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Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance.

Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein (PrP Sc ). Recent studies applied the RT-QuIC methodology to cerebrospinal fluid (CSF) for diagnosing human prion diseases. However, to date, there has not been a formal multi-centre assessment of the reproducibility, validity and stability of RT-QuIC in this context, an indispensable step for establishment as a diagnostic test in clinical practice. In the present study, we analysed CSF from 110 prion disease patients and 400 control patients using the RT-QuIC method under various conditions. In addition, “blinded” ring trials between different participating sites were performed to estimate reproducibility. Using the previously established cut-off of 10,000 relative fluorescence units (rfu), we obtained a sensitivity of 85 % and a specificity of 99 %. The multi-centre inter-laboratory reproducibility of RT-QuIC revealed a Fleiss’ kappa value of 0.83 (95 % CI: 0.40–1.00) indicating an almost perfect agreement. Moreover, we investigated the impact of short-term CSF storage at different temperatures, long-term storage, repeated freezing and thawing cycles and the contamination of CSF with blood on the RT-QuIC seeding response. Our data indicated that the PrP Sc seed in CSF is stable to any type of storage condition but sensitive to contaminations with blood (>1250 erythrocytes/μL), which results in a false negative RT-QuIC response. Fresh blood-contaminated samples (3 days) can be rescued by removal of erythrocytes. The present study underlines the reproducibility and high stability of RT-QuIC across various CSF storage conditions with a remarkable sensitivity and specificity, suggesting RT-QuIC as an innovative and robust diagnostic method.

Background The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration assays; the neutral red uptake (NRU), resazurin reduction (RES) and sulforhodamine B (SRB) assays, in comparison to the MTT assay. Interference between the MTT assay and three glycolysis inhibitors, 2-deoxyglucose, 3-bromopyruvate and lonidamine, was investigated. Results Data indicate that the NRU, RES and SRB assays showed the smallest variability across the linear range, while the largest variation was observed for the MTT assay. This implies that these assays would more accurately detect small changes in cell number than the MTT assay. The SRB assay provided the most reproducible results as indicated by the coefficient of determination after a limited number of experiments. The SRB assay also produced the lowest variance in the derived 50% inhibitory concentration (IC 50 ), while IC 50 concentrations of 3-bromopyruvate could not be detected using either the MTT or RES assays after 24 hours incubation. Interference in the MTT assay was observed for all three tested glycolysis inhibitors in a cell-free environment. No interferences were observed for the NRU, SRB or RES assays. Conclusions This study demonstrated that the MTT assay was not the best assay in a number of parameters that must be considered when a cell enumeration assay is selected: the MTT assay was less accurate in detecting changes in cell number as indicated by the variation observed in the linear range, had the highest variation when the IC 50 concentrations of the glycolysis inhibitors were determined, and interference between the MTT assay and all the glycolysis inhibitors tested were observed. The SRB assay performed best overall considering all of the parameters, suggesting that it is the most suitable assay for use in preclinical screening of novel therapeutic compounds with oxido-reductive potential.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

The parasitic mite Varroa destructor and the associated viruses it transmits are responsible for most instances of honey bee colony losses in the United States. As such, beekeepers utilize miticides to control Varroa populations. Widespread resistance has developed to the miticides fluvalinate and coumaphos. However, Varroa has largely maintained susceptibility to amitraz despite a long and extensive use history. Anecdotal reports of reduced amitraz effectiveness have been a widely discussed contemporary issue among commercial beekeepers. Amitraz resistance was measured by in vitro bioassays with technical amitraz as well as Apivar® efficacy tests. Amitraz resistance was evaluated in commercial beekeeping operations in Louisiana, New York, and South Dakota with a long history of amitraz use. This research shows that amitraz remains an effective Varroa control product in many operations. However, apiaries across operations displayed a wide range of amitraz resistance from no resistance to high resistance that resulted in Varroa control failure. The resistance ratios from in vitro amitraz bioassays were correlated with reduced Apivar® efficacy, demonstrating bona fide cases of Varroa control failures due to amitraz resistance. Therefore, amitraz resistance monitoring protocols need to be developed. A resistance monitoring network should be established to ensure the sustainability of miticide use for Varroa control.

