Multi-site clinical validation of Isothermal Amplification based SARS-COV-2 detection assays using different sampling strategies

Background: Isothermal amplification-based tests were developed as rapid, low-cost, and simple alternatives to real-time reverse transcriptase-polymerase chain reaction (RT-PCR) tests for SARS-COV-2 detection. Methods: Clinical performance of two isothermal amplification-based tests (Atila Biosystems iAMP COVID-19 detection test and OptiGene COVID-19 Direct Plus RT-LAMP test) was compared to clinical RT-PCR assays using different sampling strategies. A total of 1378 participants were tested across four study sites. Results: Compared to standard of care RT-PCR testing, the overall sensitivity and specificity of the Atila iAMP test for detection of SARS-CoV-2 were 76.2% and 94.9%, respectively, and increased to 88.8% and 89.5%, respectively, after exclusion of an outlier study site. Sensitivity varied based on the anatomic collected site. Sensitivity for nasopharyngeal was 65.4% (range across study sites:52.8%–79.8%), mid-turbinate 88.2%, saliva 55.1% (range across study sites:42.9%–77.8%) and anterior nares 66.7% (range across study sites:63.6%–76.5%). The specificity for these anatomic collection sites ranged from 96.7% to 100%. Sensitivity improved in symptomatic patients (overall 82.7%) and those with a higher viral load (overall 92.4% for ct≤25). Sensitivity and specificity of the OptiGene Direct Plus RT-LAMP test, conducted at a single study-site, were 25.5% and 100%, respectively. Conclusions The Atila iAMP COVID test with mid-turbinate sampling is a rapid, low-cost assay for detecting SARS-COV-2, especially in symptomatic patients and those with a high viral load, and could be used to reduce the risk of SARS-COV-2 transmission in clinical settings. Variation of performance between study sites highlights the need for site-specific clinical validation of these assays before clinical adoption.

and anterior nares 66.7% (range across study sites:63.6%-76.5%). The specificity for these 48 anatomic collection sites ranged from 96.7% to 100%. Sensitivity improved in symptomatic 49 patients (overall 82.7%) and those with a higher viral load (overall 92.4% for ct≤25). Sensitivity 50 and specificity of the OptiGene Direct Plus RT-LAMP test, conducted at a single study-site, were 51 25.5% and 100%, respectively. 52 for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. The Atila iAMP COVID test with mid-turbinate sampling is a rapid, low-cost assay for detecting 54 SARS-COV-2, especially in symptomatic patients and those with a high viral load, and could be 55 used to reduce the risk of SARS-COV-2 transmission in clinical settings. Variation of 56 performance between study sites highlights the need for site-specific clinical validation of these 57 assays before clinical adoption. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. ; https://doi.org/10.1101/2021.07.01.21259879 doi: medRxiv preprint Introduction 68 The COVID-19 (Coronavirus Disease of 2019) pandemic has led to major disruptions in health 69 services worldwide. In many developed nations, widespread SARS-CoV-2 (Severe acute 70 respiratory syndrome coronavirus 2) testing and mass vaccination has allowed for a return to 71 most elective health services. However, many low-and middle-income countries (LMICs) have 72 limited access to testing and vaccination and continue to struggle to contain COVID- 19 (1) (2). 73 As the COVID-19 crisis continues , considerable reductions of cancer screening, cancer control, 74 and elective clinical services remain (3). The safe return to cancer screening and elective 75 testing and procedures during the pandemic, especially in low vaccination regions, requires 76 reliable SARS-CoV-2 testing for both providers and patients. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021.  This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. ; https://doi.org/10.1101/2021.07.01.21259879 doi: medRxiv preprint was also invalid, then the that sample was considered invalid. In total, 1.0% of NP, 1.4% of 174 anterior nares, 3.9% of mid-turbinate, and 0% of saliva samples had invalid results. Less than 175 1% (0.6%) of saliva samples could not be tested secondary to the samples being predominantly This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. were negative for the subject, the subject was considered negative for that test assay. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021.  This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. Assuming that viral load would influence accuracy, we analyzed the sensitivity at different ct-243 values on RT-PCR among the positive subjects (Figure 3)  This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. ; https://doi.org/10.1101/2021.07.01.21259879 doi: medRxiv preprint 15 detection of SARS-CoV-2, excluding the outlier study site, were 88.8% and 89.5%, respectively. 284 The sensitivity, excluding the outlier study site, was 78.9% for nasopharyngeal, 88.2% for self-285 sampled mid-turbinate, 74.5% for direct saliva and 66.7% for anterior nares samples. The 286 specificity for these sites ranged from 91.8% to 100%. The sensitivity increased with higher viral 287 load (i.e., at lower ct-values) and among symptomatic as compared to asymptomatic 288 participants. The sensitivity and specificity of the OptiGene Direct Plus RT-LAMP test, conducted 289 at a single site, were 25.5% and 100%, respectively. for the assay to be 50-100 copies/reaction for ORF1-a/b gene and 1-10 for the N gene, which is 298 higher than that of RT-PCR (average range of 1-10) (13). This may explain our finding of lower 299 clinical sensitivity of the assay at higher ct-values, considering ct-values as a surrogate marker 300 for the viral load, which may not always be precise (14). In the clinical validation by the same 301 group, the assay was found to have 100% agreement with the RT-PCR. However, this validation 302 was on extracted RNA and was based on 46/50 samples that have ct≤30. Another small (n=50) 303 clinical validation (15), again on extracted RNA, showed the sensitivity and specificity of the 304 assay to be 82.8% and 100%, respectively, with all five false-negative samples to have ct≥35. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. The OptiGene Direct Plus RT-LAMP COVID assay has been previously clinically validated by the 307 NHS trust to have a sensitivity of 70% for swabs and 79% for saliva, with an increase in 308 sensitivity to 100% for swabs at ct≤25 (16). However, similar to our validation, such high 309 sensitivity was not confirmed by other groups, which showed the sensitivity in the range of It is important to note that the IFU's for both assays state the need to confirm the negative test 322 result with a more sensitive RT-PCR test, and do not claim to be the final screening answer (20). 323 However, as compared to the RT-PCR assays, which may sometimes take >24 hr of turnaround 324 time (TOT) with considerable cost, the isothermal amplification-based assay's advantage is its 325 rapid TOT (~1 hour), lower cost, and ease of performance (no nuleic acid extraction needed). 326 Thus, it can cheaply and rapidly identify high viral load subjects who are likely to be most 327 for use under a CC0 license.
This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. ; https://doi.org/10.1101/2021.07.01.21259879 doi: medRxiv preprint 17 infectious (21)(22). Moreover, there is at least some evidence to suggest that RT-PCR positivity 328 does not necessarily translate into infectivity because it can detect the shedding of post-329 infectious viral RNA particles shedding, particularly among post-symptomatic patients (23,24). 330 The Atila iAMP has similar advantages as the rapid antigen tests with regard to ease of 331 operability and quick TOT, but provides higher sensitivity (reported to be 67-73% for rapid 332 antigen test (25,26)) resulting in more reassurance of a negative test result. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) 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 6, 2021. ; https://doi.org/10.1101/2021.07.01.21259879 doi: medRxiv preprint N/A-Data not available; *All invalid runs were due to insufficient sample/mainly phlegm to process; **Three samples out of six total invalid runs were due to 470 insufficient sample/mainly phlegm to process; *** saliva, mid-turbinate, and anterior nares (at Wiconsin) were self-collected, # Type of RT-PCR data not