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Introduction/Objective COVID-19 is caused by the SARS-CoV-2 coronavirus that has led to a worldwide pandemic with an unprecedented need for fast and accurate testing under FDA Emergency Use Authorizations (EUA). Despite the promise of the Alinity-m as an upgrade from the Abbott m2000 for optimization of high throughput testing and random access for laboratory workflow, validation studies comparing Alinity-m to already used Abbott m2000 are sparse in the literature, particularly for the veteran population. Methods/Case Report The validation study for the Alinity m (Chicago IL) was performed in three parts 1) Method to method sample/patient correlation study, 2) precision study (at 250 virus copies/mL, 500 virus copies/mL and 1000 virus copies/mL versus 4 negatives), and 3) verification and confirmation of the accuracy of the assay at the lower limit of detection (LOD). For the validation study. The patient specimens were used side by side for both assays. The results from the Abbott m2000 (Chicago IL), which was already established in our lab, were used as a reference for validation. Results (if a Case Study enter NA) The validation with a method to method correlation included 157 valid specimens from which concordant results were obtained for all 157 specimens on both the Alinity-m and Abbott m2000. The precision or reproducibility of the Alinity-m was verified at all concentrations. The limit of detection verification on diluted samples determined the limit of detection to be 20 virus copies/mL (>95% of dilution samples agreed with positive results at this level), which confirmed even below the manufacturer provided LOD of 100 virus copies/mL. Conclusion Alinity m was validated for clinical use with study demonstrating that it is 1) equivalent in the method to method correlation, precision, and LOD determination. 2)The validation of Alinity m, holds great promise with its optimized throughput for testing of large numbers of specimens with less technician handling and random access, is a valuable addition to the literature of test validations under the FDA EUA. 3)The validation of tests under the FDA EUA is unprecedented and provides an important way to improve patient care during this extraordinary pandemic.

Introduction/Objective Our cardiac intensive care unit (CICU) asked the laboratory to provide plasma hemoglobin testing to monitor effects of ventricular-assist devices (VAD). Roche hemolysis index (HI) has been reported to be suitable for this purpose. Use of HI as a reportable test, however, must be validated in-house as a laboratory- developed test (LDT), according to CLIA regulations. We describe results of our evaluation, and caveats for intended use. Methods/Case Report Concentrated hemolysate was produced by osmotic lysis of packed red blood cells in water. Hemolysis in plasma was by dilution of concentrated hemolysate. Verification of HI as a measure of hemoglobin (mg/dL) used the Sigma-Aldrich spectrophotometric hemoglobin assay as a gold standard. Linearity, linear range, sensitivity, were investigated by dilution measurements; interferences by admixture experiments; reproducibility (precision, intra- and inter-assay) in each case by replicate measurements (n = 20). Results (if a Case Study enter NA) HI was confirmed to correspond to hemoglobin in mg/dL. Linearity was between 2-1000 mg/dL (r2 >0.99). Intra-assay precisions were Conclusion Analytically, HI exhibited acceptable performance for reporting of plasma hemoglobin. Deployment would require establishment of quality control (QC) and proficiency testing procedures for HI. Clinically, a caveat for use is that HI cannot distinguish between hemolysis in circulation vs. that due to sample collection. For the CICU, HI >10 mg/dL is characteristic of more than 20% of samples drawn in general, irrespective of use of VAD. Temporal patterns of HI must therefore be carefully evaluated in parallel with haptoglobin measurements to assist in VAD management.

Radial immunodiffusion (RID) is a classic methodology for antigen quantification that relies on the development of a distinct precipitin ring from precipitated antigen-antibody complex. As the precipitin ring grows, the precipitate at the inner edge of the ring constantly dissolves due to excess antigen flooding from the point of application, while new precipitate forms at the leading edge of the ring. RID plates with anti-human alpha-1-antitrypsin are prepared in our lab to measure fecal alpha-1 antitrypsin (A1AT) to aid in the diagnosis and monitoring of Protein Losing Enteropathy (PLE). The procedure has routinely produced precipitin rings with small radii and distinct edges after incubating at room temperature for 48 hours. Unexpectedly, a fecal specimen from a patient produced an extremely weak and large precipitin ring that could have been easily overlooked. Dilution studies confirmed a highly elevated A1AT level of 67 mg/g dry stool. The very weak and large precipitin ring was reproduced with a spiked specimen with similar A1AT concentration and kept expanding for several days until a distinct ring was formed eventually. Our data highlights a rare example of high-dose hook effect in RID and calls for meticulous attention and caution when reading and interpreting gel-based immunoassays with unexpected markedly elevated results to avoid additional confirmatory testing. In these cases, we recommend repeat testing with diluted specimens.

Background Bacteriophage are viruses that infect and parasitize bacteria. In the pre-antibiotic era, phage therapy was one of the few tools available against bacterial infection. After penicillin was discovered, phage therapy fell into disfavor in Western medicine, and its use was mostly limited to medical centers in the Soviet bloc. With current issues on multi-drug resistance, especially of Gram-negative bacteria, there is interest in revisiting phage therapy for use in medical and veterinary practice. This study focuses on the biofilm-forming enteroinvasive Escherichia coli (BF-EIEC) serotype O96:H19. Strain BF-EIEC 52.1 was isolated in a pediatric case–control diarrheal study in Bucaramanga, Colombia, in 2013–2014. This strain is genetically similar to a foodborne outbreak strain isolated in Italy in 2012, and in sporadic outbreaks in the UK, Spain, and Uruguay. BF-EIEC 52.1 produces biofilms in vitro, similarly to enteroaggregative E. coli, and yet invades host cells, similar to enteroinvasive E. coli and Salmonella enterica (J. Iqbal, O. Gómez-Duarte et al., manuscript in preparation), making this a potential emergent pathogen of concern. We hypothesized that lytic bacteriophage derived from wastewater sources are active against BF-EIEC 52.1. Methods Environmental wastewater samples were retrieved from the Amherst, New York, wastewater treatment facility. These were filtered against a 0.2 μm membrane and stored at 4°C. An attempt to isolate phage was done by direct plating over E. coli BF-EIEC 52.1 on solid Luria–Bertani (LB) agar. A second strategy involved enrichment for lytic phage by incubating sample water with BF-EIEC 52.1 in LB broth. Cloning was accomplished by re-filtering the preparation (to remove bacteria), then followed by serial limiting dilution of cell-free filtrate in liquid bacterial culture. Characterization of phage preparations included a demonstration of differential lytic activity against BF-EIEC 52.1 and two unrelated laboratory E. coli strains, DH5α and HB101, in both solid agar and liquid medium. Results Isolation of phage by direct plating was possible, but cloning phages proved difficult. With the alternative strategy, four of six wastewater specimens incubated with BF-EIEC 52.1 in liquid LB showed decreased turbidity compared with sterile water control. All positive specimens proved to have demonstrable lytic activity against host E. coli by plaque formation in solid medium, and in limiting dilution in liquid medium. Thirty-two putative phage preparations were cloned by repeated limiting dilution from an initial 16 independent wells. Of these, 27 preparations showed visible plaques against BF-EIEC 52.1 in solid agar, while 29 and 23 preparations showed plaques against HB101 and DH5α, respectively. All phage preparations inhibited the growth of low-inoculum BF-EIEC 52.1 in liquid medium for >6 hours. Six virus preparations inhibited growth for >72 hours. Conclusions Bacteriophage active against strain BF-EIEC 52.1 were isolated from urban wastewater samples, showing a range of activity against the parental host, as well as unrelated E. coli strains. Further study is needed to demonstrate activity in alternative clinical settings, such as in biofilm-associated infections or in animal models.

