Synchronising an IMX219 image sensor and AS7265x spectral sensor to make a novel low-cost spectral camera.

A low-cost novel spectral camera able to be used for near infrared spectroscopy was made by using a Jetson Nano to synchronize a Sony IMX219 NOIR autofocus image sensor, an AMS AS7265x 18-channel spectral sensor and Osram SFH 4737 broadband infrared LED's. Synchronizing an image sensor and spectral sensor augments a standard RGB image with light spectrum information; capturing the light distribution information normally lost in RGB image capture. Sutherland et al. [1] used this novel spectral camera to examine the dorsal surface of juvenile lobsters as a possible pre-moult detector. Having the image and spectrum in combination allowed the incomplete and unmineralized post-moult dorsal surface to be characterized with 86.7% accuracy for the first time. A proposed application for the spectral camera is to omit the local SFH 4737 light source and use the camera in daylight, effectively making a low-cost substitute hyperspectral snapshot camera. In this configuration the camera may have application for low-cost drone deployment for small scale agriculture.

Learning to Learn Independently: Guiding Students to Develop Self-Directed Learning Skills During Medical Student Independent Research Projects-Findings from an Australian University.

Introduction: Most medical schools offer students the opportunity to conduct independent research projects in order to learn about evidence-based medicine. This study aimed to explore the experience of students, graduates, and supervisors during an independent research project through the lens of self-directed learning. Methods: Students and recent graduates were asked to complete an anonymous survey about their experiences. Semi-structured interviews were also conducted with a purposeful sample of 11 students, 14 graduates, and 25 supervisors. Interviews were recorded and transcribed. An inductive thematic analysis was conducted and themes were refined through the lens of self-directed learning. Results: Most participants agreed that the independent research project could enable students to develop valuable self-directed learning skills. Participants commented on the importance of the research mentor, faculty support structures, and membership of a research team. Participants who were not well supported described feeling distressed and isolated. Discussion: Medical student involvement in independent research projects can develop self-directed learning skills in the presence of a one-to-one mentoring relationship with a research supervisor, structured guidelines and support from the faculty, and membership of a research team. The development of self-directed learning skills should be part of the learning outcomes of any independent student research project. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-024-02054-4.

A Retrospective Cohort Analysis Comparing Analytic and Holistic Marking Rubrics in Medical Research Education.

OBJECTIVES: The use of analytic rubrics in assessing and grading students' performance has become more prominent among instructors due to its reliability and validity in ensuring consistency in student evaluation. However, there is limited evidence demonstrating the consistency of examiner judgments between analytic marking rubrics and holistic marking rubrics. METHODS: Therefore, we aimed to compare the consistency of marks given using holistic marking methods and analytic rubrics at an Australian university by analyzing the mean mark differences and number of adjudications between two rubric types as well as the inter-rater reliability between two assessors. We analyzed all scores for project manuscripts between 2016 and 2021 for Honours medical students. We compared the mean mark differences graded using the Kruskal-Wallis test and Welch t-test. We used chi-squared tests to compare the frequency of adjudications for each rubric type. We assessed interrater reliability by comparing the marks between the two examiners utilizing Pearson correlation. RESULTS: We found that analytic rubrics have lower mean mark differences and fewer adjudicators are required. We showed a strong positive association between the consistency of marks given and the use of analytic rubrics when compared to holistic marking. Pearson correlation showed a low but stronger correlation between marks awarded by the two assessors when analytic rubrics were used (r = 0.36), compared to holistic marking rubrics (r = 0.24). CONCLUSIONS: Our findings suggest that the use of analytic rubrics may increase the consistency and reliability between two independent examiners in marking medical students' work.

An introduction to key ecological concepts, financial opportunities, and risks underpinning aspirations for nature positive.

