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

Abstract

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.

Figure 1:

Study site specific analysis of validity of Atila iAMP assay against PCR (Reference) test (not stratified by sample collection site) *P-value < 0.05 for McNemar’s test (continuity corrected); **Any sample collection site positive out of the total samples collected is considered positive

Figure 2:

Study site specific analysis of validity of Atila iAMP assay against PCR (Reference) test (stratified by sample collection site) *P-value < 0.05 for McNemar’s test (continuity corrected); #Samples were tested in duplicates and the test was considered positive only if both were positive; @Samples were tested in duplicates and the test was considered positive if either was positive

Figure 3:

Study site and sample site specific analysis of the sensitivity of Atila iAMP assay against PCR (Reference) test stratified by the ct-values *Sensitivity for ct<35 and ct<30 was equal

Figure 4:

Study site and sample site specific analysis of validity of Atila iAMP assay against PCR (Reference) test stratified by the Symptoms *P-value < 0.05 for McNemar’s test (continuity corrected)

Figure 5:

Study site specific analysis of validity of OptiGene Direct Plus RT-LAMP assay against PCR (Reference) test (overall and stratified by sample collection site) *P-value < 0.05 for McNemar’s test (continuity corrected); **Any sample collection site positive out of the total samples collected is considered positive; #Samples were tested in duplicates and the test was considered positive only if both were positive; @Samples were tested in duplicates and the test was considered positive if either was positive