Advantage of precision metagenomics for urinary tract infection diagnostics.

Background: Urinary tract infections (UTIs) remain a diagnostic challenge and often promote antibiotic overuse. Despite urine culture being the gold standard for UTI diagnosis, some uropathogens may lead to false-negative or inconclusive results. Although PCR testing is fast and highly sensitive, its diagnostic yield is limited to targeted microorganisms. Metagenomic next-generation sequencing (mNGS) is a hypothesis-free approach with potential of deciphering the urobiome. However, clinically relevant information is often buried in the enormous amount of sequencing data. Methods: Precision metagenomics (PM) is a hybridization capture-based method with potential of enhanced discovery power and better diagnostic yield without diluting clinically relevant information. We collected 47 urine samples of clinically suspected UTI and in parallel tested each sample by microbial culture, PCR, and PM; then, we comparatively analyzed the results. Next, we phenotypically classified the cumulative microbial population using the Explify® data analysis platform for potential pathogenicity. Results: Results revealed 100% positive predictive agreement (PPA) with culture results, which identified only 13 different microorganisms, compared to 19 and 62 organisms identified by PCR and PM, respectively. All identified organisms were classified into phenotypic groups (0-3) with increasing pathogenic potential and clinical relevance. This PM can simultaneously quantify and phenotypically classify the organisms readily through bioinformatic platforms like Explify®, essentially providing dissected and quantitative results for timely and accurate empiric UTI treatment. Conclusion: PM offers potential for building effective diagnostic models beyond usual care testing in complex UTI diseases. Future studies should assess the impact of PM-guided UTI management on clinical outcomes.

SARS-CoV-2 Next Generation Sequencing (NGS) data from clinical isolates from the East Texas Region of the United States.

The SARS-CoV-2 virus has evolved throughout the pandemic and is likely to continue evolving into new variants. Some of these variants may affect functional properties, including infectivity, interactions with host immunity, and disease severity. And compromised vaccine efficacy is an emerging concern with every new viral variant. Next-generation sequencing (NGS) has emerged as the tool of choice for discovering new variants and understanding the transmission dynamics of SARS-CoV-2. Deciphering the SARS-CoV-2 genome has enabled epidemiological survivance and forecast of altered etiologically. Clinical presentations of the infection are influenced by comorbidities such as age, immune status, diabetes, and the infecting variant. Thus, clinical management and vaccine efficacy may differ for new variants. For example, some monoclonal antibody treatments are variant-specific, and some vaccines are less efficacious against the omicron and delta variants of SARS-CoV-2. Consequently, determining the local outbreaks and monitoring SARS-CoV-2 Variants of Concern (VOC) is one of the primary strategies for the pandemic's containment. Although next-generation sequencing (NGS) is a gold standard for genomic surveillance and variant discovery, the assays are not approved for variant diagnosis for clinical decision-making. Advanta Genetics, Texas, USA, optimized Illumina COVID-seq protocol to reduce cost without compromising accuracy and validated the Illumina COVID-Seq assay as a Laboratory Developed Test (LDT) according to the guidelines prescribed by the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). The whole genome of the virus was sequenced in (n = 161) samples from the East Texas region using the Illumina MiniSeq® instrument and analyzed by using Illumina baseSpace (https://basespace.illumina.com) bioinformatics pipeline. Briefly, the library was prepared by using Illumina COVIDSeq research use only (RUO) kit, and the individual libraries were normalized using the DNA concentration measured by Qubit Flex Fluorometer, and the pooled libraries were sequenced on Illumina MiniSeq® Instrument. Illumina baseSpace application was used for sequencing QC, FASTQ generation, genome assembly, and identification of SARS-CoV-2 variants. This whole genome shotgun project (n = 161) has been deposited at GISAID.

Mentorship reconsidered: A case study of K-12 teachers' mentor-mentee relationships during the COVID-19 pandemic.

This study critically examined the impact of a crisis context (COVID-19 pandemic) on K-12 teachers by placing emphasis on the mentor-mentee dyad through the perspective of the mentee in a large United States public school system. A phenomenological case study was undertaken that used semi-structured interviews to examine 14 early career teachers (mentees) participating in a formal mentoring program during the 2020-2021 school year. The study focused on mentor-mentee relationships by accounting for the single most traumatic and transformative event of the modern era of K-12 public education. The analysis yielded three findings highlighting the impact of COVID-19 on the mentor-mentee dyadic experiences of first- and second-year teachers engaged in a mentoring relationship. The findings indicate that (a) e-mentoring allowed for avoidant behaviors from mentors (b) successful mentoring involves the development of personal relationships between a mentor and mentee, and (c) peer and reverse mentoring became commonplace during the COVID-19 pandemic. Public school systems can use these findings to help develop positive mentor and mentee relationships that go beyond the traditional dyadic roles and help reduce stress in a crisis context, while developing a culture where superiority bias is improved. Research implications offer mentoring literature a view to pay more attention to temporal influences during environments of high stress, which may provide more explanatory power on mentorship roles, cultural influences, and social interactions in the course of mentor-mentee practices.