Global biodiversity is in decline, and businesses and society are being required to urgently create new operating models to ameliorate the crisis. Among the strategies proposed to do this, implementing the concept of nature positive has captured worldwide attention. Critical to its success will be effective collaboration between ecologists and businesspeople, driven by a shared understanding of key nature positive terminology, concepts, and risks. To this end, we introduce three core aspects: the ecological concepts in the definition of nature positive (health, abundance, diversity, and resilience), a typology of financial instruments that may be applied to achieving nature positive, and an overview of risks to biodiversity and society. The pivotal findings include that ecological complexity and uncertainty belie the simplicity of the definition of nature positive and that managing risk requires embedding aspirations into existing and emerging biodiversity conservation and restoration science and policy. Although it is challenging, nature positive deserves pursuit.

Human TMEFF1 is a restriction factor for herpes simplex virus in the brain.

Most cases of herpes simplex virus 1 (HSV-1) encephalitis (HSE) remain unexplained1,2. Here, we report on two unrelated people who had HSE as children and are homozygous for rare deleterious variants of TMEFF1, which encodes a cell membrane protein that is preferentially expressed by brain cortical neurons. TMEFF1 interacts with the cell-surface HSV-1 receptor NECTIN-1, impairing HSV-1 glycoprotein D- and NECTIN-1-mediated fusion of the virus and the cell membrane, blocking viral entry. Genetic TMEFF1 deficiency allows HSV-1 to rapidly enter cortical neurons that are either patient specific or derived from CRISPR-Cas9-engineered human pluripotent stem cells, thereby enhancing HSV-1 translocation to the nucleus and subsequent replication. This cellular phenotype can be rescued by pretreatment with type I interferon (IFN) or the expression of exogenous wild-type TMEFF1. Moreover, ectopic expression of full-length TMEFF1 or its amino-terminal extracellular domain, but not its carboxy-terminal intracellular domain, impairs HSV-1 entry into NECTIN-1-expressing cells other than neurons, increasing their resistance to HSV-1 infection. Human TMEFF1 is therefore a host restriction factor for HSV-1 entry into cortical neurons. Its constitutively high abundance in cortical neurons protects these cells from HSV-1 infection, whereas inherited TMEFF1 deficiency renders them susceptible to this virus and can therefore underlie HSE.

Exploring The Impact of Acceptance and Commitment Based Cultural Humility Training on Standardized Patient Interactions: Revisiting the Measurement Process.

Recent findings in health sciences and medical education highlight the importance of training healthcare professionals to interact with their patients in a culturally humble manner (Nadal et al., in Journal of Counseling and Development 92: 57-66, 2014; Pascoe & Smart Richman, in Psychological Bulletin 135: 531, 2009; Sirois & Burg, in Behavior Modification 27: 83-102, 2003; Williams & Mohammed, in Journal of Behavioral Medicine 32: 20-47, 2009). An important piece in the progression of our ability to address training challenges is the assessment of cultural humility. As an extension of previous research (Lombardero et al., in Journal of Clinical Psychology in Medical Settings, 30: 261-273, 2023), this study implemented an evidence-based cultural humility intervention (based on Acceptance and Commitment Training) to improve medical students' interactions with standardized patients (SPs) which was assessed via direct behavioral observation. Specifically, the observational measurement system was focused on culturally humble responses to patients reporting microaggressions to the medical professional. A pre-post comparison of the results demonstrated statistically significant improvements pertaining to participants' culturally humble responses to SPs' reports of microaggressions for one of the measurement scales used (i.e., ARISE), but not the other (i.e., Responsiveness to Racial Challenges Scale). Further analyses, on the bottom quartile of performers, were conducted to assess a possible ceiling effect of the scale that did not demonstrate significant change. These results and implications for future research will be discussed.

"Excited When They See Their Name in Print": Research Outputs from an Australian Medical Program.

