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

Abstract

Background The 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 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%CI 0.487 to 0.753). We observed a positive linear relationship between SARS-CoV-2 and β-actin Ct values (slope 0.181, 95%CI 0.097 to 0.264), 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.

Longitudinal SARS-CoV-2 Testing among the Unvaccinated Is Punctuated by Intermittent Positivity and Variable Rates of Increasing Cycle Threshold Values

“…S3, Table S1), some variation in RNA detected could be due to assay differences across platforms, although our results suggest this is effect is unlikely to change an observed negative or positive test result among the assays used in our study. Additional factors, such as timing of initial presentation and specimen quality, likely contribute to RNA detection or observed decline ( 2 , 5 , 14 , 15 ). Although it was beyond the scope of this study to evaluate the likelihood of reinfection, which would require longitudinal viral sequencing, the observation of decreased RNA detection over time in the majority of patients was consistent with a single infection course, since reinfection is currently thought to be rare, especially prior to widespread VOC circulation ( 16 ).…”

“…The observation of intermittent positivity in 8% of patients with multiple SARS-CoV-2 positive tests during a time period prior to sustained VOC transmission suggests that sample quality may play a major role in C T results ( 14 ). Interestingly, prolonged-positive patients were over 3 times more likely to have intermittent positivity.…”

Investigating the optimum sample type and target genes for SARS-CoV-2 detection

“…Although there are endogenous gene quality controls, such as RNase P and β-actin, they can only help to determine whether the sampling is successful, and cannot help with strict quantification and quality control. 11, 16-18 The resulting problem is that Ct values become largely influenced by sampling. False negatives due to sampling problems and positive results in re-detections after release from quarantine based solely on pharyngeal swabs often occur.…”

Considerations regarding Interpretation of Positive SARS-CoV-2 Molecular Results with Late Cycle Threshold Values

“…Poor-quality nasopharyngeal swab specimens (as measured by amplification of a human endogenous reference gene) are associated with later SARS-CoV-2 CT values ( 32 ). The challenge of specimen quality can be compounded by inconsistent specimen collection guidance ( 33 ).…”