Quantifying the Impact of Nasopharyngeal Specimen Quality on Severe Acute Respiratory Syndrome Coronavirus 2 Test Performance

  1. Melissa Richard-Greenblatt
  2. Matthew J Ziegler
  3. Valerie Bromberg
  4. Elizabeth Huang
  5. Hatem Abdallah
  6. Pam Tolomeo
  7. Ebbing Lautenbach
  8. Laurel Glaser
  9. Brendan J Kelly

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Abstract

Background

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse-transcription polymerase chain reaction (RT-PCR) cycle threshold (Ct) has been used to estimate quantitative viral load, with the goal of targeting isolation precautions for individuals with coronavirus disease 2019 (COVID-19) and guiding public health interventions. However, variability in specimen quality can alter the Ct values obtained from SARS-CoV-2 clinical assays. We sought to define how variable nasopharyngeal (NP) swab quality impacts clinical SARS-CoV-2 test sensitivity.

Methods

We performed amplification of a human gene target (β-actin) in parallel with a clinical RT-PCR targeting the SARS-CoV-2 ORF1ab gene for 1282 NP specimens collected from patients with clinical concern for COVID-19. We evaluated the relationship between NP specimen quality, characterized by late Ct values for the human gene target β-actin Ct, and the probability of SARS-CoV-2 detection via logistic regression, as well as the linear relationship between SARS-CoV-2 and β-actin Ct.

Results

Low-quality NP swabs are less likely to detect SARS-CoV-2 (odds ratio, 0.607 [95% credible interval {CrI}, .487–.753]). We observed a positive linear relationship between SARS-CoV-2 and β-actin Ct values (slope, 0.181 [95% CrI, .097–.264]), consistent with a reduction in detection of 0.181 cycles for each additional cycle of the β-actin target. COVID-19 disease severity was not associated with β-actin Ct values.

Conclusions

Variability in NP specimen quality significantly impacts the performance of clinical SARS-CoV-2 assays, and caution should be taken when interpreting quantitative SARS-CoV-2 Ct results. If unrecognized, low-quality NP specimens, which are characterized by a low level of amplifiable human DNA target, may limit the successful application of SARS-CoV-2 Ct values to direct infection control and public health interventions.

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  1. Version published to 10.1093/ofid/ofab235
  2. ScreenIT

    SciScore for 10.1101/2020.12.09.20246520: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementConsent: A waiver of informed consent was granted by the University of Pennsylvania Institutional Review Board (IRB protocols #843085 & 106 #843274)
    IRB: A waiver of informed consent was granted by the University of Pennsylvania Institutional Review Board (IRB protocols #843085 & 106 #843274)
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    No key resources detected.

    Results from OddPub: Thank you for sharing your code and data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    However, several limitations of our analysis must be acknowledged. Oxygen saturation data and chest CT radiography reports were only available for a small percentage (32.6% and 8.5%, respectively) of our subjects. Subject demographics and medical comorbidities could not be ascertained for subjects, so unmeasured confounders may contribute to the observed association. Nevertheless, we believe that the observed association between NP specimen quality and SARS-CoV-2 RT-PCR sensitivity is an important finding. From 1311 NP specimens submitted for SARS-CoV-2 testing, we have quantified the variation in specimen quality measured by β-actin Ct value, and we have defined the impact of the observed variation on test sensitivity and SARS-CoV-2 Ct values. SARS-CoV-2 Ct values have shown promise as a means to roughly quantify viral burden and so to guide infection control and public health interventions (1, 2, 4–8). However, variability in NP specimen collection may exert large effects on observed SARS-CoV-2 Ct values, limiting these useful applications. As testing efforts expand, infrastructure to ensure quality sample collection must expand as well (9). Concurrent measurement of a β-actin human gene target may provide a means to recognize and adjust for variability in NP specimen quality.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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  3. Version published to 10.1101/2020.12.09.20246520 on medRxiv