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

Materials and Methods

The Cleveland Clinic began testing for SARS CoV-2 using the original, three-target Centers for Disease Control and Prevention (CDC) assay (ie, CDC 2019 nCoV Real-Time RT-PCR Diagnostic Panel) on March 12, 2020. Four additional assays (see below) were subsequently assessed in an effort to expand capacity and address requests for more rapid turnaround times. Nasopharyngeal (NP) or nasal swabs were collected by a trained medical practitioner, and most were submitted in universal transport medium (UTM) (Copan Diagnostics) or viral transport media (VTM), respectively; two specimens were submitted in 0.9% normal saline, which was also a validated transport medium. VTM was made by the Cleveland Clinic according to the CDC procedure (SOP #DSR-052-03; https://www.cdc.gov/coronavirus/2019-ncov/downloads/Viral-Transport-Medium.pdf). All specimens were initially tested by the CDC 2019 nCoV Real-Time RT-PCR Diagnostic Panel as part of the standard operating procedures for the laboratory. Positive and negative specimens, in an approximate 2:1 ratio, were assessed for the presence of the SARS-CoV-2 virus by the four additional assays described below. Specimens were enrolled as they were received and were not selected based on any clinical characteristics.

A specimen was considered to contain the SARS-CoV-2 virus if the results of two or more of the five tests studied were positive according to the standard operating procedure of the laboratory or the manufacturer's instructions. Otherwise stated, a positive test from any assay needed to be corroborated by a positive result from any other assay for the specimen to be characterized as containing the SARS-CoV-2 virus. The specimen was considered to not contain the SARS-CoV-2 virus if the results from all the tests were negative or if only a single test was positive (ie, a positive test that was not corroborated by any of the other four tests). Any single positive test results were characterized as false positives.

This study was approved by the Cleveland Clinic Institutional Review Board. Patient confidentiality was protected by storing data in a password-protected file on an internal electronic shared drive accessible only to study team members. This was a single-center study.

Nucleic Acid Amplification Assays

CDC SARS-CoV-2 RT-PCR Assay. We used the original, three-target SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) test that was developed at the CDC (ie, CDC 2019 nCoV Real-Time RT-PCR Diagnostic Panel).[2] This assay targets three separate loci in the SARS-CoV-2 nucleocapsid (N) gene. Commercially available plasmids that contained the N gene were used to determine the limit of detection. In brief, serial dilutions were made that contained known concentrations of the target plasmids. Multiple replicates of these were tested until the concentration wherein 95% (ie, 19/20) of the replicates were detected. This defined the limit of detection, which was found to be 20 copies/μL for upper respiratory specimens and 2 copies/μL for lower respiratory specimens. Internal validation studies, which were more extensive than those required for an FDA EUA submission, were performed prior to the introduction of this test.

For each specimen, 200 μL of clinical specimen in transport media was rendered noninfectious within a biological safety cabinet through the addition of 200 μL of Bacterial Lysis Buffer (Roche Diagnostics). A nucleic acid extract was obtained from 200 μL of the inactivated specimen using the MagNA Pure system (Roche). Then, 5 μL of eluate was added to 15 μL of PCR mastermix for each PCR well. The CDC 2019 nCoV Real-Time RT-PCR Diagnostic Panel used consisted of four separate RT-PCR assays, three of which targeted different regions of the virus nucleocapsid (N) gene. The fourth RT-PCR was an amplification control that targeted a portion of the human RNase P gene. Testing was performed on an ABI 7500 or ABI 7500 Fast Dx (Thermo Fisher) using fresh extract that was never frozen. Amplification of all three RT-PCR N gene targets (ie, N1, N2, and N3) was necessary to characterize a specimen as positive. No amplification of all N gene targets in conjunction with amplification of the human control gene was necessary to characterize a specimen as negative. Specimens with amplification of only one or two of the N gene targets were characterized as indeterminate for this study and for simplicity were excluded from further analysis.

TIB MOLBIOL/Roche z 480 Assay. The RT-PCR test used in this assay was developed by Roche Diagnostics and TIB MOLBIOL. Internal validation studies were performed before introduction of the test. Commercially available controls containing the envelope (E) gene and the RNA-dependent RNA polymerase (RdRP) gene (Exact Diagnostics; SeraCare) were used to determine the limit of detection, which was found to be 20 copies/μL for upper respiratory specimens. We also performed the other validation studies necessary for the submission of an EAU, since this was a laboratory-developed test.

For each specimen, 200 μL of clinical specimen in transport media was rendered noninfectious within a biological safety cabinet through the addition of 200 μL of Bacterial Lysis Buffer (Roche). A nucleic acid extraction was performed using 200 μL of the inactivated specimen using the MagNA Pure system (Roche). This was the same extract tested originally by the CDC assay but had undergone a single freeze-thaw cycle. The nucleic acid extracts were frozen once prior to testing with the TIB MOLBIOL/Roche z 480 Assay. Then, 10 μL of thawed and homogenized eluate was added to 10 μL of PCR mastermix for each PCR reaction. This test consisted of three separate RT-PCR assays, one that targeted the E gene, one that targeted the RdRP gene, and a third amplification control that targeted a portion of the human RNAse P gene. Testing was performed on both the Cobas Z 480 and LightCycler 480 platforms (Roche Diagnostics). Amplification of both the E gene and RdRP gene targets was necessary to characterize a specimen as positive with this assay. Both targets had to be negative with amplification of the human control to characterize a specimen as negative. Specimens with amplification of only one of the two targets were characterized as indeterminate for this study and for simplicity were excluded from further analysis.

