Benchmarking Bioinformatic Tools for Amplicon-Based Sequencing of Norovirus.

In order to survey noroviruses in our environment, it is essential that both wet-lab and computational methods are fit for purpose. Using a simulated sequencing data set, denoising-based (DADA2, Deblur and USEARCH-UNOISE3) and clustering-based pipelines (VSEARCH and FROGS) were compared with respect to their ability to represent composition and sequence information. Open source classifiers (Ribosomal Database Project [RDP], BLASTn, IDTAXA, QIIME2 naive Bayes, and SINTAX) were trained using three different databases: a custom database, the NoroNet database, and the Human calicivirus database. Each classifier and database combination was compared from the perspective of their classification accuracy. VSEARCH provides a robust option for analyzing viral amplicons based on composition analysis; however, all pipelines could return OTUs with high similarity to the expected sequences. Importantly, pipeline choice could lead to more false positives (DADA2) or underclassification (FROGS), a key aspect when considering pipeline application for source attribution. Classification was more strongly impacted by the classifier than the database, although disagreement increased with norovirus GII.4 capsid variant designation. We recommend the use of the RDP classifier in conjunction with VSEARCH; however, maintenance of the underlying database is essential for optimal use. IMPORTANCE In benchmarking bioinformatic pipelines for analyzing high-throughput sequencing (HTS) data sets, we provide method standardization for bioinformatics broadly and specifically for norovirus in situations for which no officially endorsed methods exist at present. This study provides recommendations for the appropriate analysis and classification of norovirus amplicon HTS data and will be widely applicable during outbreak investigations.

Amplicon-Based High-Throughput Sequencing Method for Genotypic Characterization of Norovirus in Oysters.

Norovirus is a highly diverse RNA virus often implicated in foodborne outbreaks, particularly those associated with shellfish. Shellfish are filter feeders, and when harvested in bays exposed to wastewater overflow or storm overflows, they can harbor various pathogens, including human-pathogenic viruses. The application of Sanger or amplicon-based high-throughput sequencing (HTS) technologies to identify human pathogens in shellfish faces two main challenges: (i) distinguishing multiple genotypes/variants in a single sample and (ii) low concentrations of norovirus RNA. Here, we assessed the performance of a novel norovirus capsid amplicon HTS method. We generated a panel of spiked oysters containing various norovirus concentrations with different genotypic compositions. Several DNA polymerases and reverse transcriptases (RTs) were compared, and performance was evaluated based on (i) the number of reads passing quality filters per sample, (ii) the number of correct genotypes identified, and (iii) the sequence identity of outputs compared to Sanger-derived sequences. A combination of the reverse transcriptase LunaScript and the DNA polymerase AmpliTaq Gold provided the best results. The method was then employed, and compared with Sanger sequencing, to characterize norovirus populations in naturally contaminated oysters. IMPORTANCE While foodborne outbreaks account for approximately 14% of norovirus cases (L. Verhoef, J. Hewitt, L. Barclay, S. Ahmed, R. Lake, A. J. Hall, B. Lopman, A. Kroneman, H. Vennema, J. Vinjé, and M. Koopmans, Emerg Infect Dis 21:592-599, 2015), we do not have standardized high-throughput sequencing methods for genotypic characterization in foodstuffs. Here, we present an optimized amplicon high-throughput sequencing method for the genotypic characterization of norovirus in oysters. This method can accurately detect and characterize norovirus at concentrations found in oysters grown in production areas impacted by human wastewater discharges. It will permit the investigation of norovirus genetic diversity in complex matrices and contribute to ongoing surveillance of norovirus in the environment.

A Qualitative Study of Spanish-Speakers' Experience with Dense Breast Notifications in a Massachusetts Safety-Net Hospital.

