Total and viable microbial cell counts are increasingly important for applications including live biotherapeutic products, food safety, and probiotics. In microbiology, cells are quantified using methods such as colony forming unit (CFU), flow cytometry, and polymerase chain reaction (PCR), but different methods measure different aspects of the cells (measurands), and results may not be directly comparable across methods. In the absence of a ground-truth reference material for cell count, one cannot quantify the accuracy of any cell counting method, which limits method performance assessments and comparisons. Herein, a modified analysis of cell counting methods based on the ISO 20391-2:2019 standard was developed and demonstrated for microbial cell samples diluted over a log-scale range of concentrations. Escherichia coli samples ranging in concentration from ~5 x 105 cells/mL to 2 x 107 cells/mL were quantified using CFU, Coulter principle, fluorescence flow cytometry, and impedance flow cytometry. Quality metrics modified from the ISO standard were calculated for each method and shown to be repeatable across replicate experiments. The quality metrics illustrate large differences in proportionality and variability across methods, with total cell counts in good agreement and viable cell count having more variability. As the ISO standard is meant to guide fit-for-purpose method selection, interpretation of the results and quality metrics can drive method choice and optimization. The framework introduced here will help researchers select fit-for-purpose counting methods for quantification of microbial total and viable cells across a range of applications.

Human untargeted metabolomics studies annotate only ~10% of molecular features. We introduce reference-data-driven analysis to match metabolomics tandem mass spectrometry (MS/MS) data against metadata-annotated source data as a pseudo-MS/MS reference library. Applying this approach to food source data, we show that it increases MS/MS spectral usage 5.1-fold over conventional structural MS/MS library matches and allows empirical assessment of dietary patterns from untargeted data.

The COVID-19 pandemic has highlighted the benefits of wastewater surveillance to supplement clinical data. Numerous online information dashboards have been rapidly, and typically independently, developed to communicate environmental surveillance data to public health offi-cials and the public. In this study, we review dashboards presenting SARS-CoV-2 wastewater data and propose a path toward harmonization and improved risk communication. A list of 127 dashboards representing 27 countries was compiled. The variability was high and encom-passed aspects including the graphics used for data presentation (e.g., line/bar graphs, maps, and tables), log versus linear scale, and 96 separate ways of labeling SARS-CoV-2 wastewater concentrations. Globally, dashboard presentations also differed by region. Approximately half of the dashboards presented clinical case data, and 25% presented variant monitoring. Only 30% of dashboards provided downloadable source data. While any single dashboard is likely useful in its own context and locality, the high variation across dashboards at best prevents optimal use of wastewater surveillance data on a broader geographical scale and at worst could lead to risk communication issues and the potential for public health miscommunication. There is a great opportunity to improve scientific communication through the adoption of uniform data presentation conventions, standards, and best practices in this field.

Wastewater surveillance is a promising approach to monitor biological and chemical contaminants on a community level in support of public health and safety decisions. Wastewater surveillance has recently been shown highly effective for the early detection of SARS-CoV-2 outbreaks within specific populations such as communities or buildings. As such, significant advances have been made in wastewater surveillance since the start of the COVID-19 pandemic. Nevertheless, many challenges remain to be addressed to establish an enduring capability for wastewater surveillance poised to respond to new targets as they emerge. The Standards for an Enduring Capability in Wastewater Surveillance for Public Health Workshop, co-sponsored by the National Institute of Standards and Technology and the Department of Homeland Security, aimed to identify these challenges and identify potential standards-based solutions to address them. This free, three-day, virtual workshop convened over 500 participants representing over 20 countries who are involved in all aspects of wastewater surveillance from sampling and testing methods to results/reporting and use of data, and represented organizations including government, public health, testing and manufacturing, academia, and non-profits. The presentations, panel discussions, and polling led to the emergence of three themes for standards for wastewater surveillance: concordance without constraints, nationally consistent, and building for the next pandemic.

