BioRssay: an R package for analyses of bioassays and probit graphs.

Dose-response relationships reflect the effects of a substance on organisms, and are widely used in broad research areas, from medicine and physiology, to vector control and pest management in agronomy. Furthermore, reporting on the response of organisms to stressors is an essential component of many public policies (e.g. public health, environment), and assessment of xenobiotic responses is an integral part of World Health Organization recommendations. Building upon an R script that we previously made available, and considering its popularity, we have now developed a software package in the R environment, BioRssay, to efficiently analyze dose-response relationships. It has more user-friendly functions and more flexibility, and proposes an easy interpretation of the results. The functions in the BioRssay package are built on robust statistical analyses to compare the dose/exposure-response of various bioassays and effectively visualize them in probit-graphs.

The within- and between-laboratories reproducibility and predictive capacity of Amino acid Derivative Reactivity Assay using 4 mM test chemical solution: Results of ring study implemented at five participating laboratories.

Amino acid derivative reactivity assay (ADRA) for skin sensitization was adopted as an alternative method in the 2019 OECD Guideline for the Testing of Chemicals (OECD TG 442C). The molar ratio of the nucleophilic reagent to the test chemicals in the reaction solution was set to 1:50. Imamura et al. reported that changing this molar ratio from 1:50 to 1:200 reduced in false negatives and improved prediction accuracy. Hence, a ring study using ADRA with 4 mM of a test chemical solution (ADRA, 4 mM) was conducted at five different laboratories to verify within- and between-laboratory reproducibilities (WLR and BLR, respectively). In this study, we investigated the WLR and BLR using 14 test chemicals grouped into three classes: (1) eight proficiency substances, (2) four test chemicals that showed false negatives in the ADRA with 1 mM test chemical solution (ADRA, 1 mM), but correctly positive in ADRA (4 mM), and (3) current positive control (phenylacetaldehyde) and a new additional positive control (squaric acid diethyl ester). The results showed 100% reproducibility and 100% accuracy for skin sensitization. Hence, it is clear that the ADRA (4 mM) is an excellent test method in contrast to the currently used ADRA (1 mM). We plan to resubmit the ADRA (4 mM) test method to the OECD Test Guideline Group in the near future so that OECD TG 442C could be revised for the convenience and benefit of many ADRA users.

Performance of a Vaginal Panel Assay Compared With the Clinical Diagnosis of Vaginitis.

OBJECTIVE: To compare the performance of vaginitis diagnosis based on clinical assessment to molecular detection of organisms associated with bacterial vaginosis, vulvovaginal candidiasis, and Trichomonas vaginalis using a vaginal panel assay. METHODS: This cross-sectional diagnostic accuracy study included 489 enrolled participants from five collection sites where those with vaginitis symptoms had a vaginal assay swab collected during their visit and a clinical diagnosis made. The swab was later sent to a separate testing site to perform the vaginal panel assay. Outcome measures include positive, negative, and overall percent agreement (and accompanying 95% CIs) of clinical assessment with the vaginal panel assay. P<.05 was used to distinguish significant differences in paired proportions between the vaginal panel assay and clinical diagnosis, using the McNemar test. Inter-rater agreement between the two diagnostic approaches was determined using Cohen's kappa coefficient. RESULTS: Clinical diagnosis had a positive percent agreement with the vaginal panel assay of 57.9% (95% CI 51.5-64.2%), 53.5% (95% CI 44.5-62.4%), and 28.0% (95% CI 12.1-49.4%) for bacterial vaginosis, vulvovaginal candidiasis, and T vaginalis, respectively. Negative percent agreement for clinical diagnosis was 80.2% (95% CI 74.3-85.2%), 77.0% (95% CI 72.1-81.4%), and 99.8% (95% CI 98.7-99.9%), respectively. Sixty-five percent (67/103), 44% (26/59), and 56% (10/18) of patients identified as having bacterial vaginosis, vulvovaginal candidiasis, and T vaginalis by assay, respectively, were not treated for vaginitis based on a negative clinical diagnosis. Compared with the assay, clinical diagnosis had false-positive rates of 19.8%, 23.0%, and 0.2% for bacterial vaginosis, vulvovaginal candidiasis, and T vaginalis, respectively. Significant differences in paired proportions were observed between the vaginal panel assay and clinical diagnosis for detection of bacterial vaginosis and T vaginalis. CONCLUSION: The vaginal panel assay could improve the diagnostic accuracy for vaginitis and facilitate appropriate and timely treatment. FUNDING SOURCE: Becton, Dickinson and Company.

