Preclinical Validation of a Novel Injection-Molded Swab for the Molecular Assay Detection of SARS-CoV-2

During the COVID-19 public health emergency, many actions have been undertaken to help ensure that patients and health care providers have timely and continued access to high-quality medical devices to respond effectively. The development and validation of new testing supplies and equipment, including collection swabs, has helped to expand the availability and capability for various diagnostic, therapeutic, and protective medical devices in high demand during the COVID-19 emergency. Here, we report the initial validation of a new injection-molded anterior nasal swab, ClearTip™, that was experimentally validated in a laboratory setting as well as in independent clinical studies in comparison to gold standard flocked swabs. We have also developed an in vitro anterior nasal tissue model which offers a novel, efficient, and clinically relevant validation tool to replicate the clinical swabbing workflow with high fidelity, while being accessible, safe, reproducible, and time- and cost-effective. ClearTip™ displayed greater inactivated virus release in the benchtop model, confirmed by its greater ability to report positive samples in a small clinical study in comparison to flocked swabs. We also quantified the detection of biological materials, as a proxy for viral material, in multi-center pre-clinical and clinical studies which showed a statistically significant difference in one study and a reduction in performance in comparison to flocked swabs. Taken together, these results emphasize the compelling benefits of non-absorbent injection-molded anterior nasal swabs for COVID-19 detection, comparable to standard flocked swabs. Injection-molded swabs, as ClearTip™, could have the potential to support future swab shortages, due to its manufacturing advantages, while offering benefits in comparison to highly absorbent swabs in terms of comfort, limited volume collection, and potential multiple usage.

Immunological status of the olfactory bulb in a murine model of Toll-like receptor 3-mediated upper respiratory tract inflammation

Background Postviral olfactory dysfunction (PVOD) following a viral upper respiratory tract infection (URI) is one of the most common causes of olfactory disorders, often lasting for over a year. To date, the molecular pathology of PVOD has not been elucidated. Methods A murine model of Toll-like receptor 3 (TLR3)-mediated upper respiratory tract inflammation was used to investigate the impact of URIs on the olfactory system. Inflammation was induced via the intranasal administration of polyinosinic–polycytidylic acid (poly(I:C), a TLR3 ligand) to the right nostril for 3 days. Peripheral olfactory sensory neurons (OSNs), immune cells in the olfactory mucosa, and glial cells in the olfactory bulb (OB) were analyzed histologically. Proinflammatory cytokines in the nasal tissue and OB were evaluated using the quantitative real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). Results In the treated mice, OSNs were markedly reduced in the olfactory mucosa, and T cell and neutrophil infiltration therein was observed 1 day after the end of poly(I:C) administration. Moreover, there was a considerable increase in microglial cells and slight increase in activated astrocytes in the OB. In addition, qPCR and ELISA revealed the elevated expression of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma both in the OB and nasal tissue. Conclusions Taken together, the decreased peripheral OSNs, OB microgliosis, and elevated proinflammatory cytokines suggest that immunological changes in the OB may be involved in the pathogenesis of PVOD.

Susceptibility of Human Airway Tissue Models Derived From Different Anatomical Sites to Bordetella pertussis and Its Virulence Factor AdenyRaylate Cyclase Toxin

To study the interaction of human pathogens with their host target structures, human tissue models based on primary cells are considered suitable. Complex tissue models of the human airways have been used as infection models for various viral and bacterial pathogens. The Gram-negative bacterium Bordetella pertussis is of relevant clinical interest since whooping cough has developed into a resurgent infectious disease. In the present study, we created three-dimensional tissue models of the human ciliated nasal and tracheo-bronchial mucosa. We compared the innate immune response of these models towards the B. pertussis virulence factor adenylate cyclase toxin (CyaA) and its enzymatically inactive but fully pore-forming toxoid CyaA-AC-. Applying molecular biological, histological, and microbiological assays, we found that 1 µg/ml CyaA elevated the intracellular cAMP level but did not disturb the epithelial barrier integrity of nasal and tracheo-bronchial airway mucosa tissue models. Interestingly, CyaA significantly increased interleukin 6, interleukin 8, and human beta defensin 2 secretion in nasal tissue models, whereas tracheo-bronchial tissue models were not significantly affected compared to the controls. Subsequently, we investigated the interaction of B. pertussis with both differentiated primary nasal and tracheo-bronchial tissue models and demonstrated bacterial adherence and invasion without observing host cell type-specific significant differences. Even though the nasal and the tracheo-bronchial mucosa appear similar from a histological perspective, they are differentially susceptible to B. pertussis CyaA in vitro. Our finding that nasal tissue models showed an increased innate immune response towards the B. pertussis virulence factor CyaA compared to tracheo-bronchial tissue models may reflect the key role of the nasal airway mucosa as the first line of defense against airborne pathogens.