The COVID-19 pandemic highlighted a wide range of public health system challenges for infectious disease surveillance. The discovery that the SARS-CoV-2 virus was shed in feces and can be characterized using PCR-based testing of sewage samples offers new possibilities and challenges for wastewater surveillance (WWS). However, WWS standardization of practices is needed to provide actionable data for a public health response. A workshop was convened consisting of academic, federal government, and industry stakeholders. The objective was to review WWS sampling protocols, testing methods, analyses, and data interpretation approaches for WWS employed nationally and identify opportunities for standardizing practices, including the development of documentary standards or reference materials in the case of SARS-CoV-2 surveillance. Other WWS potential future threats to public health were also discussed. Several aspects of WWS were considered and each offers the opportunity for standards development. These areas included sampling strategies, analytical methods, and data reporting practices. Each of these areas converged on a common theme, the challenge of results comparability across facilities and jurisdictions. For sampling, the consensus solution was the development of documentary standards to guide appropriate sampling practices. In contrast, the predominant opportunity for analytical methods was reference material development, such as PCR-based standards and surrogate recovery controls. For data reporting practices, the need for establishing the minimal required metadata, a metadata vocabulary, and standardizing data units of measure including measurement threshold definitions was discussed. Beyond SARS-CoV-2 testing, there was general agreement that the WWS platform will continue to be a valuable tool for a wide range of public health threats and that future cross-sector engagements are needed to guide an enduring WWS capability.

On September 17, 2020, NIST hosted a virtual workshop to launch the Rapid Microbial Testing Methods (RMTM) Consortium. The RMTM Consortium aims to address the need for measurements and standards to increase confidence in the use of rapid testing for microbial contaminants in regenerative medicine and advanced therapy products. The purpose of the workshop was to publicize the launch of the Consortium, recruit new members, and obtain feedback from stakeholders on both the challenges with respect to applying RMTMs and potential solutions that the Consortium could provide. Over 250 attendees from industry, government, academia, and other organizations participated in the workshop. Invited speakers and panelists provided stimulus for discussion, and feedback from the stakeholders was obtained via question submission and polling. This report summarizes the presentations, discussions, and poll results from the workshop. Overall, the stakeholders supported the three proposed topic areas for the Consortium: reference materials, testing methods, and interlaboratory studies. Based on input from the workshop and poll, the proposed scope of the reference materials topic was expanded to consider DNA and other reference materials, in addition to whole cell materials. Likewise, the testing methods scope was increased to encompass validation schema as well as the test methodologies. As a next step, the Consortium began monthly meetings in November 2020, for members to discuss and finalize the scope and proposed working groups for the Consortium. It is expected that the Consortium will lead to measurement assurance solutions and improved approaches for the community to develop, validate, and implement RMTMs.

Human untargeted metabolomics studies succeed in annotating only ~10% of molecular features. We, therefore, introduce reference data-driven analysis that uses the source data as a pseudo-MS/MS reference library to match against human metabolomics MS/MS data. We demonstrate this approach with food source data, allowing an empirical assessment of dietary patterns from untargeted data but is broadly applicable and provides an additional layer of interpretability to metabolomics data.

We aim to develop a quantitative viability method that distinguishes individual quiescent from dead cells and is measured in time (ns) as a referenceable, comparable quantity. We demonstrate that fluorescence lifetime imaging of an anionic, fluorescent membrane voltage probe fulfills these requirements for Streptococcus mutans. A random forest machine-learning model assesses whether individual S. mutans can be correctly classified into their original populations: stationary phase (quiescent), heat killed and inactivated via chemical fixation. We compare the results to intensity using three models: lifetime variables (τ1 , τ2 and p1 ), phasor variables (G, S) or all five variables, with the five variable models having the most accurate classification. This initial work affirms the potential for using fluorescence lifetime of a membrane voltage probe as a viability marker for quiescent bacteria, and future efforts on other bacterial species and fluorophores will help refine this approach.

