Development and preliminary validation of a MERS-CoV ELISA for serological testing of camels and alpacas.

This study describes the development and preliminary validation of a new serological assay using MERS-CoV S1 protein in an indirect enzyme-linked immunosorbent assay (ELISA) format. This assay has the advantage of being able to test MERS-CoV serum samples in a PC2 laboratory without the need for a high-level biocontainment laboratory (PC3 or PC4), which requires highly trained and skilled staff and a high level of resources and equipment. Furthermore, this MERS-CoV S1 ELISA enables a larger number of samples to be tested quickly, with results obtained in approximately five hours. The MERS-CoV S1 ELISA demonstrated high analytical specificity, with no cross-reactivity observed in serum of animals infected with other viruses, including different coronaviruses. We tested 166 positive and 40 negative camel serum samples and have estimated the diagnostic sensitivity (DSe) to be 99.4% (95% CI: 96.7 - 100.0%) and diagnostic specificity (DSp) to be 100% (95% CI: 97.2%-100.0%) relative to the assigned serology results (ppNT and VNT) using a S/P ratio cut-off value of >0.58. The findings of this study showed that our MERS-CoV S1 ELISA was more sensitive than the commercial EUROIMMUN ELISA (Se 99.4% vs 84.9%) and comparable to the ppNT assay, and therefore could be used as a diagnostic aid in countries in the Middle East where MERS-CoV is endemic in dromedary camels. The assay reagents and protocol were easily adapted and transferred from an Australian laboratory to a laboratory in the University of Hong Kong. Thus, the results described here show that the MERS-CoV S1 ELISA represents a cheap, rapid, robust, and reliable assay to support surveillance of MERS-CoV in camels in endemic regions.

Granting access: Development of a formal course to demystify and promote predoctoral fellowship applications for graduate students.

Strong scientific writing skills are the foundation of a successful research career and require training and practice. Although these skills are critical for completing a PhD, most students receive little formal writing instruction prior to joining a graduate program. In 2015, the University of Iowa Medical Scientist Training Program (MSTP) addressed this issue by developing the scientific writing course Grant Writing Basics (GWB). Here we describe the structure of this course and its effectiveness. GWB is an interactive, workshop-based course that uses a National Institutes of Health (NIH) F30 predoctoral fellowship proposal as a platform for building writing expertise. GWB incorporates established pedagogical principles of adult learning, including flipped classrooms, peer teaching, and reiterative evaluation. Time spent in class centers on active student analysis of previously submitted fellowship applications, discussion of writing resources, active writing, facilitated small group discussion of critiques of student writing samples, revision, and a discussion with a panel of experienced study section members and a student who completed a fellowship submission. Outcomes of GWB include a substantial increase in the number of applications submitted and fellowships awarded. Rigorous evaluation provides evidence that learning objectives were met and that students gained confidence in both their scientific writing skills and their ability to give constructive feedback. Our findings show that investment in formal training in written scientific communication provides a foundation for good writing habits, and the knowledge and skills needed to succeed in this vital aspect of a scientific research career. Furthermore, they highlight that evaluation is valuable in guiding course evolution. Strategies embedded in GWB can be adapted for use in any graduate program to advance scientific writing skills among its trainees.

Subregional analysis of striatum iron in Parkinson's disease and rapid eye movement sleep behaviour disorder.

