Abstract Background In response to the COVID-19 pandemic, bioMérieux has incorporated assays to detect SARS-CoV-2 into the BIOFIRE® Respiratory 2.1 (RP2.1) Panel, the BIOFIRE® SPOTFIRE® Respiratory (R) Panel and the BIOFIRE® SPOTFIRE® Respiratory (R) Panel Mini. All panels use two assays for SARS-CoV-2 detection, each targeting a different gene. Positive detection in only one of the two assays is required for a positive SARS-CoV-2 result. As new variants of SARS-CoV-2 emerge, in silico analysis remains crucial to ensure that both SARS-CoV-2 assays are reactive to circulating strains. Methods Sequence data (https://gisaid.org/) for SARS-CoV-2 variants deemed significant by the WHO and UKHSA are analyzed monthly. Assay primer regions are assessed for mismatches using Geneious Prime® as well as proprietary software tools. When primer mismatches with potential to affect the sensitivity of SARS-CoV-2 detection are identified (mismatches occurring in the 3’ half of a primer in both assays), and meet the testing criteria, the BIOFIRE RP2.1 Panel is used to empirically evaluate the impact by comparing synthetic templates with mismatches of concern to synthetic template without mismatches. Results As of March 21, 2023, nearly 1,000 variants and 12,744,136 sequences have been analyzed. Of these, only 481 sequences (0.0038%) contain paired mismatches of concern. Wet testing showed < 10x lower projected sensitivity for 288 (72 unique) sequences, 10-100x lower sensitivity for 16 (10 unique) sequences, and only 1 sequence had 100-1000x lower projected sensitivity. Lineage defining mutations were seen within a SARS-CoV-2 assay primer region in 27 variants. However, most lack mutations in the second assay, indicating no major risk for SARS-CoV-2 detection. Wet testing shows that the inclusion of two SARS-CoV-2 assays in the panels helps to mitigate the effect of mismatches of concern. Conclusion Based on the comprehensive in silico analysis of available sequences and variants, BIOFIRE RP2.1 Panel, SPOTFIRE R Panel and SPOTFIRE R Panel Mini continue to function as intended with >99.99% detection of SARS-CoV-2 sequences. Disclosures Eleanor K. Horrocks, bioMerieux: employee|bioMerieux: Stocks/Bonds Toma Todorov, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Alexandra Debernardi, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Usha Spaulding, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Tanner Robinson, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Jeremiah Antosch, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Zhenmei Lu, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Matthew Jones, MS, bioMerieux: employee|bioMerieux: Stocks/Bonds Joann Cloud, PhD, bioMerieux: employee|bioMerieux: Stocks/Bonds

Background Antimicrobial resistance (AMR) surveillance is critical in informing strategies for infection control in slowing the spread of resistant organisms and for antimicrobial stewardship in the care of patients. However, significant challenges exist in timely and comprehensive AMR surveillance. Methods Using BioFire Pneumonia and Blood Culture 2 Panels data from BioFire Syndromic Trends (Trend), a cloud-based population surveillance network, we described the detection rate of AMR among a US cohort. Data were included from 2019 to 2021 for Gram-positive and -negative organisms and their related AMR genomic-resistant determinants as well as for detections of Candida auris. Regional and between panel AMR detection rate differences were compared. In addition, AMR codetections and detection rate per organism were evaluated for Gram-negative organisms. Results A total of 26 912 tests were performed, primarily in the Midwest. Overall, AMR detection rate was highest in the South and more common for respiratory specimens than blood. methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus detection rates were 34.9% and 15.9%, respectively, whereas AMR for Gram-negative organisms was lower with 7.0% CTX-M and 2.9% carbapenemases. In addition, 10 mcr-1 and 4 C auris detections were observed. For Gram-negative organisms, Klebsiella pneumoniae and Escherichia coli were most likely to be detected with an AMR gene, and of Gram-negative organisms, K pneumoniae was most often associated with 2 or more AMR genes. Conclusions Our study provides important in-depth evaluation of the epidemiology of AMR among respiratory and blood specimens for Gram-positive and -negative organism in the United States. The Trend surveillance network allows for near real-time surveillance of AMR.

We evaluated the performance of the BioFire® Respiratory Panel 2.1 (RP2.1) in the detection of SARS CoV-2 in comparison against three other SARS CoV-2 EUA assays. In these studies, the RP2.1 panel had 98 % positive percent agreement (48/49) and 100 % negative percent agreement (49/49). Since 30 % of nasopharyngeal swab specimens have a SARS CoV-2 Ct >30 and thus detection of virus in low titers is clinically relevant, a sample with a high titer was diluted and each 10 fold dilution was tested in triplicate and compared against 6 other EUA approved SARS CoV-2 assays. These data suggested that the BioFire® RP2.1 panel, along with four other SARS CoV-2 assays (Roche cobas, Cepheid Xpert Xpress, BioFire® Defense COVID19, and NECoV19), consistently detected viral RNA at the 10-7 dilution. Overall, these studies suggest that the BioFire® RP2.1 assay can be used to detect acute cases of SARS CoV2 in addition to patients with low viral titer later in disease presentation.