BACKGROUND: Legislation requiring mammography facilities to notify women if they have dense breast tissue found on mammography has been enacted in 34 US states. The impact of dense breast notifications (DBNs) on women with limited English proficiency (LEP) is unknown. OBJECTIVE: This study sought to understand Spanish-speaking women's experience receiving DBNs in a Massachusetts safety-net hospital. DESIGN: Eligible women completed one audio-recorded, semi-structured interview via telephone with a native Spanish-speaking research assistant trained in qualitative methods. Interviews were professionally transcribed verbatim and translated. The translation was verified by a third reviewer to ensure fidelity with audio recordings. PARTICIPANTS: Nineteen Spanish-speaking women ages 40-74 who received mammography with a normal result and recalled receiving a DBN. APPROACH: Using the verified English transcripts, we conducted a content analysis to identify women's perceptions and actions related to receiving the notification. A structured codebook was developed. Transcripts were independently coded and assessed for agreement with a modification of Cohen's kappa. Content codes were grouped to build themes related to women's perceptions and actions after receiving a DBN. KEY RESULTS: Nineteen Spanish-speaking women completed interviews. Nine reported not receiving the notification in their native language. Four key themes emerged: (1) The novelty of breast density contributed to notification-induced confusion; (2) women misinterpreted key messages in the notification; (3) varied actions were taken to seek further information; and (4) women held unrealized expectations and preferences for follow-up. CONCLUSIONS: Not having previous knowledge of breast density and receiving notifications in English contributed to confusion about its meaning and inaccurate interpretations of key messages by Spanish speakers. Tools that promote understanding should be leveraged in seeking equity in risk-based breast cancer screening for women with dense breasts.

An in-depth evaluation of metagenomic classifiers for soil microbiomes.

BACKGROUND: Recent endeavours in metagenomics, exemplified by projects such as the human microbiome project and TARA Oceans, have illuminated the complexities of microbial biomes. A robust bioinformatic pipeline and meticulous evaluation of their methodology have contributed to the success of these projects. The soil environment, however, with its unique challenges, requires a specialized methodological exploration to maximize microbial insights. A notable limitation in soil microbiome studies is the dearth of soil-specific reference databases available to classifiers that emulate the complexity of soil communities. There is also a lack of in-vitro mock communities derived from soil strains that can be assessed for taxonomic classification accuracy. RESULTS: In this study, we generated a custom in-silico mock community containing microbial genomes commonly observed in the soil microbiome. Using this mock community, we simulated shotgun sequencing data to evaluate the performance of three leading metagenomic classifiers: Kraken2 (supplemented with Bracken, using a custom database derived from GTDB-TK genomes along with its own default database), Kaiju, and MetaPhlAn, utilizing their respective default databases for a robust analysis. Our results highlight the importance of optimizing taxonomic classification parameters, database selection, as well as analysing trimmed reads and contigs. Our study showed that classifiers tailored to the specific taxa present in our samples led to fewer errors compared to broader databases including microbial eukaryotes, protozoa, or human genomes, highlighting the effectiveness of targeted taxonomic classification. Notably, an optimal classifier performance was achieved when applying a relative abundance threshold of 0.001% or 0.005%. The Kraken2 supplemented with bracken, with a custom database demonstrated superior precision, sensitivity, F1 score, and overall sequence classification. Using a custom database, this classifier classified 99% of in-silico reads and 58% of real-world soil shotgun reads, with the latter identifying previously overlooked phyla using a custom database. CONCLUSION: This study underscores the potential advantages of in-silico methodological optimization in metagenomic analyses, especially when deciphering the complexities of soil microbiomes. We demonstrate that the choice of classifier and database significantly impacts microbial taxonomic profiling. Our findings suggest that employing Kraken2 with Bracken, coupled with a custom database of GTDB-TK genomes and fungal genomes at a relative abundance threshold of 0.001% provides optimal accuracy in soil shotgun metagenome analysis.

A Clinical Guide to Support the Implementation of Addiction Consult Services and the Value of Teaching and Technical Assistance.

ABSTRACT: People with substance use disorders (SUDs) are increasingly admitted to general hospitals; however, many hospital systems lack both formal structures and skilled staff to provide high-quality care for inpatients with SUDs. Inpatient addiction consult services (ACSs), which are increasingly being implemented around the country, are an evidence-based strategy to add focused care for people with SUDs into the general medical setting. In 2018, New York City Health + Hospitals (H + H) launched an ACS program called Consult for Addiction Care and Treatment in Hospitals in six hospitals, supported by a team of addiction consult experts to deliver teaching and technical assistance (TTA) for the Consult for Addiction Care and Treatment in Hospitals ACSs. This commentary describes the TTA, which included site visits, introductory educational lectures, case conferences, ad hoc support, implementation assistance, and the creation of an addiction care guide. Similar TTA services could be used in the future when hospitals or systems want to launch novel clinical programs.

ACR Appropriateness Criteria® Imaging of Invasive Breast Cancer.