Evaluation of the in situ assay for HBV DNA: An observational real-world study in chronic hepatitis B.

ABSTRACT: The visualization of intrahepatic hepatitis B virus (HBV) DNA by in situ hybridization (ISH) has uncovered some interesting aspects of HBV life cycle at the single-cell level. In the current study, we intend to evaluate the reliability and robustness of this assay in the real-world clinical scenario and its relationship with currently available clinical biomarkers in chronic hepatitis B (CHB) patients.In this cross-sectional study, 94 CHB patients and 10 patients with non-HBV related liver diseases were enrolled. Liver biopsies and routine histopathology analysis were performed. Intrahepatic HBV DNA and viral antigens (HBsAg and HBcAg) were detected by ISH and immunohistochemistry (IHC), respectively. The basic biochemical and virological parameters such as alanine transaminase, serum HBV DNA, and serum HBsAg were measured.The HBV DNA-ISH assay showed 55.8% (53/94 cases) positive rate in CHB patients, no false positive was found in non-HBV related hepatitis. The IHC of HBsAg and HBcAg showed a positive rate of 94.7% (89/94 cases) and 19.5% (17/87 cases), respectively. Quantification of HBV DNA-ISH signal showed a significant correlation with serum HBV DNA (rs = 0.6223, P < .0001). In addition, the staining pattern of HBV DNA in situ in the context of collagen deposition informed the histopathological progression of chronic liver disease.The application of this ISH assay in evaluating intrahepatic viral replication in real-world CHB patients showed favorable performance. It can be a complementation to conventional liver histopathology examination and IHC detection of viral antigens. This methodology provides an intuitive assessment of virological and pathological state of CHB patients, and further supports clinical diagnosis and management.

Cardiotoxicity Hazard and Risk Characterization of ToxCast Chemicals Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes from Multiple Donors.

Heart disease remains a significant human health burden worldwide with a significant fraction of morbidity attributable to environmental exposures. However, the extent to which the thousands of chemicals in commerce and the environment may contribute to heart disease morbidity is largely unknown, because in contrast to pharmaceuticals, environmental chemicals are seldom tested for potential cardiotoxicity. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes have become an informative in vitro model for cardiotoxicity testing of drugs with the availability of cells from multiple individuals allowing in vitro testing of population variability. In this study, we hypothesized that a panel of iPSC-derived cardiomyocytes from healthy human donors can be used to screen for the potential cardiotoxicity hazard and risk of environmental chemicals. We conducted concentration-response testing of 1029 chemicals (drugs, pesticides, flame retardants, polycyclic aromatic hydrocarbons (PAHs), plasticizers, industrial chemicals, food/flavor/fragrance agents, etc.) in iPSC-derived cardiomyocytes from 5 donors. We used kinetic calcium flux and high-content imaging to derive quantitative measures as inputs into Bayesian population concentration-response modeling of the effects of each chemical. We found that many environmental chemicals pose a hazard to human cardiomyocytes in vitro with more than half of all chemicals eliciting positive or negative chronotropic or arrhythmogenic effects. However, most of the tested environmental chemicals for which human exposure and high-throughput toxicokinetics data were available had wide margins of exposure and, thus, do not appear to pose a significant human health risk in a general population. Still, relatively narrow margins of exposure (<100) were estimated for some perfuoroalkyl substances and phthalates, raising concerns that cumulative exposures may pose a cardiotoxicity risk. Collectively, this study demonstrated the value of using a population-based human in vitro model for rapid, high-throughput hazard and risk characterization of chemicals for which little to no cardiotoxicity data are available from guideline studies in animals.

Voltage-sensitive sodium channel (Vssc) mutations associated with pyrethroid insecticide resistance in Aedes aegypti (L.) from two districts of Jeddah, Kingdom of Saudi Arabia: baseline information for a Wolbachia release program.

