Morphological and Phenotypic Characteristics of the Bovine Nasopharyngeal Mucosa and Associated Lymphoid Tissue.

The mammalian nasopharynx is an anatomically complex region of the upper respiratory tract that directly communicates with the nasal cavity, laryngopharynx, oesophagus and trachea. The nasopharyngeal mucosa contains moderate quantities of mucosa-associated lymphoid tissue (MALT) that is appropriately located for immunological sampling but also creates vulnerability to pathogens. In recent years, the nasopharynx has been inculpated in the pathogenesis of important diseases of cattle (foot-and-mouth disease) and humans (COVID-19), yet the tissue has never been described in detail in any species. In order to characterize the morphology and cellular composition of the bovine nasopharynx, samples of mucosa were collected from the nasopharynx of five 8-13-month-old steers and examined using light microscopy, immunohistochemistry and multichannel immunofluorescence. Morphologically, the nasopharyngeal epithelium was highly heterogeneous, with a continuum ranging from stratified squamous epithelium to highly attenuated, follicle-associated epithelium (FAE). Distribution of MALT was similarly regionally variable ranging from absent to clusters of multiple lymphoid follicles. Phenotypic characterization demonstrated dense distributions of dendritic cells and T lymphocytes surrounding lymphoid follicles, which comprised mostly B lymphocytes. The FAE overlaying the lymphoid follicles also contained higher numbers of dendritic cells and lymphocytes compared with the adjacent non-lymphoid epithelium, although cytotoxic T cells were notably scarce in the FAE. The bovine nasopharyngeal lymphoid tissue had comparable elements to other MALTs with specific differences that may help to elucidate the pathogenesis of infectious agents that have specific tropism for this tissue.

Severe Acute Respiratory Syndrome Coronavirus 2 Total and Subgenomic RNA Viral Load in Hospitalized Patients.

BACKGROUND: Previous studies demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected for weeks after infection. The significance of this finding is unclear and, in most patients, does not represent active infection. Detection of subgenomic RNA has been proposed to represent productive infection and may be a useful marker for monitoring infectivity. METHODS: We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) to quantify total and subgenomic nucleocapsid (sgN) and envelope (sgE) transcripts in 185 SARS-CoV-2-positive nasopharyngeal swab samples collected on hospital admission and to relate to symptom duration. RESULTS: We find that all transcripts decline at the same rate; however, sgE becomes undetectable before other transcripts. The median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic compared to total RNA, suggesting that subgenomic transcript copy number is dependent on copy number of total transcripts. The mean difference between total and sgN is 16-fold and the mean difference between total and sgE is 137-fold. This relationship is constant over duration of symptoms, allowing prediction of subgenomic copy number from total copy number. CONCLUSIONS: Subgenomic RNA may be no more useful in determining infectivity than a copy number threshold determined for total RNA.

Severe Acute Respiratory Syndrome Coronavirus 2 Normalized Viral Loads and Subgenomic RNA Detection as Tools for Improving Clinical Decision Making and Work Reincorporation.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse-transcription polymerase chain reaction (RT-PCR) provides a highly variable cycle threshold (Ct) value that cannot distinguish viral infectivity. Subgenomic ribonucleic acid (sgRNA) has been used to monitor active replication. Given the importance of long RT-PCR positivity and the need for work reincorporation and discontinuing isolation, we studied the functionality of normalized viral loads (NVLs) for patient monitoring and sgRNA for viral infectivity detection. METHODS: The NVLs measured through the Nucleocapsid and RNA-dependent-RNA-polymerase genes and sgRNA RT-PCRs were performed in 2 consecutive swabs from 84 healthcare workers. RESULTS: The NVLs provided similar and accurate quantities of both genes of SARS-CoV-2 at 2 different timepoints of infection, overcoming Ct-value and swab collection variability. Among SARS-CoV-2-positive samples, 51.19% were sgRNA-positive in the 1st RT-PCR and 5.95% in the 2nd RT-PCR. All sgRNA-positive samples had >4 log10 RNA copies/1000 cells, whereas samples with ≤1 log10 NVLs were sgRNA-negative. Although NVLs were positive until 29 days after symptom onset, 84.1% of sgRNA-positive samples were from the first 7 days, which correlated with viral culture viability. Multivariate analyses showed that sgRNA, NVLs, and days of symptoms were significantly associated (P < .001). CONCLUSIONS: The NVLs and sgRNA are 2 rapid accessible techniques that could be easily implemented in routine hospital practice providing a useful proxy for viral infectivity and coronavirus disease 2019 patient follow-up.

