M. Corcuera, A. Rico-Nieto, B. Loeches
Published:October 24, 2020DOI:https://doi.org/10.1016/j.jinf.2020.10.017

      Highlights

      • The amount of material collected by nasopharyngeal swabs is imprecise.
      • The determinations of SARS-CoV-2 viral loads from Cts ignore this error source.
      • SARS-CoV-2 Cts should be normalized with an internal marker to correct this errors.

      Graphical abstract

      Keywords

      Dear Editor,
      We read with interest the review by Walsh et al.
      • Walsh Kieran A.
      • Karen Jordan
      • Barbara Clyne
      • Daniela Rohde
      • Linda Drummond
      • Paula Byrne
      • et al.
      SARS-CoV-2 detection, viral load and infectivity over the course of an infection.
      summarizing data on detection patterns and viral loads of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the course of infection. We agree with them that determination of SARS-CoV-2 viral load in clinical samples will aid the interpretation of laboratory assays and in the management of isolation and contact tracing protocols, but it should be noted that currently there is no standard measure of viral load in clinical samples. It is becoming common to assume that the Ct values from real-time (quantitative) reverse transcription polymerase chain reaction (qPCR) diagnostic tests are direct measures of viral load, and the use of Ct values has been proposed as a tool to identify those patients who might not be infectious in spite of being positive
      • Tom Michael R.
      • Mina Michael J
      To interpret the SARS-CoV-2 test, consider the cycle threshold value.
      or to correlate the PCR results with infectivity in cell cultures in order to predict which samples are actually infectious.
      • Bernard La Scola
      • Marion Le Bideau
      • Julien Andreani
      • Thuan Hoang Van
      • Clio Grimaldier
      • Philippe Colson
      • et al.
      Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards.
      ,
      • Jared Bullard
      • Kerry Dust
      • Duane Funk
      • Strong James E.
      • David Alexander
      • Lauren Garnett
      • et al.
      Predicting infectious SARS-CoV-2 from diagnostic samples.
      While it is true that Cts are related to the starting amount of template in the reaction this is not a linear relation and the use of raw Ct values understates the dispersion of the measurements.
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
      Another problem is that most diagnostic SARS-CoV-2 qPCR tests are done on suspensions from nasopharyngeal swabs, and these are samples from a surface and have an intrinsic variability that depends on the operator and on the tolerance of the patients.
      • Daniela Basso
      • Ada Aita
      • Filippo Navaglia
      • Elisa Franchin
      • Paola Fioretto
      • Stefania Moz
      • et al.
      SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-analytics.
      Moreover, the concept of viral load itself is dubious in the absence of a reference mass or volume unit. Finally, the different nucleic acid extraction and amplification systems used by are additional variability sources. For these reasons the assumption that there is a direct relation between the qPCR signal, the amount of virus collected and the amount of virus in the patient's nasopharynx may be misleading and should be taken with care.
      • Binnicker Matthew J.
      Challenges and controversies related to testing for COVID-19.
      To illustrate these points, we take advantage of the design a commercial SARS-CoV-2 RT-qPCR that targets two SARS-CoV-2 genes (E and N) in two different reactions (SARS-CoV-2 Real Time PCR kit, Vircell, Granada, Spain). The tube targeting gene N includes an unrelated (and undisclosed) internal amplification control (primers, probe and a target RNA) in the reaction mix, while the tube targeting gene E includes primers and probes for human RNAse P. The housekeeping RNAse P is a ribozyme expressed in many tissues. Specific primers and probes detect both the gene (DNA) and its RNA product and are included for sample quality control in many SARS-CoV-2 and influenza virus commercial assays.
      • Ying Yan
      • Le Chang
      • Lunan Wang
      Laboratory testing of SARS-CoV, MERS-CoV, and SARS-CoV-2 (2019-nCoV): current status, challenges, and countermeasures.
      To explore the use of human RNAseP to normalize the data we collected the results of a series of 145 randomly selected positive assays from our registers (March and April 2020). In this set the internal control in the N tube had an average Ct of 30 (range 25.3 to 35.5, IQR=2.1 cycles) (Fig. 1A). The human RNAse P control in the E tube had an average Ct of 28.8 and a broader distribution: range 20.9 to 36.3 and IQR=3.5 cycles (Fig. 1B). The Ct values of the target genes were independent of those of the controls in both cases (Figs. 1A,B, r2 values not significantly different from zero), meaning that there were no interferences between the target and the control reactions. The variability of the internal controls in the N tubes must be due to experimental errors during the setting up of the PCR reactions, while the higher variability of the human RNAse P controls in the E tubes reflects, in addition, the variations in the amount of material collected with the swabs and in the nucleic acid extraction process.
      Fig 1
      Fig. 1Analysis of the SARS-CoV-2 Ct values obtained using a commercial RT-qPCR assay (Vircell) in a set of clinical samples. A) Cts of the Internal Control RNA plotted against the SARS-CoV-2 N gene Cts (r2 = 0.004). B). Cts of the human RNAse P plotted against the SARS-CoV-2 E gene Cts (r2 = 0.007). C) Normalized SARS-CoV-2 gene E Ct values (log(2−ΔCt)=log(2−Cttarget-Ctreference)) plotted against the SARS-CoV-2 E gene. The normalized Ct values are relative loads (ratios of viral target to human target) and are transformed to logarithmic scale for graphical representation. The black arrow illustrates the broad range spanned by any particular Ct value.
      To correct for sampling variability we used the human RNAse P as a reference to normalize the viral load by the comparative Ct method (ΔCt)
      • André Peinnequin
      • Catherine Mouret
      • Olivier Birot
      • Antonia Alonso
      • Jacques Mathieu
      • Didier Clarençon
      • et al.
      Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green.
      that transforms the Cts into relative loads (ratios of viral target to human target). Fig. 1C shows a plot of SARS-CoV-2 gene E Cts normalized with the human RNAse P Ct values against the gene E Cts. The plot shows an inverse linear correlation, which is expected because Ct values reflect, indeed, viral loads, but the dispersion of the data may reach up to four log units (ten thousand-fold) for any given Ct (black arrow). This is not a problem of this particular brand or PCR design, it could be observed in other commercial (TaqMan 2019-nCoV Assay Kit v1, Thermo Fisher Scientific, Waltham, MA, USA) and in-house
      • Corman Victor M.
      • Olfert Landt
      • Marco Kaiser
      • Richard Molenkamp
      • Adam Meijer
      • Chu Daniel K.W.
      • et al.
      Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.
      assays.
      Normalization is not as straightforward as suggested by this example. A full characterization of the linear ranges and a calibration using standards
      • Giannella M.
      • Alonso M.
      • de Viedma D Garcia
      • Roa P Lopez
      • Catalán P.
      • Padilla B.
      • et al.
      Prolonged viral shedding in pandemic influenza A(H1N1): clinical significance and viral load analysis in hospitalized patients.
      should be done for every different target and primer/probe design. Other reference genes might be explored as well, although human RNAseP has been widely used and might enable to exploit the huge amount of data already collected in many laboratories around the world.
      Using Ct values obtained in diagnostic PCR reactions as direct measures of SARS-CoV-2 viral loads is simple, but at the cost of introducing errors that cannot be neglected. Normalization using some marker of the cell mass or the mucosal surface sampled should be integrated into commercial diagnostic kits to make the different assays comparable and to evaluate the potential of quantitative PCR for the clinical management of COVID-19 patients.