The Standards Coordinating Body for Gene, Cell, and Regenerative Medicines and Cell-Based Drug Discovery (SCB) supports the development and commercialization of regenerative medicine products by identifying and addressing industry-wide challenges through standards. Through extensive stakeholder engagement, the implementation of rapid microbial testing methods (RMTMs) was identified as a high-priority need that must be addressed to facilitate more timely release of products. Since 2017, SCB has coordinated efforts to develop standards for this area through surveys, weekly meetings, workshops, leadership in working groups and participation in standards development organizations. This article describes the results of these efforts and discusses the current landscape of RMTMs for regenerative medicine products. Based on discussions with stakeholders across the field, an overview of traditional culture-based methods and limitations, alternative microbial testing technologies and current challenges, fit-for-purpose rapid microbial testing and case studies, risk-based strategies for selection of novel rapid microbial test methods and ongoing standards efforts for rapid microbial testing are captured here. To this end, SCB is facilitating several initiatives to address challenges associated with rapid microbial testing for regenerative medicine products. Two documentary standards are under development: an International Organization for Standardization standard to provide the framework for a risk-based approach to selecting fit-for-purpose assays primarily intended for cell and gene therapy products and an ASTM standard guide focused on sampling methods for microbial testing methods in tissue-engineered medical products. Working with the National Institute of Standards and Technology, SCB expects to facilitate the process of developing publicly available microbial materials for inter-laboratory testing. These studies will help collect the data necessary to facilitate validation of novel rapid methods. Finally, SCB has been working to increase awareness of, dialog about and participation in efforts to develop standards in the regenerative medicine field.

s and biographic information can be found in Appendix A: Speaker Abstracts and Bios. 7 Medena, G. Heijinen, L., Elsinga, G. et al., Presence of SARS-Coronavirus-2 in Sewage. MedRxiv., March 2020. DOI: https://doi.org/10.1101/2020.03.29.20045880 Experts Bharat Ramakrishna, OpenBiome Manoj Dadlani, CosmosID Katarina Papp, Water Quality R&D, Southern Nevada Water Authority Kyle Bibby, University of Notre Dame Aparna Keshaviah, Mathematica

INTRODUCTION:To date, there has been little effort to develop standards for metabolome-based gut microbiome measurements despite the significant efforts toward standard development for DNA-based microbiome measurements.OBJECTIVES:The National Institute of Standards and Technology (NIST), The BioCollective (TBC), and the North America Branch of the International Life Sciences Institute (ILSI North America) are collaborating to extend NIST's efforts to develop a Human Whole Stool Reference Material for the purpose of method harmonization and eventual quality control.METHODS:The reference material will be rationally designed for adequate quality assurance and quality control (QA/QC) for underlying measurements in the study of the impact of diet and nutrition on functional aspects of the host gut microbiome and relationships of those functions to health. To identify which metabolites deserve priority in their value assignment, NIST, TBC, and ILSI North America jointly conducted a workshop on September 12, 2019 at the NIST campus in Gaithersburg, Maryland. The objective of the workshop was to identify metabolites for which evidence indicates relevance to health and disease and to decide on the appropriate course of action to develop a fit-for-purpose reference material.RESULTS:This document represents the consensus opinions of workshop participants and co-authors of this manuscript, and provides additional supporting information. In addition to developing general criteria for metabolite selection and a preliminary list of proposed metabolites, this paper describes some of the strengths and limitations of this initiative given the current state of microbiome research.CONCLUSIONS:Given the rapidly evolving nature of gut microbiome science and the current state of knowledge, an RM (as opposed to a CRM) measured for multiple metabolites is appropriate at this stage. As the science evolves, the RM can evolve to match the needs of the research community. Ultimately, the stool RM may exist in sequential versions. Beneficial to this evolution will be a clear line of communication between NIST and the stakeholder community to ensure alignment with current scientific understanding and community needs.