Background Enterococcus faecalis have been regarded as one of the leading causes of the nosocomial infections worldwide. Tigecycline (TGC) is considered as a choice of last resort for the treatment of infections caused by multidrug-resistant E. faecalis, however, the emergence of TGC non-susceptibility has posted the therapeutic challenge. Non-susceptibility in clinical strains could be due to resistance (MIC >0.5 mg/l) or heteroresistance. Therefore, this study aimed to understand the underlying molecular mechanisms of TGC resistance and heteroresistance in E. faecalis. Methods In vitro induction experiments were carried out under TGC pressure with two TGC- sensitive E. faecalis strains. Heteroresistance was evaluated by population analysis profiling (PAP) in 270 clinical TGC- sensitive E. faecalis strains. TGC susceptibility was determined by the agar dilution method. Resistance and heteroresistance mechanisms were investigated by identifying genetic mutations in tetracycline (Tet) target sites and susceptibility testing in the presence of the efflux protein inhibitors phenylalanine-arginine-β-naphthylamide (PaβN) and carbonyl cyanide m chlorophenylhydrazine (CCCP). Comparison of single nucleotide polymorphism in the whole genome between the parental isolate and two TGC-resistant strains were investigated by next-generation sequencing. Results No mutations in Tet target sites in seven TGC heteroresistant strains were present, whereas the mutations in Tet target sites of seven TGC-resistant E. faecalis were frequently found (Table 1). TGC MICs in heteroresistant strains were reduced by CCCP (Table 2). Whole genome sequencing revealed the same non-synonymous mutations and transcoding deletions in the exons of several genes encoding for various enzymes or transfer systems (Table 3). Table 1. The characteristics of the antimicrobial susceptibility, resistance mechanism of TGC-induced resistant isolates Table 2. Characteristics of clinical heteroresistant mother E. faecalis strains and heteroresistance-derived E. faecalis clones Table 3. List of mutation-related genes, amino acids and proteins by comparison of whole genome between the parental isolate and the TGC-induced resistant strains Conclusion Our data indicated that the main mechanism of TGC heteroresistance in E. faecalis might be associated with the efflux pumps. TGC resistance in E. faecalis was associated with mutations in the 16SrRNA site or 30S ribosome protein S10. The genetic mutations in several enzymes and transfer systems might also participate in the resistance development to TGC in E. faecalis. Disclosures All Authors: No reported disclosures

Background FOF has been used in the treatment of multidrug-resistant (MDR) KP infections despite established susceptibility breakpoints. At present, agar dilution (AD) is considered the reference method for FOF while broth microdilution (BMD) is specifically recommended against despite its convenience over AD. We therefore sought to assess FOF activity against KP, along with essential and categorical agreement between AD and BMD methods to determine if BMD could be used as a reliable testing method. Methods Minimal inhibitory concentration (MIC) values were determined for a convenience collection of 69 KP isolates (59.4% MDR) from three US institutions. MIC testing was conducted in duplicate on separate days using AD and BMD methods; essential and categorical agreement were calculated using AD as the reference method. Fourteen isolates were also analyzed using high-inoculum AD (105.3-5.9 CFU/mL) similar to the BMD method. MIC values were categorized using Clinical and Laboratory Standards Institute (CLSI) interpretive criteria for Escherichia coli (≤ 64 mg/L, susceptible). ECVs were determined according to CLSI methodology. Results MIC values varied between methods, withMIC50/MIC90 values being 32/256 mg/L for AD and 128/256 mg/L for BMD. Using E. coli criteria, susceptible/intermediate/resistant rates were 82.6/2.9/14.5% (AD) and 44.9/21.7/33.3% (BMD). Essential agreement was 44.9% and categorical agreement was 60.8%. When using high-inoculum AD, MIC values were on average three-fold higher compared to standard-inoculum AD, with 10 of the 14 (71.4%) isolates brought into essential agreement with BMD. Calculated ECVs were 128 mg/L for standard-inoculum AD and 1024 mg/L for BMD. Conclusion Our collection of KP displayed high MIC values to FOF, in addition to substantial discrepancies between AD and BMD methods. Essential agreement increased with the use of high-inoculum AD testing, which better correlated with BMD results. ECV for BMD was three dilutions higher than that for standard-AD ECV. Based on these results, we recommend further investigation of BMD for FOF testing using a larger isolate collection, along with optimization of currently recommended testing methods. In light of these results, KP-specific breakpoints should also be examined. Disclosures Elizabeth B. Hirsch, PharmD, Merck (Grant/Research Support)Nabriva Therapeutics (Advisor or Review Panel member)

Introduction/Objective Significant technical issues are associated with methods used for the measurement of estradiol.The objective of this study was to qualify an electrochemiluminescent (ECL) assay for the quantification of 17β-estradiol (E2) in rat serum. Hemolysis has been identified as a factor that interferes with accurate measurement.The impact of hemolysis was also assessed. Methods Approximately 1.0 mL of whole blood was collected from male and female rats into separate red top tubes and processed to serum. The LoQ for E2 was evaluated by analyzing the low calibrator or at least 6 serum samples diluted to produce a value at the low end of the reportable range 8 times in the same run.The mean, standard deviation, and %CV were calculated for each sample.The data set was analyzed by plotting the data and determining the concentration at the intersection of the precision profile curve. Linearity of dilution was performed using commercially available calibration verification material and E2 stripped rat serum.The correlation coefficient, the slope, and the % Nominal were calculated. Intra assay precision was evaluated by analyzing 8 consecutive times in a single run one rat serum sample that was not diluted or spiked. This analysis was performed during the evaluation of the LoQ.The mean, SD and %CV were calculated. The interference of hemolysis with the E2 assay was tested by analyzing at least 5 rat serum samples/pools spiked with hemolyzed rat serum at different hemoglobin concentrations.The %RE was calculated. Results The LoQ assays were acceptable. For all samples tested, the % CV was less than or equal to 25%.The LoQ was verified to be 8.50 pg/mL. The %CV was 15.6%. For samples with estradiol concentrations below the LoQ, a value of 4.25 pg/ml was reported. Linearity of dilution for E2 was acceptable.The correlation coefficients were greater than or equal to 0.9000, the slopes were between 0.7500 and 1.2500, and the % nominals for each level were between 75-125%. The intra-assay precision was considered acceptable with a %CV of 8.6%. There was no hemolysis interference in the assay when samples were spiked with hemoglobin concentrations of up to 70 mg/dL, based on the %RE of less than or equal to 25% of non-hemolyzed samples. Conclusion Qualification of the ECL method, demonstrates the assay is suitable for the determination of E2 in serum samples from rats and absence of hemolysis interference up to 70 mg/dL of hemoglobin concentration.

Background Plazomicin (PLZ) is an aminoglycoside recently approved by the United States (US) Food and Drug Administration (FDA) for the treatment of complicated urinary tract infections, including pyelonephritis. We evaluated the susceptibility (S) rates of PLZ, amikacin (AMK), gentamicin (GEN) and tobramycin (TOB) by applying current breakpoints published by different organizations. Methods A total of 9,303 Enterobacterales (ENT) isolates (1/patient) were collected in 2018-2019 from medical centers located in the US (n=3,899; 33 centers), Europe (n=3,782; 39 centers in 19 nations), Asia-Pacific (n=795; 13 centers in 7 nations [2018 only]), and Latin America (n=827; 10 centers in 6 nations [2018 only]). PLZ and comparator agents were S tested by reference broth microdilution methods at a central laboratory. Breakpoints for the following organizations were applied when available: CLSI, EUCAST, USCAST, and US FDA. Results PLZ was active against 95.5% and 98.0% of isolates as per US FDA (≤2 mg/L) and USCAST (≤4 mg/L) criteria, respectively. S rates as per US FDA and USCAST criteria were 97.4% and 90.2% for AMK, 86.4% and 85.6% for GEN, and 83.8% and 81.1% for TOB, respectively (Table). CLSI and US FDA breakpoints were identical for these three older aminoglycosides, and EUCAST breakpoints were identical for GEN and TOB and one doubling dilution higher for AMK when compared with USCAST. Per US FDA criteria, carbapenem-resistant ENT (CRE) S rates to PLZ and AMK were 71.5% and 58.3%, respectively. Differences in S rates between PLZ and AMK were higher when applying USCAST for resistant subsets, such as CRE (72.2% versus 38.5%, respectively), ESBL-phenotype (92.7% versus 72.4%, respectively), and multidrug-resistant isolates (n=1,348; 88.6% versus 59.6%, respectively). GEN and TOB exhibited limited activity against ENT resistant subsets. Conclusion PLZ retained potent activity against ENT, including resistant subsets. The discrepancies among the S rates for aminoglycosides were greater when applying breakpoints generated using the same stringent contemporary methods applied to determine PLZ breakpoints. Table 1 Disclosures Helio S. Sader, MD, PhD, A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Melinta (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support)Pfizer (Research Grant or Support) S. J. Ryan Arends, PhD, Allergan (Research Grant or Support)Cipla Ltd. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support) Jaideep Gogtay, n/a, Cipla Ltd. (Employee) Cecilia G. Carvalhaes, MD, PhD, A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Fox Chase Chemical Diversity Center (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Pfizer (Research Grant or Support) Mariana Castanheira, PhD, 1928 Diagnostics (Research Grant or Support)A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Amplyx Pharmaceuticals (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Fox Chase Chemical Diversity Center (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support)Pfizer (Research Grant or Support)Qpex Biopharma (Research Grant or Support)