Xpert Xpress SARS-CoV-2 (Cepheid). The Xpert Xpress SARS-CoV-2 (Cepheid) assay was performed according to the manufacturer's guidelines. In brief, the fresh (ie, never frozen) specimen was mixed by vortexing for several seconds, and 300 μL of transport media was transferred to the cartridge sample chamber. The test consisted of a multiplex RT-PCR assay that targeted the SARS-CoV-2 E and N2 genes, as well as a sample processing control. Amplification of the N2 gene was necessary to characterize a sample as positive. Specimens with amplification of the E gene without amplification of the N2 gene are characterized by the manufacturer as a presumptive positive, but these were categorized as positive for the purposes of this study. Both N2 and E genes had to render negative results with amplification of the specimen processing control for a specimen to be characterized as negative.

Simplexa COVID-19 Direct Kit (DiaSorin). The Simplexa COVID-19 Direct Kit (DiaSorin) was performed according to the manufacturer's guidelines. In brief, 50 μL of fresh (ie, never frozen) clinical specimen in transport media was added to the Direct Amplification Disc sample well after 50 μL of the reaction mix was added to the reaction well. Testing was performed on the LIAISON MDX (DiaSorin). This assay used a multiplex RT-PCR that targeted SARS-CoV-2 ORF1ab and S genes along with an internal amplification control. Amplification of at least one target gene (ie, ORF1ab or S) was necessary to characterize a specimen as positive. Both target genes had to render negative results with amplification of the internal RNA control to characterize a specimen as negative.

ID Now COVID-19 (Abbott). The ID Now COVID-19 (Abbott) assay targets a portion of the RdRp gene within the SARS-CoV-2 genome (https://www.fda.gov/media/136525/download). This assay was performed according to the manufacturer's guidelines, which at the time of this study included the testing of transport media (see ID Now COVID-19 [Abbott] original FDA EUA submission). Positive and negative results were provided by the instrument and recorded as provided. In brief, fresh (ie, never frozen) specimens were allowed to reach room temperature before testing. The test base and receiver cartridges were placed in the ID Now devices, and the receiver cartridge was permitted to warm up. When prompted by the instrument, the foil seal was removed, and 200 μL of well-mixed clinical specimen in transport media was dispensed into the receiver cartridge using the disposable pipettes provided in the kit. After 10 seconds of vigorous mixing, the pipette was removed and the transfer cartridge was used to introduce sample into the test base from the receiver cartridge. The lid was closed and the isothermal amplification was initiated. Results were displayed on the ID Now screen, and the three cartridges were disposed of in accordance with instructions, which minimized the possible release of amplified products. The studies were performed in a biological safety cabinet by qualified clinical laboratory technologists. All three instruments used in this part of the study passed daily positive and negative quality control checks, and the reported results passed internal quality checks.

Clinical Parameters

Medical records were available for 208 of the 239 patients enrolled in the study. Thirty-one patients were non–Cleveland Clinic patients whose records were not available for review. Three residents in clinical pathology reviewed electronic medical records for study participants, and a subset of recorded results was checked by one author (S.M.H.). Patient identifiers were removed at the conclusion of the study. Variables assessed included the patient age, sex, patient status as a caregiver (ie, health care provider [HCP]), date of specimen collection, date of onset of symptoms, and whether the patient was evaluated in an inpatient, outpatient (including telemedicine visits), emergency department (ED), or intensive care unit (ICU) location. The presence or absence of the following clinical parameters at the time of the visit was recorded: fever, cough, nausea or vomiting, diarrhea, and dyspnea. For patients who had a chest roentgenogram or chest computed tomography scan, the presence or absence of pneumonia was recorded as interpreted by a radiologist or attending physician.

Statistical Analysis

Analyses were done using R version 4.0.0.[3] Test performance with each assay was evaluated in the entire data set, with indeterminates for each assay excluded. Viral loads (as a multiple of the minimum detectable viral load) were calculated from threshold cycle data based on a method previously described.[4] For assays with more than a 10% false-negative rate, associations with false-negative results were examined in multivariable logistic regression models, using the subset of patients with the disease. Days since onset of symptoms was missing for 14 (7%) patients. These were found to be missing completely at random on evaluation of the missing data using the R package VIM.[5] Missing values were imputed using a method of multivariate imputation by chained equations using the R package mice.[6] Initial models included all variables with univariable associations at a level of significance of .2. Variable selection was then done with stepwise backward elimination until only variables significant at a level of .05 remained. Logarithm of the viral load and transport medium was forced into the final model as there was good biological plausibility that they would influence false-negative rates. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the final model. Graphics were created using the ggplot2 package.[7]

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