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Research Letter
July 30, 2020

Age-Related Differences in Nasopharyngeal Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Levels in Patients With Mild to Moderate Coronavirus Disease 2019 (COVID-19)

Author Affiliations
  • 1Division of Infectious Diseases, Department of Pediatrics, Ann & Robert H. Lurie Children’s Hospital, Chicago, Illinois
  • 2Northwestern University Feinberg School of Medicine, Chicago, Illinois
JAMA Pediatr. Published online July 30, 2020. doi:10.1001/jamapediatrics.2020.3651

Children are susceptible to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but generally present with mild symptoms compared with adults.1 Children drive spread of respiratory and gastrointestinal illnesses in the population,2 but data on children as sources of SARS-CoV-2 spread are sparse.

Early reports did not find strong evidence of children as major contributors to SARS-CoV-2 spread,3 but school closures early in pandemic responses thwarted larger-scale investigations of schools as a source of community transmission. As public health systems look to reopen schools and day cares, understanding transmission potential in children will be important to guide public health measures. Here, we report that replication of SARS-CoV-2 in older children leads to similar levels of viral nucleic acid as adults, but significantly greater amounts of viral nucleic acid are detected in children younger than 5 years.

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    10 Comments for this article
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    Age/CT relationship between 5-17 y.o.
    S Eraly, MD, PhD |
    Thank you and your group for your important contribution to our understanding of Covid-19 epidemiology! Recognizing that the analysis might be limited by the relatively small sample size, can you comment on the relationship between age and PCR amplification cycle threshold (CT) within the age range 5 to 17 y.o.?

    It does not seem that the Age/CT relationship could be monotonic in this range, since one would in that case expect the median CT to be intermediate between those of younger children and adults. Is the lesser CT that would be expected for those at
    the young end of the 5-17 range counterbalanced by greater CT in the rest of this group?

    The most inclusive manner in which to visualize the Age/CT relationship could be to present a scatterplot of Age vs CT (both as continuous variables). Such a figure has doubtless already been generated by the research team – could it be provided here in the Comments section?
    CONFLICT OF INTEREST: None Reported
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    How Was the Adult Population Chosen?
    Marc Hainaut, MD, PhD | CHU Saint-Pierre, Université Libre de Bruxelles, Brussels, Belgium
    Thank you for this contribution to our understanding of SARS-CoV-2 infection in children. Such data are really important even if we know the transmission of a communicable disease in a group does not depend solely on the number of viruses present in the secretions (especially when counted by PCR).

    I understand the format of a « Research Letter » does not allow to put many details but the analyzed population remains unclear to me.

    In the methods section it is written "all individuals aged younger than 1 month to 65 years who tested positive for SARS-CoV-2" were included.
    As the study was conducted at a pediatric tertiary medical center in Chicago, where did the adult patients in the study come from? How were they chosen? Were they the staff members who contracted the disease?

    In all published series of SARS-CoV-2 infection, from all locations, children are significantly fewer in number than adults and have a less severe disease. How to explain that in your series no adult had a severe disease when 7 children did?

    Is it possible that the adult population of the study is not comparable to a randomly selected adult population?
    Understanding the selection of the population is critical to the analysis of these data.

    We would also have liked to know if the difference between the children aged <5 years and 5-17 years is not explained by higher viral load only in very young infants.

    It is unfortunate that data of such importance are not presented in the form of a detailed article, especially in view of the suggestion that young children could be a very important source for the spread of the epidemic, which is contrary to the majority of the data published to date.
    CONFLICT OF INTEREST: None Reported
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    Author Reply: Age vs PCR Amplification Cycle Threshold (CT)
    Larry Kociolek, MD | Ann & Robert H. Lurie Children's Hospital of Chicago
    Thank you for the comments. The sample size did not permit a robust analysis of CT by age for each pediatric age group, but we did assess age vs CT in the 5-17 year age group, and there was no relationship. R2 of that plot was 0.002. We chose to dichotomize our pediatric groups based on ages of school attendance.

    Similar to the pediatric groups, all of the adults tested had symptoms for fewer than 7 days and none had severe disease. While the adult population in our study may not be generalizable, that does not
    impact the findings of young children having higher viral loads than older children. Further, other studies have shown equivalent viral loads between children and adults, and our data are consistent when comparing older children and adults.
    CONFLICT OF INTEREST: Senior Author of Paper
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    Pediatric Incidence Rates
    Jason Bhardwaj, BSE, MBA | None
    If the general incidence rate of mild to moderate disease is lower in the younger pediatric age cohort, is it possible that an alternate conclusion from the research is that greater viral levels are required to achieve the same severity of symptoms in the younger age cohort?
    CONFLICT OF INTEREST: None Reported
    Concerns
    Aimee Renaud |
    This research letter underemphasizes the fact that this analysis relies on not just a small sample but also one primarily obtained from a hospital setting as well as from mild to moderate patients with no additional weighting for the incredibly small incidence of this population within the under-10 population. This study has been interpreted as young people are more likely to carry higher viral load and that in no way is the outcome of this dataset. Please include additional data to quantify the actual population or caveat the limitations of this analysis.
    CONFLICT OF INTEREST: None Reported
    Quantitative Assay?
    Mark Vieyra | University of Pennsylvania
    The Abbot RealTime SARS-CoV-2 Assay that used in this paper says in its package insert that it is "intended for the qualitative detection of nucleic acid from SARS-CoV-2". I understand that one would expect the CT values to be lower with higher viral loads but the assay is not really designed to draw quantitative conclusions. I get that this is potentially interesting preliminary data but shouldn't this be confirmed with an assay that is designed to quantify viral RNA before we draw the conclusion that "young children can potentially be important drivers of SARS-CoV-2 spread in the general population"?
    CONFLICT OF INTEREST: None Reported
    qPCR Not Performed State of the Art
    C X, PhD |
    Your data seem plausible, however the qPCR data are not performed how they should.