BACKGROUND: Dengue suppression often relies on control of the mosquito vector, Aedes aegypti, through applications of insecticides of which the pyrethroid group has played a dominant role. Insecticide resistance is prevalent in Ae. aegypti around the world, and the resulting reduction of insecticide efficacy is likely to exacerbate the impact of dengue. Dengue has been a public health problem in Saudi Arabia, particularly in Jeddah, since its discovery there in the 1990s, and insecticide use for vector control is widespread throughout the city. An alternative approach to insecticide use, based on blocking dengue transmission in mosquitoes by the endosymbiont Wolbachia, is being trialed in Jeddah following the success of this approach in Australia and Malaysia. Knowledge of insecticide resistance status of mosquito populations in Jeddah is a prerequisite for establishing a Wolbachia-based dengue control program as releases of Wolbachia mosquitoes succeed when resistance status of the release population is similar to that of the wild population. METHODS: WHO resistance bioassays of mosquitoes with deltamethrin, permethrin and DDT were used in conjunction with TaqMan® SNP Genotyping Assays to characterize mutation profiles of Ae. aegypti. RESULTS: Screening of the voltage-sensitive sodium channel (Vssc), the pyrethroid target site, revealed mutations at codons 989, 1016 and 1534 in Ae. aegypti from two districts of Jeddah. The triple mutant homozygote (1016G/1534C/989P) was confirmed from Al Safa and Al Rawabi. Bioassays with pyrethroids (Type I and II) and DDT showed that mosquitoes were resistant to each of these compounds based on WHO definitions. An association between Vssc mutations and resistance was established for the Type II pyrethroid, deltamethrin, with one genotype (989P/1016G/1534F) conferring a survival advantage over two others (989S/1016V/1534C and the triple heterozygote). An indication of synergism of Type I pyrethroid activity with piperonyl butoxide suggests that detoxification by cytochrome P450s accounts for some of the pyrethroid resistance response in Ae. aegypti populations from Jeddah. CONCLUSIONS: The results provide a baseline for monitoring and management of resistance as well as knowledge of Vssc genotype frequencies required in Wolbachia release populations to ensure homogeneity with the target field population. Vssc mutation haplotypes observed show some similarity with those from Ae. aegypti in southeast Asia and the Indo-Pacific, but the presence of the triple mutant haplotype in three genotypes indicates that the species in this region may have a unique population history.

Performance of 4 Automated SARS-CoV-2 Serology Assay Platforms in a Large Cohort Including Susceptible COVID-19-Negative and COVID-19-Positive Patients.

BACKGROUND: Anti-SARS-CoV-2 serological responses may have a vital role in controlling the spread of the disease. However, the comparative performance of automated serological assays has not been determined in susceptible patients with significant comorbidities. METHODS: In this study, we used large numbers of samples from patients who were negative (n = 2030) or positive (n = 112) for COVID-19 to compare the performance of 4 serological assay platforms: Siemens Healthineers Atellica IM Analyzer, Siemens Healthineers Dimension EXL Systems, Abbott ARCHITECT, and Roche cobas. RESULTS: All 4 serology assay platforms exhibited comparable negative percentage of agreement with negative COVID-19 status ranging from 99.2% to 99.7% and positive percentage of agreement from 84.8% to 87.5% with positive real-time reverse transcriptase PCR results. Of the 2142 total samples, only 38 samples (1.8%) yielded discordant results on one or more platforms. However, only 1.1% (23/2030) of results from the COVID-19-negative cohort were discordant. whereas discordance was 10-fold higher for the COVID-19-positive cohort, at 11.3% (15/112). Of the total 38 discordant results, 34 were discordant on only one platform. CONCLUSIONS: Serology assay performance was comparable across the 4 platforms assessed in a large population of patients who were COVID-19 negative with relevant comorbidities. The pattern of discordance shows that samples were discordant on a single assay platform, and the discordance rate was 10-fold higher in the population that was COVID-19 positive.

Z' Does Not Need to Be > 0.5.

The assay metric Z' has come to play a critical gatekeeping role in determining whether high-throughput assays can be performed. While Z' is commonly required to be > 0.5, this expectation is not well supported. Requiring Z' > 0.5 likely prevents many potentially useful phenotypic and cell-based screens from being conducted, and causes other assays to be conducted under extreme conditions that may prevent activity from being found. We used power analysis and a novel numerical simulation approach to determine how Z' reflects assay performance under a variety of conditions. Our results show that assays with Z' > 0.5 perform better than assays with lower Z', but when an appropriate threshold is selected, assays with Z' < 0.5 can almost always find useful compounds without generating too many false positives. We provide a method that will allow researchers to estimate how to set an appropriate threshold for their assay. We suggest that instead of always requiring Z' > 0.5, assays with Z' < 0.5 should be performed when they can be justified in terms of the importance of the target and the limitations of alternate assay formats.