ObjectivesTo identify antibacterial additives and screening/assessment approaches used to evaluate the antibacterial activity of resin-based restorative dental materials containing these additives.DataIn vitro studies that compared the antibacterial effects of resin-based restorative dental materials with and without antibacterial additives were screened. Risk bias was assessed, and the following data were extracted: antibacterial additive, parental dental material, curing mode, bacterial growth outcome assessment, samples used as a substrate for bacterial growth, inoculum complexity, and culture time as an indicator of biofilm maturity.SourceArksey and O’Malley’s five stages framework using Medline (OVID), EMBASE, and Scopus (Elsevier) databases guided this review.Study selectionFrom 6503 studies initially identified, 348 studies were considered eligible for full-text screening, and 153 were included for data extraction. Almost all studies have a high sampling bias related to both sample size and blindness. Quaternary ammonium monomers were the most investigated additive (45 %), and the most prevailing parental material was resin composite (49 %). There was extensive methodological heterogeneity among the studies for outcome assessment with the majority using resin composite disks (78 %), mono-species Streptococcus mutans as the inoculum (54 %), and a relatively short period of biofilm growth (≤24 h).ConclusionThe findings herein present the urgent need for improved biological efficacy studies in this important and exciting field. There is a need for efforts to improve study designs to mimic the oral environment in vivo and to develop standardized methods to help understand and optimize these materials.Clinical significanceMost studies that incorporate antibacterial additives into resin-based materials claim promising results by bacterial reduction. However, these results should be interpreted with caution due to significant variation in the methods applied for quantifying bacterial growth, the frequent lack of complexity in the biofilms, and the often-short duration of biofilm growth.

One common tissue engineering approach for regenerating or replacing damaged tissues involves a porous polymeric scaffold. The scaffolds serve as the mechanical framework for cell attachment and growth, and generate an environment with features that span multiple-length scales to guide cell differentiation and tissue regeneration. Laser scanning confocal microscopy (LSCM) is a useful technique to evaluate cell adhesion and spatial distribution within a scaffold since it is designed to collect image slices through the sample thickness. This chapter discusses the methods to evaluate cell distribution in the scaffolds using LSCM. Cell distribution in 3D scaffolds was characterized using new methods developed for the LSCM. Scaffold porosity and pore size are important factors for cell proliferation and distribution in 3D. Porosity affects diffusion and is vital for nutrient distribution and waste removal. All methods utilized the imaging capabilities of the LSCM while also taking advantage of the low-scattering nature of the amorphous polymers.