Background A general consensus exists that patients with inflammatory bowel disease (IBD) present compositional changes in the gut microbiota (dysbiosis), including an increase in the abundance of Enterobacteriaceae. Thiopurine drugs are commonly used in the maintenance of remission in IBD. In this context, the purpose of the project is to explore the role of candidate bacterial strains in mediating the effects of thiopurines in vitro. Methods Azathioprine (AZA), mercaptopurine (MP) and thioguanine (TG) (400 µM) were incubated in minimal salts medium (M9) in presence or not of E. coli, S. enterica and K. pneumoniae and of their growth phase broths (GPB) for 4 h at 37°C. The viability of NALM6 (B cells) and JURKAT (T cells) exposed to serial dilution of drugs (ranging from 0.2 to 15 μM of AZA, from 0.3 to 20 μM of MP, from 0.08 to 5 μM of TG) previously incubated or not with bacteria and with their GPB was determined by the MTT assay. Absorbance peaks of thiopurines were analysed by UV spectrophotometry. Statistical significance was assessed by two-way ANOVA and Bonferroni’s post-test for MTT tests and by one-way ANOVA for UV spectra. Results In NALM6 cells, the cytotoxic effects of 15 μM of AZA, 2.5 μM of MP and 1.25 μM of TG decreased significantly (p < 0.001) after incubation with K. pneumoniae (respectively 45 ± 2.9%; 34 ± 2.5%% and 21 ± 0.6%) and its GPB (respectively 41 ± 7.7%; 41 ± 5.1% and 27 ± 3.5%) compared with the drugs not previously exposed (respectively 76 ± 2.3%; 69 ± 1.7% and 43 ± 3.8%). In JURKAT cells, the cytotoxic effects of 15 μM of AZA, 2.5 μM of MP and 1.25 μM of TG decreased significantly (p < 0.001) after incubation with K. pneumoniae (respectively 46 ± 2.8%; 38 ± 1.29% and 19 ± 3.3%) and its GPB (respectively 49 ± 9.4%; 38 ± 1.5% and 26 ± 1.5%) in comparison with the drugs not exposed (respectively 75 ± 4.0%; 50 ± 3.5% and 54 ± 4.0%). E. coli and S. enterica did not affect the cytotoxicity of the thiopurines. UV analysis evidenced a reduction of absorbance peaks of AZA (21 ± 0.05%), MP (32 ± 0.015%) and TG (30 ± 0.03%) after incubation with K. pneumoniae but not with its growth phase broth (GPB). Conclusion The activity of thiopurines decreased after incubation with both K. pneumoniae and its GPB. UV analysis suggested that the lower cytotoxicity of thiopurines exposed to the bacterial strain is due to the reduction of the concentration of the drugs exposed to K. pneumoniae. Moreover, the reduction of drug availability after the exposure to GPB could be explained with a possible interaction between thiopurines and capsular polysaccharides released by the bacteria.

BackgroundIn South Africa, Neisseria gonorrhoeae (NG) is the predominant cause of male urethritis syndrome (MUS). The national MUS treatment guidelines recommend gentamicin as salvage therapy for ceftriaxone treatment failures. We ascertained and compared gentamicin MICs obtained by agar dilution and Etest for clinical isolates of NG.MethodsGentamicin MICs for NG culture isolates obtained from 272 MUS cases in 2017 were determined using agar dilution, as per CLSI agar dilution methods, and Etest® (bioMérieux, Marcy-l’Étoile, France). Previously published interpretive criteria were used: MIC ≤4 mg/L, susceptible (S); MIC 8–16 mg/L, intermediately resistant (IR); and MIC ≥32 mg/L, resistant (R). WHO 2008 NG reference strains were used as comparison standards.ResultsGentamicin agar dilution versus Etest MIC results (mg/L) were as follows: MIC50 = 16 versus 4; MIC90 = 16 versus 8; minimum MIC = 4 versus 1; and maximum MIC = 32 versus 16. Interpretive categories for agar dilution versus Etest were as follows: S, 4.4% versus 86.8%; IR, 86.0% versus 13.4%; and R, 9.6% versus 0%. The gentamicin MIC50 by agar dilution was significantly higher than by Etest (sign test P value ConclusionsThere was a significant discordance between NG gentamicin MICs by agar dilution versus Etest. NG gentamicin AST methodology must be standardized and interpretive criteria established to optimize the monitoring of susceptibility trends.

Aims All phase 3 studies in patients with acute heart failure (AHF) and HF with preserved ejection fraction (HFpEF) have failed in the last decades. We explore the likelihood that the negative results are due to chance and/or to study size and dilution of statistical power. Methods and results First, using simulations, we examined the probability that a positive finding in phase 2 would result in studying truly effective drugs in phase 3. We simulated phase 2 studies under six scenarios where the range of true relative risk (RR) for an outcome of interest varied from 0.5 (major benefit) to 1.15 (some harm). The proportion of simulated studies where the RR Figure 1. Power at two-sided 0.05 significance level to detect an effect size of hazard ratio of 0.65 (left) or 0.8 (right) with a placebo event rate of 10% (top) and 20% (bottom) at N=100 at various treatment effect dilutions with increasing sample size. Conclusion These data suggest that it is unlikely that the very high rate of negative AHF phase III trials can be explained by chance alone. Potentially, our tendency to increase sample size does not necessarily increase statistical power, due to more heterogenous populations leading to reduced event rates and treatment effects.

Background Guidance for intensified diuretic therapy in acute heart failure (AHF) is mainly based on body weight measurement, frequently leading to a short episode of dehydration with kidney failure after recompensation. In addition, patients often present immobilized due to severe health issues making weight measurement stressful. Purpose Bioelectrical impedance analysis (BIA) may be a more direct approach to guide intensified diuretic therapy analysing patient's body composition. We hypothesized that patient's weight loss during therapy correlates with loss of body water measured by BIA. Therefore, we tested if this method could be an alternative to daily weight measurement. Methods We conducted an observational, single-centre study to evaluate and monitor body composition of patients hospitalised with AHF, adjudicated according to current ESC/HFA guidelines by a cardiologist. We used an eight-electrode, segmental, multi-frequency body composition analyser, previously validated against air displacement plethysmography, whole body MRI, deuterium and sodium bromide dilution. We investigated patients until hospital discharge or latest one day after ending intensified diuretic therapy. Disease specific properties, BIA and weight measurement were assessed daily. Furthermore, we investigated BIA raw data. Results 390 BIA were applied on 76 patients (47 men; 29 women; mean age 76±11 years; mean weight 75.6±15.7 kg). 34 patients presented with global, 27 with left-heart, 8 with right-heart and 7 with not specified AHF. 44 patients presented with pleural effusion. Pearson correlations showed that total body water (r=0.737, p≤0.001) and extracellular water (r=0.69, p≤0.001) correlated each with total body weight. Changes in total body water accurately (within a range of ± 1kg) reflected changes in total body weight in 40.28% of the patients and changes in extracellular water showed a similarly accurate reflection of total body weight change in 68.06% of the patients. BIA raw-data analysis showed significant changes using Wilcoxon test between measurements at the beginning of intensified diuretic therapy and at its end. We found a significant increase of resistance (mean from 334.6±67.5 to 362.8±69.5 Ohm/m; p=0.021) and reactance (mean from 21.3±7.1 to 24.1±6.2 Ohm/m; p=0.009) standardized to patients height and a non-significant increase of phase angle (mean from 3.6±0.9 to 3.8±0.8 °; p=0.149) during hospitalisation. Conclusion BIA is able to estimate changes in total body weight by analysing changes in extracellular body water in patients under intensified diuretic therapy and raw data analysis seems even more accurate and promising. This data derive from a heterogeneous AHF patient group, needing further investigation. Once validated, wearable BIA connected to an automated device monitoring system would enable an easy to use diuretic therapy monitoring for impaired patients or outpatients and could help reducing care efforts.

OBJECTIVES: Fecal biomarkers have emerged as an important tool to assess intestinal inflammation and permeability. Commonly measured biomarkers include calprotectin (CP), myeloperoxidase (MPO), alpha-1-antitrypsin (AAT) and neopterin (NEO). We sought to develop a simple, fast and cost-effective single extraction procedure for use in determining all four biomarkers of interest. The applicability and sensitivity of this procedure for use in healthy adults was examined. METHODS: Sample extraction buffers and methods including sample weight, dilution, homogenization and centrifugation were all considered in the development of a single extraction procedure. An extraction buffer that included phosphate-buffered saline, phenylmethylsulfonyl fluoride, bovine serum albumin and Tween 20 was used to extract fecal samples. To assess the applicability and sensitivity of the single extraction procedure, concentrations of CP, MPO, AAT and NEO were measured using commercially available sandwich ELISA kits, according to manufacturer's instructions. RESULTS: CP, MPO and AAT concentrations were measured in fecal samples of healthy adults (aged 50–80 years, n = 85) and found to be comparable to findings of previously published studies in healthy populations. Mean concentrations of CP and AAT were 3.6 ± 3.8 µg/g of wet weight (range 0.14–18.0 µg/g) and 2.3 ± 0.73 µg/g (range 0.76–5.2 µg/g), respectively. Mean fecal MPO concentrations were 135 ± 24 ng/g (range 3–1290 ng/g). NEO concentrations were examined in a subset of healthy adults (n = 10), with mean concentrations of 18 ± 1 ng/g (range 17–20 ng/g). CONCLUSIONS: We demonstrated the efficacy of a single extraction procedure used to assess multiple fecal biomarkers of intestinal inflammation. This simple, fast and inexpensive extraction method will facilitate the determination of multiple fecal biomarkers which is critical in validating their use as clinical or predictive biomarkers of intestinal inflammation. FUNDING SOURCES: Supported by the U.S. Department of Agriculture – Agricultural Research Service (ARS), under Agreement No. 58–1950-4–003, the Bill & Melinda Gates Foundation and Canadian Institutes of Health Research Postdoctoral Fellowship.