As the proportion of women diagnosed with invasive breast cancer increases, the role of imaging for staging and surveillance purposes should be determined based on evidence-based guidelines. It is important to understand the indications for extent of disease evaluation and staging, as unnecessary imaging can delay care and even result in adverse outcomes. In asymptomatic patients that received treatment for curative intent, there is no role for imaging to screen for distant recurrence. Routine surveillance with an annual 2-D mammogram and/or tomosynthesis is recommended to detect an in-breast recurrence or a new primary breast cancer in women with a history of breast cancer, and MRI is increasingly used as an additional screening tool in this population, especially in women with dense breasts. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision process support the systematic analysis of the medical literature from peer reviewed journals. Established methodology principles such as Grading of Recommendations Assessment, Development, and Evaluation or GRADE are adapted to evaluate the evidence. The RAND/UCLA Appropriateness Method User Manual provides the methodology to determine the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where peer reviewed literature is lacking or equivocal, experts may be the primary evidentiary source available to formulate a recommendation.

Is Estrogen a Missing Culprit in Thyroid Eye Disease? Sex Steroid Hormone Homeostasis Is Key to Other Fibrogenic Autoimmune Diseases - Why Not This One?

Sex bias in autoimmune disease (AID) prevalence is known, but the role of estrogen in disease progression is more complex. Estrogen can even be protective in some AIDs; but in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and systemic sclerosis (SSc), estrogen, its metabolites, and its receptors have been demonstrated to play critical, localized inflammatory roles. Estrogen is instrumental to the fibrosis seen in RA, SLE, SSc and other disease states, including breast cancer and uterine leiomyomas. Fibrotic diseases tend to share a common pattern in which lymphocyte-monocyte interactions generate cytokines which stimulate the deposition of fibrogenic connective tissue. RA, SLE, SSc and thyroid eye disease (TED) have very similar inflammatory and fibrotic patterns-from pathways to tissue type. The thorough investigations that demonstrated estrogen's role in the pathology of RA, SLE, and SSc could, and possibly should, be carried out in TED. One might even expect to find an even greater role for estrogen, and sex steroid homeostasis in TED, given that TED is typically sequalae to Graves' disease (GD), or Hashimoto's disease (HD), and these are endocrine disorders that can create considerable sex steroid hormone dysregulation. This paper highlights the pathophysiology similarities in 4 AIDs, examines the evidence of sex steroid mediated pathology across 3 AIDs and offers a case study and speculation on how this may be germane to TED.

Towards Co-Design in Delivering Assistive Technology Interventions: Reconsidering Roles for Consumers, Allied Health Practitioners, and the Support Workforce.

A complexity of factors, from health and technology innovations to policy redesign to achieve consumer-directed care, are impacting traditional roles for Australian allied health practitioners (AHPs). This pilot study considers roles for AHPs in relation to assistive technology (AT) interventions. Articulating 'who does what' may serve a number of purposes including de-professionalization of the discourse; better utilization of support networks and workforces; and alignment with contemporary policy. Yet, a suitable framework to assist with collaborative AT implementation between relevant stakeholders was not identified within the existing literature. This research aimed to develop and pilot an AT collaboration tool which enables AHPs, consumers, their support networks and the support workforce, to navigate policy redesign toward ethical consumer-directed implementation of AT interventions. An AT collaboration tool was developed based upon practice-based knowledge, relevant regulatory and practice evidence and identifies relevant stakeholders, AT service steps and roles, and quality indicators to support competent practice. The tool was piloted in four separate and diverse practice analyses of AT interventions (custom prosthetics, home enteral nutrition, communication devices, and vehicle modifications) considering four allied health professions (prosthetics and orthotics, dietetics, speech pathology, occupational therapy). Pilot testing of the tool supports the feasibility of re-framing AT provision using competency-based and risk-informed approaches and enabling more inclusive roles for consumers and the support workforce. Further testing of the tool is indicated, followed by strategic actions for uptake by individuals, professions and policymakers. The AT collaboration tool has potential to enable AHPs to fulfil ethical obligations for consumer-centered practice, and to facilitate consumer choice, both in Australia and internationally.

Impact of COVID-19 Nonpharmaceutical Interventions on the Extent of Norovirus Contamination in Oyster Production Areas in Ireland during Winter 2020 to 2021.