The loss of dopamine in the striatum underlies motor symptoms of Parkinson's disease (PD). Rapid eye movement sleep behaviour disorder (RBD) is considered prodromal PD and has shown similar neural changes in the striatum. Alterations in brain iron suggest neurodegeneration; however, the literature on striatal iron has been inconsistent in PD and scant in RBD. Toward clarifying pathophysiological changes in PD and RBD, and uncovering possible biomarkers, we imaged 26 early-stage PD patients, 16 RBD patients, and 39 age-matched healthy controls with 3 T MRI. We compared mean susceptibility using quantitative susceptibility mapping (QSM) in the standard striatum (caudate, putamen, and nucleus accumbens) and tractography-parcellated striatum. Diffusion MRI permitted parcellation of the striatum into seven subregions based on the cortical areas of maximal connectivity from the Tziortzi atlas. No significant differences in mean susceptibility were found in the standard striatum anatomy. For the parcellated striatum, the caudal motor subregion, the most affected region in PD, showed lower iron levels compared to healthy controls. Receiver operating characteristic curves using mean susceptibility in the caudal motor striatum showed a good diagnostic accuracy of 0.80 when classifying early-stage PD from healthy controls. This study highlights that tractography-based parcellation of the striatum could enhance sensitivity to changes in iron levels, which have not been consistent in the PD literature. The decreased caudal motor striatum iron was sufficiently sensitive to PD, but not RBD. QSM in the striatum could contribute to development of a multivariate or multimodal biomarker of early-stage PD, but further work in larger datasets is needed to confirm its utility in prodromal groups.

Dietary Patterns Are Not Associated With Disease Activity Among Patients With Inflammatory Conditions of the Pouch in a Prospective Cohort.

Background: Evidence-based recommendations regarding the influence of diet on inflammatory conditions of the pouch after restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) are limited. Methods: We analyzed dietary patterns at enrollment in a prospective registry of patients with 1 of 4 inflammatory conditions of the pouch (acute pouchitis, chronic antibiotic-dependent pouchitis, chronic antibiotic refractory pouchitis, and Crohn's disease of the pouch). We analyzed dietary intake by disease activity at enrollment and then compared dietary patterns among patients who remained in remission throughout the 12-month follow-up to those patients who experienced a disease relapse. We also compared dietary patterns among patients with inflammatory conditions of the pouch to the United States Department of Agriculture (USDA) recommended daily goals. Results: Among 308 patients, there were no differences in dietary patterns among patients with 1 of the 4 disease states at enrollment. Additionally, among the 102 patients in remission at baseline, there were no significant differences noted among patients who went on to experience a disease flare in the 12 months after enrollment compared to those patients who remained in remission. However, patients with inflammatory conditions of the pouch demonstrated decreased intake of several food groups and macronutrients including dairy, fruits, vegetables, whole grains, and fiber when compared to USDA recommendations. Conclusions: In a prospective cohort, we demonstrated no impact of dietary patterns on disease activity. The relative deficiencies in several food groups and macronutrients among patients after IPAA indicate the potential role of targeted nutritional counseling in this population.

Potent pan huACE2-dependent sarbecovirus neutralizing monoclonal antibodies isolated from a BNT162b2-vaccinated SARS survivor.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern such as Omicron hampered efforts in controlling the ongoing coronavirus disease 2019 pandemic due to their ability to escape neutralizing antibodies induced by vaccination or prior infection, highlighting the need to develop broad-spectrum vaccines and therapeutics. Most human monoclonal antibodies (mAbs) reported to date have not demonstrated true pan-sarbecovirus neutralizing breadth especially against animal sarbecoviruses. Here, we report the isolation and characterization of highly potent mAbs targeting the receptor binding domain (RBD) of huACE2-dependent sarbecovirus from a SARS-CoV survivor vaccinated with BNT162b2. Among the six mAbs identified, one (E7) showed better huACE2-dependent sarbecovirus neutralizing potency and breadth than any other mAbs reported to date. Mutagenesis and cryo-electron microscopy studies indicate that these mAbs have a unique RBD contact footprint and that E7 binds to a quaternary structure-dependent epitope.

Emerging Minimally Invasive Percutaneous Procedures for Periacetabular Osteolytic Metastases.