    - The Abbott test is only validated for qualitative purposes. A quantitative validation with a standard curve should have been included. Furthermore, this is a multiplex, which makes quantitation even harder. Cycle quantification values (Cqs) from different targets do influence each other.
    - Getting quantitative data from such early PCR amplification cycle thresholds (Cts) is hard. Did you validate your linear range with a standard curve? I mean, at a Cq = 5 f.e. it gets really hard to correctly subtract your baseline. /> - Thirdly, Cq is not a quantitative measure. If you took a calibrator sample, you should use the values or you should work with (d)dCq taking into account efficiencies from your standard curve.
    - An analysis with normalized qPCR data would be more correct. Normalization for volume/internal control is a minimum, as extraction can be a variable factor. Even better is a human control, to assess for RT efficiency and for swabbing efficiency.

    In summary, you cannot take a quantitative assay and use in in a qualitative way.

    Kind Regards
    CONFLICT OF INTEREST: None Reported
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    Substantial Technical Issues
    Leor Weinberger, Ph.D. | University of California, San Francisco
    I agree with the critiques above; there are substantial technical issues with this report. The lack of a standard curve is a critical omission that must be corrected before conclusions can be drawn. This is standard practice and it is surprising that the reviewers did not ask for it.

    Without a standard curve, the PCR amplification cycle threshold (CT) values (median CT = 6) reported yield nonsensical viral loads: the Abbott limit of detection is 100 copies/ml carrier fluid with a cutoff CT = 39; so the viral load for a CT=6 is >100*2^(33)=8.6*10^11 copies/mL. Such viral levels
    are rarely achieved in highly idealized lab settings after concentration/ultracentrifugation (most published papers are reporting CT values > 20 for SARS-CoV-2). I also agree that the internal control to a human reference gene is needed to correct for sampling differences.

    I'd strongly caution against making conclusions until the above data are provided.
    CONFLICT OF INTEREST: None Reported
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    RE: Age-Related Differences in Nasopharyngeal SARS-CoV-2 Levels in Patients With Mild to Moderate COVID-19
    Tomoyuki Kawada, MD | Nippon Medical School
    I appreciate the authors' speculation on COVID-19 sero-epidemiology (1). Although the number of samples was limited, an appropriate statistical method was applied for the analysis.

    Dr. Eraly recommended presenting the relationship (scattergram) between age and PCR amplification cycle threshold (CT) with range of age under 17 years old. Regarding this recommendation, the authors replied that there was no significant relationship in the 5-17 years age group with square value of regression coefficient of 0.002. I think that many factors would contribute to CT values, and age might be one of the contributing factors. Although the authors emphasized that children
    < 5 years had specific biological characteristics of keeping higher amount of SARS-CoV-2 in the upper respiratory tract, there needs to be more evidence confirming their medical hypothesis. Anyway, presenting scattergram between age and CT values in children under 17 years old would present information regardless of statistical significance.

    References
    1. Heald-Sargent T, Muller WJ, Zheng X, Rippe J, Patel AB, Kociolek LK. Age-Related Differences in Nasopharyngeal Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Levels in Patients With Mild to Moderate Coronavirus Disease 2019 (COVID-19). JAMA Pediatr. 2020 doi:10.1001/jamapediatrics.2020.3651
    CONFLICT OF INTEREST: None Reported
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    Author Reply: Methodologic Questions
    William Muller, MD, PhD | Ann & Robert H Lurie Children's Hospital of Chicago; Northwestern University Feinberg School of Medicine
    We very much appreciate the attention this paper has received and the comments left by different readers.

    Several readers have left comments on technical characteristics of the assay used to generate the reported data. While it is correct that the clinical application of the assay is for qualitative detection of SARS-CoV-2 RNA, the cycle threshold data reported in this study were gathered for research purposes. There are abundant data in the literature to support the use of cycle threshold values as a proxy for the level of viral RNA in a sample, including for SARS-CoV-2 (1-4). Indeed, the commenters
    questioning the assay generally agree that cycle threshold values should correlate with the level of viral RNA in the samples. Although as many readers have noted there are caveats to doing so, some clinicians have argued that cycle threshold data could be applied in medical decision making based on correlations between these values and severity of COVID-19 disease in adults (4).

    The Abbott assay used in our clinical laboratory includes an internal control (non-viral) sequence with every sample. Additionally, every clinical run included a control sample with 1000 copies of SARS-CoV-2 RNA per well. There are known differences between the cycle threshold generated with the Abbott assay and other platforms used for clinical detection of SARS-CoV-2 RNA that lead to significantly lower cycle thresholds for the assay we used (5), which do not alter either the results of our study or their interpretation. Using a rough rule-of-thumb that a one logarithm difference in template RNA corresponds to 3.3 cycle thresholds, the reported median CT for the youngest age group of about 6 cycles would correspond to about 10^8 copies/mL, not 10^11 as one reader suggests. Such levels are not at all dissimilar to those reported from upper respiratory tract samples for other viruses (6).

    References

    1. Zou N Engl J Med 2020, 382(12): 1177
    2. Kam et al Clin Infect Dis 2020, 71(15): 847
    3. Lee et al. JAMA Intern Med doi:10.1001/jamainternmed.2020.38622020
    4. Magleby et al., Clin Infect Dis 2020; ciaa851
    5. Degli-Angeli et al., J Clin Virol 2020; 129: 104474
    6. Granados et al., J Clin Virol 2017; 86: 14
    CONFLICT OF INTEREST: Co-Author
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