In quantitative polymerase chain reaction (qPCR), multiplexing increases sample throughout, while reducing the time and cost associated with having separate reactions for each target. Furthermore, the variations that occurs between reactions are minimized when two targets of interest are measured within the same reaction. Due to these benefits, multiplexing in qPCR are widely used in different areas of research, with many applications such as diagnostics, treatment input and response monitoring, etc. While multiplexing in qPCR is advantageous, challenges such as assay design, workflow optimization, and reagent selection need to be addressed to help ensure the accuracy of the experimental output. In this webinar, the factors that impact multiplexing in qPCR are discussed and tools from Thermo Fisher Scientific that are designed for multiplexing success are presented including Applied Biosystems TaqMan Custom QSY and QSY2 based probes and TaqPath DuraPlex 1-Step RT-qPCR Master Mix.

Learning Objectives

1. Multiplexing for qPCR workflows

   • Benefits and condsiderations

   • Assay design guidelines

2. Thermo Fisher Scientific custom oligos for multiplexing needs

   • ​​​​​Applied Biosystem TaqMan Custom QSY and QSY2 based probes.

3. Features of master mixes designed for multiplexing.

   • Portfolio of master mixes with multiplexing capabilities

The effect of long-term mixed-waste contamination, particularly uranium and nitrate, on the microbial community in the terrestrial subsurface at the Oak Ridge Integrated Field Research Challenge (ORIFRC) site in Oak Ridge, TN has been extensively characterized to determine appropriate remediation strategies. In this talk, I will review combined cultivation-based and cultivation-independent molecular analyses used to understand the composition of microbial communities across a contamination gradient at the ORIFRC site. Key findings of these studies include the highly abundant presence of active, denitrifying bacteria from the genus Rhodanobacter at the contamination source zone and the presence of denitrifying fungi at lower levels. The genomics and physiology of Rhodanobacter isolates are explored to understand the physiological mechanisms by which these organisms achieve their dominance in the source zone. In addition, I examine the critical role of methodology in microbial community analyses and highlight the importance of combined cultivation-based and cultivation-independent methodologies for characterizing the microbial ecology of environmental systems.

Learning Objectives:

1. Review the critical role for cultivation in understanding cultivation-independent sequence data.

2. Discuss genomic capabilities of microorganisms tolerating a highly contaminated subsurface environment.

3. Summarize the ecological niche for bacteria from the genus Rhodanobacter.

The effects of spaceflight on those beneficial microbes that associate with animal tissues is an emerging area in space biology, however, many questions persist regarding how homeostasis between host and microbiome is maintained in the space environment. In this presentation, I address a recent space flight experiment in which the responses of both host and microbe were examined in vivo under spaceflight conditions. Specifically, we examined the onset of colonization of the bacterium Vibrio fischeri on the light organ crypt epithelial cells of the host squid Euprymna scolopes under spaceflight conditions. The discussed results suggest that maintaining healthy relationships with the natural microbiome may be critical for maintain homeostasis during spaceflight.

Learning Objectives: 

1. Review the complexities of conducting a space flight experiment.

2. Summarize how beneficial microbes can induce host morphogenesis.

3. Demonstrate understanding of how the space flight environment can alter animal physiology.

Trauma such as burn continues to be a major cause of morbidity and mortality in the United States. Gut barrier dysfunction is often implicated in post trauma complications. Intoxication at time of injury could further exacerbate these post injury complications. We will share our findings on the changes in gut barrier integrity and its role in systemic inflammation in rodent models of alcohol traumatic injury.

A study of microbiome structure and function associated with plants roots will be presented. the study showed that the microbiome structure is determined by the plant type, with root microbiome is related in cereals (corn, wheat) and in cucumber and tomato is different from that of the cereals and from each other. When examining gene expression in these communities we realized that while denitrification genes are highly expressed, the final gene (nosZ), responsible to the reduction of N2O (a potent greenhouse gas) to N2 is not. That may explain the fact that most of anthropogenic emission is from agricultural soils. N2O reducing bacteria, isolated from wheat roots, could prevent up to 40% of the emission