Abstract Background The US Food and Drug Administration (FDA) has granted Emergency Use Authorization (EUA) for multiple PCR-based tests to aid in the diagnosis and containment of COVID-19. A vast majority of these tests detect only SARS-CoV-2 which causes symptoms similar to those caused by other respiratory pathogens. Hence, other etiologies or co-infections requiring a different therapy may be missed. The prototype BioFire® Respiratory Panel 2.1 (RP2.1) continues the syndromic approach of the FDA-cleared BioFire® Respiratory Panel 2 (RP2), to provide the ability to simultaneously detect 22 common respiratory pathogens, including SARS-CoV-2, from nasopharyngeal swab (NPS) specimens. The goal of this study was to rapidly develop a RP2.1 prototype that contains high-performing SARS-CoV-2 assays and maintains the performance of assays retained from RP2. Methods Twelve assays designed for four SARS-CoV-2 genes were tested for compatibility with the RP2 assays and conditions. All retained RP2 assays were evaluated to verify established RP2 performance. The sensitivity of novel SARS-CoV-2 assays was estimated with nucleic acids at BioFire and contrived live virus NPS samples at MRIGlobal. Primer homology of SARS-CoV-2 assays to > 15,000 SARS-CoV-2 genomes from accessible databases was assessed for in silico inclusivity Results A prototype multiplexed PCR panel containing assays for 22 pathogens was developed in a 5-week period. Of the 12 SARS-CoV-2 assays, 7 were compatible with the RP2 conditions; 2 were selected for the prototype. No false positive results due to cross-reactivity with unintended analytes or non-specific amplification in negative samples were observed for any assays. All retained RP2 assays were detected at or near their established LoD. The SARS-CoV-2 LoD was estimated at 103 -102 genomes/mL with both nucleic acid and live virus spiked into NPS. Together, the assays are 100% inclusive for all 15,370 complete SARS-CoV-2 genomes assessed in silico for reactivity. Conclusion The results of this study indicate a strong potential for RP2.1 to serve as a sensitive comprehensive syndromic option to aid in the diagnosis of COVID-19 as well as respiratory syndromes caused by other pathogens, including co-infections. This study was performed with a test not cleared for diagnostic use. Disclosures All Authors: No reported disclosures

Abstract Background The BioFire® FilmArray® Blood Culture Identification 2 (BCID2) Panel is a diagnostic test that provides results for 26 bacterial, 7 fungal pathogens and 10 antimicrobial resistance (AMR) genes from positive blood culture (PBC) specimens in about an hour. The BCID2 Panel builds upon the existing BCID Panel with several additional assays that include Candida auris and an expanded AMR gene menu that provides methicillin-resistant Staphylococcus aureus (MRSA) results plus detection for mcr-1, carbapenem resistance, and ESBL. Here, we summarize studies conducted to establish clinical performance using an Investigational Use Only version of the BCID2 Panel. Methods Three studies were performed. The first involves prospective collection and testing of an expected ~1,000 residual PBCs at 7 US and 2 EU sites, which began in October 2018 and will conclude in June 2019. BCID2 Panel performance is compared with reference methods of microbial culture as well as PCR/sequencing for AMR genes. In addition, BCID2 Panel MRSA results are compared with the FDA-cleared Xpert MRSA/SA BC system (Cepheid, Inc). Relevant bacterial isolates recovered from PBCs are also evaluated by various phenotypic antimicrobial susceptibility testing (AST) methods. The prospective evaluation is supplemented with a second study that involves testing of ~300 pre-selected, archived PBCs containing rare organisms. The third study includes over 500 seeded blood cultures containing very rare organisms with an evaluation of co-spiked samples. Results With over 1,200 samples tested to date (out of an anticipated 1,800 total), the BCID2 Panel has demonstrated an overall sensitivity of >98% and specificity of >99% for identification of microorganisms compared with culture. Concordance between the BCID2 Panel and the Xpert MRSA/SA BC test is >99% for identification of MRSA. Evaluation of BCID2 Panel AMR gene detection relative to AST and PCR is ongoing. Conclusion The FilmArray® BCID2 Panel appears to be a sensitive, specific, and robust test for rapid detection of microorganisms and MRSA in PBCs. With the use of this comprehensive test, improved antimicrobial stewardship is anticipated. Disclosures All authors: No reported disclosures

