A New Methodology for the Oxygen Measurement in Lung Tissue of an Aged Ferret Model Proves Hypoxia During COVID-19.

Oxygen as a key element has a high impact on cellular processes. Infection with a pathogen such as SARS-CoV-2 and following inflammation may lead to hypoxic conditions in tissue that impact cellular responses. To develop optimized translational in vitro models for a better understanding of physiologic and pathophysiologic oxygen conditions, it is a prerequisite to determine oxygen levels generated in vivo. Our study objective was the establishment of an invasive method for oxygen measurements using a luminescence-based microsensor to determine the dissolved oxygen in the lung tissue of ferrets as animal models for SARS-CoV-2 research. In analogy to humans, aged ferrets are more likely to show clinical signs after SARS-CoV-2 infection compared to young animals. To investigate oxygen levels during a respiratory viral infection, we intratracheally infected nine aged (3-year-old) ferrets with SARS-CoV-2. The aged SARS-CoV-2 infected ferrets showed mild to moderate clinical signs associated with prolonged viral RNA shedding until 14 days post infection (dpi). SARS-CoV-2 infected ferrets showed histopathologic lung lesion scores that significantly negatively correlated with oxygen levels in lung tissue. At 4 dpi, oxygen levels in lung tissue were significantly lower (mean %O2 of 3.89 ≙ ≈ 27.78 mmHg) compared to the negative control group (mean %O2 of 8.65 ≙ ≈ 61.4 mmHg). In summary, we succeeded in determining the pathophysiologic oxygen conditions in the lung tissue of aged SARS-CoV-2-infected ferrets. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/). .

SARS-CoV-2 inactivation in laboratory animal tissues with 4% formaldehyde or 5% glutaraldehyde for transfer to biosafety level 1 laboratories.

The SARS-CoV-2 pandemic required the immediate need to transfer inactivated tissue from biosafety level (BSL)-3 to BSL-1 areas to enable downstream analytical methods. No validated SARS-CoV-2 inactivation protocols were available for either formaldehyde (FA)-fixed or glutaraldehyde (GA)-fixed tissues. Therefore, representative tissue from ferrets and hamsters was spiked with 2.2 × 106 tissue culture infectious dose 50% per ml (TCID50/ml) SARS-CoV-2 or were obtained from mice experimentally infected with SARS-CoV-2. SARS-CoV-2 inactivation was demonstrated with 4% FA or 5% GA at room temperature for 72 hours by a titer reduction of up to 103.8 TCID50/ml in different animal tissues with a maximum protein content of 100 µg/mg and a thickness of up to 10 mm for FA and 8 mm for GA. Our protocols can be easily adapted for validating the inactivation of other pathogens to allow for the transfer of biological samples from BSL-3 areas to BSL-1 laboratories.

Detrimental Role of Neutrophil Extracellular Traps during Dengue Virus Infection.

A recent article by Sung et al. identified the CLEC2 platelet receptor as an important factor of lethal dengue virus infection. Formation of neutrophil extracellular traps via crosstalk with CLEC5A and TLR2 neutrophils were ascribed a causative role in DENV infection. This provides new insights for the development of candidate broad-spectrum therapies against hemorrhagic virus infections.

Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen.

NAD+-reducing [NiFe] hydrogenases are valuable biocatalysts for H2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temperatures, the soluble NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O2-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochemical, spectroscopic, and theoretical studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallographically observed glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by density-functional-theory calculations, we identify this state as a high-valent species with a biologically unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional infrared spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, especially upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.

Outcomes and costs of ureteroscopy, extracorporeal shockwave lithotripsy, and percutaneous nephrolithotomy for the treatment of urolithiasis: an analysis based on health insurance claims data in Germany.