Optimization of the Illumina COVIDSeq™ protocol for decentralized, cost-effective genomic surveillance.

A decentralized surveillance system to identify local outbreaks and monitor SARS-CoV-2 Variants of Concern is one of the primary strategies for the pandemic's containment. Although next-generation sequencing (NGS) is a gold standard for genomic surveillance and variant discovery, the technology is still cost-prohibitive for decentralized sequencing, particularly in small independent labs with limited resources. We have optimized the Illumina COVIDSeq™ protocol for the Illumina MiniSeq instrument to reduce cost without compromising accuracy. We slashed the library preparation cost by half by using 50% of recommended reagents at each step and normalizing the libraries before pooling to achieve uniform coverage. Reagent-only cost (∼$43.27/sample) for SARS-CoV-2 variant analysis with this normalized input protocol on MiniSeq instruments is comparable to what is achieved on high throughput instruments such as NextSeq and NovaSeq. Using this modified protocol, we tested 153 clinical samples, and 90% of genomic coverage was achieved for 142/153 samples analyzed in this study. The lineage was correctly assigned to all samples (152/153) except for one. This modified protocol can help laboratories with constrained resources to contribute in decentralized COVID-19 surveillance in the post-vaccination era.

Deciphering Microbiota of Acute Upper Respiratory Infections: A Comparative Analysis of PCR and mNGS Methods for Lower Respiratory Trafficking Potential.

Although it is clinically important for acute respiratory tract (co)infections to have a rapid and accurate diagnosis, it is critical that respiratory medicine understands the advantages of current laboratory methods. In this study, we tested nasopharyngeal samples (n = 29) with a commercially available PCR assay and compared the results with those of a hybridization-capture-based mNGS workflow. Detection criteria for positive PCR samples was Ct < 35 and for mNGS samples it was >40% target coverage, median depth of 1X and RPKM > 10. A high degree of concordance (98.33% PPA and 100% NPA) was recorded. However, mNGS yielded positively 29 additional microorganisms (23 bacteria, 4 viruses, and 2 fungi) beyond PCR. We then characterized the microorganisms of each method into three phenotypic categories using the IDbyDNA Explify® Platform (Illumina® Inc, San Diego, CA, USA) for consideration of infectivity and trafficking potential to the lower respiratory region. The findings are significant for providing a comprehensive yet clinically relevant microbiology profile of acute upper respiratory infection, especially important in immunocompromised or immunocompetent with comorbidity respiratory cases or where traditional syndromic approaches fail to identify pathogenicity. Accordingly, this technology can be used to supplement current syndrome-based tests, and data can quickly and effectively be phenotypically characterized for trafficking potential, clinical (co)infection, and comorbid consideration-with promise to reduce morbidity and mortality.

A Human Oral Fluid Assay for D- and L- Isomer Detection of Amphetamine and Methamphetamine Using Liquid-Liquid Extraction.

Medical providers are increasingly confronted with clinical decision-making that involves (meth)amphetamines. And clinical laboratories need a sensitive, efficient assay for routine assessment of D- and L-isomers to determine the probable source of these potentially illicit analytes. This paper presents a validated method of D- and L-isomer detection in human oral fluid from an extract used for determination of a large oral fluid assay (63 analytes) on an older AB SCIEX 4000 instrument. Taken from the positive extract, D- and L-analytes were added. The method for extraction included addition of internal standard and a 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples. The samples were suspended in 50% MeOH in water, diluted with mobile phase, with separation and detection accomplished using LC-MS/MS to determine analyte concentration. Once samples were confirmed positive for (meth)amphetamine from the large oral fluid assay, they were further examined for the enantiomeric forms with 50 µl aliquots of the standards and samples of interest combined with 450 µl of D- and L-assay mobile phase, then analyzed using chiral column separation, and LC-MS/MS detection with standard curve spanning the range from 2.5 to 1000 ng/mL. The result is a sensitive and accurate detection of D- and L-isomers of amphetamine and methamphetamine in human oral fluid performed on an older model mass spectrometer (AB SCIEX 4000). The novelty of this assay is twofold (a) the 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples, and (b) its adoption characteristics as a reflex test from a large ODT panel without the need to invest in newer or expensive LC-MS/MS instruments. Finally, this assay also has potential to add a valuable option to high-throughput laboratories seeking a D- and L-testing alternative to urine drug testing methods.

