Dear Editor,
The use of whole genome sequencing in clinical microbiology laboratories for diagnostic purposes is expanding [1], [2]. The Clear Dx SARS-CoV-2 assay (Clear Labs, San Carlos, CA), has been approved for Emergency Use Authorization (EUA) by the Food and Drug Administration (FDA) for the diagnosis of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This assay represents a fully automated targeted next generation sequencing test intended to be performed on the Clear Dx system [3]. The purpose of this study was to evaluate the use of the Clear Dx SARS-CoV-2 test for the diagnosis of COVID-19.
From May 2020 to August 2020, 390 nasopharyngeal samples were collected from patients with COVID-19 symptoms. RNA was extracted from these specimens using the Qiagen EZ1 virus mini kit v2.0 (Qiagen, Germantown, MD) [4]. Extracted RNA was tested for the presence of SARS-CoV-2 using both the Clear Dx SARS-CoV-2 assay and CDC SARS-CoV-2 RT-PCR assay as the reference method and the results were compared. A positive COVID-19 result by the CDC assay was reported when both nucleocapsid targets (N1 and N2) had a cycle threshold (Ct) ≤ 40. Of 266 specimens positive by the CDC assay, the Clear Dx test detected 249 and of the 124 negative specimens by the CDC assay, the Clear Dx assay was negative for 123 ( Table 1). Using the CDC RT-PCR assay as the gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value of Clear Dx test were 93.6%, 99.1%, 99.6%, and 87.8% respectively with an accuracy of 95.3%. All false negative specimens by the Clear Dx assay had Ct values> 28, which suggested low quality specimens for sequencing. These findings were not surprising since the CDC recommends only specimens with Ct values< 28 be considered acceptable for testing by the national SARS-CoV-2 surveillance (NS3) program [5]. In addition to detecting for the presence or absence of SARS-CoV-2, the Clear Dx assay also detects for the presence of the S:D614G and RdRp:P314L mutations. To further evaluate this, 22 positive SARS-CoV-2 specimens were tested of which 15 encoded both mutations and 7 did not encode the mutations. These were tested using the ARTIC v3 whole genome sequencing protocol with the Minion platform and the results were compared to the Clear Dx test. All results correlated 100% between the two assays.
Table 1.
Performance of the Clear Dx SARS-CoV-2 test on individual nasopharyngeal swab specimens with the CDC SARS-CoV-2 RT-PCR assay.
| CDC SARS-CoV-2 RT-PCR assay +- | Total | |||
|---|---|---|---|---|
| Clear Dx SARS-CoV-2 Test | + | 249 | 1 | 250 |
| – | 17 | 123 | 140 | |
| Total | 266 | 124 | 390 | |
Data presented in this study showed that the Clear Dx test is accurate for the detection of SARS-CoV-2 in nasopharyngeal specimens. To the best of our knowledge, this is the first clinical report to address the use of next generation sequencing in the diagnosis of COVID-9 using direct patient specimens. This test has the advantage of short hands on time of approximately 30 for set up with results available within 24 h on one instrument. In addition, five flow cells with capacity for 192 specimens can be run per cell for up to 960 specimens within 24 h. This is significant since the US has experienced an acute shortage of certain reagents critical to perform SARS-CoV-2 detection assays at times when large-volume testing to identify infected patients was needed. Additionally, the application of group testing of specimens could also be considered to expand test capacity as well during a pandemic [4]. This test can also provide information about the unique mutations in the virus such as S:D614G and RdRp:P314L, which can help in epidemiological investigations to detect the source of an outbreak.
In conclusion, the Clear Dx SARS-CoV-2 test is a reliable and fast automated EUA-FDA approved assay to detect SARS-CoV-2 in nasopharyngeal specimens from patients suspected to have COVID-19. Additional studies are needed to evaluate test capabilities as new viral variants of the SARS-CoV-2 are identified.
Funding
This article received no funding.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgment
The authors like to acknowledge Clear Labs Company for their supply of reagents and kits.
References
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