A-376 Development of a 10-Minute Sample to Answer RT-PCR Respiratory Panel Performed on Point of Care (POC)
Authors: Peytavi R , Hambalek J , Alvarado E , Parocua Y , Abeje A , Foltz F , Pollack B , Halle P , Petrullo M , Allen K , Kido H , Martin R 
Published: September 2023

Abstract

Background
The COVID-19 pandemic has shown the importance of fast and sensitive PCR testing to identify SARS-CoV-2 (SC2) infected patients. Identifying infected individuals with a fast sample to answer test provides the opportunity to limit the spread of the virus and prescribe antimicrobial treatment in the same visit. Additionally, the post pandemic world has seen simultaneous outbreaks of RSV, influenza, and SC2, the so-called tridemic. These higher risk respiratory viruses can appear against a background of lower risk viruses such as rhinovirus, adenovirus, enterovirus, other corona viruses, and bacterial infections that present with similar symptoms and make triage challenging. Fast turnaround molecular testing along with multiplex detection of all key pathogens associated with flu-like symptoms is important for timely selection of treatment, counseling to contain the spread of disease, and provide public health officials information on prevalence of outbreaks. AMDI is developing a cloud-based, ultrafast, fully automated POC molecular technology that enables detection of up to 32 microbial targets in less than 10 min from sample to answer. A 15-second extraction-free hyperbaric heating (HBH) step is performed on the input swab sample to release nucleic acid and neutralize PCR inhibitors in the sample in seconds. The sample is directly transferred without dilution to a chamber where it mixes with dry RT-PCR reagents and then is dispatched to eight thin (&lt;250 µm thick) PCR reaction cuvettes where rapid heat transfer can occur. This is followed by thermocycling on an instrument designed to achieve 10 sec per cycle and detect four fluorescent signals per cuvette. This study first evaluates the HBH method against nucleic acid purification assays using SC2 positive clinical samples. Next, a preliminary LoD on the AMDI system (HBH and fast thermocycling) was performed using inactivated SC2 and RSV whole organism in a nasal swab matrix.

Methods
To evaluate HBH as a sample prep method, 12 known SC2 positive remnant clinical samples in VTM or saline were diluted 1:1 with AMDI collection buffer, treated by HBH, tested by RT-PCR on QuantStudioTM and compared with the Roche LIAT. A preliminary SC2 and RSV LoD on the full AMDI system was determined by spiking dilutions of inactivated viruses into pooled nasal swab matrix, followed by treatment with HBH, and testing by RT-PCR using the ultra-fast thermocycler.

Results
In clinical sample testing HBH shows equal or greater sensitivity than LIAT, even for high Ct samples. HBH followed by RT-PCR detected 12/12 (100% sensitivity) samples while Roche LIAT detected 11/12 (92% sensitivity). When HBH is coupled with AMDI ultra-fast PCR thermocycling technology, an SC2 preliminary LoD of 500 copies/mL and an RSV preliminary LoD of 250 copies/mL was obtained in &lt;9 min including a 60 second RT step.

Conclusion
HBH is an efficient and effective sample prep method and when coupled with ultrafast thermocycling provides sensitive PCR results in 10 minutes sample to answer.