Hospital physicians' experiences with procalcitonin - implications for antimicrobial stewardship; a qualitative study.

BACKGROUND: Procalcitonin is an inflammatory biomarker that is sensitive for bacterial infections and a promising clinical decision aid in antimicrobial stewardship programs. However, there are few studies of physicians' experiences concerning the use of PCT. The objective of this study was to investigate whether hospital physicians' experience with procalcitonin after 18 months of use can inform the PCT implementation in antimicrobial stewardship programs. MATERIALS/METHODS: We deployed a qualitative approach using semi-structured interviews with 14 hospital physicians who had experience with procalcitonin in clinical practice. Interviews were audio-taped, transcribed verbatim and analysed using thematic analysis. RESULTS: Physicians reported a knowledge gap, which made them uncertain about the appropriate procalcitonin use, interpretation, and trustworthiness. Simultaneously, the physicians experienced procalcitonin as a useful clinical decision aid but emphasised that their clinical evaluation of the patient was the most important factor when deciding on antibiotic treatment. CONCLUSIONS: Procalcitonin was regarded a helpful clinical tool, but the physicians called for more knowledge about its appropriate uses. Active implementation of unambiguous procalcitonin algorithms and physician education may enhance the utility of the test as an antimicrobial stewardship adjunct.

Accounting for Multiple Comparisons in Statistical Analysis of the Extensive Bioassay Data on Glyphosate.

Glyphosate is a widely used herbicide worldwide. In 2015, the International Agency for Research on Cancer (IARC) reviewed glyphosate cancer bioassays and human studies and declared that the evidence for carcinogenicity of glyphosate is sufficient in experimental animals. We analyzed 10 glyphosate rodent bioassays, including those in which IARC found evidence of carcinogenicity, using a multiresponse permutation procedure that adjusts for the large number of tumors eligible for statistical testing and provides valid false-positive probabilities. The test statistics for these permutation tests are functions of p values from a standard test for dose-response trend applied to each specific type of tumor. We evaluated 3 permutation tests, using as test statistics the smallest p value from a standard statistical test for dose-response trend and the number of such tests for which the p value is less than or equal to .05 or .01. The false-positive probabilities obtained from 2 implementations of these 3 permutation tests are: smallest p value: .26, .17; p values ≤ .05: .08, .12; and p values ≤ .01: .06, .08. In addition, we found more evidence for negative dose-response trends than positive. Thus, we found no strong evidence that glyphosate is an animal carcinogen. The main cause for the discrepancy between IARC's finding and ours appears to be that IARC did not account for the large number of tumor responses analyzed and the increased likelihood that several of these would show statistical significance simply by chance. This work provides a more comprehensive analysis of the animal carcinogenicity data for this important herbicide than previously available.

A curve similarity approach to parallelism testing in bioassay.

Potency determination via bioassay is a relative measure that requires an evaluation of parallelism between the dose-response relationships of a reference standard and a sample material. Typical approaches for assessing parallelism include difference ([Formula: see text]-value) and equivalence tests. These traditional methods rely on a statistical assessment of model parameters as opposed to direct evaluation of the similarity of the dose-response curves. We propose a simple curve similarity approach that tests the hypothesis that the sample material is a dilution or concentration of the reference standard. The test is achieved by quantifying and normalizing the total area between the two curves and provides a single composite measure of parallelism. Both a frequentist and a Bayesian approach to the test are provided. We show through a simulation study that the curve similarity approach overcomes the drawbacks of the traditional methods and is effective at detecting parallelism and non-parallelism for bioassays.

Interleukin-6 signaling requires only few IL-6 molecules: Relation to physiological concentrations of extracellular IL-6.