To promote evidence-based practice, medical schools offer students opportunities to undertake either elective or mandatory research projects. One important measure of the research program success is student publication rates. In 2006, UNSW Medicine implemented a mandatory research program in the 4th year of the undergraduate medical education program. This study identified student publication rates and explored student and supervisor experiences with the publication process. A retrospective audit of student publications from the 2007, 2011, and 2015 cohorts was undertaken to look at trends over time. Data collected included type of publication and study methodology. Semi-structured interviews were conducted with a sample of undergraduate students (n = 11), medical graduates (n = 14), and supervisors (n = 25) and analysed thematically. Student publication rates increased significantly (P = 0.002) from 28% in 2007 to 50.2% in 2015. Students able to negotiate their own project were more likely to publish (P = 0.02). Students reported personal affirmation and development of research skills from publishing their research findings, while graduates noted improved career opportunities. Supervisors expected students to publish but identified the time to publications and student motivation as key factors in achieving publication(s). A high publication rate is possible in a mandatory research program where students can negotiate their own topic and are given protected time. Publications happen after the research project has finished. Critical factors in successful publication include supervisor support and student motivation. Given the importance of the supervisor's role, staff development and faculty support to train and develop a body of skilled supervisors is required.

Enzyme-Linked Immunosorbent Assay Using Henipavirus-Receptor EphrinB2 and Monoclonal Antibodies for Detecting Nipah and Hendra Viruses.

The Nipah virus (NiV) and the Hendra virus (HeV) are highly pathogenic zoonotic diseases that can cause fatal infections in humans and animals. Early detection is critical for the control of NiV and HeV infections. We present the development of two antigen-detection ELISAs (AgELISAs) using the henipavirus-receptor EphrinB2 and monoclonal antibodies (mAbs) to detect NiV and HeV. The NiV AgELISA detected only NiV, whereas the NiV/HeV AgELISA detected both NiV and HeV. The diagnostic specificities of the NiV AgELISA and the NiV/HeV AgELISA were 100% and 97.8%, respectively. Both assays were specific for henipaviruses and showed no cross-reactivity with other viruses. The AgELISAs detected NiV antigen in experimental pig nasal wash samples taken at 4 days post-infection. With the combination of both AgELISAs, NiV can be differentiated from HeV. Complementing other henipavirus detection methods, these two newly developed AgELISAs can rapidly detect NiV and HeV in a large number of samples and are suitable for use in remote areas where other tests are not available.

Economic impact analysis of a minimally invasive temperature-controlled radiofrequency device versus nasal surgery for the treatment of nasal airway obstruction in the United States.

OBJECTIVE: To determine the economic impact of a minimally invasive temperature-controlled radiofrequency (TCRF) device for treating nasal airway obstruction (NAO). METHODS: A budget impact model was developed for two scenarios: a reference scenario of functional rhinoplasty surgery with concomitant septoplasty and inferior turbinate reduction (ITR) performed in the hospital outpatient department where TCRF is not an available treatment option and a new scenario consisting of in-office TCRF treatment of the nasal valve and ITR. A payor perspective was adopted with a hypothetical population plan size of one million members. Costs were estimated over a time horizon of 4 years. The eligible population included patients with severe/extreme NAO and nasal valve collapse (NVC) as the primary cause or significant contributor. Data inputs were sourced from targeted literature reviews. Uncertainty within the model structure and input parameters was assessed using one-way sensitivity analysis. RESULTS: The introduction of a TCRF device resulted in population-level cost savings of $20,015,123 and per-responder average cost savings of $3531 through a 4-year time horizon due to lower procedure costs and complication rates of the device relative to the surgical comparator. Results were robust when varying parameter values in sensitivity analyses, with cost savings being most sensitive to the prevalence of NAO and estimated response rates to functional rhinoplasty and TCRF. CONCLUSIONS: In patients with severe/extreme NAO, with NVC as the primary or major contributor, introducing TCRF with ITR as a treatment option demonstrates the potential for significant cost savings over functional rhinoplasty with septoplasty and ITR.