The formation of marine snow is an important part of the biological carbon pump as carbon is exported to depth. These particles are composed of cells, minerals, and other sources of organic carbon that can be held together by transparent exopolymeric particles (TEP) acting as a glue-like matrix. Due to their small size, heterotrophic bacteria were previously expected to not contribute appreciably to aggregation. However, heterotrophic bacteria have been found to influence the aggregation of phytoplankton and in some cases result in an increase in TEP production, but it is unclear exactly how they contribute to aggregation. We determined the aggregation potential of Pseudoalteromonas carrageenovora, Vibrio thalassae, and Marinobacter adhaerens HP15 through growth experiments with axenic cultures to determine growth, TEP production, and aggregation. The bacteria were also inoculated into roller tanks to determine their ability to form macroaggregates. M. adhaerens HP15 had the lowest cell-normalized TEP production at all growth stages and the lowest microaggregate formation. The cell-normalized TEP production and microaggregate formation was not significantly different between P. carrageenovora and V. thalassae. All bacteria formed visible macroaggregates in roller tanks with clay addition and exhibited high sinking velocities (150-1200 m d-1) that are comparable to those of aggregates formed by large mineral ballasted phytoplankton. Experiments involving the simulation of ambient and future ocean conditions using natural seawater in mesocosms have revealed a stark difference in macroaggregate formation between treatments, potentially indicating a difference in microbial community and TEP composition. These findings expand our understanding of how heterotrophic bacteria can contribute to export flux in the ocean.

In recent past respiratory seasons, the world witnessed a “tripledemic” of Influenza (flu), SARS-CoV-2 (COVID-19), and Respiratory Syncytial Virus (RSV). In this presentation, I will discuss the primary microbiology, epidemiology, control, prevention, vulnerable populations, the importance of laboratory validation, and the benefits of molecular-based testing of these three primary respiratory season pathogens. The 2024-25 landscape will likely be different, with new virus variants, new vaccines and treatments, like the game-changing antibody that protects children from RSV. The healthcare and public health community have also learned some valuable lessons going forward with the ever-evolving respiratory agents. How can we all best prepare for the upcoming season?

Bacterial vaginosis is a common cause of vaginal complaints, mainly of odor and discharge in women of reproductive age. BV is poorly understood and recurrence is frequent. More questions than answers remain, but new molecular studies are beginning to provide some additional insights into this condition which could in the future lead to better management strategies. This presentation will discuss new insights into BV based primarily on microbiome-related research.

Antipsychotic (AP) medications are increasingly recognised for their association with a heightened risk of infections. Clozapine, an atypical AP reserved for treatment-resistant schizophrenia (TRS), has been particularly linked to an elevated risk of respiratory infections. AP that exert antimicrobial properties are known to cause microbiome imbalances, fostering an environment for pathobiont colonisation. In a dysbiotic oral microbiome, colonisation with opportunistic pathogens could heighten respiratory infection risks. An increased respiratory infection trend is observed amongst patients prescribed clozapine “on” and "off-label". Our work aims to characterise the transient oral pathobiont colonisation rate linked to clozapine usage.

Saliva samples were collected from patients diagnosed with TRS prescribed clozapine and a control group not on AP or diagnosed with schizophrenia or schizoeffective disorder. Participants' background medical history and oral health were also surveyed. Identified transient pathobiont isolates underwent phenotypic and genomic analysis, including antimicrobial susceptibility testing (AST), genome sequencing, and typing of virulence factors (VFs) and antimicrobial resistance genes (ARGs).

Transient pathobionts were found in the saliva of 74% of clozapine users, with 43% showing multi-colonisation. The control group had a less than 10% colonisation rate. Sixteen species of Enterobacteriaceae and three species of Moraxellaceae were identified. All isolate genomes had genotypes of increased virulence potential, averaging 77 VF genes per genome. Notably virulent Escherichia coli and Kalamiella piersonii have been was identified encoding VF facilitating blood-brain barrier penetration. Possible patient-to-patient transmission of Enterobacter hormaechei was also noted.

Clozapine use is linked to oral transient pathobiont colonization, which may contribute to the increased risk of respiratory infections observed in antipsychotic users.

The skin epidermis is the body's main defense against dehydration and harmful substances. We used germ-free mice to show that the microbiota is essential for proper differentiation and repair of the epidermal barrier. This effect is mediated by the aryl hydrocarbon receptor (AHR), a regulator of epidermal differentiation. We hypothesized that the skin microbiota activates AHR to promote barrier repair. Tryptophan metabolites, which are potent AHR ligands, are enriched in the skin's outermost layer (stratum corneum) and can be produced by microbial metabolism. We constructed metabolic enzyme profiles and mined them against healthy human skin metagenomes. These analyses revealed motif enrichment for enzymes that metabolize tryptophan to indole and its derivatives. To identify microbially regulated tryptophan metabolites in vivo, we established a gnotobiotic model with 50 skin commensals from healthy humans and performed targeted mass spectrometry on murine skin. We found three novel indole-related metabolites that are regulated by microbes and tested therapeutic efficacy in a murine model of atopic dermatitis. We provide a novel ecological framework that demonstrates that microbiota regulates skin barrier formation and repair via tryptophan metabolism. This knowledge can guide the development of precise microbe-based therapies for various skin disorders characterized by impaired epidermal barrier function.

