Development or improvement of clinical diagnostic tests for SARS-CoV-2 to increase the sensitivity, specificity and ability to provide rapid results

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Abstract Unmet Need: q-rtPCR technology has dominated COVID-19 diagnostics and public health screening. Independent of the test developer, q-rtPCR has been shown to have an unusually high false negative rate: 15% up to 48% (1). According to the Covid Tracking Project. as of May 16th, 2020, 11 Million COVID-19 tests had been administered in the US (2). With 15% false negative rate, approximately 1.65M people would be falsely classified as free of infection. As might be expected, meta-analysis has shown that the false negative rate for q-rtPCR “explodes” before day 7 of infection (3) when viral load is still low, to render q-rtPCR ineffective as a tool for detecting weakly symptomatic carriers early, while also lessening its value in epidemiology (4). The Solution: PathogenDx has invented, patented and developed a microarray-based test, DetectX-Rv, and has submitted it for FDA-EUA review to screen for COVID-19 in NP swabs. The microarray has the capacity to test for multiple viral analytes in parallel with [SARS-CoV-2] as the primary analyte under FDA submission. Content Enhancement. We propose here the addition of a newly identified COVID-19 clade variant, which has been hypothesized by others to be more infective (5). DetectX-Rv already contains content needed to test SARS-CoV-2 plus multiple other coronavirus [SARS-CoV, MERS-CoV, CoV 229E,CoV OC43, CoV NL63, CoV HKU1] plus influenza + [PanA & Pan B] which are defined as a set as a “Pan-Respiratory” Virus Test. However for the development proposed in this RO1, this “extra” coronavirus content will be rationally modified and used instead as a large set of specificity controls. Other sources of funding outside this RO1 will be used to develop the full Pan-Respiratory virus content, as a separate product. Based on the work completed thus far, including April 15, 2020 filing to the FDA, we propose that with RO1 funding, the new test variant (DetectX-Rv-v2) can be made ready (by Q2) for deployment with NP swab collection as an automated 96 array/SBS plate COVID-19 test (@576 tests/shift). Sensitivity Improvement. The DetectX-Rv test is based on two Tandem Endpoint PCR reactions in series [Enrichment + Labeling] coupled to microarray hybridization. This technical approach gives rise to detection at single nucleotide resolution over a 6-log sample input dynamic range. Most importantly, the [Tandem PCR + Hybridization] assay routinely generates a Lowest Limit of Detection (LLOD) <1 genome per reaction. We anticipate that with this approach, we will routinely detect COVID-19 (signal/noise >20x background) at only 1 viral genome per reaction. Preliminary data, including that submitted to the FDA EUA program, suggests that the LLOD for DetectX-Rv will be roughly 10x lower than for an optimized q-RT-PCR reaction. Thus, with the DetectX-Rv test, COVID-19 should be routinely detected at 100 virus particles/swab. Specificity Enhancement. DetectX-Rv (144 tests) has enormous test capacity relative to q-rtPCR, which has allowed DetectX-Rv to monitor 3 different sites in the SARS-CoV-2 genome and a human RNA control, concurrently (N1,N2,N3,P) along with a panel of 8 viral controls all performed in parallel and in triplicate, thus allowing confirmation of COVID-19 signals with experimental specificity >10x than attainable with q-rtPCR. In the proposed RO1 program, that redundant COVID-19 content will be amended (DetectX-Rv-v2) to include a recently-identified clade variant S-D614G (5) so that initial and variant clades can be measured concurrently. Throughput Enhancement. We are near completion of a program to deploy full automation of DetectX-Rv function. As a short term RO1 deliverable, we will develop, by Sept 1, 2020, an integrated suite of technology for processing 576 NP swab or saliva tests/shift, in a 96 microarray/SBS plate format, based on the labor burden of 1 technician. The 96 well format employs the same printing technology already in use and the same (patented) microarray fabrication chemistry and is thus low risk. However, by completing the transition to the 96 well format, we will have achieved 12,000 arrays/day manufacturing scale, which with support from equity other sources, could be immediately scaled to 48,000 per day. Based the requirements of PAR-20-178, we propose 4 Specific Aims to support this 2 year RO1 program. SA1. Q1. Add Clade & Coronavirus Content Enabling Analysis of SARS-CoV-2 & variants. SA2. Q2. Sensitivity/specificity analysis of DetectX-Rv-v2 vs qRT-PCR predicate: Clinical Specimens SA3. Q3-5. Simplify DetectX-Rv-v2 Throughput via One pot RT-PCR & Hybridization Rate Enhancement. SA4. Q5-8. Enhance & Validate DetectX-Rv Performance to Diagnose Asymptomatic Transmission