PURPOSE: Comparisons of ureteroscopy (URS), extracorporeal shockwave lithotripsy (SWL), and percutaneous nephrolithotomy (PCNL) for urolithiasis considering long-term health and economic outcomes based on claims data are rare. Our aim was to analyze URS, SWL, and PCNL regarding complications within 30 days, re-intervention, healthcare costs, and sick leave days within 12 months, and to investigate inpatient and outpatient SWL treatment as the latter was introduced in Germany in 2011. METHODS: This retrospective cohort study based on German health insurance claims data included 164,203 urolithiasis cases in 2008-2016. We investigated the number of complications within 30 days, as well as time to re-intervention, number of sick leave days and hospital and ambulatory health care costs within a 12-month follow-up period. We applied negative binomial, Cox proportional hazard, gamma and two-part models and adjusted for patient variables. RESULTS: Compared to URS cases, SWL and PCNL had fewer 30-day complications, time to re-intervention within 12 months was decreased for SWL and PCNL, SWL and PCNL were correlated with a higher number of sick leave days, and SWL and particularly PCNL were associated with higher costs. SWL outpatients had fewer complications, re-interventions and lower costs than inpatients. This study was limited by the available information in claims data. CONCLUSION: URS cases showed benefits in terms of fewer re-interventions, fewer sick leave days, and lower healthcare costs. Only regarding complications, SWL was superior. This emphasizes URS as the most frequent treatment choice. Furthermore, SWL outpatients showed less costs, fewer complications, and re-interventions than inpatients.

Ferrets are valuable models for SARS-CoV-2 research.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in an ongoing pandemic with millions of deaths worldwide. Infection of humans can be asymptomatic or result in fever, fatigue, dry cough, dyspnea, and acute respiratory distress syndrome with multiorgan failure in severe cases. The pathogenesis of COVID-19 is not fully understood, and various models employing different species are currently applied. Ferrets can be infected with SARS-CoV-2 and efficiently transmit the virus to contact animals. In contrast to hamsters, ferrets usually show mild disease and viral replication restricted to the upper airways. Most reports have used the intranasal inoculation route, while the intratracheal infection model is not well characterized. Herein, we present clinical, virological, and pathological data from young ferrets intratracheally inoculated with SARS-CoV-2. Infected animals showed no significant clinical signs, and had transient infection with peak viral RNA loads at 4 days postinfection, mild to moderate rhinitis, and pulmonary endothelialitis/vasculitis. Viral antigen was exclusively found in the respiratory epithelium of the nasal cavity, indicating a particular tropism for cells in this location. Viral antigen was associated with epithelial damage and influx of inflammatory cells, including activated neutrophils releasing neutrophil extracellular traps. Scanning electron microscopy of the nasal respiratory mucosa revealed loss of cilia, shedding, and rupture of epithelial cells. The currently established ferret SARS-CoV-2 infection models are comparatively discussed with SARS-CoV-2 pathogenesis in mink, and the advantages and disadvantages of both species as research models for zoonotic betacoronaviruses are highlighted.

Modification of Skin Flaps for Single-Stage Reconstruction of the Nasal Ala Using All-Layer Suture: A Prospective Study.

BACKGROUND AND OBJECTIVES: After local flaps, it may be necessary to reconstruct the contour of the nasal ala. This is possible with a single-stage all-layer shaping suture. In the present study, the functional and aesthetic results after single-stage reconstruction of the nasal ala were prospectively evaluated. PATIENTS AND METHODS: Patients who underwent surgery for skin tumors of the nose between 06/2019 and 06/2020 who required reconstruction of the nasal ala as part of the defect closure and had an all-layer suture used were prospectively included in the study. A standardized evaluation of aesthetic and functional outcome was conducted by the patient and a physician at discharge as well as 4 weeks later. Patients additionally underwent a follow-up survey 6 months later. RESULTS: Thirty-seven patients were included in the study. Four weeks postoperatively, all flaps were found to be fully healed and vital. Aesthetic outcome at 4 weeks was rated as very good or good by physicians in 73% and by patients in 78.4%. Persistent complications due to reduced blood flow were not observed. CONCLUSION: The reshaping of the nasal ala as part of the defect reconstruction with an all-layer suture demonstrates very good aesthetic as well as functional results and can be performed in a single-stage procedure. .

SARS-CoV-2-Specific Antibodies in Domestic Cats during First COVID-19 Wave, Europe.

We conducted a severe acute respiratory syndrome coronavirus 2 antibody seroprevalence study among >2,000 domestic cats from 4 countries during the first coronavirus disease wave in Europe. We found 4.4% seroprevalence using a virus neutralization test and 4.3% using a receptor-binding domain ELISA, demonstrating probable human-to-cat transmission.