We describe the use of in silico approaches to improve the process of molecular assay development and reduce time and cost by utilizing available databases of whole genome pathogen sequences combined with modern bioinformatics and physical modeling tools. Well-characterized assays are needed for accurately detecting pathogens in environmental and patient samples and also for evaluation of the efficacy of a medical countermeasure that may be administered to patients. The polymerase chain reaction (PCR) remains the gold standard for pathogen detection due to the simplicity of its instrumentation, low cost of reagents, and outstanding limit of detection (LOD), sensitivity, and specificity. However, creation of such PCR assays often involves iterations of design, preliminary testing, and thorough validation with clinical isolates and testing in relevant matrices, which can be time consuming, costly, and result in suboptimal assays. Since formal validation (e.g., for Emergency Use Authorization [EUA] or Food and Drug Administration [FDA] licensure) of an infectious disease assay can be very expensive and can require extensive time of development, having a well-designed assay up front is a critical first step. Yet, many assays described in the literature utilized limited design capabilities and many initially promising assays fail the validation process, resulting in increased costs and timelines for successful product development. While the computational approaches outlined in this document by no means obviate the need for wet lab testing, they can reduce the amount of effort wasted on empirical optimization and iterative redesigns and also guide validation studies. The proposed computational approaches also result in higher performing assays with better sensitivity, specificity, and lower LOD and reduce the possibility of assay failure due to signature erosion. To provide clarity, an extensive glossary of defined terms is provided. Received: 19 March 2020; Accepted: 19 March 2020 VC AOAC INTERNATIONAL 2020. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 882 Journal of AOAC INTERNATIONAL, 103(4), 2020, 882–899 doi: 10.1093/jaoacint/qsaa045 Technical Communication 1 Background and Rationale Nucleic acid-based assays, such as real-time PCR, are the mainstay of clinical diagnostics and biosurveillance. A typical PCR assay design begins with computational (“in silico”) identification of a unique region (signature) that can support the binding of primer and probe sequences for target-specific amplification as a means of detecting the presence of the target organism. This step is followed by wet lab testing of the primers and probes using genomic deoxyribonucleic acid (DNA) or reverse transcribed ribonucleic acid (RNA) and performance-optimization of selected assays. In addition, extensive testing of the assay in the intended clinical matrix is required to evaluate assay parameters, such as LOD, sensitivity (probability of detection), and specificity (see glossary for definitions). The sensitivity and specificity of the assay are experimentally determined using a set of target (inclusivity) strains, near-neighbor (exclusivity) strains, and matrix-relevant (background) organisms. Assay performance also needs to be measured in assay-specific matrices (i.e., blood, stool, water, soil, etc.). Often, assays are computationally designed using a set of available genomic/gene sequences at that time and then experimentally validated for signature presence in all available samples of the target organism (inclusivity panel) and validated for signature absence in many other samples that do not contain the target (exclusivity panel and matrix panel). In an ideal scenario, a laboratory routinely engaged in assay development could complete this process within 6 to 12 months. Detection assays are typically designed using all sequences available at that time. Many of the biodefense assays were designed and tested at least a decade ago when available sequences were limited. Thanks to recent advances in modern sequencing technologies, there is a sharp increase in the availability of whole genome sequences (Figure 1). Hence, these older assays have the potential to fail if evaluated against currently available sequences. Moreover, publicly available sequence databases (e.g., GenBank) typically contain only a small fraction of naturally occurring sequence diversity. As a result, detection assays are vulnerable to “overfitting”: correctly differentiating known (i.e., sequenced) targets and nontargets but failing to detect novel target variants or falsely detecting novel nontargets. Knowledge of the true genetic diversity is limited for some biodefense agents and their near neighbors, as often only several geographical and temporal representatives are fully characterized while other geographic locations have been ignored or significantly undersampled and hence are under-represented. In addition, while some agents, such as the bacterium Bacillus anthracis, are monomorphic (i.e., highly conserved), other agents, especially RNA viruses, are very diverse [e.g., Lymphocytic choriomeningitis virus (LCMV), Lassa virus, and Crimean-Congo hemorrhagic fever virus (CCHFV)]. In general, detection assays targeting highly conserved targets tend to fail due to unsequenced near-neighbor cross-reactivity, while assays targeting diverse targets tend to fail due to false negatives against unsequenced target variants. While the recent revolution in next-generation sequencing technologies combined with decreasing sequencing costs has increased knowledge of population genomic structure, the capability for laboratory-based evaluation of newly sequenced strains has not kept pace. In this scenario, replacing or redesigning older assays to incorporate new knowledge of the target genomic landscape is critical. However, wet lab testing may not be feasible due to limitations on the timely availability of samples/strains. This problem is further exacerbated by policy decisions, such as the 2015 Department of Defense (DoD) moratorium that decreased access to live/inactivated biodefense pathogens for various applications, including assay development and validation (1). 1.1 Additional Considerations with the Status Quo Testing of Assays Against Inclusivity/Exclusivity Panels The AOAC Stakeholder Panel on Agent Detection Assays (SPADA) inclusivity/exclusivity panels for the biodefenserelevant bacterial pathogens, such as Bacillus anthracis, Yersinia pestis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis, comprise a total of approximately 100 strains. These strains are used to validate the inclusivity/exclusivity criteria for the respective detection assays. Most of the inclusivity strains, and some exclusivity strains, are considered Biosafety Level 3 (BSL3) agents and, as a result, are limited to laboratories that are registered and certified for such work. Moreover, extensive laboratory testing adds cost and time to the assay development effort. Many whole genome sequences of these bacterial strains are available now (2–6), which allows the in silico evaluation of assays. An example set of assays developed prior to the nextgeneration sequencing revolution with representative analyses is illustrated in Figure 2. As expected, the majority of the evaluated assay signatures had perfect sequence matches to the target inclusivity genome sequences, and much less (0 to 40%) sequence identity to the exclusivity panel genome sequences. However, for all the assays evaluated, there was no “perfect” assay (i.e., no false positives and no false negatives). Some assays were computationally predicted to have both false negatives (e.g., Bacillus anthracis assay 1 against strain 10 in the inclusivity panel) and false positives (e.g., Bacillus anthracis assay 1 against strain 8 in the exclusivity panel). Many of these predicted assay failures correspond to expected deviations based on the genotypes of these strains. There are other assays that simply fail the inclusivity and/or exclusivity criteria (e.g., Bacillus anthracis assay 7 or Yersinia pestis assay 15) and are therefore not reliable diagnostics due to low specificity. However, given the high conservation of the assay signatures to the target strains in the inclusivity panel and their low conservation in the exclusivity panel, the “brute force” testing of all available strains is not cost Figure 1. Availability of whole genome sequences for representative bacteria. Black bar represents the assay design time frame. SantaLucia et al.: Journal of AOAC INTERNATIONAL Vol. 103, No. 4, 2020 | 883

