key: cord-1040909-ea1r90io
authors: Pham, J.; Meyer, S.; Nguyen, C.; Williams, A.; Hunsicker, M.; McHardy, I.; Gendlina, I.; Goldstein, D. Y.; Fox, A. S.; Hudson, A.; Darby, P.; Hovey, P.; Morales, J.; Mitchell, J.; Harrington, K.; Majlessi, M.; Moberly, J.; Shah, A.; Worlock, A.; Walcher, M.; Eaton, B.; Getman, D.; Clark, C.
title: Performance characteristics of a high throughput automated transcription mediated amplification test for SARS-CoV-2 detection
date: 2020-07-10
journal: nan
DOI: 10.1101/2020.07.06.20143719
sha: 87e07b2eb2685537171a5ed058aa976bce7912a9
doc_id: 1040909
cord_uid: ea1r90io

The COVID19 pandemic caused by the new SARSCoV2 coronavirus has imposed severe challenges on laboratories in their effort to achieve sufficient diagnostic testing capability for identifying infected individuals. In this study we report the analytical and clinical performance characteristics of a new, high throughput, fully automated nucleic acid amplification test system for the detection of SARSCoV2. The assay utilizes target capture, transcription mediated amplification, and acridinium ester labeled probe chemistry on the automated Panther System to directly amplify and detect two separate target sequences in the ORF1ab region of the SARSCoV2 RNA genome. The probit 95% limit of detection of the assay was determined to be 0.004 TCID50/ml using inactivated virus, and 25 c/ml using synthetic in vitro transcript RNA targets. Analytical sensitivity (100% detection) was confirmed to be 83 to 194 c/ml using three commercially available SARSCoV2 nucleic acid controls. No cross reactivity or interference was observed with testing six related human coronaviruses, as well as 24 other viral, fungal, and bacterial pathogens, at high titer. Clinical nasopharyngeal swab specimen testing (N=140) showed 100%, 98.7%, and 99.3% positive, negative, and overall agreement, respectively, with a validated reverse transcription PCR NAAT for SARSCoV2 RNA. These results provide validation evidence for a sensitive and specific method for pandemic-scale automated molecular diagnostic testing for SARSCoV2.

Coronavirus disease-19 (COVID19) is a novel respiratory illness caused by severe acute respiratory 47 syndrome coronavirus 2 (SARS-CoV-2), a novel Sarbecovirus that emerged from the region of Wuhan, 48

China in late 2019 (1) . People with COVID-19 experience mild to severe respiratory symptoms including 49 fever, cough and shortness of breath or difficulty breathing (2), although some individuals experience no 50 symptoms at all (3). 51

The COVID-19 pandemic has occurred across all continents, adding more than 100,000 new SARS-CoV-52 2 cases globally each day (4,5). As communities begin reopening and relaxing quarantine measures, there 53 is the potential risk for an upsurge in cases and rates of viral transmission. The availability of validated 54 high-throughput diagnostic tests is therefore essential for rapidly and efficiently informing patient 55 management decisions, implementing hospital infection prevention practices, and for guiding public 56 health responses to wide-scale infection control measures to reduce transmission in populations. 57 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. . https://doi.org/10.1101/2020.07.06.20143719 doi: medRxiv preprint

To meet the need for pandemic-scale diagnostic testing, we have developed and validated a high-58 throughput, fully automated nucleic acid amplification test (NAAT) for direct amplification and detection 59 of SARS-CoV-2 RNA from specimens of infected individuals. The assay employs target capture, 60 transcription mediated amplification (TMA) and acridinium ester-labeled probe chemistries to enable a 61 sample-to-result solution for detection of two different conserved target regions within the ORF1ab 62 region of the SARS-CoV-2 genome. Herein, we describe the analytical and clinical performance 63 characteristics of the assay. 64 65

Transcription Mediated Amplification Test for SARS-CoV-2. The Aptima® SARS-CoV-2 assay 67 utilizes magnetic bead-based target capture, isothermal TMA of RNA, and dual kinetic acridinium ester-68 labeled probe hybridization for the isolation, amplification, and detection of an internal process control 69 RNA, and two unique sequences within the ORF1ab region of the SARS-CoV-2 viral genome. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 10, 2020. . https://doi.org/10.1101/2020.07.06.20143719 doi: medRxiv preprint