Prolonged SARS-CoV-2 RNA Shedding from Therapy Cat after Cluster Outbreak in Retirement Home.

We report a therapy cat in a nursing home in Germany infected with severe acute respiratory syndrome coronavirus 2 during a cluster outbreak in the home residents. Although we confirmed prolonged presence of virus RNA in the asymptomatic cat, genome sequencing showed no further role of the cat in human infections on site.

Scent dog identification of SARS-CoV-2 infections in different body fluids.

BACKGROUND: The main strategy to contain the current SARS-CoV-2 pandemic remains to implement a comprehensive testing, tracing and quarantining strategy until vaccination of the population is adequate. Scent dogs could support current testing strategies. METHODS: Ten dogs were trained for 8 days to detect SARS-CoV-2 infections in beta-propiolactone inactivated saliva samples. The subsequent cognitive transfer performance for the recognition of non-inactivated samples were tested on three different body fluids (saliva, urine, and sweat) in a randomised, double-blind controlled study. RESULTS: Dogs were tested on a total of 5242 randomised sample presentations. Dogs detected non-inactivated saliva samples with a diagnostic sensitivity of 84% (95% CI: 62.5-94.44%) and specificity of 95% (95% CI: 93.4-96%). In a subsequent experiment to compare the scent recognition between the three non-inactivated body fluids, diagnostic sensitivity and specificity were 95% (95% CI: 66.67-100%) and 98% (95% CI: 94.87-100%) for urine, 91% (95% CI: 71.43-100%) and 94% (95% CI: 90.91-97.78%) for sweat, 82% (95% CI: 64.29-95.24%), and 96% (95% CI: 94.95-98.9%) for saliva respectively. CONCLUSIONS: The scent cognitive transfer performance between inactivated and non-inactivated samples as well as between different sample materials indicates that global, specific SARS-CoV-2-associated volatile compounds are released across different body secretions, independently from the patient's symptoms. All tested body fluids appear to be similarly suited for reliable detection of SARS-CoV-2 infected individuals.

Local Recurrence Rates of Skin Tumors After Resection With Complete Circumferential Peripheral and Deep Margin Assessment-Identification of High-Risk Entities.

BACKGROUND: The local recurrence rates of malignant skin tumors after micrographic controlled surgery using complete circumferential peripheral and deep margin assessment (CCPDMA) are reported to be low. However, in daily practice, tumor entities with a significantly higher recurrence rate are found. OBJECTIVE: The aim of the investigation was to identify these high-risk tumors to develop approaches for risk stratification. PATIENTS AND METHODS: We included different malignant tumors that were surgically treated and examined with CCPDMA using paraffin sections and H&E staining. Re-excisions were performed until the tumor was completely removed. RESULTS: Ninety-nine thousand three hundred seventy-two tumors were included in the study; the follow-up period was 4 years (median). Eight tumor entities were identified as high-risk entities, showing a significantly higher local recurrence rate of over 20%: desmoplastic squamous cell carcinoma, desmoplastic melanoma, Merkel cell carcinoma, microcystic adnexal carcinoma, pleomorphic sarcoma, liposarcoma as well as angiosarcoma, and extramammary Paget's disease. CONCLUSION: Complete circumferential peripheral and deep margin assessment allows complete control of the resection margins and enables skin-sparing resections with low recurrence rates for basal cell carcinomas, nondesmoplastic squamous cell carcinomas, lentiginous melanomas, and dermatofibrosarcoma protuberans. However, other tumors show significantly higher recurrence rates and therefore need to be classified as high-risk tumors.

Post-operative follow-up of 150 patients with sentinel lymph node biopsy under tumescence local anesthesia.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is useful for staging of patients with melanoma. Although SLNB is mostly performed under general anesthesia (GA), tumescence local anesthesia (TLA) can also be used. However, less data are available regarding feasibility of SLNB under TLA. Here we present a post-operative follow-up of 150 patients. PATIENTS AND METHODS: We prospectively analyzed data from 150 patients with primary cutaneous malignant melanoma. We assessed pain, post-operative complications and patients' satisfaction after SLNB under TLA. RESULTS: 32 % of the patients reported post-operative pain within the first 48 h after SLNB. Seroma was the most frequent complication, as 29 seromas after SLNB were observed. Wound infection was observed in 3.3 % of the patients. 98.7 % of the patients were satisfied with SLNB under TLA. CONCLUSIONS: SLNB under TLA is a safe and feasible option and should be considered for patients with melanoma. Especially with multimorbid or elderly patients, the risks of GA can be avoided.