Innovation in mass spectrometry-based methods to both quantify and perform discovery has blurred the lines between targeted and untargeted assays of biospecimens. Continuous data-concentrations or intensity values generated from both methods-can be used in statistical analysis to determine associations with health outcomes, but concentration values are needed to compare measurements from one study to another to inform policy making decisions and to develop clinically relevant thresholds. As a single solution for discovery and quantitation, new hybrid-type assays derive concentration values for chemicals or metabolites but with varying degrees of uncertainty that may be greater than traditional quantitative assays. There is no current single standard to guide reporting bioassay concentrations or their uncertainty in concentration values from hybrid assays. Even when measures are robust, obtained with high scientific rigor, and provide valuable data toward risk assessment, unknown uncertainty can lead to bias in interpretation of reported data or omission of reported data that does not meet the strict criteria for absolute quantitation. The objective of this commentary is to articulate a scheme that enables investigators across bioanalytical fields to easily report analyte measurement assurance on the same scale from quantitative, untargeted, or hybrid assays that include a range of concentration confidences. We propose a simple scheme to report concentrations for targeted and untargeted analytes. Level 1 is a confirmed concentration following established tolerances in a fully quantitative assay while level 5 is a tentative intensity from a typical untargeted assay. This framework enables easy communication of uncertainty in concentration measurements to aid cross-validation, meta-analysis, and extrapolation across studies. It will facilitate interpretation while supporting analytical advancement and allow clear and concise measurement reporting across a broad range of confidences. https://doi.org/10.1289/EHP15465.

One quantitative liquid handling method in conventional assay processes is pipetting, which delivers a precise volume of one sample at a time. As this process becomes laborious and time-consuming as the number of samples increases, researchers in individual laboratories need a way to conduct large-scale assays in a reasonable amount of time and at an affordable cost. Here we report a novel handling technique of chemical substances termed ‘partipetting’, which allows the one-step pipetting of various chemical-laden hydrogels. We pipette and assemble various types of encoded chemical-laden microparticles in microwell arrays in parallel. The combination of this heterogeneous particle chip and a cell chip induces the release of the chemicals from the hydrogels and, eventually, the chemicals treat the targets. Based on bioassay applications using partipetting, we show its capability in large-scale bioassays, without the need for high-throughput bioassay resources, owing to a reduction in the assay costs and time. High-throughput screening allows for the rapid assessment of biochemical compounds and processes, but with increasing scale comes increasing costs. Here, the authors use an array of lithographically encoded hydrogel microparticles as a more accessible screening technique.

Selected reaction monitoring (SRM) is a powerful mass spectrometry technology to reliably detect selected protein targets, even those at very low abundance, but requires tedious assay development for each protein of interest. High-throughput SRM assay development is now possible by using crude synthetic peptide libraries without purification to represent each protein target. Selected reaction monitoring (SRM) uses sensitive and specific mass spectrometric assays to measure target analytes across multiple samples, but it has not been broadly applied in proteomics owing to the tedious assay development process for each protein. We describe a method based on crude synthetic peptide libraries for the high-throughput development of SRM assays. We illustrate the power of the approach by generating and applying validated SRM assays for all Saccharomyces cerevisiae kinases and phosphatases.

Chemiluminescence-based bioassays have become increasingly important in clinical, pharmaceutical, environmental, and food safety fields owing to their high sensitivity, wide linear range, and simple instrumentation. During the past decade, it has been found that metal nanoparticles can initiate various liquid-phase chemiluminescence reactions as catalysts, reductants, energy acceptors, and nanosized reaction platforms owing to their unique optical, catalytic, and surface properties and chemical reactivity, which are very important for chemiluminescence bioassays based on metal nanoparticles as nanoprobes or a nanointerface. In this article, we summarize recent progress in metal-nanoparticle-initiated liquid-phase chemiluminescence, including reaction systems, mechanisms, and their applications in chemiluminescence-based bioassays, especially for immunoassays, DNA assays, aptamer-based assays, high-performance liquid chromatography or capillary electrophoresis analysis, and flow injection analysis. Figure Comprehensive summary of metal nanoparticle (NP)-involved chemiluminescence (CL) systems and their applications. CE capillary electrophoresis, HPLC highperformance liquid chromatography