(H-296) In-clinic feasibility testing of a point-of-care low-cost COVID-19 RT-PCR test: a case study at MedStar Georgetown University Hospital Pediatric clinic

Introduction::

During the initial phase following the peak of the COVID-19 pandemic in 2020, the District of Columbia was one of the top five states in the US with the greatest effort in testing per capita. However, most testing at that time required sending samples to process in the laboratories, and the results were available in 1-5 days and about 7 days during the peak of the 2020 pandemic. The delay in results could impact care providers’ decisions in providing access to care to an individual. The prolonged uncertainty caused anxiety and inconvenience for many individuals, hindering them from fulfilling essential responsibilities while complying with restrictions. To enhance the accessibility of real-time COVID-19 molecular testing, we developed a modified US CDC SARS-CoV-2 assay, consisting of reverse transcription polymerase chain reaction (RT-PCR) and visual detection of SARS-CoV-2 viral RNA through end-point fluorescence signal under a low-cost reader [1]. Furthermore, this assay allows for lyophilized reagents to be added directly to samples – ultimately simplifying the standard extraction workflow and allowing for a greater sample volume. Here we present the feasibility testing of this test at a pediatric clinic in DC and share lessons learned from this investigation. 

Materials and Methods::

Our study was approved by the MedStar Georgetown University Hospital Pediatric Clinics Ethics Review Committee. The study was conducted in two phases. During phase I, healthcare workers (HCWs) without experience were recruited to participate in this study without compensation. They were given the opportunity to test contrived swab specimens on this simplified RT-PCR assay. During this period, we investigated the failed modes and determined the root causes in order to upgrade the test kits. During phase II, participating HCWs were asked to perform the test on a new set of blinded specimens. A combination of instructional videos and guidebooks was provided, guiding the HCWs to complete a workflow to demonstrate the feasibility of this method. Each HCW conducted the tests blindly on three swab specimens, each containing varying amounts of deactivated SARS-CoV-2 virus, specifically 0, 2000, and 5000 copies/mL. Briefly, the testing process involved eluting swabs in provided buffers and using disposable plastic pipettes to transfer the eluate into three tubes containing in-house lyophilized RT-PCR reagents for three genes: SARS-CoV-2 N1, SARS-CoV-2 N2, and Human control RP. Then the tubes were subjected to a mini-PCR thermal cycler for RT-PCR. Upon completing the RT-PCR process, the HCWs used a newly developed mobile phone app to capture images of the reaction tubes. These images were then automatically analyzed by the app, and the tubes were classified as positive or negative results.

Results, Conclusions, and Discussions:: The underlying objective was to evaluate a point-of-care configuration that uses low-cost, commercially available consumables and equipment as an alternative to existing options. We conducted a case study outside laboratories to explore its potential contribution in routine clinical COVID-19 testing. In our study, seven HCW volunteers participated, and they tested a total of 21 blinded swabs (equivalent to 63 RT-PCR reactions tested). Our assay demonstrated 93.6% (59/63) concordance with expected results, comprising 45 true positives, 14 true negatives, 1 false negative, and 3 false positive results. The false positives were attributed to a volume issue in the RT-PCR tube. We found that these tubes had approximately half of the volume in the RT-PCR tubes, and this drastic variation from the optimal reaction volume could result in a significant increase in salt concentrations and cause non-specific amplification. Regardless, compared to the performance during phase I, this performance of HCWs had significantly improved. Most HCWs had initially reported being unable to see clear elution buffers when transferring the elute into the PCR tubes. Thus, we adjusted our rehydration buffer to include Aurora Red dye for better visibility without interfering with the assay performance and post-run analysis. Though the study was limited by the number of recruited HCWs, it holds promise as a pioneering attempt to transfer a laboratory-based RT-PCR assay with minimal modifications to a clinical setting. This assay could be valuable in future scenarios like ‘tridemics,’ where multiple viruses (Influenza, RSV, and COVID) coexist during winter, especially for vulnerable populations such as infants and young children.

Acknowledgements (Optional): :

We thank Dr. Lisa Frenkel, Ingrid Beck, and Lutz laboratory members: Dr. Amy Oreskovic, Dr. Ian Hull, and Enos Kline, who contributed to the development and evaluation of the simplified CDC RT-PCR workflow for SARS-CoV-2 RNA detection. We thank the healthcare worker volunteers at MedStar Georgetown University Hospital Pediatric Clinics for participating in this study.

References (Optional): : [1] Panpradist N, Wang Q, Ruth PS, Kotnik JH, Oreskovic AK, Miller A, Stewart SWA, Vrana J, Han PD, Beck IA, Starita LM, Frenkel LM, Lutz BR. Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays. EBioMedicine. 2021 Feb;64:103236. doi: 10.1016/j.ebiom.2021.103236. Epub 2021 Feb 12. Erratum in: EBioMedicine. 2021 Apr;66:103296. PMID: 33582488; PMCID: PMC7878117.