key: cord-0790849-ihg0ymkr
authors: Hogan, Catherine A.; Garamani, Natasha; Lee, Andrew S.; Tung, Jack K.; Sahoo, Malaya K.; Huang, ChunHong; Stevens, Bryan; Zehnder, James; Pinsky, Benjamin A.
title: Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens
date: 2020-05-13
journal: bioRxiv
DOI: 10.1101/2020.05.12.092379
sha: 79514ecc2f30b0f600f61608fb43d92464e58a32
doc_id: 790849
cord_uid: ihg0ymkr

Background Several point-of-care (POC) molecular tests have received emergency use authorization (EUA) from the Food and Drug Administration (FDA) for diagnosis of SARS-CoV-2. The test performance characteristics of the Accula (Mesa Biotech) SARS-CoV-2 POC test need to be evaluated to inform its optimal use. Objectives The aim of this study was to assess test performance of the Accula SARS-CoV-2 test. Study design The performance of the Accula test was assessed by comparing results of 100 nasopharyngeal swab samples previously characterized by the Stanford Health Care EUA laboratory-developed test (SHC-LDT) targeting the envelope (E) gene. Assay concordance was assessed by overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohen’s kappa coefficient. Results Overall percent agreement between the assays was 84.0% (95% confidence interval [CI] 75.3 to 90.6%), PPA was 68.0% (95% CI 53.3 to 80.5%) and the kappa coefficient was 0.68 (95% CI 0.54 to 0.82). Sixteen specimens detected by the SHC-LDT were not detected by the Accula test, and showed low viral load burden with a median cycle threshold value of 37.7. NPA was 100% (95% CI 94.2 to 100%). Conclusion Compared to the SHC-LDT, the Accula SARS-CoV-2 test showed excellent negative agreement. However, positive agreement was low for samples with low viral load. The false negative rate of the Accula POC test calls for a more thorough evaluation of POC test performance characteristics in clinical settings, and for confirmatory testing in individuals with moderate to high pre-test probability of SARS-CoV-2 who test negative on Accula.

The aim of this study was to evaluate the test performance characteristics of the Accula SARS-67

CoV-2 test in a clinical setting against a high complexity reference standard. 68 69 Study design 70 Nasopharyngeal (NP) swabs were collected in viral transport medium or saline from adult 71 patients from SHC, and from pediatric and adult patients from surrounding hospitals in the Bay 72

Area. Testing for this study was performed at the SHC Clinical Virology Laboratory using 73 samples collected between April 7, 2020 and April 13, 2020. The same NP specimen was used 74 for both the reference assay and Accula test for comparison. 75 76

The reference assay for this study was the Stanford Health Care Clinical Virology Laboratory 78 real-time reverse transcriptase polymerase chain reaction LDT (SHC-LDT) targeting the E gene 79 (11-13). The Accula SARS-CoV-2 POCT (Mesa Biotech, Inc., San Diego, CA) is a sample-to-80 answer nucleic acid amplification test that can yield a diagnostic result within 30 minutes of 81 specimen collection. This test uses RT-PCR to target the nucleocapsid protein (N) gene, and is 82 read out via lateral flow (Figure 1) (14) . The manufacturer's instructions are comprised of the 83 following steps: collection of nasopharyngeal (NP) swab, lysis of viral particles in SARS-CoV-2 84 buffer, transfer of nucleic acid solution to a test cassette which contains internal process positive 85 and negative controls, reverse transcription of viral RNA to cDNA, nucleic acid amplification, 86 and detection by lateral flow. Due to biosafety regulations and hospital-mandated protocols for 87 sample collection at SHC, NP swabs were directly placed into VTM or saline at the patient 88 bedside after collection. Each test was performed at the laboratory, where a volume of 10 µL of 89 VTM or saline was transferred to 60 µL of SARS-CoV-2 buffer and added to the test cassette. 90

These steps were performed within a biosafety cabinet to protect against aerosolization. All 91 remaining steps were followed as per the manufacturer's instructions (14). Testing was repeated 92 once for invalid results on initial testing, and the second result was interpreted as final if valid. Although SARS-CoV-2 testing capacity has improved in many countries, a global shortage of 124 diagnostic infrastructure and consumable reagents has limited testing efforts. Point-of-care tests 125 offer the potential advantages of improved access to testing and reduced turnaround time of 126 results. Of the multiple EUA assays for diagnosis of SARS-CoV-2, only the Xpert Xpress, the ID 127 NOW, and the Accula are CLIA-waived (6). Recent data support the test performance of the 128 Cepheid Xpert SARS-CoV-2 assay, with agreement over 99% compared to high-complexity 129 EUA assays (8, 16, 17). In contrast, some studies have raised concern regarding the diagnostic 130 accuracy of the ID NOW, with positive percent agreement ranging from 75-94% compared to 131 reference assays (8-10, 18). Given the poor diagnostic performance of the ID NOW, and 132 uncertainty regarding availability of Xpert Xpress cartridges, the Accula system has been tauted 133 as an interesting POCT alternative but data were previously lacking on its clinical performance. 134

In this study, we showed that similar to ID NOW, the Accula SARS-CoV-2 test has a lower 135 sensitivity for diagnosis of COVID-19 compared to an EUA LDT. The false negatives obtained 136 from the Accula SARS-CoV-2 test were predominantly observed with low viral load specimens. 137 138 Given the accumulating evidence on lower diagnostic performance with 2 of the 3 CLIA-waived 139 SARS-CoV-2 assays, it is now important to consider how best to integrate these tests in 140 diagnostic workflows and to identify groups of individuals for whom POCT use should be 141 prioritized. Furthermore, reagents and kits have been limited, which limits POCT capacity. 142

Certain groups such as individuals requiring urgent pre-operative assessment including 143 transplantation, patient-facing symptomatic healthcare workers, and individuals waiting for 144 enrollment in a SARS-CoV-2 therapeutic trial have been identified as key groups in whom to 145 prioritize POCT. However, for each of these scenarios and depending on the POCT used, the risk 146 of missing a case due to low sensitivity must be considered. In individuals with moderate to high 147 pre-test probability of SARS-CoV-2, reflex testing of negative samples on a separate EUA assay 148 should be performed. Education of health care professionals on the limitations of SARS-CoV-2 149 POCT should also be implemented to ensure optimal interpretation and management of negative 150 results. 151

Our study has several limitations. First, NP swabs were placed in VTM or saline at the patient 153 bedside before loading the Accula test cassette, which may have decreased sensitivity by diluting 154 the viral inoculum. Although this is discordant with the best recommended practice by the 155 manufacturer, it is in line with the practice at multiple institutions with clinical laboratories that 156 have assessed SARS-CoV-2 POCT due to biosafety concerns from risk of aerosolization (8-10, 157 18, 19). Second, it is possible that the use of saline instead of VTM led to poorer performance of 158 the Accula. However, aliquots from the same sample were used for parallel testing with the EUA 159 method, which minimizes sources of variation, and represents a pragmatic comparison given 160 widespread VTM shortages. Finally, the lateral-flow read-out of the Accula test is generally easy 161 to interpret; however, faint lines may be more challenging to interpret 

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