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

Discussion

Accurate results for SARS-CoV-2 testing are extremely important since they affect not only decision-making in the clinical setting but also have public health implications for the community. In addition, turnaround time for results can be critical when treating an isolated patient suspected of having COVID-19. Use of personal protective equipment required by health care professionals might be spared along with the use of limited space in isolation rooms and other vital resources such as personnel to care for those patients. The consequences of a false-negative result can be serious since the virus is so transmissible and can have devastating effects in vulnerable patient populations. A false-positive result is also undesirable, as it may have a negative impact on the health of patients (eg, not seeking care for another disease) or that of health care workers, as they will have to abstain from their duties when they are most needed.

A number of different studies have been published regarding the clinical sensitivity of various RT-PCR COVID-19 assays.[14–16] Published reports have demonstrated 94% PPA of the Abbott ID NOW and 96% PPA of the DiaSorin Simplexa assays compared to a modified CDC assay,[17] while another study comparing nasal and nasopharyngeal specimens tested on Abbott ID NOW and mAbbott 2000 showed 75% PPA between the two assays.[18] In our study we compared the PPA and NPA of the Abbott ID NOW to the DiaSorin Simplexa and the Roche cobas assays. In our area, the positivity rate based on reporting of positive tests was 7% at the time of testing.[19] The variability in PPA observed in different reports likely depends on the amount of virus present in samples at the time of testing. This suggests the importance of choosing the right assay for each patient population.

In our institution only the DiaSorin Simplexa platform was available initially, followed soon after by the Abbott ID NOW and then the Roche cobas. Because of questions surrounding the use of UVT on the Abbott ID NOW assay we performed an additional study to evaluate the equivalency of the nasopharyngeal swab in UVT to that of dry swabs (either oropharyngeal or nasal). This was critical, as Abbott modified the assay instructions for use to include only dry swabs. In our study the use of UVT was equivalent to that of dry swabs when using the Abbott ID NOW. During the same time period, a large educational effort was underway by our nursing educators for proper nasopharyngeal specimen collections. All the specimen collectors in our system were reeducated and their competency reverified by direct observation. Given the emphasis put on properly collected specimens, we believe the difference observed in the PPA between assays was not due to specimen collection but rather due to the analytical limits of the assays.

Due to the lack of verified reference material to evaluate the LOD across the assays available in our institution, we examined the relative LOD using real patient specimens. By performing serial dilutions on patient samples with varying degrees of positivity (moderately to low positive samples as indicated by the cycle threshold values obtained by the DiaSorin Simplexa assay), we directly tested the LOD relative to one another. Roche cobas was the most sensitive assay as expected, since it uses a larger volume of sample for extraction (600 μL), and an RNA extraction step takes place followed by the RT-PCR assay. DiaSorin Simplexa appears to have a higher LOD than the Roche cobas assay; this test utilizes a smaller sample volume (50 μL) than Roche cobas and does not have a separate extraction step. Abbott ID NOW has an LOD approximately 10- to 100-fold higher than the PCR assays in our study. The assay uses 200 μL of UVT sample or uses a dry swab placed into the sample receiver buffer. Also, the Abbott ID NOW assay only amplifies one target for detection of SARS-CoV-2 compared to the other assays where 2 targets are used. However, its overall clinical sensitivity was about 10% lower when compared to DiaSorin Simplexa and Roche cobas. This suggests that the performance of the assay will suffer when it is used in patient populations that have low viral loads.

High testing demand coupled with limited availability of testing kits makes a multiplatform approach an attractive method for laboratories to meet the high demands for testing. Laboratories are in need of every testing option available but they must gain an understanding of the limitations of each. It is crucial to optimize utilization within the patient populations that can benefit most from each platform's unique capabilities and limitations. Our institution is a large, multihospital network, and based on our experience with a variety of patient populations, turnaround time demands, and instrument/reagent availability, we propose a model for the use of each type of assay Table 3. Currently, none of the assays are FDA EUA approved for testing of asymptomatic individuals. However, a highly sensitive test is needed to assess patients as hospitals open up for elective and other procedures, particularly those that may involve aerosolization. In this scenario, a highly sensitive assay with a longer turnaround time may be most appropriate. For asymptomatic patients being admitted to the hospital, or for inpatients that had previously tested negative but have clinical symptoms consistent with COVID-19, a fast and sensitive test is needed to appropriately identify and stratify them in hospital units. Finally, for symptomatic patients with high clinical suspicion and high pretest probability presenting to the emergency department, a rapid point-of-care option may be appropriate.

This is a single center study with a relatively small sample size and low positivity rate in our population. However, these studies were performed after a large educational effort was put in place in our system regarding specimen collection, therefore minimizing the impact of sample quality in our study. Additionally, all the matched samples were tested within 2 hours of collection so as not to compromise sample integrity for the dry collected swabs as defined by the manufacturer. A dry paired nasopharyngeal swab was not collected since the patients were already contributing 3 different specimens for this study. Finally, all samples were collected from patients meeting testing criteria for COVID-19 as described by the CDC recommendations. Due to limited availability of inactivated viral material, a formal evaluation of the limits of detection was not possible so the assessment of these assays was restricted to the relative limit of detection experiment.

In summary, we evaluated the Abbott ID NOW point-of-care testing system using nasopharyngeal samples collected in UVT and compared its analytical sensitivity and clinical performance to 2 other molecular assays. Also, by analyzing matched specimens we found no difference in the performance of the Abbott ID NOW between swabs collected in UVT and dry swab collection. Finally, the data suggest that the molecular point-of-care testing using isothermal amplification is not as sensitive as other molecular assays included in the study that utilize PCR amplification. DiaSorin Simplexa and Roche cobas assays appear to have LODs at least 10× and 100× lower than Abbott ID NOW, respectively. Laboratories are faced with the challenge of limited testing supplies yet immense testing needs. Taking into consideration all parameters for each testing platform and patient care needs, laboratories should assess what the best test is for the diagnosis of each patient.

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