The ongoing surge of clade 2.3.4.4b H5N1 viruses in avian species has resulted in numerous spillover events into mammalian species including dairy cows and humans, posing a potential pandemic threat of this H5N1 clade in human population. We have previously shown that inhalation of small-particle aerosols containing clade 1 H5N1 A/Vietnam/1203/2004 (VN04) by cynomolgus macaques triggered fever and severe respiratory disease and death, and that the severity of disease was dependent on the inhaled dose of virus. Here, we investigated whether a clade 2.3.4.4b virus isolate from a human case in Chile would trigger severe respiratory disease in cynomolgus macaques. Macaques were implanted with radiotelemetry devices to continuously monitor body temperature and infected with A/Chile/25945/2023 by small-particle aerosol. On days 2, 4, 7, 11, and 15 respiratory function was assessed using a head-out plethysmography chamber. At doses equivalent to the LD50 (5.1 log10 pfu) for VN04, two out of two macaques were febrile but only had mild-moderate respiratory disease unlike VN04 virus at this dose level. At 6 log10 pfu, macaques developed respiratory disease characterized by a sharp increase in respiratory rate with one of the two succumbing on day 7 post-infection. At 6.7 log10 pfu, two out of two macaques developed severe acute respiratory distress syndrome and succumbed on day 4 post-infection. Chest radiography and gross pathology indicated severe pneumonia in euthanized macaques. This demonstrates that akin to well-characterized VN04 infection, A/Chile/25945/2023 can be lethal in cynomolgus macaques through aerosolized virus exposure although a somewhat higher dose is required to achieve the same disease outcome and lethality. Together, our macaque model of clade 2.3.4.4b H5N1 virus would foster evaluation of potential medical countermeasures against this pandemic potential virus in a pathophysiologically relevant model to human severe influenza.

Apolipoprotein E epsilon 4 (APOE4) is the primary genetic risk factor for late-onset Alzheimer's disease (AD). APOE4 carriers, especially females, tend to develop gut dysbiosis earlier than non-carriers, which may elevate their AD risk. Early intervention to address this dysbiosis is critical for risk mitigation. In this presentation, I will show that dysbiosis from AD donors significantly exacerbates AD pathology in young, healthy APOE4 mice, while a healthy donor’s microbiome may provide protective benefits against AD. I will also present data indicating that a prebiotic diet can protect brain metabolism and reduce neuroinflammation in these mice. Furthermore, I will discuss the development of precision probiotics tailored to combat gut dysbiosis in female APOE4 carriers.

Human respiratory syncytial virus (hRSV) infection is a significant global health and economic burden. Lower respiratory tract hRSV infections predominantly affect young children, the elderly, and those with compromised immune systems. Worldwide each year, there are over 3.6 million hRSV hospital admissions in children under age 5. Though 3 new vaccines were recently approved, none of these are approved for use in young children. Thus, new strategies are needed to combat hRSV. hRSV is a negative sense single-stranded RNA virus that targets the host transcriptional machinery upon infection to modulate gene expression, including host immune response genes. We previously demonstrated that hRSV nonstructural protein 1 (NS1) partitions to the host nucleus and binds to chromatin, the proteins associated with host genomic DNA. Using biochemical and chromatin mapping studies, we demonstrated that NS1 associates with the Mediator complex. The human Mediator complex is comprised of approximately 30 subunits, many of which bind to multiple transcription factors and relay signals that control gene transcription. These Mediator subunits bind to transcription factors at gene regulatory regions, which are distant from each other, via chromatin looping. In this way, the Mediator complex bridges the physical gap between gene regulatory control regions to modulate and refine the level and timing of gene transcription. We performed chromatin mapping and transcriptome sequencing that revealed colocalization of NS1 with Mediator complex near regulatory elements of genes differentially expressed during hRSV infection, particularly at interferon-induced viral response genes. Together, our results identify hRSV NS1 as a viral interactor of MED25 that modulates Mediator complex associated transcriptional activity in hRSV infection. Our ongoing work will further elucidate viral gene regulatory mechanisms and reveal targets for development of therapeutic strategies for hRSV infections.

Curious about how engineered polymerases are transforming the landscape of infectious disease diagnostics?

Our speaker, Dr. Maja Studencka-Turski, will guide you through a comprehensive discussion on:
- The fundamentals of engineered polymerases and their critical role in infectious disease diagnostics.
- An overview of MedixMDx's specialized products tailored for a variety of infectious disease applications.
- The significant enhancements these innovations bring to the accuracy and speed of disease detection.

This webinar is perfectly suited for researchers, healthcare practitioners, lab staff, and anyone keen on the latest developments in medical diagnostics.

Join this year's poster presenters in the Poster Hall during the Poster Discussion, Tuesday, September 3rd, from 10:00 –11:00 AM PDT, to chat live about their posters and latest research and findings.