Online water bioburden analyzers (OWBAs) can provide real-time feedback on viable bacteria in high-purity water (HPW) systems for pharmaceutical manufacturers. To calibrate and validate OWBAs, which detect bacteria using scattered light and bacterial autofluorescence, standards are needed that mimic the characteristics of bacteria in HPW. To guide selection of potential standards, e.g., fluorescent microspheres, a relevant bacterial contaminant, Ralstonia pickettii, was characterized for size, count, viability, and autofluorescence after exposure for 24 h to HPW or a nutrient environment. The cells exposed to HPW showed smaller sizes, with lower counts and autofluorescence intensities, but similar spectral features. The cell characteristics are discussed in comparison with a set of fluorescent microspheres, considering factors relevant to OWBAs. These studies suggest that fluorescent microspheres should be relatively small (< 1 µm diameter) and dim, while covering a broad emission range from ≈ (420 to 600) nm to best mimic the representative R. pickettii.

The antimicrobial properties of silver nanomaterials (AgNM) have been exploited in various consumer applications, including in textiles for use as wound dressings. Understanding how these materials chemically transform throughout their use is necessary to predict their efficacy during use and their behavior after disposal. The aim of this work was to evaluate chemical and physical transformations occurring to a commercial AgNM-containing wound dressing during modeled human exposure in synthetic sweat (SW) or simulated wound fluid (WF), which model human exposure scenarios. Scanning electron microscopy with energy dispersive X-ray spectroscopy (EDS) revealed the formation of micrometer-sized structures on the wound dressing surface after SW exposure while WF resulted in a largely featureless surface. Measurements by X-ray photoelectron spectroscopy (XPS) revealed a AgCl surface (consistent with EDS) while X-ray diffraction (XRD) found a mixture of zero valent silver and AgCl, suggesting the AgNM wound dressings surface formed a passivating AgCl surface layer after SW and WF exposure. For WF exposed wound dressings, XPS based findings revealed protein adsorption based on the nitrogen marker which adsorbed released silver at prolonged exposures. Silver release was evaluated by inductively coupled - plasma mass spectrometry which revealed a factor of 36 times more released silver in WF than in SW. Analysis suggests that the protein in WF sequestered a fraction of the released silver in the solution suggesting additional processing at the wound dressing surface occurred after the initial transformation to AgCl. To evaluate the impact on antimicrobial efficacy, zone of inhibition (ZOI) testing was conducted which found no significant change after modeled human exposure compared to the pristine wound dressing. The results presented here suggest AgNM-containing wound dressings transform chemically in simulated human fluids resulting in a material with comparable antimicrobial properties to the pristine wound dressings. Ultimately, knowing the resulting chemical properties of the AgNM wound dressings will allow more predictive models for fate.