OBJECTIVES: Animal milk can be contaminated with mycotoxins (secondary metabolites of fungi) through poor quality feed and may be a source of human exposure. Our objective was to develop and optimize a method to detect biologically relevant concentrations of 8 mycotoxins (Aflatoxins B1, B2, G1, G2, M1, M2; Ochratoxins A, B) in animal milk. METHODS: We used ultra-high performance liquid chromatography/tandem mass spectrometry using selected reaction monitoring (UHPLC/MS-SRM) to quantify mycotoxins in animal milk samples (total N = 38; n = 10 each from cow and commercial milk and n = 9 from buffalo and goat) from the southern Indian states of Karnataka and Tamil Nadu. Method development was conducted and stable isotope dilution employed, using AFB1-D3 for aflatoxins and OTA-D5 for ochratoxins. We validated the method and examined matrix effects, freeze-thaw and auto-sampler stability. Our dynamic ranges from quantification were between 7.8–5000 pg/mL. RESULTS: Among samples collected from Southern India, 8 of 10 cow [median 103.35 pg/mL; n = 3 > 500 pg/mL]; 0 of 9 buffalo and 10 of 10 commercial [median: 151.5 pg/mL], milk samples were above the LOQ. AFM2 was also seen in samples from both regions, but in lower quantities when compared to AFM1 [median (north): 25.8 pg/mL; median (south): 70.95 pg/mL]. All except 3 samples were below the LOQ (31.3 pg/mL) for OTA, however we detected a sodium adduct of OTA above LOQ, across samples. We found [Na-OTA] in goat milk [median: 5.9 ng/mL] > buffalo [median: 2.2 ng/mL] > commercial [median: 2.04 ng/mL] > cow [median: 0.8 ng/mL]. Other mycotoxins were seen in concentrations close to or below LOQ. We did not identify significant stability issues. CONCLUSIONS: We developed a highly sensitive method with biologically relevant dynamic ranges for detection of mycotoxins in milk samples. We found AFM1, AFM2, and Na-OTA in milk samples from Southern India. Further studies with larger sample sizes are warranted to establish the extent of mycotoxin contamination in milk. FUNDING SOURCES: Funded by University Research Committee, Emory University and International Society for Research in Human Milk and Lactation.

Background Several studies have reported an association between ustekinumab serum concentrations and clinical outcomes, suggesting a potential role of therapeutic drug monitoring (TDM) for guiding clinical decision-making and treatment optimisation in ustekinumab-treated patients with inflammatory bowel diseases. Usually, ustekinumab concentrations are measured with an enzyme-linked immunosorbent assay (ELISA). Disadvantages of using ELISA for TDM are the rather long time-to-result and the necessity of collecting multiple samples in order to reduce the price per determination. The performance of TDM could be further enhanced by using a rapid point-of-care assay, which would allow immediate and individualised dose optimisation based on real-time pharmacokinetic information instead of awaiting dose adjustments at the subsequent administration. Methods A fiber-optic surface plasmon resonance (FO-SPR) assay was developed on a White FOx 1.0 device (FOx Biosystems), using a monoclonal antibody (MA)/MA sandwich approach. MA-UST56A2D11 was covalently immobilised to a gold-sputtered fiber surface to capture ustekinumab. After incubation with the serum sample, biotinylated MA-UST56C1H12 was used for detection of bound ustekinumab, and the signal was amplified using a polyclonal anti-biotin antibody conjugated to gold nanoparticles. A calibration curve was constructed by spiking ustekinumab in 1/1000 diluted human serum. Recovery and imprecision were assessed, and the performance of the FO-SPR assay was compared with that of our previously developed in-house ELISA using twelve serum samples of ustekinumab-treated patients. Results A dose-response curve ranging from 1.25 – 40 ng/mL was obtained, allowing quantification of ustekinumab concentrations from 0.31 µg/mL (using a 1/250 dilution) up to 80 µg/mL (using a 1/2000 dilution) in serum samples. Measurement of ustekinumab-spiked samples (1 – 25 µg/mL) showed an average intra-assay recovery of 111% (range: 95-124%) and imprecision of 10% (range: 8-15%), and an average inter-assay recovery of 109% (range: 100-122%) and imprecision of 5% (range: 1-8%). Comparison of measurements between FO-SPR and ELISA revealed a very good correlation (Pearson r coefficient of 0.980, 95% CI 0.928-0.995, p Conclusion A rapid FO-SPR assay for quantification of ustekinumab concentrations in serum samples was established and showed a very good correlation with ELISA. The reduced assay time and the possibility of measuring a single sample are advantages compared to ELISA, and may allow implementation of FO-SPR as a point-of-care diagnostic tool.

Electronic cigarette (e-cigarette) use is increasing worldwide and is highest among both daily and nondaily smokers. E-cigarettes are perceived as a healthier alternative to combustible tobacco products, but their health risk factors have not yet been established, and one of them is lack of data on aerosol size generated by e-cigarettes.We applied a real-time, high-resolution aerosol differential mobility spectrometer to monitor the evolution of aerosol size and concentration during puff development. Particles generated by e-cigarettes were immediately delivered for analysis with minimal dilution and therefore with minimal sample distortion, which is critically important given the highly dynamic aerosol/vapor mixture inherent to e-cigarette emissions.E-cigarette aerosols normally exhibit a bimodal particle size distribution: nanoparticles (11-25nm count median diameter) and submicron particles (96-175nm count median diameter). Each mode has comparable number concentrations (10(7)-10(8) particles/cm(3)). "Dry puff" tests conducted with no e-cigarette liquid (e-liquid) present in the e-cigarette tank demonstrated that under these conditions only nanoparticles were generated. Analysis of the bulk aerosol collected on the filter showed that e-cigarette emissions contained a variety of metals.E-cigarette aerosol size distribution is different from that of combustible tobacco smoke. E-cigarettes generate high concentrations of nanoparticles and their chemical content requires further investigation. Despite the small mass of nanoparticles, their toxicological impact could be significant. Toxic chemicals that are attached to the small nanoparticles may have greater adverse health effects than when attached to larger submicron particles.The e-cigarette aerosol size distribution is different from that of combustible tobacco smoke and typically exhibits a bimodal behavior with comparable number concentrations of nanoparticles and submicron particles. While vaping the e-cigarette, along with submicron particles the user is also inhaling nano-aerosol that consists of nanoparticles with attached chemicals that has not been fully investigated. The presence of high concentrations of nanoparticles requires nanotoxicological consideration in order to assess the potential health impact of e-cigarettes. The toxicological impact of inhaled nanoparticles could be significant, though not necessarily similar to the biomarkers typical of combustible tobacco smoke.