In Vitro Nasal Tissue Model for the Validation of Nasopharyngeal and Mid-turbinate Swabs for SARS-CoV-2 Testing

Large-scale population testing is a key tool to mitigate the spread of respiratory pathogens, as in the current COVID-19 pandemic, where swabs are used to collect samples in the upper airways (e.g. nasopharyngeal and mid-turbinate nasal cavities) for diagnostics. However, the high volume of supplies required to achieve large-scale population testing has posed unprecedented challenges for swab manufacturing and distribution, resulting in a global shortage that has heavily impacted testing capacity world-wide and prompted the development of new swabs suitable for large-scale production. Newly designed swabs require rigorous pre-clinical and clinical validation studies that are costly and time consuming (i.e. months to years long); reducing the risks associated with swab validation is therefore paramount for their rapid deployment. To address these shortages, we developed a 3D-printed tissue model that mimics the nasopharyngeal and mid-turbinate nasal cavities, and we validated its use as a new tool to rapidly test swab performance. In addition to the nasal architecture, the tissue model mimics the soft nasal tissue with a silk-based sponge lining, and the physiological nasal fluid with asymptomatic and symptomatic viscosities of synthetic mucus. We performed several assays comparing standard flocked and injection-molded swabs. We quantified the swab pick-up and release, and determined the effect of viral load and mucus viscosity on swab efficacy by spiking the synthetic mucus with heat-inactivated SARS-CoV-2 virus. By molecular assays, we found that injected molded swabs performed similarly or superiorly in comparison to standard flocked swabs and we underscored a viscosity-dependent difference in cycle threshold values between the asymptomatic and symptomatic mucus for both swabs. To conclude, we developed an in vitro nasal tissue model, that corroborated previous swab performance data from clinical studies, with the potential of providing researchers with a clinically relevant, reproducible, safe, and cost-effective validation tool for the rapid development of newly designed swabs.

Alteration of blood monocyte subsets in chronic rhinosinusitis with regard to anti-inflammatory 1,8-Cineol treatment

Background: Chronic rhinosinusitis (CRS) affects about 10% of the european population causing considerable disease burden. The inflammatory microenvironment is mainly Th2 driven, but the impact of monocytes is still poorly understood. Aim of this study was to comprehensively investigate the composition of circulating monocytes and T cells in CRSwNP and CRSsNP patients, particularly with regard to the therapeutic herbal monoterpene 1,8-Cineol. Methodology: We analyzed the distribution of CD14 and CD16 classified monocyte subsets and the T-cell subset composition with respect to their PD-1 and PD-L1 expression in the peripheral blood of CRS patients using flow cytometry. Additionally, the M1/M2 like macrophage infiltration in nasal tissue and polyps was examined by immunofluorescence staining. Results: Data revealed a decrease of classical monocytes accompanied by a significant increase of intermediate CD16+ monocytes in CRSwNP and CRSsNP patients compared to healthy donors. PD-L1 expression on overall monocytes was also significantly increased in CRSwNP and CRSsNP patients. CRS patients with a severe drop of the proportion of classical monocytes showed a significant restoration of this subset in response to two-week 1,8-Cineol treatment. Conclusions: Our data indicate a CRS-induced shift of peripheral monocyte subsets to more inflammatory phenotypes that might be reversed by the herbal drug 1,8-Cineol.

Primary Cutaneous Cryptococcosis of Nasal Ala with Extensive Tissue Destruction

Introduction/Objective Primary cutaneous cryptococcosis (PCC) is rare and shows skin lesion(s) confined to a circumscribed body region mostly in immunocompromised host, with no sign of simultaneous dissemination condition. PCC usually presents as non-specific skin lesions such as cellulitis, nodules, and ulcers, and can be misdiagnosed in biopsy. We present a case of PCC with extensive tissue destruction. Methods/Case Report A 43-year-old male, with a history of human immunodeficiency virus infection 17 years ago, presented with complaint of his nose slowly “being eaten away” over the past 5 years after a bike accident. Physical examination showed most of the left nasal ala was completely destroyed, with visualizable septum. The biopsy of the left nasal ala showed the dermis has numerous yeasts with marked variation in size and shape, in foamy stroma with little inflammation. The capsules of the yeasts were highlighted by Mucicarmine stain. Grocott methenamine silver stain showed budding yeasts. The diagnosis of cutaneous cryptococcosis, gelatinous type, was rendered. Primary cutaneous cryptococcosis was considered based on no disseminated disease found, positive serum cryptococcus antigen with low titer (1:20), the culture of the nasal lesion positive for Crptococcus Neoformans, and the history of skin injury. The patient received appropriate treatment for PCC subsequently. Results (if a Case Study enter NA) NA Conclusion We have demonstrated a very rare case of undiagnosed/untreated PCC causing extensive destruction of skin and underlying nasal tissue. Identification of the histological features of cutaneous cryptococcosis, shown in this case, is the key for making the correct diagnosis.