Rapid diagnosis of causative agents of bloodstream infections improves patient outcomes and antibiotic stewardship. BioFire Diagnostics, LLC, is developing the BioFire® Blood Culture Identification 2 (BCID2) Panel, increasing the coverage of the BioFire® FilmArray® Blood Culture Identification (BCID) Panel for key pathogens and antimicrobial resistance (AMR) markers in aerobic and anaerobic positive blood culture (PBC). This revision expands the menu from 27 to 42 targets, with 26 bacterial (14 revised, six new) and seven fungal analytes (two revised, three new), as well as nine AMR markers (one revised, six new). Notable additions include the anaerobe Bacteroides fragilis, the emerging fungus Candida auris, and the mobile colistin resistance gene, mcr-1. This study details the reactivity and specificity of an RUO BioFire BCID2 panel. The prototype was tested with fungal and bacterial isolates, some carrying AMR markers, at two sites by multiple operators. Reactivity was assessed at 106 CFU/mL for 301 analytes, and specificity at 108 CFU/mL for 43 on-panel and 93 off-panel strains. Evaluation included multiple strains for species level and AMR marker assays, as well as multiple species for family/genus level assays. Concordance with standard of care (SoC) results was examined for 126 archived PBC. Testing against 136 on-panel organisms, phylogenetic-neighbors, and normal cutaneous flora, showed 100% specificity for 41/42 targets. Reactivity was confirmed for 346/351 target analytes, and comprehensive detection was observed for the revised family-level Enteric assay (90/90) and genus-level Staphylococcus spp. (51/51), Streptococcus spp. (17/17), and Candida spp. (67/71) assays. The prototype showed excellent sensitivity (97.1%) and specificity (99.7%) compared with SoC with archived PBC. Performance of this RUO BioFire BCID2 Panel indicates that many key pathogens implicated in bloodstream infections can be identified with high sensitivity and specificity, and highlights the utility of the expanded menu to provide actionable information. Future panel versions will address observed deficiencies. RUO products used in this study have not been evaluated by the FDA or other regulatory agencies for In Vitro Diagnostic use. J. Antosch, BioFire Diagnostics, LLC: Employee, Salary. U. Spaulding, BioFire Diagnostics, LLC: Employee, Salary. J. Stone, BioFire Diagnostics, LLC: Employee, Salary. C. Later, BioFire Diagnostics, LLC: Employee, Salary. K. Koch, BioFire Diagnostics, LLC: Employee, Salary. I. Kavetska, BioFire Diagnostics, LLC: Employee, Salary. H. Ton, BioFire Diagnostics, LLC: Employee, Salary. C. Alberti-Segui, bioMérieux: Employee, Salary. A. Grange, bioMereiux, Inc.: Employee, Salary. C. Dubost, bioMérieux: Employee, Salary. M. Rogatcheva, BioFire: Employee, Salary.

Rapid identification of causative agents from positive blood culture (PBC) can aid earlier targeted therapy, as well as reduce mortality, length of stay, and costs associated with systemic infections. The BioFire® Blood Culture Identification 2 (BCID2) Panel being developed by BioFire Diagnostics, LLC, aims to maintain or improve the performance of the BioFire® FilmArray® Blood Culture Identification (BCID) Panel with updated and novel assays (15 new analytes: six antimicrobial resistance (AMR), six bacterial, and three fungal analytes). The performance of an RUO BioFire BCID2 Panel during a prospective pilot study is compared with standard of care (SoC), as well as independent PCR comparator assay (compPCR) results. Two pilot sites enrolled de-identified PBC (<24 hours post-positivity) for which clinician-ordered SoC tests had been performed. Aliquots of residual PBC and isolates were frozen for compPCR testing of AMR markers and discrepancy resolution. 100 aerobic PBC (A-PBC) and 85 anaerobic PBC (AN-PBC) were tested with the BioFire BCID2 Panel; 70 A-PBCs and 56 AN-PBCs were concurrently tested on BioFire BCID Panel. Also, isolates from PBCs positive for AMR markers were tested using compPCR. The BioFire BCID2 Panel results matched SoC results in 176/177 detections from 100 A-PBC, and in 167/168 detections from 85 AN-PBC. Both BioFire panels detected Candida glabrata and Candida parapsilosis from an A-PBC with only C. parapsilosis SOC result; interestingly, C. glabrata was detected by SoC in the paired AN-PBC. False-positive Bacteroides fragilis detection in an AN-PBC was resolved favorably by compPCR. Two patient samples, positive in both A-PBC and AN-PBC by SoC, were not detected by either the Staphylococcus epidermidis or the Staphylococcus spp. assays on the BioFire BCID2 Panel. All 26 AMR marker detections in both types of PBC were concordant with either SoC or compPCR results. With an expanded menu, >99% specificity, and >97% sensitivity, the BioFire BCID2 Panel is expected to provide rapid and accurate results for key pathogens associated with systemic infections, as well as important AMR markers. RUO products used in this study have not been evaluated by the FDA or other regulatory agencies for In Vitro Diagnostic use. U. Spaulding, BioFire Diagnostics, LLC: Employee, Salary. J. Stone, BioFire Diagnostics, LLC: Employee, Salary. K. Koch, BioFire Diagnostics, LLC: Employee, Salary. J. Antosch, BioFire Diagnostics, LLC: Employee, Salary. M. Jones, BioFire Diagnostics, LLC: Employee, Salary. Z. Lu, BioFire Diagnostics, LLC: Employee, Salary. T. Todorov, BioFire Diagnostics, LLC: Employee, Salary. S. Kerr, BioFire Diagnostics, LLC: Employee, Salary. K. Holmberg, BioFire Diagnostics, LLC: Employee, Salary. M. Rogatcheva, BioFire: Employee, Salary.