Background Ebolaviruses cause viral hemorrhagic fever with high mortality rates. Nearly all Ebola vaccines in development use Ebola glycoprotein (GP) as the immunizing antigen. GP is present on the viral membrane and functions in cell entry by binding the cellular receptor and mediating membrane fusion; antibodies to GP induce protective immunity. Ebola also produces sGP: a smaller, secreted form of GP containing the receptor-binding domain; it is also able to induce protective immunity. sGP naturally refolds after thermal denaturation and thus may be more stable than GP, and may also be more cost effective as it is produced easily in high quantities. sGP is a homodimer that is covalently linked by a cysteine near its C-terminus. In this work, we explored how modifications to sGP that affect its ability to dimerize also alter its immunogenicity. Methods sGP mutants were generated in the pCAGGS mammalian expression plasmid, and injected into mice as a DNA vaccine. Mouse sera was tested by ELISA against sGP and GP proteins, and in a neutralization assay against GP-typed pseudovirions. Results We generated 4 different mutants of sGP that had altered abilities to form inter-protomer disulfide bonds. All had a mutated or deleted cysteine at position 306; two had disulfide-bonding restored by introduction of an engineered inter-protomer disulfide bond. Mice were immunized with a DNA vaccine encoding either an sGP mutant or wild-type sGP, and sera were collected. We found that sera from sGP mutants with reduced interprotomer disulfide bonds had significantly higher antibody titers to sGP and GP than sera from our wild-type sGP and engineered-disulfide sGP immunized mice. Antibody titers were similar between sGP and GP; these titers correlated with neutralization ability. Relative binding to sGP & GP (by ELISA OD) & relative % neutralization of pseudovirions at 1:10 dilution Conclusion Immunogenicity of Ebola sGP was enhanced significantly when mutations were introduced to reduce its ability to covalently dimerize. Immunogenicity correlated with induction of neutralizing antibodies, implying that our mutants may outperform wild-type sGP when used as a vaccine in vivo. This work helps paves the way for an alternative Ebola vaccine that has the potential to be more cost-effective and heat-stable than the currently-licensed vaccine. Disclosures Samuel D. Stampfer, MD/PhD, Gilead (Shareholder)

Background In the United States, interpretive criteria for FOF are established only for EC, yet those criteria are often extrapolated to KP. Recent studies have highlighted both inferior clinical outcomes after FOF treatment and difficulties in interpretation of inner colony subpopulations, the presence of which may affect clinical efficacy. We sought to compare FOF activity against EC and KP and to determine the prevalence of inner colony subpopulations following disk diffusion (DD) testing of the two species. Methods A convenience collection of 73 KP and 42 EC isolates from 3 U.S. institutions were included. Minimal inhibitory concentration (MIC) testing was performed in duplicate on separate days using agar dilution (AD) and DD as recommended by the Clinical and Laboratory Standards Institute guidelines, with application of EC susceptibility (≤ 64mg/L) breakpoints. The frequency and counts of inner colonies observed during DD testing was calculated, and colonies were subcultured for use in future studies. Results MIC50/90 values were 1/16 mg/L and 32/256 mg/L for EC and KP respectively. All EC isolates were considered susceptible and therefore categorical agreement was 100%. The majority of KP isolates were considered susceptible (83.6% with AD and 86.3% with DD) and categorical agreement between the methods was 84.9%. Inner colonies were observed during DD testing in 88.1% of EC isolates and 80.8% of KP isolates during at least one replicate, with 47.6% of EC isolates and 39.7% of KP isolates showing inner colony growth during both DD test replicates. More than 10 inner colonies were observed in 50% of EC isolates compared to 12.3% of KP isolates. Conclusion KP isolates demonstrated considerably higher FOF MIC values compared to EC, as evidenced by MIC50/90 values 4-5 dilutions higher than those for EC. The categorical agreement rate was higher among EC than KP, highlighting concerns regarding the practice of extrapolating FOF susceptibility breakpoints for EC to KP. The high frequency of inner colonies observed in DD for both species necessitates further studies to determine best practices for interpreting their relevance, fitness, and resistance in order to identify potential impacts to clinical efficacy of FOF. Disclosures Elizabeth B. Hirsch, PharmD, Merck (Grant/Research Support)Nabriva Therapeutics (Advisor or Review Panel member)

Background C. jejuni is the leading cause of bacterial gastroenteritis worldwide. It has been classified as a serious antibiotic resistant threat, causing 13,000 hospitalizations and 120 deaths annually. Our goal was to describe the diversity of clinical C. jejuni using phylogenomics and classify resistance mechanisms. Methods Isolates were collected via sentinel surveillance at four hospitals, and demographic and clinical data were obtained. DNA was extracted and sequenced. Raw reads were processed with Trimmomatic and quality checked with FastQC. De novo genome assembly was performed in Spades. Assembled genomes were filtered for quality and completeness; samples of 1.4-2.1MB were annotated in Prokka followed by pangenome and phylogenetic analyses. Multilocus sequence typing loci and virulence and antibiotic resistance genes were extracted from each genome. Results Among the 214 C. jejuni isolates recovered, 86 unique sequence types (STs) were identified; five were novel STs with unique allele combinations. ST353 (8.3%: n=18), ST982 (7.4%: n=16), ST50 (5.1 %: n=11) and ST48 (5.1%: n=11) were the most prevalent STs identified, while the majority (50.1%: n=50) of STs were singletons. The pangenome analysis identified 8781, 615, and 1169 total, core, and shell core genes, respectively, which grouped the isolates into three major clades. Most isolates belonged to clade 1. A neighbor-net analysis detected significant recombination among all 86 STs (pairwise homoplasy index p=< 0.00001) and evidence of horizontal gene transfer across clades. The beta-lactamase gene, blaOXA-605, was the most common resistance gene identified (58.8%: n=125) followed by tet(O) (56.0%: n=121), which mediate resistance to beta-lactams and tetracyclines, respectively. Resistance phenotypes were confirmed using microbroth dilution. Conclusion: Together, these data demonstrate that the C. jejuni population is highly diverse and carries important resistance determinants. The phylogenomic analyses also provide insight into the evolution of this major foodborne pathogen. Future work will focus on identifying molecular and epidemiological factors associated with specific strain types and resistance and virulence profiles circulating in Michigan. Disclosures All Authors: No reported disclosures

OBJECTIVES: To characterize the intestinal, hepatic, muscle, and renal release and uptake of xenometabolites (non-host derived metabolites) and endogenously modified xenometabolites by measuring their across-organ net release and uptake in a post-absorptive conscious pig model. METHODS: Twelve 2–3 months old female pigs (25.6 ± 2.2 kg) had surgically-implanted catheters across portal drained viscera (PDV), splanchnic area (SPL), liver, kidney, and hindquarter muscle. Ten days after catheter implantation, overnight fasted arterial and venous plasma was collected in a conscious state and stored at -80°C. Thawed samples were analyzed by liquid chromatography-mass spectrometry. Xenometabolites were identified by retention time and MS2 spectra from in-house authentic standards and expressed as peak areas. Plasma flow measurements determined with para-aminohippuric acid dilution technology. Net organ balance rate (peak area/kg body weight/min) was calculated. Significant (P

OBJECTIVES: The objective of this study was to investigate differences in live and dead bacteria counts a month before the expiration date and right after the expiration date and observe any differences in bacteria counts between types of yogurt, brands, and stores tested. METHODS: Two yogurt containers with the same expiration date were collected for each brand at three stores. The yogurts collected were three national brands of Greek yogurts and five regular yogurts. The first container was tested one month prior to expiry. The second container was tested at the expiration date. After being thoroughly mixed, triplicate 50–75 mg of yogurt were sampled from each carton. Yogurt samples were diluted 9-fold with freshly prepared peptone water. A second dilution was prepared by mixing 25 µL of the yogurt peptone mix with 975 µL of freshly prepared sterile PBS. The samples were incubated for 15 min. at 37°C with 1 µL SYBR green and 5 µL propidium iodine and iced for 1 hour in the dark. 200 µL of dyed yogurt were diluted with 800 µL of PBS and analyzed by flow cytometer (BD Accuri™ C6 Cytometer). Each yogurt sample was run in triplicate on slow speed setting. Flow cytometer parameters were set to differentiate live and dead bacteria based on fluorescence as well as yogurt matrix artifacts. The data was analyzed by a least-squares fit model to test for significance among variables. RESULTS: Greek yogurts had significantly higher live and dead bacteria counts compared to regular yogurts. The Greek yogurts differed amongst themselves. Brand 3 had significantly lower live counts than brand 2 and 1. Brand 1 had significantly higher counts of dead bacteria compared to the other two. While differences between Greek yogurt are statistically significant, they are most likely not clinically significant. For the regular yogurt live and dead counts there were no significant differences. No significant differences were observed based on the timing of yogurt sampling or the store the yogurt came from. CONCLUSIONS: Preparing Greek yogurt involves more straining and whey removal and yields a higher yogurt concentration, so it is no surprise that Greek yogurts have higher bacteria counts. This study demonstrates that when considering yogurt for its probiotic qualities, the most relevant factor is the type of yogurt. Yogurt brand, store, or when it is sampled have little effect on the probiotics count. FUNDING SOURCES: USU.