INTRODUCTION: The aim of this study was to give quantitative insight into the number of cytokine molecules needed to activate a target cell and relate this to the physiological consequences of the amounts of cytokines typically detectable in humans. As a model system blood interleukin-6 (IL-6) was chosen since this cytokine is one of the most studied and clinically monitored cytokines, and because of the tools for the present investigations such as fully bioactive iodinated recombinant IL-6, cellular cytokine binding assays, and bioassays have been thoroughly validated. METHODS: The key intermediates of the basic equilibrium principles that govern cytokine binding and exchange were deduced and applied on concrete estimations of cellular and extracellular IL-6 binding in the bloodstream based on experimental binding data and data from the literature. In parallel, in vitro cellular IL-6 binding data was substantiated by paired measurements of IL-6 bioactivity on IL-6 sensitive B9 hybridoma cells. RESULTS: Blood leucocytes and B9 cells expressed 50 to 300, 10 to 20 picomolar affinity, IL-6 binding sites per cell and at physiological concentrations of IL-6 less than 10 IL-6 molecules seemed to be bound to blood cells. Nonetheless, binding off as few as four IL-6 molecules per cell seemed to result in statistically significant bioactivity, whereas binding of 16 IL-6 molecules triggered extensive cellular responses. CONCLUSION: Together, the estimations and the measurements support the notion that target cells with more than 100 bioactive cytokine receptors per cell, such as T cells and hepatocytes, are likely to be under steady and substantial cytokine-induced endocrine activation.

Clinical Significance of Low Serum Cryptococcal Antigen Titers by Lateral Flow Assay in Immunocompromised Patients: a Retrospective Case-Control Study.

Cryptococcus species are associated with invasive fungal infections in immunosuppressed individuals. The clinical significance of low-titer cryptococcal antigen (CrAg) by lateral flow assay is frequently uncertain. We investigated the correlation of low CrAg titers with disease in an immunocompromised patient population. Patients with first-time positive CrAg results with low serum titers (≤1:10) at two medical centers (Los Angeles, CA) from April 2014 to July 2018 were included. Age-matched controls with high (≥1:20) and negative titers were selected. We extracted medical records for pertinent clinical, radiologic, and laboratory data for cryptococcal disease. From 2,196 serum samples submitted for CrAg testing, 96 cases were included (32 each in low-titer, high-titer, and negative-titer groups). One or more immunocompromising condition was identified in 95% of patients, including HIV infection (45%), solid organ transplant (26%), and cirrhosis (22%). Pulmonary cryptococcosis was diagnosed in 9 (28%) low-titer and 8 (25%) high-titer patients (P = 1.00). Disseminated cryptococcosis occurred in 7 (22%) low-titer and 15 (47%) high-titers cases (P = 0.064). Titers ≤1:10 more frequently represented isolated antigenemia in HIV-positive than non-HIV, immunocompromised patients (P < 0.001). Follow-up testing in patients with ≤1:5 titers (n = 21) showed persistently low titers in 6 of 12 instances and increased titers in 2 cases. Twenty-seven patients with low CrAg titers were treated with antifungal therapy and 22 (81%) responded well clinically. Low-serum CrAg titers (≤1:10) correlated with cryptococcal disease in a substantial proportion of non-HIV immunocompromised patients and should prompt careful clinical workup for cryptococcal infection.

Optimal designs for asymmetric sigmoidal response curves in bioassays and immunoassays.

The 5-parameter logistic (5PL) model is frequently used to model and analyze responses from bioassays and immunoassays which can be skewed. Various types of optimal experimental designs for 2, 3 and 4-parameter logistic models have been reported but not for the more complicated 5PL model. We construct different types of optimal designs for studying various features of the 5PL model and show that commonly used designs in bioassays and immunoassays are generally inefficient for statistical inference. To facilitate use of such designs in practice, we create a user-friendly software package to generate various tailor-made optimal designs for the 5PL model and evaluate robustness properties of a design under a variation of criteria, model forms and misspecification in the nominal values of the model parameters.

Comparisons of outlier tests for potency bioassays.

Potency bioassays are used to measure biological activity. Consequently, potency is considered a critical quality attribute in manufacturing. Relative potency is measured by comparing the concentration-response curves of a manufactured test batch with that of a reference standard. If the curve shapes are deemed similar, the test batch is said to exhibit constant relative potency with the reference standard, a critical requirement for calibrating the potency of the final drug product. Outliers in bioassay potency data may result in the false acceptance/rejection of a bad/good sample and, if accepted, may yield a biased relative potency estimate. To avoid these issues, the USP<1032> recommends the screening of bioassay data for outliers prior to performing a relative potency analysis. In a recently published work, the effects of one or more outliers, outlier size, and outlier type on similarity testing and estimation of relative potency were thoroughly examined, confirming the USP<1032> outlier guidance. As a follow-up, several outlier detection methods, including those proposed by the USP<1010>, are evaluated and compared in this work through computer simulation. Two novel outlier detection methods are also proposed. The effects of outlier removal on similarity testing and estimation of relative potency were evaluated, resulting in recommendations for best practice.