➤: Periacetabular osteolytic skeletal metastases are frequently associated with pain and impaired ambulatory function. Minimally invasive techniques allow for the restoration of ambulation without interrupting critical systemic cancer therapy. ➤: The open surgical management of massive periacetabular osteolytic lesions, such as by curettage, internal fixation, or complex total hip reconstruction, is associated with blood loss, hospitalization, rehabilitation, and complications such as infection or delayed wound-healing. ➤: Minimally invasive percutaneous procedures have become increasingly popular for the management of periacetabular osteolytic metastases by interventional oncologists and orthopaedic surgeons before complex open surgical procedures are considered. ➤: Minimally invasive procedures may include various methods of cancer ablation and reinforcement techniques. Minimally invasive procedures may entail cancer ablation, polymethylmethacrylate (PMMA) cement reinforcement, balloon osteoplasty, percutaneous screw fixation, or combinations of the aforementioned techniques (e.g., ablation-osteoplasty-reinforcement-internal fixation [AORIF]).

A platform technology for generating subunit vaccines against diverse viral pathogens.

The COVID-19 pandemic response has shown how vaccine platform technologies can be used to rapidly and effectively counteract a novel emerging infectious disease. The speed of development for mRNA and vector-based vaccines outpaced those of subunit vaccines, however, subunit vaccines can offer advantages in terms of safety and stability. Here we describe a subunit vaccine platform technology, the molecular clamp, in application to four viruses from divergent taxonomic families: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), Lassa virus (LASV) and Nipah virus (NiV). The clamp streamlines subunit antigen production by both stabilising the immunologically important prefusion epitopes of trimeric viral fusion proteins while enabling purification without target-specific reagents by acting as an affinity tag. Conformations for each viral antigen were confirmed by monoclonal antibody binding, size exclusion chromatography and electron microscopy. Notably, all four antigens tested remained stable over four weeks of incubation at 40°C. Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge.

Treatment Patterns and Standardized Outcome Assessments Among Patients With Inflammatory Conditions of the Pouch in a Prospective Multicenter Registry.

Background: Much of our understanding about the natural history of pouch-related disorders has been generated from selected populations. We designed a geographically diverse, prospective registry to study the disease course among patients with 1 of 4 inflammatory conditions of the pouch. The primary objectives in this study were to demonstrate the feasibility of a prospective pouch registry and to evaluate the predominant treatment patterns for pouch-related disorders. Methods: We used standardized diagnostic criteria to prospectively enroll patients with acute pouchitis, chronic antibiotic-dependent pouchitis (CADP), chronic antibiotic refractory pouchitis (CARP), or Crohn's disease (CD) of the pouch. We obtained detailed clinical and demographic data at the time of enrollment, along with patient-reported outcome (PRO) measures. Results: We enrolled 318 patients (10% acute pouchitis, 27% CADP, 12% CARP, and 51% CD of the pouch). Among all patients, 55% were on a biologic or small molecule therapy. Patients with CD of the pouch were more likely to use several classes of therapy (P < .001). Among patients with active disease at the time of enrollment, 23% with CARP and 40% with CD of the pouch were in clinical remission at 6 months after enrollment. Conclusions: In a population where most patients had refractory inflammatory conditions of the pouch, we established a framework to evaluate PROs and clinical effectiveness. This infrastructure will be valuable for long-term studies of real-world effectiveness for pouch-related disorders.

Detection of filovirus-reactive antibodies in Australian bat species.

Bats have been implicated as the reservoir hosts of filoviruses in Africa, with serological evidence of filoviruses in various bat species identified in other countries. Here, serum samples from 190 bats, comprising 12 different species, collected in Australia were evaluated for filovirus antibodies. An in-house indirect microsphere assay to detect antibodies that cross-react with Ebola virus (Zaire ebolavirus; EBOV) nucleoprotein (NP) followed by an immunofluorescence assay (IFA) were used to confirm immunoreactivity to EBOV and Reston virus (Reston ebolavirus; RESTV). We found 27 of 102 Yinpterochiroptera and 19 of 88 Yangochiroptera samples were positive to EBOV NP in the microsphere assay. Further testing of these NP positive samples by IFA revealed nine bat sera that showed binding to ebolavirus-infected cells. This is the first report of filovirus-reactive antibodies detected in Australian bat species and suggests that novel filoviruses may be circulating in Australian bats.

Serological Hendra Virus Diagnostics Using an Indirect ELISA-Based DIVA Approach with Recombinant Hendra G and N Proteins.