Objectives To describe the distributions of bedaquiline and linezolid MIC values for the Mycobacterium tuberculosis WT population and to define the corresponding epidemiological cut-offs (ECOFFs) in three Latin American countries. Methods MICs of bedaquiline and linezolid were determined by the resazurin microtitre assay (REMA). In phase 1, interlaboratory reproducibility was assessed using a panel of 10 fully susceptible M. tuberculosis strains. Phase 2 involved MIC determination for 248 clinical isolates from Argentina (n = 58), Brazil (n = 100) and Peru (n = 90) from patients who were treatment-naive for bedaquiline and linezolid. We then determined the ECOFFs for bedaquiline and linezolid by the eyeball method and the ECOFFinder statistical calculator. Results Phase 1: REMA MIC values in the three sites were either identical to each other or differed by one 2-fold dilution from the consensus value with the exception of a single value. Phase 2: The bedaquiline MIC range was 0.0039-0.25 mg/L for pan-susceptible and drug-resistant isolates combined. The linezolid MIC range was 0.062-0.5 mg/L for pan-susceptible isolates and 0.031-4 mg/L for drug-resistant isolates. ECOFFs were 0.125 mg/L for bedaquiline and 0.50 mg/L for linezolid. Conclusions REMA is reproducible and robust for the determination of bedaquiline and linezolid MIC distributions and ECOFF values when applied in laboratories of medium/low-resource countries. We suggest that WT MIC distributions for both drugs should be used as a monitoring tool to control the possible rapid emergence of resistance. Fil: Lopez, Beatriz. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Siqueira De Oliveira, Rosangela. Instituto Adolfo Lutz; Brasil Fil: Pinhata, Juliana M. W.. Instituto Adolfo Lutz; Brasil Fil: Chimara, Erica. Instituto Adolfo Lutz; Brasil Fil: Pacheco Ascencio, Edson. Instituto Nacional de Salud; Perú Fil: Puyén Guerra, Zully M.. Instituto Nacional de Salud; Perú Fil: Wainmayer, Ingrid. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Símboli, Norberto Fabián. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Del Granado, Mirtha. Pan American Health Organization; Estados Unidos Fil: Palomino, Juan Carlos. University of Ghent; Bélgica Fil: Ritacco, Gloria Viviana. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Martin, Anandi. Université Catholique de Louvain; Bélgica

Background Environmental contamination in outpatient clinics is poorly understood. Methods We performed a microbiological analysis of surfaces in wound and pulmonary outpatient clinics at a tertiary care center. Cultures were obtained with pre-moistened cellulose sponges from three locations (Exam bed/chair, patient chair, physician area/chair) before and after clinic days. Sponges were combined with 1% Tween20-PBS and mixed in the Seward Stomacher. The homogenate was centrifuged and all but 5 mL of the supernatant was discarded. Samples were plated on Sheep’s blood agar and selective media for S. aureus, Enterococcus spp. and Gram-negative bacteria. CFU was determined by counting the number of colonies on each plate and using dilution calculations to calculate the CFU of the original ~5 mL homogenate. The total sample areas in the wound and pulmonary clinic were 12,735 cm2 and 16,400 cm2, respectively. Results A total of 300 samples were obtained over 90-days. Median total room CFU was 7,918 (IQR 2,939–18,855) (Figure 1). Median CFU for the examination area, patient area and physician areas were 2090 (537–10508), 1524 (573–4605) and 960 (371–2183), respectively (Figure 2). The proportions of samples positive for S. aureus, Enterococcus spp. and Gram-negative bacteria were 5.0, 3.3 and 7.7%, respectively (Table 1). In general, median total CFU increased during the clinic day (median CFU before the clinic day 6883 (2937–14983) vs. median after clinic day=10351 (3484–21263) (Figure 3). Environmental bioburden was higher in the wound clinic than the pulmonary clinic (median 18206 CFU [IQR 10048–25037) vs. 3764 [IQR 1452–6671], P < 0.001) (Figure 1). The average number of patients seen in the wound clinic per clinic day was greater than the pulmonary clinic (3.8 vs. 2.2, P < 0.001). Conclusion Outpatient clinic rooms were contaminated with clinically important pathogens. Contamination varied by environmental location and increased as the clinic day progressed. Higher contamination was seen in the wound clinic possibly due to higher patient volume vs. increased environmental contamination that occurs while giving wound care. Wound care clinics may need to focus on more detailed cleaning to reduce environmental contamination and the risk of pathogen transmission in at-risk patients. Disclosures All authors: No reported disclosures.

Background To prevent the spread of drug-resistant bacteria, a rapid and accurate antimicrobial susceptibility test (AST) is necessary. Recently, morphokinetic microscopy approaches have been reported as a rapid AST method. However, these still require several hours to obtain a minimum inhibitory concentration (MIC). Adenosine triphosphate (ATP) luminescence has also been reported as a rapid AST method that can detect bacterial growth more rapidly than morphokinetic approaches, since ATP in bacteria increases prior to bacterial division. In this study, we designed a new machine learning-based algorithm that predicts MIC rapidly, using a dataset that contains ATP luminescence patterns and conventional MICs determined by turbidity. Essential agreement (EA) rates between rapid and conventional MIC were then evaluated. Methods Sixty-three strains of E. coli (ATCC 25922 and clinical isolates from Toyama University Hospital) were tested. Bacterial suspensions were diluted 500-fold in Mueller–Hinton broth from 0.5 McF solutions, and the final concentration of bacteria was 3×105 CFU/mL. The suspensions were dispensed into a 96-well microplate, which had 12 antimicrobials in two-fold dilution series, and the microplate was incubated at 35°C. At each measurement time point, the amount of ATP in a 10 μL aliquot from each well was evaluated by our original measurement system, which can sensitively detect ATP luminescence equivalent to a single bacterium. After 22 hours, MIC was determined conventionally by measuring turbidity. A rapid MIC for each bacterium was estimated by the algorithm based on the dataset consisting of the rest of the 62 strains (leave-one-out cross validation). Results Table 1 shows the EA rate for the 12 antimicrobials; EA rates > 90% were achieved for 7 antimicrobials in 2 hours and for 12 antimicrobials in 3 hours. In 6 hours, an average EA rate > 97% was achieved. Conclusion Using the dataset, our new machine learning-based algorithm predicted MIC rapidly within 2 hours with an EA rate > 90% for 7 antimicrobials. The rapid AST detected by the ATP luminescence method will contribute toward both appropriate antimicrobial treatment and reduction in medication and admission charges. In the future, other species of bacteria will be evaluated by our ATP method. Disclosures All authors: No reported disclosures.

Background Aerococcus urinae is frequently identified by MALDI-TOF in urinary specimens. It is generally susceptible to β-lactams, but its susceptibility pattern to fluoroquinolones (FQ) remains unpredictable. The goal of this study was to evaluate the performance of the gradient diffusion method (Etest®) to determine FQ susceptibility compared with broth microdilution (BMD) and agar dilution (AD). Methods Prospectively collected isolates of A. urinae from urinary tract specimens originating from 5 hospitals in Quebec city and Montreal were identified by MALDI-TOF (Vitek-MS). All isolates were tested using BMD according to CLSI guidelines, and also with Etest® strips on MH agar w/ 5% sheep blood. Isolates showing trailing, insufficient growth or discordance between both methods were further tested by agar dilution (MH agar w/5% horse blood + β-NAD) according to EUCAST guidelines. Breakpoints were interpreted using CLSI M45-A3. Combined results of BMD and AD were then compared with Etest. Results Of the 207 isolates of A. urinae tested, 37 showed trailing (17,8%) and 19 (9,2%) insufficient growth with the BMD method and were retested using AD. Moreover, 38 isolates (ciprofloxacin) and 13 isolates (levofloxacin) showed either lack of categorical or essential agreement between Etest and BMD and were also retested using AD to arbitrate discrepancies. Susceptibility profiles combining BMD and AD are presented in Table 1. As suggested in EUCAST guidelines, readings were much clearer and growth was better with AD compared with BMD. The categorical agreement of the Etest® with BMD+AD was 95% for ciprofloxacin and 97% for levofloxacin. Essential agreement was 95% for ciprofloxacin and 97% for levofloxacin. No very major errors were identified. Two major errors were identified for levofloxacin (1,2%) and one for ciprofloxacin (0.6%). Conclusion Gradient diffusion method using Etest® strips on MH agar w/ sheep blood is a valid method to determine susceptibility to FQ for urinary tract isolates. As a reference method, AD provides clearer endpoints and better growth than BMD for FQ susceptibility testing. Disclosures All authors: No reported disclosures.