SARS CoV-2 VIRUS Vs OPPORTUNISTIC MYCOSES

Purpose: SARS CoV 2 virus a novel coronavirus, which is a single stranded positive sense RNA virus. It causes severe pneumonia in susceptible individuals. In individuals with predisposing factors, like diabetes, neutropenia, corticosteroids therapy, and etc., the virus causes opportunistic fungal infections. This study aims the correlation between COVID 19 disease and fungal infections. Methodology: The present study includes 202 specimens collected from patients with symptoms suspected of fungal infections. The collected specimens were subjected to direct microscopy and fungal culture. Results: Nasal tissue scrapings were the most common specimen collected followed by sputum. Among the 202 specimens collected, 106 were positive for direct microscopy and 81 were positive for fungal culture. Conclusion: The fungal infections are more common among the patients with comorbidities, and appropriate guidelines should be followed in the management of such infections in order to prevent the morbidity and the mortality.

Immunohistochemical and qPCR Detection of SARS-CoV-2 in the Human Middle Ear Versus the Nasal Cavity: Case Series

Viral infections have already been implicated with otitis media and sudden sensorineural hearing loss. However, the pathophysiology of COVID-19 as it relates to otologic disorders is not well-defined. With the spread of SARS-CoV-2, it is important to evaluate its colonization of middle ear mucosa. Middle ear and nasal tissue samples for quantitative RT-PCR and histologic evaluations were obtained from post-mortem COVID-19 patients and non-diseased control patients. Here we present evidence that SARS-CoV-2 colonizes the middle ear epithelium and co-localizes with the primary viral receptor, angiotensin-converting enzyme 2 (ACE2). Both middle ear and nasal epithelial cells show relatively high expression of ACE2, required for SARS-CoV-2 entry. The epithelial cell adhesion molecule (EpCAM) was use as a biomarker of epithelia. Furthermore, we found that the viral load in the middle ear is lower than that present in the nasal cavity.

Dermal Olfactory Receptor OR51B5 Is Essential for Survival and Collagen Synthesis in Human Dermal Fibroblast (Hs68 Cells)

Skin dermis comprises extracellular matrix components, mainly collagen fibers. A decrease in collagen synthesis caused by several factors, including ultraviolet (UV) irradiation and stress, eventually causes extrinsic skin aging. Olfactory receptors (ORs) were initially considered to be specifically expressed in nasal tissue, but several ORs have been reported to be present in other tissues, and their biological roles have recently received increasing attention. In this study, we aimed to characterize the role of ORs in cell survival and collagen synthesis in dermal fibroblasts. We confirmed that UVB irradiation and dexamethasone exposure significantly decreased cell survival and collagen synthesis in Hs68 dermal fibroblasts. Moreover, we demonstrated that the mRNA expression of 10 ORs detectable in Hs68 cells was significantly downregulated in aged conditions compared with that in normal conditions. Thereafter, by individual knockdown of the 10 candidate ORs, we identified that only OR51B5 knockdown leads to a reduction of cell survival and collagen synthesis. OR51B5 knockdown decreased cAMP levels and dampened the downstream protein kinase A/cAMP-response element binding protein pathway, downregulating the survival- and collagen synthesis-related genes in the dermal fibroblasts. Therefore, OR51B5 may be an interesting candidate that plays a role in cell survival and collagen synthesis.

Vocal Creativity in Elephant Sound Production

How do elephants achieve their enormous vocal flexibility when communicating, imitating or creating idiosyncratic sounds? The mechanisms that underpin this trait combine motoric abilities with vocal learning processes. We demonstrate the unusual production techniques used by five African savanna elephants to create idiosyncratic sounds, which they learn to produce on cue by positive reinforcement training. The elephants generate these sounds by applying nasal tissue vibration via an ingressive airflow at the trunk tip, or by contracting defined superficial muscles at the trunk base. While the production mechanisms of the individuals performing the same sound categories are similar, they do vary in fine-tuning, revealing that each individual has its own specific sound-producing strategy. This plasticity reflects the creative and cognitive abilities associated with ‘vocal’ learning processes. The fact that these sounds were reinforced and cue-stimulated suggests that social feedback and positive reinforcement can facilitate vocal creativity and vocal learning behavior in elephants. Revealing the mechanism and the capacity for vocal learning and sound creativity is fundamental to understanding the eloquence within the elephants’ communication system. This also helps to understand the evolution of human language and of open-ended vocal systems, which build upon similar cognitive processes.