ABSTRACT: A significant decrease in norovirus prevalence and concentration was observed in oyster production areas in Ireland during winter 2020 to 2021. Oyster production areas impacted by human wastewater discharges that had been undergoing norovirus surveillance since 2018 were investigated. Samples collected in the winter seasons of 2018 to 2019 and 2019 to 2020, prior to when the COVID-19 pandemic interventions were applied, showed a prevalence of 94.3 and 96.6%, respectively, and geometric mean concentrations of 533 and 323 genome copies per g, respectively. These values decreased significantly during the winter of 2020 to 2021 (prevalence of 63.2% and geometric concentration of below the limit of quantification), coinciding with the control measures to mitigate the transmission of severe acute respiratory syndrome coronavirus 2 of the genus Betacoronavirus. Divergence between norovirus GI and GII prevalence and concentrations was observed over the 3-year monitoring period. Norovirus GII was the dominant genogroup detected in winter 2020 to 2021, with over half of samples positive, although concentrations detected were significantly lower than prepandemic winters, with a geometric mean concentration of below the limit of quantification.

Examining the Content and Outcomes of Training in Dysphagia and Mealtime Management: A Systematic Review Informing Co-Design of New Training.

PURPOSE: Dysphagia (swallowing difficulty) impacts physical health, quality of life, and mealtime enjoyment. Staff who provide mealtime assistance to people with dysphagia require adequate training to help ensure that the mealtimes are safe and enjoyable. This systematic review examined literature relating to training in dysphagia (e.g., recognizing signs and symptoms) and mealtime assistance, its components, and benefits for people with dysphagia. METHOD: In July 2020, five scientific databases were searched for papers meeting the inclusion criteria relating to mealtime assistance training. The quality of the studies was evaluated using the Quality Assessment Tool for Studies of Diverse Design, with scores ranging from 38.1% to 83.3%. We completed a qualitative synthesis using the data extracted from the included studies. RESULTS: Twenty-four studies met the inclusion criteria. Participants in these studies benefited from both group training and one-on-one training. Training programs had many formats including computer-based, face-to-face, individual training, and group training. Each included study demonstrated some level of positive impact to the learners, such as improved knowledge and skills in mealtime management for people with dysphagia. No studies reported negative outcomes. Training duration ranged from 30 min to 5 days. CONCLUSIONS: The benefits of different components of mealtime training (e.g., group training, or face-to-face training) for mealtime assistance for people with dysphagia were reviewed. Further research is needed to compare the effectiveness of different training formats, involving not only the assistant but also people with dysphagia as both trainers and trainees, and determine the health outcomes of training programs for people with dysphagia.

High Throughput Sequencing for the Detection and Characterization of RNA Viruses.

This review aims to assess and recommend approaches for targeted and agnostic High Throughput Sequencing of RNA viruses in a variety of sample matrices. HTS also referred to as deep sequencing, next generation sequencing and third generation sequencing; has much to offer to the field of environmental virology as its increased sequencing depth circumvents issues with cloning environmental isolates for Sanger sequencing. That said however, it is important to consider the challenges and biases that method choice can impart to sequencing results. Here, methodology choices from RNA extraction, reverse transcription to library preparation are compared based on their impact on the detection or characterization of RNA viruses.

Evaluation of Norovirus Reduction in Environmentally Contaminated Pacific Oysters During Laboratory Controlled and Commercial Depuration.

Norovirus contamination of oysters is the lead cause of non-bacterial gastroenteritis and a significant food safety concern for the oyster industry. Here, norovirus reduction from Pacific oysters (Crassostrea gigas), contaminated in the marine environment, was studied in laboratory depuration trials and in two commercial settings. Norovirus concentrations were measured in oyster digestive tissue before, during and post-depuration using the ISO 15216-1 quantitative real-time RT-PCR method. Results of the laboratory-based studies demonstrate that statistically significant reductions of up to 74% of the initial norovirus GII concentration was achieved after 3 days at 17-21 °C and after 4 days at 11-15 °C, compared to 44% reduction at 7-9 °C. In many trials norovirus GII concentrations were reduced to levels below 100 genome copies per gram (gcg-1; limit of quantitation; LOQ). Virus reduction was also assessed in commercial depuration systems, routinely used by two Irish oyster producers. Up to 68% reduction was recorded for norovirus GI and up to 90% for norovirus GII reducing the geometric mean virus concentration close to or below the LOQ. In both commercial settings there was a significant difference between the levels of reduction of norovirus GI compared to GII (p < 0.05). Additionally, the ability to reduce the norovirus concentration in oysters to < LOQ differed when contaminated with concentrations below and above 1000 gcg-1. These results indicate that depuration, carried out at elevated (> 11 °C) water temperatures for at least 3 days, can reduce the concentration of norovirus in oysters and therefore consumer exposure providing a practical risk management tool for the shellfish industry.