Enhanced numeracy skills following team-based learning in United States pharmacy students: a longitudinal cohort study

PURPOSE: The literature suggests that the ability to numerate cannot be fully understood without accounting for the social context in which mathematical activity is represented. Team-based learning (TBL) is an andragogical approach with theoretical links to sociocultural and community-of-practice learning. This study aimed to quantitatively explore the impact of TBL instruction on numeracy development in 2 cohorts of pharmacy students and identify the impact of TBL instruction on numeracy development from a social perspective for healthcare education. METHODS: Two cohorts of students were administered the Health Science Reasoning Test-Numeracy (HSRT-N) before beginning pharmacy school. Two years after using TBL as the primary method of instruction, both comprehensive and domain data from the HSRT-N were analyzed. RESULTS: In total, 163 pharmacy student scores met the inclusion criteria. The students' numeracy skills measured by HSRT-N improved after 2 years of TBL instruction. CONCLUSION: Numeracy was the most significantly improved HSRT-N domain in pharmacy students following two years of TBL instruction. Although a closer examination of numeracy development in TBL is warranted, initial data suggest that TBL instruction may be an adequate proxy for advancing numeracy in a cohort of pharmacy students. TBL may encourage a social practice of mathematics to improve pharmacy students' ability to numerate critically.

Confirming Multiplex RT-qPCR Use in COVID-19 with Next-Generation Sequencing: Strategies for Epidemiological Advantage.

Rapid identification and tracking of emerging SARS-CoV-2 variants are critical for understanding the transmission dynamics and developing strategies for interrupting the transmission chain. Next-Generation Sequencing (NGS) is an exceptional tool for whole-genome analysis and deciphering new mutations. The technique has been instrumental in identifying the variants of concern (VOC) and tracking this pandemic. However, NGS is complex and expensive for large-scale adoption, and epidemiological monitoring with NGS alone could be unattainable in limited-resource settings. In this study, we explored the application of RT-qPCR-based detection of the variant identified by NGS. We analyzed a total of 78 deidentified samples that screened positive for SARS-CoV-2 from two timeframes, August 2020 and July 2021. All 78 samples were classified into WHO lineages by whole-genome sequencing and then compared with two commercially available RT-qPCR assays for spike protein mutation(s). The data showed good concordance between RT-qPCR and NGS analysis for specific SARS-CoV-2 lineages and characteristic mutations. RT-qPCR assays are quick and cost-effective and thus can be implemented in synergy with NGS for screening NGS-identified mutations of SARS-CoV-2 for clinical and epidemiological interest. Strategic use of NGS and RT-qPCR can offer several COVID-19 epidemiological advantages.

The Student Engagement Effect of Team-Based Learning on Student Pharmacists.

Objective. To expand our understanding of student engagement by qualitatively examining how student pharmacists experienced the psychological state of engagement when applying team-based learning (TBL) pedagogy.Methods. A qualitative case study was conducted. Data were obtained through semi-structured interviews with a purposeful and convenience sample of student pharmacists (n=14). Our initial data analysis identified common themes for student engagement in TBL. We then characterized each common theme by deductively coding the themes into predetermined focal concepts of engagement based upon Kearsley and Shneiderman's 1 previous characterization of student engagement as either relate, create, or donate components.Results. Seven common themes arose from this research: accountability, communication, conflict, learning, preparation, purpose, and teamwork. Results indicated that student pharmacists engaged in TBL pedagogy mostly experience the psychological state of student engagement through a relate (41%) component by drawing on team support and trust, followed by the donate (32%) and create (27%) components.Conclusion. Findings in this study are consistent with other research on TBL pedagogy which concluded that, at least in part, this type of learning was a conduit for building student pharmacists' engagement skills. The novelty of this research is that it deductively characterized how student pharmacists perceive, comprehend, and interpret the psychological state of engagement in TBL. Specifically, our findings concluded student pharmacists mostly identify with a relate component of engagement by drawing on team support and trust developed from TBL tenets that encourage communication, conflict resolution, and teamwork.

COVIDSeq as Laboratory Developed Test (LDT) for Diagnosis of SARS-CoV-2 Variants of Concern (VOC).

Rapid classification and detection of SARS-CoV-2 variants have been critical in comprehending the virus's transmission dynamics. Clinical manifestation of the infection is influenced by comorbidities such as age, immune status, diabetes, and the infecting variant. Thus, clinical management may differ for new variants. For example, some monoclonal antibody treatments are variant-specific. Yet, a U.S. Food and Drug Administration (FDA)-approved test for detecting the SARS-CoV-2 variant is unavailable. A laboratory-developed test (LDT) remains a viable option for reporting the infecting variant for clinical intervention or epidemiological purposes. Accordingly, we have validated the Illumina COVIDSeq assay as an LDT according to the guidelines prescribed by the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). The limit of detection (LOD) of this test is Ct<30 (~15 viral copies) and >200X genomic coverage, and the test is 100% specific in the detection of existing variants. The test demonstrated 100% precision in inter-day, intra-day, and intra-laboratory reproducibility studies. It is also 100% accurate, defined by reference strain testing and split sample testing with other CLIA laboratories. Advanta Genetics LDT COVIDSeq has been reviewed by CAP inspectors and is under review by FDA for Emergency Use Authorization.