The presence of hemolysis or icterus can interfere with chromogenic anti-Xa assays for unfractionated heparin monitoring (heparin activity), resulting in falsely lower estimates of heparin activity. Dilution is a common method for mitigating interference in spectroscopic assays that measure a single analyte by endpoint analysis and has been proposed as a solution for hemolysis interference in heparin activity assays. Heparin activity assays measure heparin/antithrombin complex activity using a chromogenic rate of absorbance change method. Heparin activity is a complex equilibrium of heparin binding to multiple proteins, including antithrombin, heparin cofactor II, platelet factor 4, histidine-rich glycoprotein, vitronectin, fibronectin, and others; some enhance while others neutralize heparin activity. The objective of this study was to determine how dilution affects heparin activity with different starting levels of antithrombin. First samples were diluted without interference (no hemolysis). Samples with 1 U/mL heparin in pooled normal plasma (PNP) and 50% PNP (to simulate 50% antithrombin levels) were diluted 1:1 with PNP versus OK buffer. Dilution resulted in nonlinear changes. Samples with 100% antithrombin recovered 88% of expected activity with PNP versus 74% with OK buffer; samples with 50% antithrombin recovered 111% with PNP versus 77% with OK buffer. Samples were then spiked with varying concentrations of hemolysate and 1:1 dilutions with PNP demonstrated nonlinear recoveries ranging from 41% to 84%. We were unable to recover the original heparin activity with or without hemolysis due to the complex interaction of initial heparin, antithrombin, and other binding protein concentrations with diluent antithrombin and other diluent heparin binding protein levels. When PNP is used as the diluent, the recovery will be lower when the initial antithrombin level is normal due to the addition of other heparin binding proteins; when the initial antithrombin level is low, the recovery is higher due to the addition of antithrombin from the diluent plasma. The recovery is always lower when buffer is used due to dilution of sample antithrombin. Careful consideration should be taken in evaluating the utility of sample dilution to mitigate interference from hemolysis and icterus in heparin activity assays, particularly in patients with fluctuating levels of antithrombin, such as those on extracorporeal life support or with liver failure.

The diagnosis of infective endocarditis is based on positive blood cultures. Modern microbiological techniques can isolate the aetiological agent in 90–95% of cases. The rapidity of detection has been improved by inoculation of 10 ml of blood, adequate dilution and media and systematic subcultures. Lysis-centrifugation has yreatly improved the detection of fungi in blood.

The doubly labeled water (DLW) method is considered the reference method for the measurement of energy expenditure under free-living conditions. However, the reproducibility of the DLW method in longitudinal studies is not well documented. This study was designed to evaluate the longitudinal reproducibility of the DLW method using 2 protocols developed and implemented in a multicenter clinical trial—the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE). To document the longitudinal reproducibility of the DLW method, 2 protocols, 1 based on repeated analysis of dose dilutions over the course of the clinical trial (dose-dilution protocol) and 1 based on repeated but blinded analysis of randomly selected DLW studies (test-retest protocol), were carried out. The dose-dilution protocol showed that the theoretical fractional turnover rates for 2 H and 18 O and the difference between the 2 fractional turnover rates were reproducible to within 1% and 5%, respectively, over 4.5 y. The Bland-Altman pair-wise comparisons of the results generated from 50 test-retest DLW studies showed that the fractional turnover rates and isotope dilution spaces for 2 H and 18 O, and total energy expenditure, were highly reproducible over 2.4 y. Our results show that the DLW method is reproducible in longitudinal studies and confirm the validity of this method to measure energy expenditure, define energy intake prescriptions, and monitor adherence and body composition changes over the period of 2.5–4.4 y. The 2 protocols can be adopted by other laboratories to document the longitudinal reproducibility of their measurements to ensure the long-term outcomes of interest are meaningful biologically. This trial was registered at clinicaltrials.gov as NCT00427193. J. Nutr. 144: 777–783, 2014.

Are the new Ansh Labs Ultra-Sensitive anti-Müllerian hormone (AMH) and picoAMH ELISA assays suitable for clinical use and is the Ultra-Sensitive assay comparable to the Beckman Coulter AMH Gen II assay?The Ultra-Sensitive assay appears to have different calibration to the Gen II assay, but has performance characteristics generally suitable for clinical use.The Gen II assay is the most commonly used AMH assay in routine biochemistry at present, but persistent calibration/interference problems have been reported.Serum from patients referred for AMH measurement was assayed (in duplicate) using the Gen II assay in Glasgow Royal Infirmary between January and February 2013. We randomly selected 193 stored serum samples to re-run (in duplicate) using the Ultra-Sensitive AMH Ansh Labs assay, blinded to the original result. Samples that returned low results were run on the picoAMH Ansh Labs assay. Performance characteristics and linearity of the new assays were also assessed.All serum samples from patients referred for AMH at Glasgow Royal Infirmary between January and February 2013 were eligible for inclusion. Investigators were blinded to any identifiable information regarding the patients, including sex, age and reason for AMH measurement.Intra-assay and inter-assay coefficients of variation of the Ultra-Sensitive and picoAMH assays were ≤6.0 and ≤10.7%, respectively, over a range of concentrations. The assays had mean linearity of 98 and 97% over the dilution range of 1:2-1:16 and 1:2-1:8, respectively. The limit of detection of the ultrasensitive assay was calculated to be 0.34 pmol/l. For 166 samples which provided a quantitative result on the Gen II and Ultra-Sensitive Ansh Labs assays, the median (interquartile range) was 12.2 (3.4-29.3) pmol/l and 20.0 (6.6-36.8) pmol/l, respectively (P0.0001). The Passing-Bablok regression equation (in pmol/l) was y (Ultra-Sensitive) = 1.7 + 1.4 × Gen II. More samples were below the clinical cut-off of 5.4 pmol/l using the Gen II assay (a difference between paired proportions of 15.0%, P0.001). Fifteen of the 22 undetectable samples yielded a measurable concentration result on the picoAMH assay.The present study is a pragmatic assessment of the new assay under ideal conditions. Lot-to-lot variation could not be assessed. Demographics and outcomes of patients referred for AMH measurement were not known.The Ansh Labs Ultra-Sensitive assay performance characteristics are similar to the Gen II assay and may be suitable for clinical and epidemiological use. Enhanced sensitivity of the Ansh Labs picoAMH assay enables measurement of low AMH concentrations. These results re-emphasize the need for an AMH international standard.Ansh Labs provided kits for this study free of charge. The manufacturer played no part in conducting assays or data analysis. S.M.N. has received speaker's fees and participated in advisory boards for Beckman Coulter, Merck Serono, MSD and Ferring regarding AMH. P.W. is supported by British Heart Foundation fellowship FS/12/62/29889. We declare no other financial relationship or competing interests.

Background Aerococcus urinae is an emerging urinary pathogen frequently identified by MALDI-TOF. It is generally susceptible to β-lactams, however, its susceptibility pattern to fluoroquinolones (FQ) remains variable. The goals of this study were (i) to evaluate the performance of the gradient diffusion method (Etest®) to determine FQ resistance compared with broth microdilution (BMD) and (ii) to estimate the resistance rate of A. urinae toward FQ in Quebec hospitals. Methods Two hundred seven consecutive isolates of A. urinae from urinary tract specimens originating from five hospitals in Quebec and Montreal were identified by MALDI-TOF (Vitek-MS and Bruker). All isolates were tested with the BMD and gradient diffusion methods. BMD was carried out in triplicate and was conducted in accordance with CLSI guidelines (M45-A3). Isolates with insufficient growth at 24 hours were reincubated and evaluated at 48 hours. The gradient diffusion method was carried out using Etest® strips on MH agar with 5% sheep blood. Results Of the 207 isolates of A. urinae, 52 (25%) gave uninterpretable results using the BMD method (insufficient growth = 20; trailing = 32). We obtained the following results for the remaining 155 isolates: Susceptible, n (%) Intermediate, n (%) Resistant, n (%) Ciprofloxacin 105 (67%) 16 (10%) 35 (23%) Levofloxacin 114 (74%) 6 (4%) 35 (23%) BMD readings were often complicated by noticeably poor growth. The categorical agreement of the Etest® was 83% for ciprofloxacin and 95% for levofloxacin. Four very major errors were identified in a preliminary manner on 11% (4/35) of the ciprofloxacin-resistant isolates and 11%(4/35) of the levofloxacin-resistant isolates. Agar dilution will be done to confirm these results. Conclusion In our experience, the method recommended by the CLSI for A. urinae susceptibility testing of FQ presented several problems, including insufficient growth and difficult reading. The Etest® appears to be a promising method for susceptibility testing of FQ for urinary tract isolates, but will first require a further comparison with agar dilution methods. In our study, the rate of FQ non-susceptibility of A. urinae was 27% for levofloxacin and 33% for ciprofloxacin. Therefore, FQ cannot be empirically recommended for the treatment of urinary tract infections caused by A. urinae. Disclosures J. M. Leduc, Biomérieux: Investigator, Research grant.