Since the identification of Hendra virus (HeV) infections in horses in Australia in 1994, more than 80 outbreaks in horses have been reported, and four out of seven spillover infections in humans had a fatal outcome. With the availability of a subunit vaccine based on the HeV-Glycoprotein (HeV-G), there is a need to serologically Differentiate the Infected from the Vaccinated Animals (DIVA). We developed an indirect ELISA using HeV-G expressed in Leishmania tarentolae and HeV-Nucleoprotein (HeV-N) expressed in recombinant baculovirus-infected insect cells as antigens. During evaluation, we tested panels of sera from naïve, vaccinated and infected horses that either originated from a Hendra-virus free region, or had been pre-tested in validated diagnostic tests. Our data confirm the reliability of this approach, as HeV-N-specific antibodies were only detected in sera from infected horses, while HeV-G-specific antibodies were detected in infected and vaccinated horses with a high level of specificity and sensitivity. Given the excellent correlation of data obtained for German and Australian HeV-negative horses, we assume that this test can be applied for the testing of horse serum samples from a variety of geographical regions.

Characterisation and natural progression of SARS-CoV-2 infection in ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the infectious disease COVID-19, which has rapidly become an international pandemic with significant impact on healthcare systems and the global economy. To assist antiviral therapy and vaccine development efforts, we performed a natural history/time course study of SARS-CoV-2 infection in ferrets to characterise and assess the suitability of this animal model. Ten ferrets of each sex were challenged intranasally with 4.64 × 104 TCID50 of SARS-CoV-2 isolate Australia/VIC01/2020 and monitored for clinical disease signs, viral shedding, and tissues collected post-mortem for histopathological and virological assessment at set intervals. We found that SARS-CoV-2 replicated in the upper respiratory tract of ferrets with consistent viral shedding in nasal wash samples and oral swab samples up until day 9. Infectious SARS-CoV-2 was recovered from nasal washes, oral swabs, nasal turbinates, pharynx, and olfactory bulb samples within 3-7 days post-challenge; however, only viral RNA was detected by qRT-PCR in samples collected from the trachea, lung, and parts of the gastrointestinal tract. Viral antigen was seen exclusively in nasal epithelium and associated sloughed cells and draining lymph nodes upon immunohistochemical staining. Due to the absence of clinical signs after viral challenge, our ferret model is appropriate for studying asymptomatic SARS-CoV-2 infections and most suitable for use in vaccine efficacy studies.

Knee Reconstruction following Sarcoma Resection Utilizing Pedicled Anterolateral Thigh Propeller Flap: A Case Series.

Coverage of knee wounds with exposure or violation of the joint capsule has long been a challenge to plastic surgeons. Wide resection and radiation treatment for soft-tissue sarcomas further this difficulty due to resultant diminished vascularity and soft tissue fibrosis. Traditional muscle flaps such as the gastrocnemius may be within the radiated field, limiting their arc of rotation to the knee. We present a series of exposed knee joint reconstructions using pedicled propeller flaps after sarcoma resection. Three patients diagnosed with soft tissue sarcomas underwent neoadjuvant radiation followed by wide local resection by orthopedic oncology. All patients had underlying knee joint exposure and underwent successful soft tissue reconstruction utilizing pedicled anterolateral thigh (ALT) propeller flaps. The ALT flap is widely used in plastic surgery for reconstruction of soft tissue defects due to its reliable vascularity, long pedicle, versatility, low donor-site morbidity, and large size. As a propeller flap, we demonstrate this is a viable alternative for reconstruction when the vascular plexus around the knee is unreliable after neoadjuvant radiation. Extending the ALT propeller flap with a large proximal skin paddle provides a nonmicrosurgical alternative to traditional muscle flaps at this location. The ALT propeller flap is an excellent option for reconstruction of large defects of the knee, especially in the setting of a radiated wound bed with unpredictable vascularity. In our case series, all three patients underwent successful reconstruction of exposed knee joints after resection of soft tissue sarcoma utilizing ALT propeller flaps.