Limit of Detection. The analytical sensitivity of the SARS-CoV-2 TMA assay was assessed using 2 lots 85 of reagents to test 60 replicates each of dilution panels containing cultured SARS-CoV-2 virus strain 86 USA-WA1/2020 (BEI Resources, Manassas, VA) and diluted in Aptima specimen transport medium 87 (STM) matrix to a range of 0.03 to 0.0003 tissue culture infectious dose 50 per ml (TCID50/ml). Also 88 tested were replicates (n = 43 to 60) of panels consisting of two in vitro transcribed (IVT) RNA targets, 89 corresponding to two unique target sequences within the ORF1ab region of the SARS-CoV-2 RNA 90 genome, diluted in STM. Assay positivity for both studies was determined using a pre-defined cutoff 91 value of 560 kilo relative light units (kRLU). Results were analyzed by probit analysis (normal model) to 92 determine the 95% limit of detection (LOD). Analytical sensitivity was confirmed by testing 20 replicates 93 each of SARS-CoV-2 virus diluted in four specimen matrices (pooled NP swab, STM, saline and Liquid 94

Amies transport medium (Copan, Murrieta, CA)) at 0.003 TCID50/ml for NP swab, STM and saline, and 95 0.003 and 0.01 TCID50/ml for Liquid Amies. 96

Analytical Specificity/Interference. Analytical specificity of the SARS-CoV-2 TMA assay was 97 determined by evaluating assay cross-reactivity and interference using 30 non-target microorganisms (17 98 viral species, including 6 non -SARS-CoV-2 coronaviruses, 11 bacterial species, and 2 fungal species; 99 N=3 replicates each) at the highest titer achievable. Thirty NP swab specimens obtained from consented 100 asymptomatic donors were also tested to represent diverse microbial flora in the human respiratory tract. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 10, 2020. . https://doi.org/10.1101/2020.07.06.20143719 doi: medRxiv preprint ERC0831042) were diluted in STM to 6 concentrations ranging from 833 to 8 copies per ml (c/ml) and 109 multiple replicates (n = 20 to 40) were tested with the SARS-CoV-2 TMA assay. The control panels were 110 also tested with the SARS-CoV-2 RT-PCR assay on the automated Panther Fusion platform. for 3 to 5 seconds and placing the swab into a tube of STM. OP swab samples were collected immediately 120 following NS samples by swabbing the posterior pharynx for 3-5 seconds and placing the swab into a 121 specimen tube. Samples were frozen and shipped to Hologic (San Diego, CA) for testing. 122

Data for the analytical sensitivity determination of the SARS-CoV-2 TMA assay are shown in Figure 1 . 124

Using a pre-determined cutoff value of 560 kRLU, the assay yielded 100% positivity at a concentration of 125 0.01 TCID50/ml of SARS-CoV-2 virus and at 100 c/ml of SARS-CoV-2 IVT RNA targets. Using probit 126 analysis, the 95% limit of detection was determined to be 0.004 TCID50/ml (95% CI: 0.003 -0.007) for 127 SARS-CoV-2 virus, and 25.4 c/ml (95% CI: 16.9 -50.5) for ORF1ab IVT RNA targets. Analytical 128 sensitivity for the assay was confirmed by testing SARS-CoV-2 virus in 4 specimen matrices (pooled NP 129 swab specimens, STM, saline, and Liquid Amies transport medium) at 0.003 TCID50/ml. All specimen 130 matrices yielded 95% positivity (19/20 replicates detected) at this concentration, except Liquid Amies 131 transport medium, which was 85% (17/20) positive at 0.003 TCID50/ml and 100% (20/20) positive at 0.01 132 TCID50/ml ( Table 1) . 133 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. . https://doi.org/10.1101/2020.07.06.20143719 doi: medRxiv preprint Analytical specificity of the SARS-CoV-2 TMA assay was determined by evaluating assay cross-134 reactivity and interference using 30 non-target viral, bacterial and fungal microorganisms at the highest 135 titer achievable, as well as in 30 NP swab specimens obtained from consented asymptomatic donors at 136 low risk of SARS-CoV-2 infection. As shown in the Supplemental Table, none of the microorganisms or 137 NP swab specimens tested caused cross-reactivity in the absence of SARS-CoV-2 target or interfered with 138 TMA detection in the presence of SARS-CoV-2 spiked at 0.03 TCID50/ml. 139

