Multi-site Technical Performance and Concordance of Optical Genome Mapping: Constitutional Postnatal Study for SV, CNV, and Repeat Array Analysis

Background The standard of care (SOC) cytogenetic testing methods, such as chromosomal microarray (CMA) and Fragile-X syndrome (FXS) testing, have been employed for the detection of copy number variations (CNVs), and tandem repeat expansions/contractions that contribute towards a sizable portion of genetic abnormalities in constitutional disorders. However, CMA is unable to detect balanced structural variations (SVs) or determine the precise location or orientation of copy number gains. Karyotyping, albeit with lower resolution, has been used for the detection of balanced SVs. Other molecular methods such as PCR and Southern blotting, either simultaneously or in a tiered fashion have been used for FXS testing, adding time, cost, and complexity to reach an accurate diagnosis in affected individuals. Optical genome mapping (OGM), innovative technology in the cytogenomics arena enables a direct, high-resolution view of ultra-long DNA molecules (>150 kbp), which are then assembled de novo to detect germline SVs ranging from 500 bp insertions and deletions to complex chromosomal rearrangements. The purpose of this study was to evaluate the performance of OGM in comparison to the current SOC methods and assess the intra- and inter-site reproducibility of the OGM technique. We report the largest retrospective dataset to date on OGM performed at five laboratories (multi-site) to assess the robustness, QC performance, and analytical validation (multi-operator, and multi-instrument) in detecting SVs and CNVs associated with constitutional disorders compared to SOC technologies. Methods This multi-center IRB-approved, double-blinded, study includes a total of 331 independent flow cells run (including replicates), representing 202 unique retrospective samples, including but not limited to pediatric-onset neurodevelopmental disorders. This study included affected individuals with either a known genetic abnormality or no known genetic diagnosis. Control samples (n=42) were also included. Briefly, OGM was performed on either peripheral blood samples or cell lines using the Saphyr system. The OGM assay results were compared to the human reference genome (GRCh38) to detect different types of SVs (CNV, insertions, inversions, translocations). A unique coverage-based CNV calling algorithm was also used to complement the SV calls. Analysis of heterozygous SVs was performed to assess the absence of heterozygosity (AOH) regions in the genome. For specific clinical indications of FSHD1 and FXS, the EnFocus FXS and FSHD1 tools were used to generate the region-specific reports. OGM data was analyzed and visualized using Access software (version 1.7), where the SVs were filtered using an OGM specific internal control database. The samples were analyzed by laboratory analysts at each site in a blinded fashion using ACMG guidelines for SV interpretation and further reviewed by expert geneticists to assess concordance with SOC testing results. Results Of the first 331 samples run between five sites, 99.1% of sample runs were completed successfully. Of the 331 datasets, 219 were assessed for concordance by the time of this publication; these were samples that harbored known variants, of which 214/219 were detected by OGM resulting in a concordance of 97.7% compared to SOC testing. 47 samples were also run in intra- and inter-site replicate and showed 100% concordance for pathogenic CNVs and SVs and 100% concordance for pathogenic FMR1 repeat expansions. Conclusion The results from this study demonstrate the potential of OGM as an alternative to existing SOC methods in detecting SVs of clinical significance in constitutional postnatal genetic disorders. The outstanding technical performance of OGM across multiple sites demonstrates the robustness and reproducibility of the OGM technique as a rapid cytogenomics testing tool. Notably, OGM detected all classes of SVs in a single assay, which allows for a faster result in cases with diverse and heterogeneous clinical presentations. OGM demonstrated 100% concordance for pathogenic variants previously identified including FMR1 repeat expansions (full mutation range), pathogenic D4Z4 repeat contractions (FSHD1 cases), aneuploidies, interstitial deletions, interstitial duplications, intragenic deletions, balanced translocations, and inversions. Based on our large dataset and high technical performance we recommend OGM as an alternative to the existing SOC tests for the rapid detection and diagnosis of postnatal constitutional disorders.

