149. Extraction-free RT-PCR to Detect SARS-CoV-2 Variants of Concern

Abstract Background SARS-CoV-2 variants of concern (VOC) have challenged real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods for the diagnosis of COVID-19. Methods The CDC 2019-Novel Coronavirus real-time RT-PCR panel was modified to create a single-plex extraction-free proxy RT-PCR assay, VOCFast™. This assay uses the nucleocapsid N1 as well as novel primer/probe pairs to target VOC mutations in the Orf1a and spike (S) genes. For analytical validation of VOCFast, synthetic controls for the Wuhan, alpha/B.1.1.7, beta/B.1.351, and gamma/P.1 strains were tested at various concentrations. Clinical validation was performed using patient anterior nares swab and saliva specimens collected in the Denver, CO area between Nov 2020 and Feb 2021 or in March 2021. Orthogonal next-generation sequencing (NGS) was also performed. Results Similar N1 quantification cycle (Cq) values corresponding to viral load were observed for all strains, suggesting that VOC mutations do not affect performance of the N1 primer/probe. Orf1a-mut and S1-mut primer/probes generated a stable high Cq value for the Wuhan strain. Conversely, Orf1a-mut Cq values were inversely correlated with viral load for all VOC. The S1-mut Cq was inversely correlated with viral load of the alpha strain, but did not reliably amplify beta/gamma VOC. The limit of detection was 8 copies/uL. The first set of COVID-19 patient specimens revealed no amplification using Orf1a-mut whereas 53% of specimens collected in Mar 2021 demonstrated amplification by Orf-1a. Orthogonal testing by the SARS-CoV-2 NGS Assay and COVID-DX software demonstrated that 12/12 alpha strains, 2/2 beta/gamma strains, and 33/33 Wuhan strains were correctly identified by VOCFast. Detection of VOC in clinical specimens and validation by NGS Conclusion The combination of the N1, Orf1a-mut, and S1-mut primers/probes in VOCFast can distinguish the Wuhan, alpha, and beta/gamma strains and it consistent with NGS results. Testing of clinical samples revealed that VOC emerged in Denver, CO in March 2021. Future work to discriminate beta, gamma, and emerging VOC is ongoing. In summary, VOCFast is an extraction-free RT-PCR assay for nasal swab and saliva specimens that can identify VOC with a turnaround time suitable for clinical testing. Disclosures Brian L. Harry, MD PhD, Summit Biolabs Inc. (Grant/Research Support, Shareholder) Mara Couto-Rodriguez, MS, Biotia (Employee) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder) Shi-Long Lu, MD PhD, Summit Biolabs Inc. (Grant/Research Support, Shareholder)

. Distribution of negative to positive test pairs by 4 hour time intervals Table 1. Multivariate regression predicting a negative to positive test pair Conclusion. The likelihood of detecting early infection is dependent on PCR platform and body site of sampling. A range of time intervals between 16 to 36 hours after the initial test were likely to identify positive cases.
Disclosures. Sanjat Kanjilal, MD, MPH, GlaskoSmithKline (Advisor or Review Panel member) Background. Detection and surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is of great public health importance. Broadly accessible and inexpensive assays are needed to enhance variant surveillance and detection globally. We developed and validated a single-reaction multiplex real-time RT-PCR (the Spike SNP assay) to detect specific mutations associated with variants of concern (VOC).