SARS-CoV-2 Impairs Dendritic Cells and Regulates DC-SIGN Gene Expression in Tissues.

The current spreading coronavirus SARS-CoV-2 is highly infectious and pathogenic. In this study, we screened the gene expression of three host receptors (ACE2, DC-SIGN and L-SIGN) of SARS coronaviruses and dendritic cells (DCs) status in bulk and single cell transcriptomic datasets of upper airway, lung or blood of COVID-19 patients and healthy controls. In COVID-19 patients, DC-SIGN gene expression was interestingly decreased in lung DCs but increased in blood DCs. Within DCs, conventional DCs (cDCs) were depleted while plasmacytoid DCs (pDCs) were augmented in the lungs of mild COVID-19. In severe cases, we identified augmented types of immature DCs (CD22+ or ANXA1+ DCs) with MHCII downregulation. In this study, our observation indicates that DCs in severe cases stimulate innate immune responses but fail to specifically present SARS-CoV-2. It provides insights into the profound modulation of DC function in severe COVID-19.

Identification of COVID-19 prognostic markers and therapeutic targets through meta-analysis and validation of Omics data from nasopharyngeal samples.

BACKGROUND: While our battle with the COVID-19 pandemic continues, a multitude of Omics data have been generated from patient samples in various studies. Translation of these data into clinical interventions against COVID-19 remains to be accomplished. Exploring host response to COVID-19 in the upper respiratory tract can unveil prognostic markers and therapeutic targets. METHODS: We conducted a meta-analysis of published transcriptome and proteome profiles of respiratory samples of COVID-19 patients to shortlist high confidence upregulated host factors. Subsequently, mRNA overexpression of selected genes was validated in nasal swabs from a cohort of COVID-19 positive/negative, symptomatic/asymptomatic individuals. Guided by this analysis, we sought to check for potential drug targets. An FDA-approved drug, Auranofin, was tested against SARS-CoV-2 replication in cell culture and Syrian hamster challenge model. FINDINGS: The meta-analysis and validation in the COVID-19 cohort revealed S100 family genes (S100A6, S100A8, S100A9, and S100P) as prognostic markers of severe COVID-19. Furthermore, Thioredoxin (TXN) was found to be consistently upregulated. Auranofin, which targets Thioredoxin reductase, was found to mitigate SARS-CoV-2 replication in vitro. Furthermore, oral administration of Auranofin in Syrian hamsters in therapeutic as well as prophylactic regimen reduced viral replication, IL-6 production, and inflammation in the lungs. INTERPRETATION: Elevated mRNA level of S100s in the nasal swabs indicate severe COVID-19 disease, and FDA-approved drug Auranofin mitigated SARS-CoV-2 replication in preclinical hamster model. FUNDING: This study was supported by the DBT-IISc partnership program (DBT (IED/4/2020-MED/DBT)), the Infosys Young Investigator award (YI/2019/1106), DBT-BIRAC grant (BT/CS0007/CS/02/20) and the DBT-Wellcome Trust India Alliance Intermediate Fellowship (IA/I/18/1/503613) to ST lab.

Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19.

SARS-CoV-2 infection can cause severe respiratory COVID-19. However, many individuals present with isolated upper respiratory symptoms, suggesting potential to constrain viral pathology to the nasopharynx. Which cells SARS-CoV-2 primarily targets and how infection influences the respiratory epithelium remains incompletely understood. We performed scRNA-seq on nasopharyngeal swabs from 58 healthy and COVID-19 participants. During COVID-19, we observe expansion of secretory, loss of ciliated, and epithelial cell repopulation via deuterosomal cell expansion. In mild and moderate COVID-19, epithelial cells express anti-viral/interferon-responsive genes, while cells in severe COVID-19 have muted anti-viral responses despite equivalent viral loads. SARS-CoV-2 RNA+ host-target cells are highly heterogenous, including developing ciliated, interferon-responsive ciliated, AZGP1high goblet, and KRT13+ "hillock"-like cells, and we identify genes associated with susceptibility, resistance, or infection response. Our study defines protective and detrimental responses to SARS-CoV-2, the direct viral targets of infection, and suggests that failed nasal epithelial anti-viral immunity may underlie and precede severe COVID-19.

Performing nasopharyngeal swabs-Guidelines based on an anatomical study.