Nasal valve dysfunction is a common cause of nasal airway obstruction (NAO) that has a significant impact on heath and quality of life for affected individuals. Previously, patients were offered temporary measures or a type of surgery called functional rhinoplasty which is a highly complex surgery that can be costly, requires recovery time, and in rare cases, not be successful. Recently, a new minimally invasive treatment alternative for NAO called temperature-controlled radiofrequency (TCRF) that may be performed in a surgery center or a doctor's office has become available. This paper provides the results of budget impact analysis performed to assess whether adding the TCRF procedure in place of surgery as a choice for patients with NAO will result in cost savings to an insurance payer with 1 million covered individuals in the United States over a period of 4 years. Results show that TCRF may result in an average of 9,416 fewer rhinoplasty surgeries, provide an average 4-year cost-savings of $3,531 for every patient that responds to TCRF treatment, and a savings of $20,015,123 over 4 years for the insurance provider. These potential cost savings over 4 years would likely be due to reduced procedure costs and complication rates compared to surgery.

Natural capital approaches for the optimal design of policies for nature recovery.

By embedding a spatially explicit ecosystem services modelling tool within a policy simulator we examine the insights that natural capital analysis can bring to the design of policies for nature recovery. Our study is illustrated through a case example of policies incentivising the establishment of new natural habitat in England. We find that a policy mirroring the current practice of offering payments per hectare of habitat creation fails to break even, delivering less value in improved flows of ecosystem services than public money spent and only 26% of that which is theoretically achievable. Using optimization methods, we discover that progressively more efficient outcomes are delivered by policies that optimally price activities (34%), quantities of environmental change (55%) and ecosystem service value flows (81%). Further, we show that additionally attaining targets for unmonetized ecosystem services (in our case, biodiversity) demands trade-offs in delivery of monetized services. For some policy instruments it is not even possible to achieve the targets. Finally, we establish that extending policy instruments to offer payments for unmonetized services delivers target-achieving and value-maximizing policy designs. Our findings reveal that policy design is of first-order importance in determining the efficiency and efficacy of programmes pursuing nature recovery. This article is part of the theme issue 'Bringing nature into decision-making'.

Evaluation of a Combined Live Attenuated Vaccine against Lumpy Skin Disease, Contagious Bovine Pleuropneumonia and Rift Valley Fever.

The use of effective vaccines is among the most important strategies for the prevention and progressive control of transboundary infectious animal diseases. However, the use of vaccine is often impeded by the cost, a lack of cold chains and other factors. In resource-limited countries in Africa, one approach to improve coverage and reduce cost is to vaccinate against multiple diseases using combined vaccines. Therefore, the objective of this study was to evaluate a combined vaccine for the prevention and control of Lumpy Skin Disease (LSD), Contagious Bovine Pleuropneumonia (CBPP) and Rift Valley fever (RVF). The LSD and CBPP were formulated as a combined vaccine, and the RVF was formulated separately as live attenuated vaccines. These consisted of a Mycoplasma MmmSC T1/44 strain that was propagated in Hayflick-modified medium, RVF virus vaccine, C13T strain prepared in African green monkey cells (Vero), and the LSDV Neethling vaccine strain prepared in primary testis cells. The vaccines were tested for safety via the subcutaneous route in both young calves and pregnant heifers with no side effect, abortion or teratogenicity. The vaccination of calves induced seroconversions for all three vaccines starting from day 7 post-vaccination (PV), with rates of 50% for LSD, 70% for CBPP and 100% for RVF, or rates similar to those obtained with monovalent vaccines. The challenge of cattle vaccinated with the LSD/CBPP and the RVF vaccine afforded full protection against virulent strains of LSDV and RVFV. A satisfactory level of protection against a CBPP challenge was observed, with 50% of protection at 6 months and 81% at 13 months PV. A mass vaccination trial was performed in four regions of Burkina Faso that confirmed safety and specific antibody responses induced by the vaccines. The multivalent LSD/CBPP+RVF vaccine provides a novel and beneficial approach to the control of the three diseases through one intervention and, therefore, reduces the cost and improves vaccination coverage.

Whole-genome sequencing of SARS-CoV-2 from the initial cases of domestic cat infections in Canada.

