Abstract and Introduction
Objectives: We conducted an analytic and clinical comparison of a novel high-definition polymerase chain reaction PCR (HDPCR) assay to traditional real-time PCR (RT-PCR) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in upper respiratory specimens.
Methods: Analytic performance of RT-PCR, HDPCR, and extraction-free HDPCR was established through replicate testing of a serially diluted clinical specimen containing SARS-CoV-2. A clinical comparison of all 3 assays was conducted using 351 prospectively collected upper respiratory swab specimens obtained from symptomatic and asymptomatic individuals collected in various transport media.
Results: RT-PCR and HDPCR assays using extracted nucleic acid demonstrated similar analytic limits of detection (LoD) and clinical performance, with 100% positive and negative agreement. Extraction-free HDPCR demonstrated a 1.5 to 2.0 log10 increase in LoD based on cycle threshold values. However, clinical performance of extraction-free HDPCR remained high, demonstrating 97.8% positive and 99.6% negative agreement with RT-PCR. An overall increase in "invalid" and "presumptive" results was observed when using the extraction-free method, but this was highly variable based on transport medium used.
Conclusions: HDPCR performs similar to RT-PCR for the detection of SARS-CoV-2. The use of an extraction-free HDPCR protocol maintained high clinical performance despite reduced analytic LoD, with the benefit of reduced hands-on time and cost of reagents associated with nucleic acid extraction.
Identification of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), symptomatically or asymptomatically, is essential for appropriate management of the acutely ill and for enabling appropriate infection prevention, quarantine, and contact tracing for those that may be infectious. Nucleic acid amplification tests (NAATs) are considered the gold standard method for detection of SARS-CoV-2 viral RNA in clinical specimens. Currently, over 200 NAATs have received emergency use authorization (EUA) status from the US Food and Drug Administration. These tests can be broadly classified as on-demand sample-to-answer cartridge-based tests (eg, Xpert Xpress SARS-CoV-2, BioFire RP2.1, ID NOW COVID-19), high-throughput sample-to-answer tests (eg, Aptima SARS-CoV-2, cobas SARS, Alinity m SARS-CoV-2), and high-complexity manual batch-based tests (eg, TaqPath COVID-19, Quidel Lyra SARS-CoV-2, CDC 2019 nCoV RT-PCR). Clinical laboratories frequently offer each of these test types to accommodate the needs of different patient populations: acutely ill emergency room or inpatient, preadmission or presurgical screening, symptomatic ambulatory, and asymptomatic exposure or population screening.
Manual batch-based tests consist of polymerase chain reaction (PCR) reagents that can be utilized on various existing open-platform real-time PCR (RT-PCR) thermocyclers present in most clinical laboratories. These thermocyclers are often capable of accommodating 96 or 384-well PCR plates, and when used in conjunction with SARS-CoV-2 RT-PCR reagents have the potential to provide the highest throughput and lowest cost per specimen among laboratory-based NAATs. However, these tests require extraction of nucleic acids from specimens prior to RT-PCR amplification and detection. In addition to hands-on time required to manually set up extraction and RT-PCR plates, the additional reagents and disposables required increase the total cost per result and present increased potential for supply chain gaps. Further, many automated nucleic acid extraction platforms can process only 16 to 24 specimens per batch, which can lead to workflow bottlenecks and extended turnaround time. Given these barriers, adaptation of batch-based tests using a direct or extraction-free protocol would be desirable and provide several potential benefits to cost, hands-on time, turnaround time, throughput, and reduced risk of downtime due to supply chain shortages.
Prior studies have examined an extraction-free approach using either chemical or heat-based lysis steps and noted decreases in analytic limit of detection (LoD) as well as clinical sensitivity.[2–4] Differences in sensitivity between extracted and nonextracted specimens can be attributed to multiple variables, including (1) volume and type of transport medium put into the direct RT-PCR reaction, (2) genomic target and length of amplicon, (3) lack of specimen concentration in nonextracted specimens, and (4) stability of the polymerase enzyme utilized. These factors resulted in median increases of up to 6.7 cycle threshold (CT), which is equivalent to an approximately 2 log10 decrease in analytic sensitivity. Importantly, in these studies the clinical sensitivity also fell to 72% to 81% when compared to extracted specimens, with false-negative results weighted toward those specimens with high CT values.[2–4] While these data involve laboratory-developed protocols, the decreased analytic sensitivity associated with extraction-free protocols is also observed when comparing tests that have received EUA. Specifically, the Lyra SARS-CoV-2 Direct assay (Quidel) demonstrates an approximately 3 log10 decrease in analytic LoD when compared to the Lyra SARS CoV-2 assay that uses extracted viral RNA as template.
The EUA high-definition PCR (HDPCR) SARS-CoV-2 assay (ChromaCode) is a multiplexed molecular test that targets 2 regions of the SARS-CoV-2 nucleocapsid gene (N1 and N2), as well as the human RNase P gene (RP) as an internal control, in a single well. HDPCR utilizes standard RT-PCR instrumentation and well-established hydrolysis probe chemistry; however, in addition to differentiation of each target based on unique fluorophores, HDPCR employs a limiting probe design. The use of limited probe does not impact detection of a specific target (ie, CT value); however, the maximal fluorescence signal (ie, amplitude) reached during plateau phase of PCR can be modulated based on the amount of probe included in the reaction. This design enables differentiation of multiple unique targets using the same fluorophore based on the endpoint fluorescent signal or plateau associated with each probe. In addition to differentiation of multiple targets in a single fluorescent channel, HDPCR may also increase specificity of target detection. This HDPCR technology has successfully been applied to multiplex molecular tests targeting multiple respiratory viruses in nasopharyngeal specimens as well as multiple tickborne pathogens in whole-blood samples with high sensitivity and specificity.[6–8]
The primary aim of our study was to provide an analytic and clinical performance comparison between a traditional RT-PCR assay, TaqPath COVID-19 Combo Kit (ThermoFisher), and the HDPCR SARS-CoV-2 Assay (ChromaCode). Further, we examined the feasibility, performance, and potential impact of a research use only extraction-free protocol using the HDPCR SARS-CoV-2 reagents.
Am J Clin Pathol. 2021;156(1):24-33. © 2021 American Society for Clinical Pathology