Scent dog identification of samples from COVID-19 patients - a pilot study.

BACKGROUND: As the COVID-19 pandemic continues to spread, early, ideally real-time, identification of SARS-CoV-2 infected individuals is pivotal in interrupting infection chains. Volatile organic compounds produced during respiratory infections can cause specific scent imprints, which can be detected by trained dogs with a high rate of precision. METHODS: Eight detection dogs were trained for 1 week to detect saliva or tracheobronchial secretions of SARS-CoV-2 infected patients in a randomised, double-blinded and controlled study. RESULTS: The dogs were able to discriminate between samples of infected (positive) and non-infected (negative) individuals with average diagnostic sensitivity of 82.63% (95% confidence interval [CI]: 82.02-83.24%) and specificity of 96.35% (95% CI: 96.31-96.39%). During the presentation of 1012 randomised samples, the dogs achieved an overall average detection rate of 94% (±3.4%) with 157 correct indications of positive, 792 correct rejections of negative, 33 incorrect indications of negative or incorrect rejections of 30 positive sample presentations. CONCLUSIONS: These preliminary findings indicate that trained detection dogs can identify respiratory secretion samples from hospitalised and clinically diseased SARS-CoV-2 infected individuals by discriminating between samples from SARS-CoV-2 infected patients and negative controls. This data may form the basis for the reliable screening method of SARS-CoV-2 infected people.

Treatment utilization and treatment barriers in individuals with body dysmorphic disorder.

BACKGROUND: Although effective treatments are available, most individuals with body dysmorphic disorder (BDD) do not receive an appropriate diagnosis or treatment. We aimed to examine treatment utilization and barriers to treatment, and to identify associated socio-demographic and clinical characteristics. METHODS: German individuals completed an online self-report survey of appearance concerns. A sample of N = 429 individuals met criteria for BDD. We examined the frequency of treatment utilization and barriers, analyzed comparisons between treated and untreated individuals and assessed the relationships of socio-demographic and clinical features with mental health treatment utilization and treatment barriers, respectively. RESULTS: Only 15.2% of the individuals with BDD had been diagnosed with BDD, and lifetime rates of mental health treatment were low (39.9%). Individuals endorsed multiple barriers to mental health treatment, especially shame, low perceived need and a preference for cosmetic and medical treatments. Associated features were identified, including age, a BDD diagnosis, body dysmorphic symptom severity, a likely major depressive disorder, prior cosmetic surgery, and insight. CONCLUSIONS: The results of this largest study to date highlight that BDD is still underrecognized and undertreated even in a country with extensive mental health care and few financial barriers. We discuss modifiable factors and strategies to foster awareness of BDD in sufferers and professionals to improve treatment dissemination and to reduce treatment barriers.

Impact of morbidity on care need increase and mortality in nursing homes: a retrospective longitudinal study using administrative claims data.

BACKGROUND: A growing number of older people are care dependent and live in nursing homes, which accounts for the majority of long-term-care spending. Specific medical conditions and resident characteristics may serve as risk factors predicting negative health outcomes. We investigated the association between the risk of increasing care need and chronic medical conditions among nursing home residents, allowing for the competing risk of mortality. METHODS: In this retrospective longitudinal study based on health insurance claims data, we investigated 20,485 older adults (≥65 years) admitted to German nursing homes between April 2007 and March 2014 with care need level 1 or 2 (according to the three level classification of the German long-term care insurance). This classification is based on required daily time needed for assistance. The outcome was care level change. Medical conditions were determined according to 31 Charlson and Elixhauser conditions. Competing risks analyses were applied to identify chronic medical conditions associated with risk of care level change and mortality. RESULTS: The probability for care level change and mortality acted in opposite directions. Dementia was associated with increased probability of care level change compared to other conditions. Patients who had cancer, myocardial infarction, congestive heart failure, cardiac arrhythmias, renal failure, chronic pulmonary disease, weight loss, or recent hospitalization were more likely to die, as well as residents with paralysis and obesity when admitted with care level 2. CONCLUSION: This paper identified risk groups of nursing home residents which are particularly prone to increasing care need or mortality. This enables focusing on these risk group to offer prevention or special treatment. Moreover, residents seemed to follow specific trajectories depending on their medical conditions. Some were more prone to increased care need while others had a high risk of mortality instead. Several conditions were neither related to increased care need nor mortality, e.g., valvular, cerebrovascular or liver disease, peripheral vascular disorder, blood loss anemia, depression, drug abuse and psychosis. Knowledge of functional status trajectories of residents over time after nursing home admission can help decision-makers when planning and preparing future care provision strategies (e.g., planning of staffing, physical equipment and financial resources).