In vitro characterisation of SARS-CoV-2 and susceptibility of domestic ferrets (Mustela putorius furo).

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS-CoV-2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS-CoV-2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS-CoV-2 infection following intranasal challenge.

Reciprocal SH2-SH3 Domain Contacts between ITK Molecules Limit T Cell Receptor Signaling in Th2-type CD4+ T Cells.

The nonreceptor tyrosine kinase ITK is a key component of the T cell receptor (TCR) signaling pathway and is required for cytokine production by CD4+ T cells that have differentiated into Th2 cells. Structural and biochemical studies suggest that contacts between the SH2 and SH3 domains of ITK mediate intermolecular self-association, forming a structure that restrains ITK activity by interfering with interactions between ITK and other components of the TCR signaling pathway. Wild-type (WT) ITK and a panel of ITK mutants containing amino acid substitutions in the SH2 and SH3 domains were tested for self-association and for binding to the adaptor protein SLP76, a key ligand for the ITK SH2 domain. WT and ITK mutants were also expressed in Itk-deficient CD4+ T cells via retroviral-mediated gene delivery to analyze their ability to support TCR signaling and cytokine production by Th2 cells. Specific amino acid substitutions in the ITK SH2 or SH3 domains impaired self-association, with the greatest effects being seen when both intermolecular SH2-SH3 domain contacts were disrupted. Two of the SH2 domain substitutions tested reduced ITK self-association but had no effect on binding to SLP-76. When their function was analyzed in Th2 cells, ITK proteins with diminished self-association activity supported greater IL-4 production and calcium flux in response to TCR stimulation compared to WT ITK. Our findings indicate that intermolecular contacts between ITK molecules can restrain the amplitude of TCR signaling, suggesting ITK is a limiting factor for responses by CD4+ T cells.

Incidence and Recovery of Vocal Fold Immobility Following Pediatric Cardiac Operations.

BACKGROUND: Loss of laryngeal function after congenital cardiac surgery causes morbidity and prolongs hospitalization. Early diagnosis of vocal fold immobility (VFI) and referral to pediatric otolaryngology (pOTO) aids in laryngeal rehabilitation. Understanding the incidence and recovery rates of VFI enables counseling for families of infants undergoing high-risk surgery. METHODS: A retrospective chart review from November 2014 to July 2019 of infants postcardiac surgery where the aortic arch or surrounding structures were manipulated and were screened via flexible fiberoptic laryngoscopy (FFL) at a single institution was performed. Patients were divided into five surgical categories: Norwood procedure, aortic arch augmentation via median sternotomy, arterial switch operation, coarctation repair via lateral thoracotomy, and cardiac surgeries including ligation of a patent ductus arteriosus (PDA). Patients undergoing isolated PDA ligation were excluded. RESULTS: One hundred ninety-nine qualifying operations occurred during this period; 28 patients did not undergo FFL before discharge and were excluded from the analysis. Immediately following cardiac surgery, 34% (58 of 171 patients) had VFI. Follow-up was completed by 38 of 58 patients with VFI. Complete recovery was demonstrated in 63% (24 of 38) of patients by 6 months and in 86% (33 of 38) within 18 months. The highest risk occurred with the Norwood procedure and arch augmentation via median sternotomy. CONCLUSIONS: Infants undergoing surgery involving the aortic arch and surrounding structures have high rates of VFI. Follow-up by pOTO is recommended to optimize laryngeal rehabilitation. Most patients have spontaneous recovery within 18 months of cardiac surgery.

Type I Hypersensitivity in Ferrets Following Exposure to SARS-CoV-2 Inoculum: Lessons Learned.