Transforming Prescription Opioid Practices in Primary Care With Change Theory.

The opioid epidemic continues to be an ongoing public health crisis. Many primary health care providers aptly serve as the gatekeeper to opioid prescriptions. The opioid epidemic has challenged the primary care profession whilst many of these providers have opted out of opioid prescribing altogether. This unintended consequence affirms erosion to primary care that is vital to the ecosystem of opioid management. The purpose of this study was to understand strategies to deliver opioids safely and effectively. Results indicate primary care providers are uniquely positioned to make a positive opioid impact through focused change initiatives. Five common themes arose from the inductive analysis: (1) provide leadership support; (2) define standard of work; (3) conduct pre-visit reviews; (4) conduct post-visit reviews; and (5) measure progress. Then, each common theme was deductively analyzed through a view of Kotter's change theory to support an effective proxy for implementing and sustaining chronic opioid therapy in a primary care context. These finding have potential to provide actionable implications for health care management professionals and primary care organizations such as hospitals and group practices.

Working Against the Clock: A Model for Rural STEMI Triage.

Residents in rural communities have a higher incidence of cardiac death and risk factors associated with cardiac disease. Living in a rural region can add precious time that amplifies cardiac death during an ST-elevated myocardial infarction (STEMI) episode. The consensus is that improved efficiencies can increase myocardial salvage and decrease STEMI mortality rates. This article identifies issues that may impact pre-hospital STEMI triage of patients in a rural region of the United States (U.S.). A qualitative research design was chosen to gain insight into emergency personnel perceptions of pre-hospital STEMI triage. The participants (n = 18) were obtained from a convenience sample in rural Northeast Texas, U.S. Data were gathered by individual and group semi-structured interviews. Themes were identified, synthesized, and oriented to offer a basis for understanding opportunities to improve the delivery of rural STEMI care. This study demonstrated that quality improvement initiatives aimed at achieving pre-hospital STEMI triage efficiencies have dependencies on teamwork, technology, and training in the context of 3 stages (a) pre-transport, (b) door-to-door, and (c) post-transport. A pre-hospital STEMI triage model is offered based on the findings. By incorporating this model, emergency medical coordinators in rural communities have a better opportunity to facilitate timely reperfusion therapy for this high-risk population.

The impact of team-based learning on the critical thinking skills of pharmacy students.

INTRODUCTION: Critical thinking is an important ability for pharmacists, but few studies have found improvements in pharmacy student critical thinking skills as a consequence of their education. Team-based learning (TBL) is an active learning strategy that encourages students to think critically to solve problems. The purpose of this study was to evaluate the impact of TBL on the critical thinking skills of pharmacy students. METHODS: One hundred ninety students from the first two cohorts at a pharmacy school were invited to participate. The Health Science Reasoning Test (HSRT) was administered prior to the first semester and after two years of the TBL-based pharmacy curriculum. Student's t-test was used for a pairwise analysis along with Welch's t-test for unequal variances when comparing HSRT score modulation. RESULTS: There was an overall increase in mean HSRT score. However, some participants (29%) with initially higher mean HSRT scores did not demonstrate an increase. Nearly all (99%) participants demonstrated improvements of one of the eight domains of critical thinking evaluated in the HSRT. This corresponded with an improvement in score of the majority of participants (n = 115). CONCLUSION: This study provides evidence that TBL improves critical thinking skills. More research is needed to identify the specific aspects of TBL that influence critical thinking.

Expression of 25-hydroxyvitamin D-1-alpha-hydroxylase, and vitamin D receptor mRNA in normal and malignant breast tissue.

Vitamin D has anti-proliferative and proapoptotic effects on several cell types, including breast cancer cells. There have been no studies quantifying the expression of the enzyme 25-hydroxyvitamin D-1-alpha-hydroxylase (1alphaOHase), which converts 25-hydroxyvitamin D to its active metabolite, in breast tissue. We performed real-time RT-PCR to quantity 1alphaOHase and vitamin D receptor (VDR) mRNA in samples of breast cancer, adjacent non-cancerous tissue and normal breast tissue. 1alphaOHase and VDR mRNA were universally expressed, however, 1alphaOHase expression was significantly down-regulated in adjacent non-cancerous tissue from women with breast cancer in comparison to individuals without cancer. VDR was also up-regulated in breast tumours. The vitamin D axis expression in the breast suggests a role for its actions in normal tissue homeostasis and breast cancer pathogenesis. The decreased expression of 1alphaOHase in normal tissue from women with breast cancer may be important in their predisposition to the development of cancer.