      Acknowledgments

      E. D. has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Individual Fellowship grant agreement No. 796084 .

      References

        • Walsh Kieran A.
        • Karen Jordan
        • Barbara Clyne
        • Daniela Rohde
        • Linda Drummond
        • Paula Byrne
        • et al.
        SARS-CoV-2 detection, viral load and infectivity over the course of an infection.
        J Infect. 2020; 81: 357-371https://doi.org/10.1016/j.jinf.2020.06.067
        • Tom Michael R.
        • Mina Michael J
        To interpret the SARS-CoV-2 test, consider the cycle threshold value.
        Clin Infect Dis. 2020; : 1-19https://doi.org/10.1093/cid/ciaa619
        • Bernard La Scola
        • Marion Le Bideau
        • Julien Andreani
        • Thuan Hoang Van
        • Clio Grimaldier
        • Philippe Colson
        • et al.
        Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards.
        Eur J Clin Microbiol Infect Dis. 2020; 39: 1059-1061https://doi.org/10.1007/s10096-020-03913-9
        • Jared Bullard
        • Kerry Dust
        • Duane Funk
        • Strong James E.
        • David Alexander
        • Lauren Garnett
        • et al.
        Predicting infectious SARS-CoV-2 from diagnostic samples.
        Clin Infect Dis. 2020; 954162: 1-4https://doi.org/10.1093/cid/ciaa638
        • Livak K.J.
        • Schmittgen T.D.
        Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
        Methods. 2001; 25: 402-408https://doi.org/10.1006/meth.2001.1262
        • Daniela Basso
        • Ada Aita
        • Filippo Navaglia
        • Elisa Franchin
        • Paola Fioretto
        • Stefania Moz
        • et al.
        SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-analytics.
        Clin Chem Lab Med. 2020; : 1-8https://doi.org/10.1515/cclm-2020-0749
        • Binnicker Matthew J.
        Challenges and controversies related to testing for COVID-19.
        J Clin Microbiol. 2020; : 1-16https://doi.org/10.1128/JCM.01695-20
        • Ying Yan
        • Le Chang
        • Lunan Wang
        Laboratory testing of SARS-CoV, MERS-CoV, and SARS-CoV-2 (2019-nCoV): current status, challenges, and countermeasures.
        Rev Med Virol. 2020; 30: 1-14https://doi.org/10.1002/rmv.2106
        • André Peinnequin
        • Catherine Mouret
        • Olivier Birot
        • Antonia Alonso
        • Jacques Mathieu
        • Didier Clarençon
        • et al.
        Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green.
        BMC Immunol. 2004; 5: 3https://doi.org/10.1186/1471-2172-5-3
        • Corman Victor M.
        • Olfert Landt
        • Marco Kaiser
        • Richard Molenkamp
        • Adam Meijer
        • Chu Daniel K.W.
        • et al.
        Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.
        Eurosurveillance. 2020; 25: 1-8https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045
        • Giannella M.
        • Alonso M.
        • de Viedma D Garcia
        • Roa P Lopez
        • Catalán P.
        • Padilla B.
        • et al.
        Prolonged viral shedding in pandemic influenza A(H1N1): clinical significance and viral load analysis in hospitalized patients.
        Clin Microbiol Infect. 2011; 17: 1160-1165https://doi.org/10.1111/j.1469-0691.2010.03399.x