665. Clinical and Financial Impact of Next Generation Sequencing (NGS) in addition to Conventional Microbiology Testing in our Urban Referral Health Center

Background Clinical microbiology traditionally relies on culture methodology and serological testing, that have inherent limitations. Newer diagnostic techniques such as Next Generation Sequencing (NGS) have shown promise to improve microbial identification. In select scenarios, we send clinical specimens to reference laboratories for NGS testing in addition to current standard of care (SOC) diagnostics. We wanted to determine how this diagnostic approach has impacted patient care. We also wanted to review the financial burden through cost-benefit analysis for these ‘send-out’ tests. Methods We performed a retrospective chart review of all cases over a 3-year period in which NGS was submitted. Data, including demographics, comorbidities, antimicrobial use, and diagnosis (by SOC and NGS) were gathered. We delineated how often there was concordance or discordance between SOC and NGS. We also obtained information on financial cost (direct and indirect) and turnaround time (TAT) for NGS results. Results A total of 33 clinical specimens from 25 patients were sent for NGS. The majority of specimens comprised joint tissue/fluid, organ tissue and CSF. Concordance occurred between SOC and NGS testing in 75.8% (25/33) of samples; of those, 88% excluded infection. NGS identified a pathogen in 20% (5/25) patients in which concomitant SOC testing was negative. A subsequent change in antimicrobial management occurred in 16% (4/25) of patients. The mean TAT was 14 days and average cost per specimen was &821.52 (range: &573-&1590). Table 1. Pathogens identified by NGS with negative traditional microbiological test results Figure 1. Distribution of specimen site (in %) sent for NGS Conclusion NGS can provide additional diagnostic sensitivity in infectious diseases, which at our institution identified a new pathogen in 20% and a resultant treatment change in 16% of our patients. This testing may also allow physicians to reaffirm the absence of an infection diagnosis. A larger NGS testing population may reveal more significant benefits. While the attributable cost of NGS was substantial, it should be measured against the costs of administration of unnecessary antibiotics, inaccurate diagnosis, and adverse patient outcomes that may result from SOC testing alone. Considering its financial cost and extended TAT, in-house NGS testing may be warranted to facilitate a higher volume of testing. Disclosures All Authors: No reported disclosures

Assessing cost-effectiveness of hepatitis C testing pathways in Georgia using the Hep C Testing Calculator

The cost of testing can be a substantial contributor to hepatitis C virus (HCV) elimination program costs in many low- and middle-income countries such as Georgia, resulting in the need for innovative and cost-effective strategies for testing. Our objective was to investigate the most cost-effective testing pathways for scaling-up HCV testing in Georgia. We developed a Markov-based model with a lifetime horizon that simulates the natural history of HCV, and the cost of detection and treatment of HCV. We then created an interactive online tool that uses results from the Markov-based model to evaluate the cost-effectiveness of different HCV testing pathways. We compared the current standard-of-care (SoC) testing pathway and four innovative testing pathways for Georgia. The SoC testing was cost-saving compared to no testing, but all four new HCV testing pathways further increased QALYs and decreased costs. The pathway with the highest patient follow-up, due to on-site testing, resulted in the highest discounted QALYs (123 QALY more than the SoC) and lowest costs ($127,052 less than the SoC) per 10,000 persons screened. The current testing algorithm in Georgia can be replaced with a new pathway that is more effective while being cost-saving.