The diagnostic performance of rapid diagnostic tests and microscopy for malaria diagnosis in eastern Sudan using a nested polymerase chain reaction assay as a reference standard.

BACKGROUND: Accurate diagnosis of malaria infection is essential for successful control and management of the disease. Both microscopy and rapid diagnostic tests (RDTs) are recommended for malaria diagnosis, however, RDTs are more commonly used. The aim of the current study was to assess the performance of microscopy and RDTs in the diagnosis of Plasmodium falciparum infection using a nested polymerase chain reaction (PCR) assay as the gold standard. METHODS: A cross-sectional study was carried out in Kassala Hospital, eastern Sudan. A total of 341 febrile participants of all ages were recruited. Blood specimens were collected and malaria testing was performed using an RDT (SD Bioline Malaria Ag Pf), microscopy and nested PCR. The sensitivity, specificity, positive and negative predictive values (PPV and NPV, respectively) of microscopy and the RDT were investigated. RESULTS: The prevalence of P. falciparum malaria infections in this study was 22.9%, 24.3% and 26.7% by PCR, microscopy and RDT, respectively. Compared with microscopy, the RDT had slightly higher sensitivity (80.7% vs 74.3%; p=0.442), equivalent specificity (89.3% vs 90.4%), a similar PPV (69.2% vs 69.8%) and a higher NPV (94.0% vs 92.2%). CONCLUSIONS: The diagnostic performance of the RDT was better than that of microscopy in the diagnosis of P. falciparum malaria when nested PCR was used as the gold standard.

Factors affecting salivary α-amylase levels measured with a handheld biosensor and a standard laboratory assay.

OBJECTIVES: A handheld biosensor for measuring salivary α-amylase (sAA) was developed for convenient on-site measurement. Previous studies reported some discrepancies in sAA levels measured with a biosensor and a standard assay. This study aimed to compare sAA levels measured with three different methods and the factors affecting its levels. METHODS: Thirty-eight participants collected saliva two times for three measurements. First, the collector strip was placed under the tongue for 2 minutes, then the strip was used to measure sAA level on-site immediately (intraoral biosensor; method 1). Then, a participant pooled the saliva for 4 minutes and collected the saliva into the tube which was aliquoted to measure in a laboratory with a handheld biosensor (extraoral biosensor; method 2) and with a standard enzyme kinetic assay (EKA; method 3). Additional experiments were carried out to compare the levels of sAA measured with differences in pooling time and positioning of the collector strip. RESULTS: A high correlation of sAA levels between an extraoral and an EKA measurement (r = 0.989) was observed, while sAA levels measured with an intraoral method showed a significant but weaker correlation with either an EKA (r = 0.475) or an extraoral method (r = 0.436). Saliva pooling time and positioning of the collector strip significantly affected sAA levels. CONCLUSIONS: A handheld biosensor is valid to measure sAA levels extraorally. For an intraoral measurement, pooling time and positioning of the collector strip need to be taken into account.

An open-source software analysis package for Microspheres with Ratiometric Barcode Lanthanide Encoding (MRBLEs).

Multiplexed bioassays, in which multiple analytes of interest are probed in parallel within a single small volume, have greatly accelerated the pace of biological discovery. Bead-based multiplexed bioassays have many technical advantages, including near solution-phase kinetics, small sample volume requirements, many within-assay replicates to reduce measurement error, and, for some bead materials, the ability to synthesize analytes directly on beads via solid-phase synthesis. To allow bead-based multiplexing, analytes can be synthesized on spectrally encoded beads with a 1:1 linkage between analyte identity and embedded codes. Bead-bound analyte libraries can then be pooled and incubated with a fluorescently-labeled macromolecule of interest, allowing downstream quantification of interactions between the macromolecule and all analytes simultaneously via imaging alone. Extracting quantitative binding data from these images poses several computational image processing challenges, requiring the ability to identify all beads in each image, quantify bound fluorescent material associated with each bead, and determine their embedded spectral code to reveal analyte identities. Here, we present a novel open-source Python software package (the mrbles analysis package) that provides the necessary tools to: (1) find encoded beads in a bright-field microscopy image; (2) quantify bound fluorescent material associated with bead perimeters; (3) identify embedded ratiometric spectral codes within beads; and (4) return data aggregated by embedded code and for each individual bead. We demonstrate the utility of this package by applying it towards analyzing data generated via multiplexed measurement of calcineurin protein binding to MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding) containing known and mutant binding peptide motifs. We anticipate that this flexible package should be applicable to a wide variety of assays, including simple bead or droplet finding analysis, quantification of binding to non-encoded beads, and analysis of multiplexed assays that use ratiometric, spectrally encoded beads.