Two cat nasal swabs from Canada's earliest confirmed SARS-CoV-2 positive domestic cats were sequenced to over 99% SARS-CoV-2 genome coverage. One cat had lineage A.23.1 SARS-CoV-2 not reported before in animals. Both sequences have multiple spike gene mutations and clustered closely with human-derived sequences in the global SARS-CoV-2 phylogenetic tree.

Distinguishing host responses, extensive viral dissemination and long-term viral RNA persistence in domestic sheep experimentally infected with Crimean-Congo haemorrhagic fever virus Kosovo Hoti.

Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is a tick-borne, risk group 4 pathogen that often causes a severe haemorrhagic disease in humans (CCHF) with high case fatality rates. The virus is believed to be maintained in a tick-vertebrate-tick ecological cycle involving numerous wild and domestic animal species; however the biology of CCHFV infection in these animals remains poorly understood. Here, we experimentally infect domestic sheep with CCHFV Kosovo Hoti, a clinical isolate representing high pathogenicity to humans and increasingly utilized in current research. In the absence of prominent clinical signs, the infection leads to an acute viremia and coinciding viral shedding, fever and markers for potential impairment in liver and kidney functions. A number of host responses distinguish the subclinical infection in sheep versus fatal infection in humans. These include an early reduction of neutrophil recruitment and its chemoattractant, IL-8, in the blood stream of infected sheep, whereas neutrophil infiltration and elevated IL-8 are features of fatal CCHFV infections reported in immunodeficient mice and humans. Several inflammatory cytokines that correlate with poor disease outcomes in humans and have potential to cause vascular dysfunction, a primary hallmark of severe CCHF, are down-regulated or restricted from increasing in sheep. Of particular interest, the detection of CCHFV RNA (including full-length genome) in a variety of sheep tissues long after the acute phase of infection indicates a widespread viral dissemination in the host and suggests a potentially long-term persisting impact of CCHFV infection. These findings reveal previously unrecognized aspects of CCHFV biology in animals.

Domestic pigs are susceptible to experimental infection with non-human primate-derived Reston virus without the need for adaptation.

Domestic pigs are a critical component of the food supply and one of the most commonly raised production animals. Pork consumption has driven the intensification of pig production expanding into environments conducive to increased emergence and spread of infectious diseases, including the spillover of pathogens into human populations. One of these emerging viruses, Reston virus (RESTV), is an enigma among the Orthoebolavirus genus in that its lack of human pathogenicity is in stark contrast to the high virulence associated with most other ebolaviruses. RESTV is, however, associated with outbreaks of highly lethal hemorrhagic disease in non-human primates (NHP), as well as poorly understood clinical manifestations of mixed virulence and lethality in naturally and experimentally infected domestic pigs. Our results show it is possible for RESTV derived from an NHP to infect domestic pigs resulting in a spectrum of disease, from asymptomatic to severe respiratory distress. Further, we report on the first experimental transmission of RESTV between infected pigs and a co-housed, naïve animal, as well as the first report of the successful use of group oral fluids for the detection of RESTV RNA and virus-specific IgA antibodies.

Head-to-head comparison of BAM15, semaglutide, rosiglitazone, NEN, and calorie restriction on metabolic physiology in female db/db mice.

Metabolic disorders such as type 2 diabetes, fatty liver disease, hyperlipidemia, and obesity commonly co-occur but clinical treatment options do not effectively target all disorders. Calorie restriction, semaglutide, rosiglitazone, and mitochondrial uncouplers have all demonstrated efficacy against one or more obesity-related metabolic disorders, but it currently remains unclear which therapeutic strategy best targets the combination of hyperglycaemia, liver fat, hypertriglyceridemia, and adiposity. Herein we performed a head-to-head comparison of 5 treatment interventions in the female db/db mouse model of severe metabolic disease. Treatments included ∼60 % calorie restriction (CR), semaglutide, rosiglitazone, BAM15, and niclosamide ethanolamine (NEN). Results showed that BAM15 and CR improved body weight and liver steatosis to levels superior to semaglutide, NEN, and rosiglitazone, while BAM15, semaglutide, and rosiglitazone improved glucose tolerance better than CR and NEN. BAM15, CR, semaglutide, and rosiglitazone all had efficacy against hypertriglyceridaemia. These data provide a comprehensive head-to-head comparison of several key treatment strategies for metabolic disease and highlight the efficacy of mitochondrial uncoupling to correct multiple facets of the metabolic disease milieu in female db/db mice.