Several cellular pathways control the fine balance between self-renewal and differentiation to maintain leukemia-initiating cell (LIC) function. To identify cellular dependencies with relevance for oncogenic fusion proteins, we performed global proteome profiling. Acute myeloid leukemia (AML) was induced by retroviral expression of either MLL-AF9 (MA9) or AML1-ETO9a (AE) in murine hematopoietic stem and progenitor cells (HSPCs) (Lineage-Sca1+Kit+, LSK) which were subsequently transplanted into irradiated syngeneic recipients. After onset of leukemia, LIC-enriched (GFP+ Kithigh) cells isolated from 4 different primary recipients (per oncogene) were analyzed by in-depth quantitative proteomic analysis using high-resolution mass spectrometry (MS). More than 3,000 proteins were quantified with 868 proteins being differentially expressed between MA9 and AE LIC-enriched populations. In MLL-rearranged (MLLr) cells, gene set enrichment analysis (GSEA) revealed significant enrichment of cellular functions related to protein degradation and proteasome function. As this enrichment is present in MLLr-leukemia but not AE-driven LICs, may indicate an oncogene specific vulnerability. Expression of proteasome subunits is highly heterogeneous between different cell types and therefore may also be influenced by the underlying differentiation stage or oncogenic fusion. In published AML gene-expression datasets, immunoproteasome (IP) subunits PSMB8/LMP7 (p=0.0003***), PSMB9/LMP2 (p=0.0007***) and PSMB10/MECL1 (p Disclosures Heidel: Celgene: Consultancy; Novartis: Consultancy, Research Funding; CTI: Consultancy.

Introduction: The ability to detect and quantitate low frequency T-cell clones enables numerous hematology/oncology research applications, including identification and assessment of biomarkers associated minimal residual disease (MRD). Rare clone detection via NGS requires highly efficient library preparation and accurate sequencing methodologies, because single nucleotide substitution sequencing errors mimic the natural variation in the T-cell repertoire, resulting in detection of artifactual low frequency clones. Here we present an experimental framework and corresponding performance of rare clone detection utilizing the OncomineTM TCR Beta short read (TCRb-SR) assay, using Ampliseq-based library preparation targeting the highly variable CDR3 region of TCRb using either DNA or RNA as input, with sample-to-result in 2 days. Methods: To evaluate detection sensitivity of the TCRB-SR assay, we utilized Jurkat cell line DNA and RNA because the presence of a single T cell clone enables precise control of dilution studies. Commercially procured Jurkat gDNA or RNA was spiked into peripheral blood leukocyte gDNA or RNA from 10-1 to 10-6 absolute clone frequency to create specimens with a known T-cell clone at frequencies commonly observed in MRD research applications. Peripheral blood leukocyte gDNA or RNA was used as the background for spike in studies due to its high T-Cell diversity. Six to twenty technical replicates were analyzed per dilution point, with DNA inputs ranging from 100ng to 1ug and RNA inputs ranging from 25ng to 100ng to evaluate the minimum detectable clone frequency as a function of nucleic acid input. Libraries were prepared following the TCRB-SR manufacturer's instructions for both DNA and RNA, followed by templating and sequencing using Ion Chef and S5 systems. Data processing was performed in Torrent Suite software (v5.10) followed by read alignment to the IMGT database of variable, diversity, and joining genes using Ion Reporter software (v5.10). For the 10-6 target frequency with gDNA as the input, four 1ug libraries were combined for analysis in Ion Reporter. Analytical sensitivity was calculated at each target clone frequency by detection of the Jurkat clone as defined by V-gene, Joining gene, and CDR3 nucleotide sequence. Results: Detection sensitivity was dependent on the amount gDNA or RNA input. For gDNA inputs, we observed 100% sensitivity at 10-3 with 100ng input, 100% sensitivity at 10-4 with 250ng input, 95% sensitivity at 10-5 with 1ug input, and 100% sensitivity at 10-6 with 4ug input. For RNA inputs, we observed 100% sensitivity at 10-5 with 25ng input and 100% sensitivity at 10-6 with 100ng input. In addition, we observe a strong linearity of observed clone frequencies at each dilution level, with an r-squared of 0.97. Conclusions: Here we demonstrate the ability to detect T-cell clones down to 10-6 from gDNA or RNA inputs with high sensitivity and linearity utilizing the OncomineTM TCR Beta short read assay. We present data demonstrating detection of clones with absolute frequencies of 10-6 utilizing 4ug gDNA input or 100ng RNA input, highlighting strong performance at nucleic acid input levels typically seen in clinical research samples. Taken together, we show feasibility for rare clone detection in either gDNA or RNA enabling research and development for T-cell minimal residual disease applications. For research use only, not for use in diagnostic procedures. Disclosures Williamson: Thermo Fisher Scientific: Employment. Looney:Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Sidhu:Thermo Fisher Scientific: Employment. Solano:Thermo Fisher Scientific: Employment. Salazar:Thermo Fisher Scientific: Employment. Tian:Thermo Fisher Scientific: Employment. Ramsamooj:Thermo Fisher Scientific: Consultancy.

ATP-dependent chromatin remodeling complexes, including BAF (BRG1 associated factor) and PBAF (Polybromo-associated BRG1 associated factor), play major roles in development and disease. These complexes contain multiple subunits and utilize ATP to remodel nucleosomes and, as a result, affect downstream processes. Subunits of the BAF and PBAF complexes are frequently mutated in various cancers, including in acute myeloid leukemia. However, the role of distinct members of these complexes in leukemogenesis is poorly understood. BAF and PBAF complexes are distinguished from each other by unique AT-rich interacting domain (ARID) subunits, ARID1A/B and ARID2, respectively. Recently, Arid2 was suggested to have tumor suppressive functions in MLL-AF9 leukemogenesis, however the role of Arid1b is unknown. In this study, we further evaluated the tumor suppressor roles of Arid2 and Arid1b in MLL-AF9 leukemogenesis. To study the effect of the loss of Arid2 and Arid1b on MLL-AF9 leukemia in vitro, hematopoietic stem and progenitor cells from Arid1bfl/fl, Arid2fl/fl and wild type (control) mice were transformed by retroviral expression of MLL-AF9. Arid1b or Arid2 was then deleted by retroviral delivery of Cre-recombinase and cellular phenotypes were characterized by flow cytometry. In vitro characterization revealed Arid1b-/-and Arid2-/-MLL-AF9 cells have similar growth rates to control cells and no changes in cell cycle status and apoptosis. To study leukemogenesis in vivo, two hematopoietic specific conditional knockout mouse models, Vav1Cre, a constitutively active Cre-recombinase, and Mx1Cre, an inducible Cre-recombinase, were used. Primary and secondary transplant recipient mice were assessed for survival, disease burden, and leukemia stem cell frequency. In primary transplantation, leukemic initiation was not altered by the loss of the Arid2. Overall survival of primary recipients and disease burden in moribund mice (percentage of GFP positive MLL-AF9 cells in bone marrow/spleen and spleen weight) were similar to control counterparts. However, in limiting dilution secondary transplantation of Arid2fl/flVav1Cre (Arid2-/-) and wild type Vav1Cre (control) MLL-AF9, median survival of the Arid2-/- cohort decreased 1 to 2 weeks in comparison to the control cohort and there was a two-fold increase in leukemia initiating cell frequency upon deletion of Arid2. Together these results support the previous indication that Arid2 is a tumor suppressor in MLL-AF9 leukemic initiation. To gain insight into the function of Arid1b in MLL-AF9 leukemogenesis, we performed primary transplantation of Arid1bfl/flcells transduced with MLL-AF9. Our data showed that loss of Arid1b enhanced leukemic initiation in the MLL-AF9 leukemia model. One-month post induction with pIpC, Arid1bfl/fl Mx1Cre MLL-AF9 cells accumulated faster in the peripheral blood and primary recipients exhibited decreased median survival compared with wild type Mx1Cre controls. Percentage of GFP+ leukemia cells was higher in the spleen of moribund mice in the Arid1bfl/fl Mx1Cre cohort, compared with the control cohort. Taken together, our data suggests Arid1b, in addition to Arid2, acts as a potential novel tumor suppressor in MLL-AF9 leukemogenesis. To study the mechanism of Arid1b and Arid2's function in MLL-AF9 leukemogenesis, RNA-sequencing of leukemic bone marrow cells was conducted. Consistent with the previous study, Arid2 null leukemia cells had increased Gata2 expression and decreased p57 expression. Gene Set Enrichment Analysis of Arid1b null cells showed enrichment of oncogenic signatures including ATM, Cyclin D and macrophage development as potentially contributing to the underlying tumor suppressive mechanism of Arid1b. In summary, our study uncovered Arid1b, in addition to Arid2, as a potential novel tumor suppressor in MLL-AF9 leukemia. Our study, together with published studies on the requirement of other subunits (SMARCA4, SMARCD2 and DPF2) of the BAF and PBAF complexes in MLL-AF9 leukemia, reveal opposing roles of the components of the BAF and PBAF complexes in leukemogenesis, the mechanism of which require further investigation. Disclosures No relevant conflicts of interest to declare.