Analytical and Clinical Comparison of Three Nucleic Acid Amplification Tests for SARS-CoV-2 Detection

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was first identified in December 2019 and has quickly become a worldwide pandemic. In response, many diagnostic manufacturers have developed molecular assays for SARS-CoV-2 under the Food and Drug Administration (FDA) Emergency Use Authorization (EUA) pathway. This study compared 3 of these assays: the Hologic Panther Fusion SARS-CoV-2 assay (Fusion), the Hologic Aptima SARS-CoV-2 assay (Aptima) and the BioFire Defense COVID-19 test (BioFire), to determine analytical and clinical performance, as well as workflow. All 3 assays showed a similar limit of detection (LOD) using inactivated virus, with 100% detection ranging from 500-1,000 genome equivalents/ml, whereas use of a quantified RNA transcript standard showed the same trend, but had values ranging from 62.5 to 125 copies/ml, confirming variability in absolute quantification of reference standards. The clinical correlation found that the Fusion and BioFire assays had a positive percent agreement (PPA) of 98.7%, followed by the Aptima assay at 94.7% when compared to the consensus result. All three assays exhibited 100% negative percent agreement (NPA). Analysis of discordant results revealed that all 4 samples missed by the Aptima assay had Ct values >37 on the Fusion assay. In conclusion, while all three assays showed similar relative LODs, we showed differences in absolute LODs depending on which standard was employed. In addition, the Fusion and BioFire assays showed better clinical performance, while the Aptima assay showed a modest decrease in overall PPA. These findings should be kept in mind when making platform testing decisions.

Performance Characteristics of a High-Throughput Automated Transcription-Mediated Amplification Test for SARS-CoV-2 Detection

“…These analytical sensitivity values correlate with previously reported LOD values by Smith et al (16). However, using LOD values as determined by external control material to assess or compare assay performance warrants some caution, as different control materials may give considerably different results for the absolute LOD value (16), and the reported RNA or DNA stock concentrations of these materials may differ from true concentrations (17). The limitations of this study include the small number of clinical specimens available for testing and the absence of discordant result resolution by testing with a third assay.…”

“…Our evaluation of three different external RNA control materials demonstrated comparable analytical sensitivity with both the SARS-CoV-2 TMA assay and the SARS-CoV-2 RT-PCR assay, with LOD values of between 83 c/ml of 194 c/ml. These analytical sensitivity values correlate with previously reported LOD values by Smith et al (16). However, using LOD values as determined by external control material to assess or compare assay performance warrants some caution, as different control materials may give considerably different results for the absolute LOD value (16), and the reported RNA or DNA stock concentrations of these materials may differ from true concentrations (17).…”

Fully automated detection and differentiation of pandemic and endemic coronaviruses (NL63, 229E, HKU1, OC43 and SARS-CoV-2) on the Hologic Panther Fusion

“…Although the intended use of the Aptima SARS-CoV-2 is limited to diagnostic materials from the upper respiratory tract, SARS-CoV-2 RNA was detected with 100 % PPA and 99.0 % NPA. These results compared favorably to other studies, which had only included nasopharyngeal swabs (94.7 % to 100 % PPA and 98.7 % to 100 % NPA) (28)(29)(30). However, detection of SARS-CoV-2 in materials from the LRT is crucial as it was shown that nasopharyngeal swabs may turn negative in the course of infection prior to specimens from the LRT (16,(31)(32)(33).…”

Prevalence and Clinical Profile of Severe Acute Respiratory Syndrome Coronavirus 2 Infection among Farmworkers, California, USA, June–November 2020

“…Testing for SARS-CoV-2 infection at CSVS clinics began on June 15, 2020, and was offered to all persons at clinics during weekday business hours. Medical personnel collected oropharyngeal specimens for detection of SARS-CoV-2 RNA via the qualitative Aptima SARS-CoV-2 Assay (Hologic, https://www.hologic.com ), a nucleic acid transcription-mediated amplification (TMA) assay with an analytical sensitivity of 62.5 RNA transcript copies/mL ( 17 ) and clinical specificity of 99.9% ( 18 ). Patients receiving care from CSVS for any reason were encouraged by their healthcare providers to receive SARS-CoV-2 testing, regardless of symptoms; testing also was made available to persons who were not CSVS patients.…”