A Comparison of Five SARS-CoV-2 Molecular Assays With Clinical Correlations

Gary W. Procop, MD, MS; Jay E. Brock, PhD; Edmunds Z. Reineks, MD; Nabin K. Shrestha, MD, MPH; Ryan Demkowicz, MD; Eleanor Cook, MD; Emad Ababneh, MD; Susan M. Harrington, PhD

Disclosures

Am J Clin Pathol. 2021;155(1):69-78. 

In This Article

Results

A total of 239 specimens were tested by all methods, with the exception of one specimen that could not be performed on the Xpert Xpress SARS-CoV-2 (Cepheid) because of inadequate specimen volume. Composite analysis demonstrated that 168 specimens contained SARS-CoV-2, whereas 71 specimens did not. The performance characteristics of the five tests are summarized in Table 1.

Test Performance

A total of 234 specimen results were available for assessment of the CDC 2019 nCoV Real-Time RT-PCR Diagnostic Panel; there were five indeterminate results. There were no false negatives and three false positives when this assay was assessed against a composite standard. In total, 208 specimen results were available for assessment of the TIB MOLBIOL/Roche z 480 assay because of 31 indeterminate results. There were five false-negative results and one false-positive result when this assay was assessed against a composite standard. A total of 238 specimen results were available for assessment of the Xpert Xpress SARS-CoV-2 (Cepheid) assay since one of the specimens in this collection could not be tested due to inadequate volume. There were four false negatives and five false positives when this assay was assessed against a composite standard. In total, 239 specimen results were available for assessment of the Simplexa COVID-19 Direct Kit (DiaSorin) assay. There were 20 false negatives and one false positive when this assay was assessed against a composite standard. Finally, 239 specimen results were available for assessment of the ID Now COVID-19 (Abbott) assay. There were 28 false negatives and two false positives when this assay was assessed against a composite standard. The sensitivities, specificities, and false-negative rates for the five assays are shown (Table 1).

Scatterplots of mean Ct values from the three N gene RT-PCRs from the CDC 2019 nCoV Real-Time RT-PCR Diagnostic Panel vs days since onset of symptoms with results as determined for each assay indicated with colored circles are shown in Figure 1. For each assay, there was a trend of lower Ct values (higher viral load) closer to the time of onset of symptoms. As days since onset increased, Ct value tended to increase (ie, viral load decreases). False-negative results for the Simplexa COVID-19 Direct Kit (DiaSorin) and ID Now COVID-19 (Abbott) assays tended to occur more frequently as time from onset of symptoms increased and Ct values increased (Figure 1).

Figure 1.

Scatterplots of crossing threshold (Ct), as a surrogate for viral load, vs the days since onset of symptoms for the five molecular severe acute respiratory syndrome coronavirus 2 assays studied. A, Centers for Disease Control and Prevention assay. B, Cepheid assay. C, Roche assay. D, Abbott assay. E, DiaSorin assay. Performance characteristics (eg, TP, TN) are listed and color coded. The y-axis is on a reversed scale. FN, false negative; FP, false positive; Neg, negative; Pos, positive; TN, true negative; TP, true positive.

Clinical Characteristics

Clinical characteristics of the 208 patients for which clinical information was available are shown in Table 2. The mean age was 49.3 years, and 45.2% were male. Most patients were seen in the outpatient setting (63.9%), followed by those seen in the ED (25.0%). Fewer specimens were collected from patients in the ICU (2.4%) or another inpatient unit (8.7%). More specimens were collected with a nasal swab in VTM (60.9%) compared with an NP swab in UTM (39.1%) due to a national shortage of UTM. Ten (4.8%) patients were asymptomatic. The mean days from onset of symptoms to specimen collection was 8.6, although the standard deviation was large. HCPs represented 38.9% of patients; 96% of specimens collected from HCPs were nasal swab/VTM. Most patients were not severely ill. Only 13.0% were characterized as having pneumonia; 19.2% were admitted to the hospital in a non-ICU setting, and 4.8% were admitted to the ICU. The proportion of patients with other symptoms is given (Table 2).

Associations With False-negative Test Results

The Simplexa COVID-19 Direct Kit (DiaSorin) and ID Now COVID-19 (Abbott) assays had false-negative rates exceeding 10%. Because the testing of HCPs occurred at a particular location, the swab type and transport medium used at that location were very strongly correlated with each other; of these, only swab/transport medium was used in multivariable analysis to avoid multicollinearity. On multivariable analysis, log10 viral load (OR, 0.46; 95% CI, 0.29–0.67; P < .001) was the only variable significantly associated with false-negative results for the Simplexa COVID-19 Direct Kit (DiaSorin) assay Table 3. For the ID Now COVID-19 (Abbott) assay, both log10 viral load (OR, 0.32; 95% CI, 0.18–0.50; P < .001) and swab/transport medium (OR, 4.95; 95% CI, 1.30–25.30; P = .018) remained significantly associated with false-negative results in a multivariable analysis Table 4.

Comments

3090D553-9492-4563-8681-AD288FA52ACE

processing....