Host-microbiome interactions in nicotinamide mononucleotide (NMN) deamidation.

The nicotinamide adenine dinucleotide (NAD+ ) precursor nicotinamide mononucleotide (NMN) is a proposed therapy for age-related disease, whereby it is assumed that NMN is incorporated into NAD+ through the canonical recycling pathway. During oral delivery, NMN is exposed to the gut microbiome, which could modify the NAD+ metabolome through enzyme activities not present in the mammalian host. We show that orally delivered NMN can undergo deamidation and incorporation in mammalian tissue via the de novo pathway, which is reduced in animals treated with antibiotics to ablate the gut microbiome. Antibiotics increased the availability of NAD+ metabolites, suggesting the microbiome could be in competition with the host for dietary NAD+ precursors. These findings highlight new interactions between NMN and the gut microbiome.

Tools for deprescribing in severe dementia: A scoping review.

OBJECTIVES: Identification of inappropriate medications in people living with severe dementia is a complex task which has the potential to reduce avoidable adverse events and increase quality of life. This scoping review (i) identifies published tools intended to aid deprescribing in people living with severe dementia and (ii) describes evaluations of their usefulness in clinical practice. METHODS: A scoping review was undertaken, with Medline, Medline in Process, EMBASE, Cochrane Library, CINAHL, Scopus and Web of Science databases, from inception to April 2023, identifying tools for deprescribing in severe dementia. A tool was considered as any resource for deprescribing, including clinical study, scientific publication, health guideline, website, algorithm, model or framework. Two reviewers assessed the eligibility of articles through abstract and full text review. Data extracted from included studies were summarized through narrative synthesis. RESULTS: Twelve studies were identified from 18,633 articles screened. Tools were categorized into three groups: deprescribing interventions (n = 2), consensus-based deprescribing criteria (n = 5), and medication-specific recommendations (n = 5). Six studies developed tools using expert opinion and ten tools were tested in people living with severe dementia. Only one of the four studies that evaluated patient outcomes (cognitive change and adverse events) identified clear clinical benefit from medication withdrawal. CONCLUSIONS: Clinical application of current deprescribing tools is limited due to the lack of evidence-based research on the clinical effects of individual medication deprescribing in people with severe dementia. Further research on patient outcomes, including cognitive change and adverse events, will help clarify the role of these tools in clinical care.

A Survey of Industry Perceptions of Facilitated Regulatory Pathways in Drug Development in Australia.

BACKGROUND: In Australia, facilitated regulatory pathways (FRPs) became available with the introduction of priority review (PR) in 2017 and provisional approval (PA) in 2018, which aim to facilitate expedited review and approval for novel medicines. The pathways were developed in consultation with a wide range of stakeholders and have since been utilised by pharmaceutical companies for various therapeutic products. However, the perceptions of the firsthand users of these pathways have not been evaluated in Australia. OBJECTIVES: We have conducted a survey of Australian regulatory professionals aiming to solicit the perceived benefits, barriers to utilisation, shortcomings and proposed modifications to utilising these pathways. We have also solicited the users' perspective on key aspects of the pathways, including overall satisfaction, regulatory burden, availability and ease of use of guidelines, regulator support, impact on company strategy and recommendations for improvement. METHODS: A survey was developed and distributed to Australian regulatory professionals from the pharmaceutical industry who had submission experience of new medicine applications via either PR, PA or the standard registration pathway to the Therapeutic Goods Administration (TGA). The questionnaire consisted of 44 questions with a skip logic and the option for free text comments. RESULTS: We received responses from 16/42 companies that had utilised these new pathways. Nine respondents had experience with the PR pathway and ten with the PA pathway. The respondents were generally satisfied with the effectiveness of the PR process in expediting registration approvals, but they were ambivalent towards the PA pathway in terms of overall satisfaction and timelines. Respondents expressed a desire for further improvements in the speed of approval, earlier access for patients across various pathways and introduction of new Health Technology Assessment processes for medicines approved under PA. CONCLUSION: While the FRPs have been an important and positive development in the Australian regulatory landscape, there remain opportunities for further improvements, some of which have been highlighted by this study and may help inform future regulatory decisions.