This case report discusses Type I hypersensitivity in ferrets following exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inoculum, observed during a study investigating the efficacy of candidate COVID-19 vaccines. Following a comprehensive internal root-cause investigation, it was hypothesized that prior prime-boost immunization of ferrets with a commercial canine C3 vaccine to protect against the canine distemper virus had resulted in primary immune response to fetal bovine serum (FBS) in the C3 preparation. Upon intranasal exposure to SARS-CoV-2 virus cultured in medium containing FBS, an allergic airway response occurred in 6 out of 56 of the ferrets. The 6 impacted ferrets were randomly dispersed across study groups, including different COVID-19 vaccine candidates, routes of vaccine candidate administration, and controls (placebo). The root-cause investigation and subsequent analysis determined that the allergic reaction was unrelated to the COVID-19 vaccine candidates under evaluation. Histological assessment suggested that the allergic response was characterized by eosinophilic airway disease; increased serum immunoglobulin levels reactive to FBS further suggested this response was caused by immune priming to FBS present in the C3 vaccine. This was further supported by in vivo studies demonstrating ferrets administered diluted FBS also presented clinical signs consistent with a hyperallergic response, while clinical signs were absent in ferrets that received a serum-free SARS-CoV-2 inoculum. It is therefore recommended that vaccine studies in higher order animals should consider the impact of welfare vaccination and use serum-free inoculum whenever possible.

ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-CoV-2 shedding in ferrets.

Vaccines against SARS-CoV-2 are likely to be critical in the management of the ongoing pandemic. A number of candidates are in Phase III human clinical trials, including ChAdOx1 nCoV-19 (AZD1222), a replication-deficient chimpanzee adenovirus-vectored vaccine candidate. In preclinical trials, the efficacy of ChAdOx1 nCoV-19 against SARS-CoV-2 challenge was evaluated in a ferret model of infection. Groups of ferrets received either prime-only or prime-boost administration of ChAdOx1 nCoV-19 via the intramuscular or intranasal route. All ChAdOx1 nCoV-19 administration combinations resulted in significant reductions in viral loads in nasal-wash and oral swab samples. No vaccine-associated adverse events were observed associated with the ChAdOx1 nCoV-19 candidate, with the data from this study suggesting it could be an effective and safe vaccine against COVID-19. Our study also indicates the potential for intranasal administration as a way to further improve the efficacy of this leading vaccine candidate.

Multifocal Xanthoma of Bone.

We report a case of a 40-year-old woman with hyperlipidemia and associated multifocal xanthoma of bone requiring prophylactic fixation of her bilateral femurs. Although xanthomas of bone are themselves a benign process, their presence may indicate that the patient has poorly controlled lipids and is at an increased risk of cardiovascular disease. Lytic lesions may require prophylactic fixation to prevent pathologic fracture.

Exploring how differently patients and clinical tutors see the same consultation: building evidence for inclusion of real patient feedback in medical education.

BACKGROUND: Undergraduate medical education recognises that patient feedback is potentially valuable for student learning and development as a component of multi-source feedback. However greater exploration of how patient feedback perspectives differ to clinical educators is required for curriculum development and improving student feedback literacy. This study aimed to determine how two sources of feedback, patients and clinical tutors, compare on the same patient-centred, interpersonal criteria. METHODS: A patient feedback instrument designed for the undergraduate medical education setting was used to compare patients' feedback with clinical tutors' feedback following a student-patient consultation in the learning context. Assessments from 222 learning consultations involving 40 medical students were collected. Descriptive statistics for tutors and patients for each question were calculated and correlations between patient and tutor were explored using Spearman's rank-order correlation. Mixed effects ordered logistic regression was used to compare each question with an overall rating for tutor and patients in addition to comparing patient with tutor ratings. RESULTS: Clinical tutor and patient assessments had a weak but significant positive correlation in all areas except questions related to respect and concern. When making judgements compared with overall assessment, patients' ratings of respect, concern, communication and being understood in the consultation have a greater effect. After eliminating the effect of generally higher ratings by patients compared with tutors using comparative ordered logistic regression, patients rated students relatively less competent in areas of personal interaction. CONCLUSION: This study provides insight about patient feedback, which is required to continue improving the use and acceptability of this multisource feedback to students as a valuable component of their social learning environment. We have revealed the different perspective-specific judgement that patients bring to feedback. This finding contributes to building respect for patient feedback through greater understanding of the elements of consultations for which patients can discriminate performance.