Uptake of an exercise class and use of bone density measurement after advice by the health insurance fund: results from the osteoporotic fracture prevention program in rural areas (OFRA).

BACKGROUND: OFRA is a large health insurance fund-driven program which aims to reduce the risk of falls and fractures in older people living in rural areas. The programme offered specific mobility and falls prevention classes and bone density measurement by a DXA scan free of charge to more than 10,000 people, and was promoted by staff of the health insurance fund either by a visit at home, or a phone call, or a visit at home and a subsequent phone call. The aim of this study was to analyse the uptake of an exercise class and the use of a DXA scan after advice. METHODS: Telephone interviews were conducted in a randomly selected subgroup of 780 persons 9 months after first contact. Rates of uptake of an exercise class or use of a DXA scan were calculated. Predictors of uptake and use were analysed applying logistic regression models. RESULTS: The rate of uptake after advice for specific mobility and fall prevention class was 29.6%. For DXA scan, the rate of use after advice was 16.7%. Rates of uptake and use increased if the first contact by a visit at home or a phone call was followed by an additional subsequent phone call. CONCLUSION: A direct motivational approach in older people by a health insurance fund is feasible and results in relevant participation and utilization rates in exercise classes and DXA scans.

[Surgical techniques in the treatment of basal cell carcinoma-a prospective investigation]. / Chirurgische Techniken in der Therapie des Basalzellkarzinoms ­ eine prospektive Untersuchung.

BACKGROUND: Basal cell carcinomas are the most frequent epithelial skin tumors and a frequent indication for dermatological interventions. Despite the establishment of drug treatment options, surgery is still the treatment of choice. Various options are available ranging from curettage to complex dermatosurgical procedures. In addition to the main aspect of low local recurrence rates, esthetic factors and the number of procedures are also important for the choice of treatment. METHODS: In this study 347 patients with 398 basal cell carcinomas (nodular type, diameter up to 10 mm) were prospectively examined. The patients were randomized into two treatment arms: in one group the tumor was treated by ring curettage and in the other group the tumor was excised. Patients who underwent 3D histologically controlled surgery due to basal cell carcinoma during the same investigation period served as controls. RESULTS: The highest local recurrence rate was observed after curettage (14.0%), whereas the group with 3D histology had the lowest recurrence rate (0.9%, p < 0.001). In the 3D group, more re-excisions were required to achieve complete tumor clearance compared to the group who underwent excisions with serial section histology. Patients rated the esthetic outcome best after curettage. The median follow-up was 3.9 years. CONCLUSION: The choice of surgical treatment for small nodular basal cell carcinomas depends on individual circumstances. Excisions controlled by 3D histology with wound closure after complete tumor clearance showed the lowest recurrence rate in our study; however, curettage is also a possible surgical treatment option with minimal effort and an acceptable recurrence rate, which can lead to good esthetic results.

Neglected Hosts of Small Ruminant Morbillivirus.

Eradication of small ruminant morbillivirus (PPRV) is targeted for 2030. PPRV lineage IV is found in much of Asia and Africa. We used PPRV lineage IV strain Kurdistan/2011 in transmission trials to investigate the role of pigs, wild boar, and small ruminants as PPRV reservoirs. Suids were a possible source of infection.

Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. IMPORTANCE: Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce highly attenuated, safer vaccines was evident after the development of the BTV reverse-genetics system that allows the introduction of targeted mutations in the virus genome. We targeted an essential gene to develop disabled virus strains as vaccine candidates. The results presented in this report further substantiate our previous evidence and support the suitability of these virus strains as the next-generation BTV vaccines.