In the rapidly expanding landscape of molecular diagnostics, the focus is shifting from top to bottom, with a redirection from post-pandemic respiratory testing to developments in gastrointestinal (GI) diagnostics. This presentation delves into the journey of building a molecular GI test from the ground up and enhancing the laboratory's test menu with focused panels. Through a personal case study, attendees will gain insights into the decision-making process, strategic considerations, and collaborative efforts that have propelled one lab to its current standing.

Demonstrating the important relationship between laboratory and industry expertise, this presentation showcases how the collaboration with Thermo Fisher Scientific’s Validation and Sample Preparation teams provided a custom research solution for a GI molecular test. Outlining the rationale behind creating focused panels, this presentation emphasizes the necessity for optimized workflows, pathogen relevance and anticipation of population needs.  From sample preparation to panel launch, this presentation traverses the sample prep journey and elaborates on the thought process behind choosing Thermo Fisher's GI OpenArray real-time PCR workflow solution.

Learning Objectives

• Understand key considerations when selecting a new panel 
• Discover the significance of collaborative partnerships between client and industry
• Describe use cases for syndromic and focused panels in GI testing

The global demand for point-of-care testing (POCT) for diagnosing infectious diseases is growing. Bringing the test directly to the person needing the result greatly reduces the turn-around-time between sample acquisition to result reporting. This can have a positive impact on preventing the spread of infection, correctly and adequately treating the infection, as well as provide critically needed surveillance data for your community. However, performing POCT outside of clinical or public health laboratories where highly trained personnel working in an environment steeped in quality control/quality assurance and proficiency testing requirements can be challenging. Working with lab directors and personnel with the goals of generating helpful training materials and increase the awareness around quality and test accuracy are key elements to the success of POCT. These challenges and their solutions will be discussed along with the criteria to keep in mind when designing POCT that can be successfully implemented. Lastly, we will discuss some of the newest designs and technological advances in POCT for infectious disease diagnosis, including molecular-based platforms and the impact these devices can have on global health.

Learning Objectives: 

1. Discuss applications available for infectious disease POCT.

2. Review WHO's ASSURED criteria that should be used in developing POCT devices.

3. Identify the newest technologies being used in designing POCT.

bacteriovorus is a predatory bacterium that preys on Gram-negative bacteria. B. bacteriovorus invades the prey-cell periplasm – the space between the inner and outer membrane – leaches host nutrients to replicate, and ultimately lyses the infected cell to exit and find new prey. The infectious life cycle of B. bacteriovorus is, in many ways, comparable to a bacteriophage. However, unlike phage resistance, defense against B. bacteriovorus has not been widely investigated and until recently, was not thought to exist. At this conference I will present my work uncovering a “molecular suit of armor” which blocks infection before it can occur. Curli fibers are polymers of the functional amyloid protein CsgA formed on the outer membrane of many bacteria. Using genetic and microscopy-based assays, we suggest that curli fibers form a barrier that shields susceptible cells. Bioinformatic analysis of bacterial amyloid proteins demonstrates previously unappreciated diversity in amyloid encoding genes and that production of these fibers is selectively enriched in bacteria with two membranes, the obligatory targets of B. bacteriovorus. In support of these findings, functional amyloid proteins from Pseudomonas aeruginosa [AW1] also blocked B. bacteriovorus infection. These observations suggest an unappreciated role for curli-like fibers. Indeed, we found that amyloid barriers block not only B. bacteriovorus but limit infection by other predatory bacteria too. In total, this work establishes that functional amyloids, and extracellualr barriers, are an important new dimension for the bacterial immune system.

Infectious diseases have been a major threat to human health throughout history, causing significant economic damage worldwide. In modern medicine, quickly and accurately identifying the germs causing these diseases is crucial for timely treatment and preventing their spread. In Western countries, this job is typically done by highly trained professionals using advanced technology in well-equipped labs. However, in low to middle-income countries, the lack of these skills and equipment can lead to disastrous outcomes during outbreaks.

It's estimated that by 2050, microbial infections will be the leading cause of death globally, with a significant financial burden and associated morbidity. To improve diagnosis and treatment, especially in places with fewer resources, innovative solutions are needed for point of care testing (POCT), which means testing patients on the spot.

Innovation is required to drive forward point of care testing in microbial infections particularly in the ever-evolving medical landscape. A specific example being the use of highly effective modulator therapies (HEMT) in people with Cystic Fibrosis (pwCF) whereby production of sputum is vastly or completely reduced complicating microbial surveillance in pwCF. Novel areas in detection are being explored from medical detection dogs, analyzing compounds in breath, and finding biomarkers in other body fluids. Recent advances in lateral flow technology, clinical and patient acceptability have also paved the way for lower cost rapid testing that can be performed in the community, which may also find application in low to medium income countries.

Discussion of chikungunya arthritis studies and dengue vascular permeability studies including translation science.