Single-amplicon, Multiplex Real-time RT-PCR with Tiled Probes to Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern
Methods. A single primer pair was designed to amplify a 348 bp region of spike. Probes were initially designed with locked nucleic acids (LNAs) to increase probe melting temperature, shorten probe length, and specifically detect 417K, E484K, and N501Y ( Figure). The assay was optimized and evaluated using characterized variant sample pools. Clinical evaluation was performed on a convenience set of residual nasopharyngeal swabs, and variant calls were confirmed by SARS-CoV-2 genomic sequencing in a subset of samples. Following the initial evaluation, unmodified probes (without LNAs) were designed to detect L452R, L452Q, and E484Q. Representative results of variant detection a single Spike SNP run are shown for mutations in the codons for 4177K (A) and mutations that encode 484K (B) and 501Y (C). Curves show dilutions of the following variants: blue, BEI 52286 (wild type); pink B.1.1.7; purple, B1.525; and green, P.1. Variant pools were used for B.1.17, B.1.525, and P.1 strains. Curves are displayed for a given dilution in each channel and result interpretation is shown (D).
Results. The lower limit of 95% detection was 2.46 to 2.48 log 10 GE/mL for the three targets (~1-2 GE/reaction). Among 253 nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%), including all samples with Ct values < 30 (220/220) for the N2 target and 18/33 samples with N2 Ct values ≥ 30. Results were confirmed by SARS-CoV-2 genomic sequencing in 50/50 samples (100%). Subsequent addition of the 452R probe did not affect performance for the original targets, and probes for 452Q and 484Q performed similarly to LNAmodified probes.
Conclusion. The Spike SNP assay provides fast, inexpensive and sensitive detection of specific mutations associated with SARS-CoV-2 VOCs, and the assay can be quickly modified to detect new mutations in the receptor binding domain. Similar analytical performance of LNA-modified and unmodified probes presents options for future assay customization that balance the shorter probe length (LNAs) and increased accessibility (unmodified). The Spike SNP assay, if implemented across laboratories offering SARS-CoV-2 testing, could greatly increase capacity for variant detection and surveillance globally.
Disclosures. Colleen S. Kraft, MD, MSc, Rebiotix (Individual(s) Involved: Self): Advisor or Review Panel member Background. In the US, the burden of multidrug resistant bacterial infections, including carbapenem-resistant P. aeruginosa (CRPA) and ESBL-producing Enterobacterales (ESBL-E), is substantial. These resistant pathogens may affect the delivery of timely effective therapy. The aim of this study is to evaluate beta-lactam (BL) susceptibility trends based on the aggregate frequency of CRPA and a combined ESBL-E phenotype (K. pneumoniae (KPn) + E. coli (EC)) observed in critically ill patients with lower respiratory tract infections (LRTI).
Methods. In 2016-2019, ~20 US institutions per year submitted up to 250 gram-negative pathogens as part of the Study for Monitoring Antimicrobial Resistance Trends. A total of 871 PA, 380 KPn, and 336 EC isolates were collected from ICU patients with LRTI. MICs were determined using broth microdilution and interpreted using 2021 CLSI breakpoints. ESBL-E phenotype was defined as: ceftriaxone MIC ≥ 2 mcg/mL. Institutions were stratified into two groups based on frequency of CRPA and combined ESBL-E phenotype: Group 1: CRPA ≤ 15% and ESBL-E ≤ 15%; Group 2: CRPA > 15% and ESBL-E > 15%. Based on CLSI guidance, an empiric antibiotic susceptibility threshold of ≥90% was deemed optimal.
Results. Overall, CRPA and ESBL-E phenotypes were identified in 28.4% and 21.2% of isolates, respectively. Aggregate BL susceptibility in group 1 was above the 90% threshold for cefepime (FEP), piperacillin/tazobactam (TZP), meropenem (MEM), ceftolozane/tazobactam (C/T), and imipenem/relebactam (I/R) (Table 1). However, as frequency of CRPA and ESBL-E exceeded 15%, aggregate BL susceptibility declined to 77.3%, 79.3%, and 86.2% for FEP, TZP, and MEM, respectively. In contrast, C/T and I/R maintain susceptibility above the empiric susceptibility threshold. Table 1. Aggregate susceptibility of P. aeruginosa, E. coli, and K. pneumoniae ICU LRTI isolates stratified by resistance frequency: Best-(Group 1) and worst-case (Group 2) scenarios Conclusion. In ICU patients, exceeding CRPA and combined ESBL-E phenotype frequency of 15% for both classifications, impacts susceptibility to 1 st line BL's resulting in a failure to achieve empiric susceptibility thresholds. This stratification could serve as a decision point for triggering earlier susceptibility testing or modifying empiric therapy recommendations for LRTI to include newer agents pending microbiology results. Disclosures