Strategies to Improve Multi-enzyme Compatibility and Coordination in One-Pot SHERLOCK.

While molecular diagnostics generally require heating elements that supply high temperatures such as 95 °C in polymerase chain reaction and 60-69 °C in loop-mediated isothermal amplification, the recently developed CRISPR-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can operate at 37 °C or a similar ambient temperature. This unique advantage may be translated into highly energy-efficient or equipment-free molecular diagnostic systems with unrestricted deployability. SHERLOCK is characterized by ultra-high sensitivity when performed in a traditional two-step format. For RNA sensing, the first step combines reverse transcription with recombinase polymerase amplification, while the second step consists of T7 transcription and CRISPR-Cas13a detection. The sensitivity drops dramatically, however, when all these components are combined into a single reaction mixture, and it largely remains an unmet need in the field to establish a high-performance one-pot SHERLOCK assay. An underlying challenge, conceivably, is the extremely complex nature of a one-pot formulation, crowding a large number of reaction types using at least eight enzymes/proteins. Although previous work has made substantial improvements by serving individual enzymes/reactions with accommodating conditions, we reason that the interactions among different enzymatic reactions could be another layer of complicating factors. In this study, we seek optimization strategies by which inter-enzymatic interference may be eliminated or reduced and cooperation created or enhanced. Several such strategies are identified for SARS-CoV-2 detection, each leading to a significantly improved reaction profile with faster and stronger signal amplification. Designed based on common molecular biology principles, these strategies are expected to be customizable and generalizable with various buffer conditions or pathogen types, thus holding broad applicability for integration into future development of one-pot diagnostics in the form of a highly coordinated multi-enzyme reaction system.

Comparative characterization of Crimean-Congo hemorrhagic fever virus cell culture systems with application to propagation and titration methods.

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is a biosafety level 4 and World Health Organization top priority pathogen. Infection leads to an often fatal hemorrhagic fever disease in humans. The tick-borne virus is endemic in countries across Asia, Europe and Africa, with signs of spreading into new regions. Despite the severity of disease and the potential of CCHFV geographic expansion to cause widespread outbreaks, no approved vaccine or treatment is currently available. Critical for basic research and the development of diagnostics or medical countermeasures, CCHFV viral stocks are commonly produced in Vero E6 and SW-13 cell lines. While a variety of in-house methods are being used across different laboratories, there has been no clear, specific consensus on a standard, optimal system for CCHFV growth and titration. In this study, we perform a systematic, side-by-side characterization of Vero E6 and SW-13 cell lines concerning the replication kinetics of CCHFV under different culture conditions. SW-13 cells are typically cultured in a CO2-free condition (SW-13 CO2-) according to the American Type Culture Collection. However, we identify a CO2-compatible culture condition (SW-13 CO2+) that demonstrates the highest viral load (RNA concentration) and titer (infectious virus concentration) in the culture supernatants, in comparison to SW-13 CO2- and Vero E6 cultures. This optimal viral propagation system also leads to the development of two titration methods: an immunostaining-based plaque assay using a commercial CCHFV antibody and a colorimetric readout, and an antibody staining-free, cytopathic effect-based median tissue culture infectious dose assay using a simple excel calculator. These are anticipated to serve as a basis for a reproducible, standardized and user-friendly platform for CCHFV propagation and titration.