Lab Matters Summer 2019 - Page 72

APHL 2019 POSTER ABSTRACTS Use of Galileo™ AMR for the Automated Annotation of Antimicrobial and Antiviral Resistance Genes in GenBank Evaluation of Leading Commercial Assays for the Early Diagnosis of Lyme Disease in US Populations G. Tsafnat, A. Watts and T. Watson, Arc Bio LLC J. Hahm, J. Breneman, J. Liu, L. Rabkina, W. Zheng, S. Zhou, R. Walker and R. Kaul, Bio-Rad laboratories Introduction: Galileo™ AMR is a cutting-edge software system for the comprehensive annotation of antimicrobial resistance (AMR) genes and associated elements that allow their mobilization. Galileo™ AMR supersedes the Multiple Antimicrobial Resistance Annotator [1] developed at the New South Wales Univ and Westmead Hosp, Sydney Australia and adds important features like high-throughput (HT) analysis. Galileo™ AMRlets users upload bacterial sequences. In a few minutes, Galileo™ AMR accurately annotates all known Gram- negative resistance genes and associated mobile elements, including resistance gene cassettes, transposons etc. The High- Throughput (HT) version of Galileo AMR™ can additionally annotate SCCMec regions in Staphylococcus aureus and antiviral resistance (AVR) markers in human cytomegalovirus (CMV). Results: Over 99% of the elements in the Galileo™ AMR database were found at least once in GenBank. Gram-negatives: Galileo™ AMR found over 17,282 sequences containing resistance genes and/or associated mobile elements. Approximately half the sequences (n=8,948;51.8%) contained an insertion sequence and over a quarter (n=4,737;27.4%) included a transposon. Resistance genes were found for all classes of antibiotics with beta-lactamase genes being the most common. S. aureus: Approximately 1.6 million S. aureus sequences were identified. Less than 6% showed evidence of the presence of SCCMec. CMV: All CMV sequences of containing the genes UL54 (n=399) and UL97 (n=630) were annotated. All genes included at least one AVR markers (range [1:56] each gene). Conclusion: GenBank provided a rich catalogue of AMR sequences in the studied organisms. Although the frequencies of exemplars do not reflect frequencies in the real world, the database represents drug resistance in the studied organisms. These results demonstrate the ability of Galileo™ AMR to detect antimicrobial resistance markers. Powered by multiple state-of-the-art databases and a next generation genomic detection engine, Galileo™ AMR is designed to empower those who perform AMR sequence research and analysis. Disclaimer: Galileo™ is a trademark of Arc Bio, LLC. For Research Use Only. Galileo™ AMR HT is currently under development References: 1. Partridge SR, Tsafnat G, et al. FEMS Microbiol Rev. 2009 Jul;33(4):757-84. Presenter: Guy Tsafnat, Arc Bio LLC, Menlo Park, CA 70 LAB MATTERS Summer 2019 Methods: An externally-validated Lyme ‘Premarketing’ panel from the Centers for Disease Control consisting of 280 well-characterized Lyme disease and non-disease controls was used to evaluate the sensitivity and specificity of the BioPlex 2200 Lyme Total Assay. In addition, 105 vendor provided Lyme disease positive samples and 200 endemic and non-endemic hospital normals were evaluated during the course of this investigation. Results obtained were compared to LIAISON Borrelia burgdorferi (DiaSorin, Inc.); Vidas II IgM and IgG (BioMerieux, Inc.); Lyme PepC10/VlsE1 IgM/IgG (Zeus, Inc.); Lyme C6 B. burgdorferi ELISA (Immunetics, Inc.). Positive or equivocal samples were subsequently tested by both IgM and IgG Western blot (MarDx, Inc.) assay. Results: Using the CDC Lyme Premarketing panel, overall clinical sensitivities of 91.1% (82/90), 84.4% (76/90), 88.9% (80/90), and 85.6% (77/90) with specificities of 96.8% (184/190), 97.9% (186/190), 89.5% (170/190), and 84.7% (161/190) were respectively measured for the BioPlex 2200 Lyme Total assay, Immunetics C6, Zeus PepC10/VlsE1, and BioMerieux VIDAS Lyme II. Notably, sensitivities of 84.6% (33/39), 71.8% (28/39), 82.1% (32/39), and 79.5% (31/39) were respectively observed among an early infection category of “Acute” samples. Of the vendor-tested Lyme positive samples, positive agreements of 68.6% (72/105), 52.4% (55/105), 61.9% (65/105), 64.8% (68/105), and 70.5% (74/105) were observed for the BioPlex 2200 Lyme Total, DiaSorin LIAISON, Immunetics C6, Zeus PepC10/VlsE1, and BioMerieux VIDAS Lyme II, respectively. Additionally, positive agreements of 69.4% (50/72), 55.6% (40/72), 58.3% (42/72), 62.5% (45/72), and 73.6% (53/72) were respectively observed for the acute group of vendor samples. In parallel, non-endemic prevalence values of 3%, 4%, 4%, 7%, 10% and endemic values of 5%, 3%, 8%, 6%, and 5% were observed for the BioPlex 2200 Lyme Total, DiaSorin LIAISON, Immunetics C6, Zeus PepC10/VlsE1, and BioMerieux VIDAS Lyme II. Conclusion: Overall, the BioPlex 2200 Lyme Total assay exhibited an unmatched combination of both relatively high sensitivity and specificity that was not observed among commercially available Lyme kits. Importantly, the improved sensitivity of the BioPlex 2200 Lyme assay was primarily from early Lyme disease samples observed in both the CDC Premarketing panel as well as vendor- supplied samples. Presenter: Johnnie Hahm, Bio-Rad Laboratories, Hercules, CA, PublicHealthLabs @APHL Methods: Galileo AMR™ HT was used to annotate all of GenBank. The nucleotide database from NCBI was downloaded (Jan 28, 2018). BLAST was performed for all sequences in the Galileo AMR™ HT database except for very common elements such as IS1. The flat file for accessions that had one or more matches was subsequently downloaded[1] and species names extracted. All synthetic, eukaryotic and non-CMV viruses were removed. The sequences of the remaining ~22,000 sequences were converted to FASTA format and annotated using Galileo™ AMR. Annotations in the flat file were ignored. Background: Lyme disease is a growing public health concern as the number of US counties reporting its incidence has increased substantially over the past decade. Early detection of Lyme disease is of high clinical value for treatment efficacy and the prevention of more serious, long-term health consequences. Bio-Rad Laboratories has developed a multiplexed Lyme assay that detects both IgM and IgG antibodies using a unique combination of synthetic peptides and recombinant protein antigens for an improved early detection without sacrificing high specificity.