High-Density Poplar SRC Accumulates More Soil Organic Carbon Than Very-High-Density SRC

Short rotation coppice (SRC) systems play an important role in producing renewable energy and contributing to soil organic carbon storage while providing potential mitigation for climate change. Our chronosequence study assessed the influence of 6 years of high-density (H) and very-high-density (VH) SRCs on soil organic carbon (SOC) accumulation in an alluvial area of Piedmont (Italy) by investigating the effects of agronomic management (fertilization, irrigation, fertilization and irrigation, no treatment) using a spatial survey approach. A first sampling was performed at 40 points to characterize the variability of the initial SOC and other soil properties; 6 years after, a total of 80 samplings were carried out to verify changes in SOC. A mixed effect model procedure was used to evaluate the impact of 6 years of SRC and agronomic management on SOC, testing for autocorrelation among the model residuals. The results showed: (i) a higher accumulation potential of H-SRC (0.076 kg m−2 year−1) compared to VH-SRC (0.037 kg m−2 year−1); (ii) a significant positive influence of the initial SOC value and of fertilization associated with irrigation on the SOC sequestration; (iii) the importance of considering spatial variability at sites with high particle-size diversity in evaluating the SOC changes.

285. Evaluation of the ePlex® Blood Culture Identification (BCID) Panels for Gram-positive/Gram-negative bacteria and yeasts

Background Multiple methods used for blood culture identification create inconsistent to reporting of critical results. Study aim was to evaluate performance characteristics of the ePlex BCID panels compared to current standard of care (SOC) methods used in our lab. Methods Identification sensitivity and specificity were assessed across all targets detected by the ePlex as well as time to final identification (from time of bottle positive Gram stain) between ePlex and SOC testing. SOC included Xpert MRSA/SA or latex agglutination for Gram-positive cocci in clusters (GPCC), Vitek MS + Accelerate Pheno for Gram-negative rods (GNRs), serotyping or optochin disk ± Vitek MS for Gram-positive cocci in chains (GPC chains), Vitek MS or Vitek-2 for Gram-positive rods (GPR), and PNA-FISH or Vitek MS for yeasts. Results 313 unique prospective blood culture specimens were tested with ePlex BCID panels during a 3-month period (January-March 2020). The positive percent agreement was 100% for GNR (n= 98), S. aureus (n= 42), coagulase-negative staphylococci (n= 38), Group A Streptococcus (n= 3), Group B Streptococcus (n= 5), S. pneumoniae (n= 10), GPR (n= 21), and yeasts (n= 20). There was 1 false negative, (S.mutans) which should have been detected. The negative percent agreement was 100% across all targets except for 1 false positive Corynebacterium spp. In total, 6.7% of blood cultures had an off-panel organism which ePlex did not detect. The median time to final identification was 3 (2 – 4) hrs. for ePlex and calculated for all other SOC methods. Compared to SOC molecular methods, the ePlex reduced time to identification 0.5 h compared to Xpert MRSA/SA, 6.7 h compared to Accelerate Pheno for GNR (but Accelerate Pheno provides susceptibilities), and 3 h compared to PNA-FISH for yeasts (p< 0.05). ePlex compared to non-molecular techniques (MALDI-TOF), SOC for Streptococcus spp. and Enteroococcus spp., the time to final identification was reduced by 24 – 30 hours (p< 0.05). Workflow chart comparison eplex to SOC Time to results eplex vs SOC Conclusion The ePlex BCID system provided highly accurate identification results for GP and GN bacteria as well as for yeasts. Our evaluation showed that this system significantly reduced time to final identification compared to SOC testing methods. Disclosures Kimberle Chapin, MD, genmark (Scientific Research Study Investigator) Giannoula Tansarli, MD, GenMark (Grant/Research Support)

Randomized Trial Evaluating Clinical Impact of RAPid IDentification and Susceptibility Testing for Gram-negative Bacteremia: RAPIDS-GN

