Clinical Evaluation and Utilization of Multiple Molecular in Vitro Diagnostic Assays for the Detection of SARS-CoV-2

Kendall Cradic, PhD; Marie Lockhart, PhD; Patrick Ozbolt, MLS(ASCP); Lisa Fatica, MT(ASCP); Lorie Landon, SM(ASCP); Michael Lieber, MS; David Yang, MT(ASCP); Juanita Swickard, MBA; Nicholas Wongchaowart, MD; Susan Fuhrman, MD; Stella Antonara, PhD, D(ABMM)

Disclosures

Am J Clin Pathol. 2020;154(2):201-207. 

In This Article

Materials and Methods

Specimen Collection and Storage

Flocked swabs were used to collect nasopharyngeal specimens from symptomatic patients suspected of COVID-19 that met criteria for testing, either presenting to the emergency department or as inpatients at OhioHealth Riverside Methodist Hospital. After collection, the swabs were placed into 3 mL of sterile UVT (Becton Dickinson). Specimens were tested as soon as possible after collection, or if testing was delayed, were stored for up to 72 hours at 2°C to 8°C. Following routine testing, samples were stored frozen (≤–80°C) until comparator testing with the Roche cobas assay could be completed.

Study Design

Clinical performance of the 3 assays was evaluated using a total of 184 prospective nasopharyngeal specimens collected from patients of all ages and both genders presenting with signs and/or symptoms of COVID-19 infection. The samples were originally submitted for routine COVID-19 testing at OhioHealth Laboratories on the DiaSorin Simplexa assay. Subsequent testing on the additional platforms was based on availability of samples and meeting storage requirement criteria for testing on each platform according to the package insert requirements at the time of the study.

To compare the equivalence of nasopharyngeal, oropharyngeal, and nasal swabs using the Abbott ID NOW assay, a quality improvement study approved by the OhioHealth Research Institute was conducted where 3 specimens were collected from patients that presented to the emergency department. A total of 182 prospective specimen sets were collected. The nasopharyngeal swab was collected as part of standard of care testing, while oropharyngeal and nasal swabs were collected upon consent from the patient. Nasal swabs were collected according to the CDC instructions using a single swab to sample both nares. All 3 samples were tested using the Abbott ID NOW assay, while the nasopharyngeal samples collected in UVT were also tested using the DiaSorin Simplexa assay.

DiaSorin Molecular Simplexa COVID-19 Direct EUA

The DiaSorin Molecular Simplexa COVID-19 Direct EUA assay (DiaSorin Molecular) was performed according to the manufacturer's instructions for use. Briefly, 50 μL of Simplexa COVID-19 Direct Kit reaction mix (MOL4150) was added to the "R" well of the 8-well Direct Amplification Disc followed by adding 50 μL of nonextracted nasopharyngeal swab sample (collected in approximately 3 mL of UVT) to the "SAMPLE" well. Tests were run on the LIAISON MDX system and data collection and analysis was performed with LIAISON MDX Studio software. The assay targets 2 different regions of the SARS-CoV-2 genome, the S gene and ORF1ab, differentiated with FAM and JOE fluorescent probes. An RNA internal control (Q670 probe) is used to detect reverse transcription polymerase chain reaction (RT-PCR) failure and/or inhibition. The result interpretation algorithm for reporting a positive specimen requires only 1 of the 2 targets to be detected (S or ORF1ab gene).

Abbott ID NOW COVID-19 EUA

The Abbott ID NOW COVID-19 assay (Abbott Diagnostics) was performed according to the manufacturer's instructions for use. Briefly, the orange test base was inserted into the appropriate orange color-coded receptacle, followed by placing the sample receiver into the corresponding blue color-coded receptacle. The nasopharyngeal swab was placed into UVT, while the oropharyngeal and nasal dry swabs were placed directly into the sample receiver. The swab was vigorously mixed in the sample receiver buffer for 10 seconds for direct testing. For specimens in UVT, 200 μL was pipetted into the sample receiver and mixed. The assay uses isothermal nucleic acid amplification and targets the RdRp region of the SARS-CoV-2 genome. Abbott ID NOW contains an internal control that has been designed to control for sample inhibition, amplification, and assay reagent function.

Roche cobas 6800 SARS-CoV-2

The Roche cobas 6800 SARS-CoV-2 assay (Roche Diagnostics) was performed according to the manufacturer's instructions for use. The test uses a minimum required sample volume of 600 μL. The sample preparation is fully automated (nucleic acid extraction and purification) followed by PCR amplification and detection. The assay targets the ORF1 a/b nonstructural region that is unique to SARS-CoV-2. Additionally, the assay targets a conserved region in the structural protein envelope E-gene with pan-sarbecovirus detection that will also detect SARS-CoV-2 virus. The result was interpreted as positive if both targets were detected and presumptive positive if 1 of 2 targets was detected.

Analytical Sensitivity

Relative analytic sensitivity was determined by evaluating serial dilutions from 10 clinical specimens containing SARS-CoV-2. Ten-fold dilutions of each clinical sample were tested on all 3 platforms. The relative limit of detection (LOD) was evaluated qualitatively using all 10 clinical samples.

Statistical Methods

Positive percent agreement (PPA), and negative percent agreement (NPA) between all assays were calculated with two-sided (upper/lower) 95% confidence intervals (CIs) using the Evidence-Based Medicine Toolbox, Knowledge Translation Program. The PPA was calculated as the fraction and percentage of cases that agree with a consensus standard of 2 out of 3 results when the specified technique produced a positive test. The NPA was similarly calculated among cases for which the specified technique produced a negative test.

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