Insufficient harmonization of antibiotics assays - Polish experience with an external quality assessment program in the years 2011-2018.

INTRODUCTION: Treatment with vancomycin and gentamycin requires strict monitoring of its serum concentration for proper dosage optimization. This study aimed to assess the quality and the harmonization of antibiotics assays in Polish laboratories. MATERIALS AND METHODS: 413 results of vancomycin and 148 results of gentamycin assays obtained from Polish laboratories in 30 international external quality assessment (EQA) surveys carried out from March 2011 to May 2018 were analyzed. RESULTS: Interlaboratory robust coefficients of variation (rCVs) in particular surveys comprised between 1.3 and 47.2% for vancomycin, and between 1.8 and 34.2% for gentamycin. The percentage of the results with the difference above acceptable limit ±10% from the target value established for own method group was 25.7% for vancomycin and 25.6% for gentamycin. When the difference was established according to target value for all methods, the percentage of results outside the acceptable limit was 2-fold higher on average (54.8% for vancomycin and 43.2% for gentamycin). The comparison of target values for methods revealed statistically significant differences between analytical systems used (p < .0001). The highest difference was 40% for vancomycin and 12% for gentamycin. CONCLUSIONS: The present analysis revealed high dispersion of the antibiotics assays results in Polish laboratories. Moreover, vancomycin and gentamycin results differed significantly in a way dependent on the analytical system used. There appear to be an urgent need for harmonization of methods used for vancomycin and gentamycin measurement.

Impact of an electronic decision support rule on ESR/CRP co-ordering rates in a community health system and projected impact in the tertiary care setting and a commercially insured population.

INTRODUCTION: Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are common laboratory assays used as markers of inflammation. ESR suffers from higher false positive and false negative rates than CRP. To that end, the American Board of Internal Medicine's (ABIM's) Choosing Wisely campaign has recommended against ESR testing for those with undiagnosed conditions in favor of CRP testing. This study describes the impact of a computerized provider order entry (CPOE) decision support rule against ESR/CRP co-ordering within a community health system that predates the ABIM's Choosing Wisely national guidance. To demonstrate the potential impact of such a CPOE rule within other healthcare settings, ESR/CRP ordering data from a multi-site tertiary care practice and from the commercially insured population in the OptumLabs® Data Warehouse (OLDW) were analyzed and the relative reduction in ESR/CRP co-ordering achieved within the community health system was projected onto these populations. MATERIALS AND METHODS: ESR and/or CRP orders from a community health system were assessed from 2012 to 2016. Co-ordering and test concordance rates between ESR and CRP were compared before and after CPOE decision support rule launch. Similarly, ESR/CRP co-ordering across three tertiary care sites from 2015 to 2016 and the OLDW from 2009 to 2013 were assessed and the co-ordering rate reduction achieved in the community health system was mathematically projected onto these populations. Estimated payer savings from the rule's effect were calculated within each population using Medicare reimbursement rates. RESULTS: The CPOE decision support rule realized an unadjusted 42% relative rate reduction in ESR/CRP co-ordering within the community health system yielding an annual payer savings of $15,000 with a modest increase in ESR/CRP concordance rates. Projecting a 40% relative reduction in ESR/CRP co-ordering rates from a similarly effective CPOE rule, annual payer cost reductions exceeding $100,000 within a multi-site tertiary care setting and $1,000,000 within the OLDW would be expected. CONCLUSION: ESR/CRP co-ordering represents an opportunity to eliminate testing waste and reduce payer costs. A CPOE decision support rule stably reduces ESR/CRP co-ordering rates. Similar results may occur as one component of new commercially available decision support platforms.