Background Rapid blood culture diagnostics are of unclear benefit for patients with gram-negative bacilli (GNB) bloodstream infections (BSIs). We conducted a multicenter, randomized, controlled trial comparing outcomes of patients with GNB BSIs who had blood culture testing with standard-of-care (SOC) culture and antimicrobial susceptibility testing (AST) vs rapid organism identification (ID) and phenotypic AST using the Accelerate Pheno System (RAPID). Methods Patients with positive blood cultures with Gram stains showing GNB were randomized to SOC testing with antimicrobial stewardship (AS) review or RAPID with AS. The primary outcome was time to first antibiotic modification within 72 hours of randomization. Results Of 500 randomized patients, 448 were included (226 SOC, 222 RAPID). Mean (standard deviation) time to results was faster for RAPID than SOC for organism ID (2.7 [1.2] vs 11.7 [10.5] hours; P < .001) and AST (13.5 [56] vs 44.9 [12.1] hours; P < .001). Median (interquartile range [IQR]) time to first antibiotic modification was faster in the RAPID arm vs the SOC arm for overall antibiotics (8.6 [2.6–27.6] vs 14.9 [3.3–41.1] hours; P = .02) and gram-negative antibiotics (17.3 [4.9–72] vs 42.1 [10.1–72] hours; P < .001). Median (IQR) time to antibiotic escalation was faster in the RAPID arm vs the SOC arm for antimicrobial-resistant BSIs (18.4 [5.8–72] vs 61.7 [30.4–72] hours; P = .01). There were no differences between the arms in patient outcomes. Conclusions Rapid organism ID and phenotypic AST led to faster changes in antibiotic therapy for gram-negative BSIs. Clinical Trials Registration NCT03218397.

640. Randomized Clinical Trial Evaluating Clinical Impact of RAPid IDentification and Antimicrobial Susceptibility Testing for Gram-Negative Bacteremia (RAPIDS-GN)

Background Rapid blood culture diagnostics increase cost and have unclear benefit for patients with Gram-negative bacilli (GNB) bloodstream infections (BSIs). We conducted a multicenter, prospective randomized controlled trial (RAPIDS-GN), comparing outcomes of patients with GNB BSI who had blood culture testing with standard of care (SOC) culture and antibiotic susceptibility testing (AST) vs. rapid organism identification (ID) and phenotypic AST using the Accelerate Pheno System (AXDX). Methods Subjects with blood culture Gram stain showing GNB were randomized to receive SOC testing with antimicrobial stewardship review (AS) or AXDX plus SOC testing with AS, at two academic medical centers between October 2017 and October 2018. SOC testing included rapid MALDI-TOF mass spectrometry ID and agar dilution or broth microdilution AST. In a modified intention to treat analysis, subjects were excluded if: Gram stain was erroneous, culture was positive during off-hours, blood culture in the prior week had GNB, they were deceased/on comfort care, or admitted to a nonparticipating hospital. The primary outcome was time to first antibiotic modification within 72 hours after randomization. Subjects without antibiotic modifications were assigned a time of 72 hours. No censoring was observed. T-tests and Wilcoxon rank-sum tests were used for statistical analyses. Results Of 500 randomized subjects, 448 were included (226 SOC, 222 AXDX). Groups did not differ in baseline characteristics (Table 1). Median (IQR) hours to first antibiotic modification was faster in the AXDX vs. SOC group [8.6 (2.6, 27.6) vs. 14.9 (3.3, 41.1)], P = 0.02 (Figure 1). Median (IQR) hours to first Gram-negative antibiotic modification (including escalation and de-escalation) was faster in the AXDX than SOC group [17.4 (4.9, 72) vs. 42.1 (10.1, 72)], P < 0.001 (Figure 2). Groups did not differ in clinical outcomes (Table 2). Mean (S.D.) time to results was faster for AXDX than SOC for organism ID [2.7 (1.2) h vs. 15.6 (20.3) h, P < 0.001] and AST [13 (55.7) h vs. 54.6 (45.5) h, P < 0.001]. Conclusion In the largest trial to evaluate the clinical impact of a blood culture diagnostic for GNB BSI, we found that rapid organism ID and phenotypic AST led to faster changes in antibiotic therapy for Gram-negative bacteremia. Disclosures Ritu Banerjee, MD, PhD, Accelerate Diagnostics: Grant/Research Support; BioFire: Research Grant; Biomerieux: Research Grant; Roche: Research Grant Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.