Nasopharyngeal swabs are performed to collect material for diagnosing diseases affecting the respiratory system, such as Covid-19. Yet, no systematic anatomical study defines concrete prerequisites for successfully targeting the nasopharyngeal mucosa. We therefore aim at simulating nasopharyngeal swabs in human body donors to characterize parameters allowing and supporting to enter the nasopharynx with a swab, while avoiding endangering the cribriform plate. With the aid of metal probes and commercial swabs a total of 314 nasopharyngeal swabs in anatomical head/neck specimens stemming from 157 body donors were simulated. Important anatomical parameters were photo-documented and measured. We provide information on angles and distances between prominent anatomical landmarks and particularly important positions the probe occupies during its advancement through the nares to the upper and lower parts of the nasopharynx and cribriform plate. Based on these data we suggest a simple and safe three-step procedure for conducting nasopharyngeal swabs. In addition, we define easily recognizable signals for its correct performance. Evaluations prove that this procedure in all specimens without deformations of the nasal cavity allows the swab to enter the nasopharynx, whereas a widespread used alternative only succeeds in less than 50%. Our data will be the key for the successful collection of nasopharyngeal material for detecting and characterizing pathogens, such as SARS-CoV-2, which have a high affinity to pharyngeal mucosa. They demonstrate that the danger for damaging the cribriform plate or olfactory mucosa with swabs is unlikely, but potentially higher when performing nasal swabs.

Evaluation of Nasopharyngeal and Conjunctival Swab Samples of Hospitalised Patients with Confirmed COVID-19.

PURPOSE: To evaluate the results of conjunctival and nasopharyngeal swab tests in patients with confirmed COVID-19. METHODS: This prospective study included 45 patients who were hospitalized for confirmed COVID-19. Nasopharyngeal swab samples were obtained from the patients before hospitalization. Only one eye of each patient was randomly selected for-conjunctival sampling. All participants underwent a complete slit-lamp examination. Conjunctival and nasopharyngeal swab samples were analyzed by reversetranscriptase-polymerase-chain reaction (RT-PCR). RESULTS: Twenty seven (60%) of the patients were male and 18 (40%) were female. Conjunctival swab was positive in only one (2.22%) patient. None of the COVID-19 patients showed ocular changes and symptoms. There were no abnormalities of the ocular surface, anterior chamber or posterior segment at slit-lamp examination. CONCLUSIONS: The RT-PCR was not high positive in the conjunctiva as in nasopharyngeal swabs. Ocular changes were not common in COVID-19 patients.

Persistent SARS-CoV-2 RNA Shedding Without Evidence of Infectiousness: A Cohort Study of Individuals With COVID-19.

BACKGROUND: To better understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shedding and infectivity, we estimated SARS-CoV-2 RNA shedding duration, described participant characteristics associated with the first negative rRT-PCR test (resolution), and determined if replication-competent viruses was recoverable ≥10 days after symptom onset. METHODS: We collected serial nasopharyngeal specimens from 109 individuals with rRT-PCR-confirmed COVID-19 in Utah and Wisconsin. We calculated viral RNA shedding resolution probability using the Kaplan-Meier estimator and evaluated characteristics associated with shedding resolution using Cox proportional hazards regression. We attempted viral culture for 35 rRT-PCR-positive nasopharyngeal specimens collected ≥10 days after symptom onset. RESULTS: The likelihood of viral RNA shedding resolution at 10 days after symptom onset was approximately 3%. Time to shedding resolution was shorter among participants aged <18 years (adjusted hazards ratio [aHR], 3.01; 95% confidence interval [CI], 1.6-5.6) and longer among those aged ≥50 years (aHR, 0.50; 95% CI, .3-.9) compared to participants aged 18-49 years. No replication-competent viruses were recovered. CONCLUSIONS: Although most patients were positive for SARS-CoV-2 for ≥10 days after symptom onset, our findings suggest that individuals with mild to moderate COVID-19 are unlikely to be infectious ≥10 days after symptom onset.

Postmortem Swabs in the Severe Acute Respiratory Syndrome Coronavirus 2 Pandemic: Report on 12 Complete Clinical Autopsy Cases.