The clinical accuracy of the SARS-CoV-2 TMA assay was compared to the SARS-CoV-2 RT-PCR assay 140 using 140 patient NP swab specimens ( Table 2 ). This analysis resulted in positive, negative, and overall 141 agreements of 100% (95%CI: 94.3% -100%), 98.7% (95%CI: 92.9% -99.8%), and 99.3% (95%CI: 142 96.1% -99.3%), respectively. Clinical performance of the SARS-CoV-2 TMA assay was also assessed by 143 testing sets of NP swabs, OP swabs, and nasal swabs co-collected from 35 symptomatic patients 144 suspected of being infected with SARS-CoV-2. Figure 2 shows the SARS-CoV-2 TMA assay had 100% 145 positive and negative agreements of the NP swab specimens with the co-collected OP swab and nasal 146 swab specimens. Similar results were obtained for the paired specimen sets using the SARS-CoV-2 RT-147 PCR assay (Supplemental Figure) . 148 SARS-CoV-2 control panel materials from three commercial suppliers (Exact Diagnostics, SeraCare, 149 ZeptoMetrix) were evaluated by building dilution panels of each control material and testing multiple 150

replicates with both the SARS-CoV-2 TMA assay and the SARS-CoV-2 RT-PCR assay ( Table 3) . Both 151 assays yielded similar results. For the Exact Diagnostics SARS-CoV-2 control, the TMA and RT-PCR 152 assays each had 100% detection down to 83 c/ml (N=40 each). For the SeraCare SARS-CoV-2 control, 153 the TMA assay was 100% at 83 c/ml (N=20) while the RT-PCR assay was 90% at 83 c/ml and 100% at 154 194 c/ml (N=20 for both). The SARS-CoV-2 TMA assay (N=37) and the SARS-CoV-2 RT-PCR assay 155 (N=40) were both 100% reactive at 194 c/ml using the ZeptoMetrix control material. 156 157 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. infection (9) as it has been reported that nasal swab or OP swab samples may have a slightly lower 177 sensitivity compared to NP swabs (10, 11). However, given the challenges of collection device shortages 178 for SARS-CoV-2 diagnosis, particularly NP swabs and VTM, the use of nasal swab and OP swab as 179 alternate samples for diagnosis of SARS-CoV-2 has been evaluated. The CDC recently removed NP swab 180 as the "preferred" sample type from their sample collection guidelines (7) and others have reported 181 comparable performance of NS and OP swabs for the diagnosis of SARS-CoV-2 (12-15). Our data shows 182 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. . https://doi.org/10.1101/2020.07.06.20143719 doi: medRxiv preprint strong agreement between SARS-CoV-2 detection from NS and OP swabs compared to paired NP swab 183 samples for the SARS-CoV-2 TMA and SARS-CoV-2 RT-PCR assays. We did observe for one patient 184 positive NP and nasal swab results, but negative results in the paired OP swab, however overall the data 185 indicate that both NS and OP swab specimens are adequate sample types for diagnosis of SARS-CoV-2. 186

The performance of other SARS-CoV-2 NAATs that have received Emergency Use Authorization has 187 been characterized using commercially available inactivated virus preparations (e.g., BEI Resources, Additional clinical data should be collected to more fully assess and compare performance between these 201 sample types. 202

In summary, the SARS-CoV-2 TMA assay is highly sensitive and specific and provides an automated 203 high-throughput testing solution for large-scale diagnostic testing for the virus. The assay system is able 204 to process and generate results for > 1,000 samples per day enabling medical centers, reference 205 laboratories and public health laboratories to efficiently process and analyze very high volumes of 206 specimens for the detection of SARS-CoV-2 RNA (18). 207 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. . https://doi.org/10.1101/2020.07.06.20143719 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. Comparison of Three Nucleic Acid Amplification Tests for SARS-CoV-2 Detection. bioRxiv 256 https://doi.org/10.1101/2020.05.14.097311. Accessed 10Jun2020. 257 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. . . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. . https://doi.org/10.1101/2020.07.06.20143719 doi: medRxiv preprint Table 2 . Agreement analysis (%, (95% confidence interval) between the Aptima SARS-CoV-2 TMA assay and the Panther Fusion SARS-CoV-2 RT-PCR assay for nasopharyngeal swab specimens. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. . https://doi.org/10.1101/2020.07.06.20143719 doi: medRxiv preprint Table 3 . Performance of Aptima SARS-CoV-2 TMA and Panther Fusion SARS-CoV-2 RT-PCR assays for detection of commercially available SARS-CoV-2 controls. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 10, 2020. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 10, 2020. Each NP swab specimen was tested in triplicate for a total of 90 replicates in the absence and presence of spiked SARS-CoV-2 inactivated virus.

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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Agreement (A) between 35 sets of co-collected nasopharyngeal swab, oropharyngeal swab, and nasal swab clinical specimens with positive (+) and negative (-) Panther Fusion SARS-CoV-2 RT-PCR assay results. Scatter plot (B) of RT-PCR Ct values corresponding to Panther Fusion SARS-CoV-2 RT-PCR assay positive samples for each swab type