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. 2021 May 12;8(6):ofab235.
doi: 10.1093/ofid/ofab235. eCollection 2021 Jun.

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

Melissa Richard-Greenblatt  1 , Matthew J Ziegler  2   3 , Valerie Bromberg  2 , Elizabeth Huang  2 , Hatem Abdallah  2 , Pam Tolomeo  2 , Ebbing Lautenbach  2   3 , Laurel Glaser  1 , Brendan J Kelly  2   3
Affiliations

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

Melissa Richard-Greenblatt et al. Open Forum Infect Dis. .

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.

Keywords: SARS-CoV-2; cycle threshold; test sensitivity.

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Figures

Figure 1.
Figure 1.
Relationship between β-actin and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection probability. Binomial logistic regression relating SARS-CoV-2 detection to β-actin cycle threshold (Ct) value reveals a negative association, with high β-actin Ct (ie, low quality) nasopharyngeal specimens less likely to detect SARS-CoV-2. The absolute probability of SARS-CoV-2 detection is presented in relation to the observed range of β-actin Ct values.
Figure 2.
Figure 2.
Relationship between β-actin and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection probability, stratified by illness severity. A mixed-effects binomial logistic regression relating SARS-CoV-2 detection to β-actin cycle threshold (Ct) value reveals a negative association, with high β-actin Ct (ie, low-quality) nasopharyngeal specimens less likely to detect SARS-CoV-2, across all strata of disease severity as assessed by chest radiography (A) or oxygen saturation (B). The absolute probability of SARS-CoV-2 detection is presented in relation to the observed range of β-actin Ct values. Abbreviations: Ct, cycle threshold; CT, computed tomography; O2, oxygen; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
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