CONTEXT.­: Clinical autopsies have historically provided a fundamental contribution in the definition of the clinicopathologic basis of infectious diseases. Even though we are witnessing the decline of the clinical autopsy, its importance remains unchanged as it is the most exhaustive way to investigate diseases. The identification of the virus in postmortem tissues is a fundamental step in the definition of its clinical features. OBJECTIVE.­: To investigate the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in postmortem examination with swabs. DESIGN.­: We performed postmortem swabs in 12 autopsy cases of patients with a clinical diagnosis of SARS-CoV-2-related pneumonia. Our protocol consisted of a rhinopharyngeal and a tracheal swab in order to search for the virus in the upper airways, and of 2 swabs on the parenchyma of each lung. We also performed a fifth swab on the parenchyma of both lungs in order to search for other viruses that could evolve in a clinical picture of interstitial pneumonia. RESULTS.­: Overall, we found 9 of 12 cases had at least 1 postmortem swab positive for SARS-CoV-2. Moreover, we evaluated the time between the antemortem and postmortem swabs, the time between death and the postmortem swabs, and the time between the postmortem swabs and acceptance to the microbiology laboratory. Of note, we did not find a relationship between the results of the swabs and either the time elapsed from their collection or the time elapsed before their acceptance in the microbiology laboratory. CONCLUSIONS.­: A thorough knowledge of the eventual persistence of pathogens in deaths related to infectious diseases is fundamental for the safety of the operators during the autopsy practice, especially when referring to emergent pathogens, such as SARS-CoV-2. Our study highlights the importance in performing multiple swabs in the postmortem examination, because SARS-CoV-2 swab positivity can be limited to only a single swab.

Commercial Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Molecular Assays: Superior Analytical Sensitivity of cobas SARS-CoV-2 Relative to NxTAG CoV Extended Panel and ID NOW COVID-19 Test.

CONTEXT.­: We implemented multiple nucleic acid amplification test platforms because of the limited availability of test kits for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the early stages of the pandemic. Interpretation of results generated by different platforms and prioritization for testing algorithms required cross-comparison. OBJECTIVE.­: To compare the analytical sensitivity of 3 commercial SARS-CoV-2 molecular assays, selected samples were studied in parallel with Cobas SARS-CoV-2 test, NxTAG CoV Extended Panel, and ID NOW COVID-19 assays. DESIGN.­: A total of 8043 SARS-CoV-2 tests performed from March 22 to April 19, 2020, were included in this study. For all 1794 positive specimens detected by the cobas SARS-CoV-2 assay, the cycle threshold (Ct) values were manually tracked and plotted to demonstrate the distribution of sample viral levels. Additionally, 50 and 63 low-positive specimens (Ct values >32) as well as 50 and 61 consecutive positive specimens by the cobas assay were tested with NxTAG and ID NOW, respectively, to estimate their relative sensitivities. RESULTS.­: The Ct values of cobas SARS-CoV-2-positive samples were evenly distributed throughout ranges of 13.32 to 39.50 (mean, 25.06) and 13.60 to 42.49 (mean, 26.45) for ORF1 and E gene targets, respectively. NxTAG reliably detected only specimens with E gene Ct values lower than 33, and is estimated to detect 89.4% of positive specimens detected by cobas assay. ID NOW had performance variation independent of Ct value and is estimated to detect 83.5% of cobas positives. CONCLUSIONS.­: Clinical specimens exhibit a wide range of viral burden, with a significant portion at low levels. Analytical sensitivity of testing platforms is critical for reliable detection of SARS-CoV-2 and uniform care to patients.

Interferon gamma, TGF-ß1 and RANTES expression in upper airway samples from SARS-CoV-2 infected patients.

Upper respiratory tract is the primary site of SARS-CoV-2 replication. Releasing of pro and anti-inflammatory mediators plays an important role in the immunopathogenesis of Coronavirus Disease 2019 (COVID-19). The aim of this study was to evaluate the early inflammatory response in upper airway by measuring of IFN-γ, TGF-ß1 and RANTES at mRNA level. Forty five SARS-CoV-2 infected patients were enrolled, whose were divided in two groups: asymptomatic and symptomatic. Twenty healthy persons, SARS-CoV-2 negative were included as controls. Higher IFN-γ expression was detected in SARS-CoV-2 infected patients in comparison with controls (p = 0.0393). IFN-γ expression was increased in symptomatic patients (p = 0.0405). TGF-ß1 and RANTES expressions were lower in SARS-CoV-2 infected patients than controls (p < 0.0001; p = 0.0011, respectively). A significant correlation between IFN-γ and TGF-ß1 was observed in SARS-CoV-2 asymptomatic patients (r = +0.61, p = 0.0014). The findings suggest that imbalance between IFN-γ and TGF-ß1 expression could be an impact in clinical expression of SARS-CoV-2 infection.

3D-Printing to Address COVID-19 Testing Supply Shortages.

The recent SARS-CoV-2 outbreak has placed immense pressure on supply chains, including shortages in nasopharyngeal (NP) swabs. Here, we report our experience of using 3D-printing to rapidly develop and deploy custom-made NP swabs to address supply shortages at our healthcare institution.