Complications of Nasopharyngeal Swabs and Safe Procedures for COVID-19 Testing Based on Anatomical Knowledge.

Nasopharyngeal swabs have been widely to prevent the spread of coronavirus disease 2019 (COVID-19). Nasopharyngeal COVID-19 testing is a generally safe and well-tolerated procedure, but numerous complications have been reported in the media. Therefore, the present study aimed to review and document adverse events and suggest procedural references to minimize preventable but often underestimated risks. A total of 27 articles were selected for the review of 842 related documents in PubMed, Embase, and KoreaMed. The complications related to nasopharyngeal COVID-19 testing were reported to be rarely happened, ranging from 0.0012 to 0.026%. Frequently documented adverse events were retained swabs, epistaxis, and cerebrospinal fluid leakage, often associated with high-risk factors, including severe septal deviations, pre-existing skull base defects, and previous sinus or transsphenoidal pituitary surgery. Appropriate techniques based on sufficient anatomical knowledge are mandatory for clinicians to perform nasopharyngeal COVID-19 testing. The nasal floor can be predicted by the line between the nostril and external ear canal. For safe testing, the angle of swab insertion in the nasal passage should remain within 30° of the nasal floor. The swab was gently inserted along the nasal septum just above the nasal floor to the nasopharynx and remained on the nasopharynx for several seconds before removal. Forceful insertion should be attempted, and alternative examinations should be considered, especially in vulnerable patients. In conclusion, patients and clinicians should be aware of rare but possible complications and associated high-risk factors. The suggested procedural pearls enable more comfortable and safe nasopharyngeal COVID-19 testing for both clinicians and patients.

TLR activation, immune response and viral protection elicited in cattle by a commercial vaccine against Bovine Herpesvirus-1.

The innate and acquired immune response induced by a commercial inactivated vaccine against Bovine Herpesvirus-1 (BoHV-1) and protection conferred against the virus were analyzed in cattle. Vaccination induced high levels of BoHV-1 antibodies at 30, 60, and 90 days post-vaccination (dpv). IgG1 and IgG2 isotypes were detected at 90 dpv, as well as virus-neutralizing antibodies. An increase of anti-BoHV-1 IgG1 in nasal swabs was detected 6 days post-challenge in vaccinated animals. After viral challenge, lower virus excretion and lower clinical score were observed in vaccinated as compared to unvaccinated animals, as well as BoHV-1-specific proliferation of lymphocytes and production of IFNγ, TNFα, and IL-4. Downregulation of the expression of endosome Toll-like receptors 8-9 was detected after booster vaccination. This is the first thorough study of the immunity generated by a commercial vaccine against BoHV-1 in cattle.

Metabolic Reprogramming of Nasal Airway Epithelial Cells Following Infant Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is a seasonal mucosal pathogen that infects the ciliated respiratory epithelium and results in the most severe morbidity in the first six months of life. RSV is a common cause of acute respiratory infection during infancy and is an important early-life risk factor strongly associated with asthma development. While this association has been repeatedly demonstrated, limited progress has been made on the mechanistic understanding in humans of the contribution of infant RSV infection to airway epithelial dysfunction. An active infection of epithelial cells with RSV in vitro results in heightened central metabolism and overall hypermetabolic state; however, little is known about whether natural infection with RSV in vivo results in lasting metabolic reprogramming of the airway epithelium in infancy. To address this gap, we performed functional metabolomics, 13C glucose metabolic flux analysis, and RNA-seq gene expression analysis of nasal airway epithelial cells (NAECs) sampled from infants between 2-3 years of age, with RSV infection or not during the first year of life. We found that RSV infection in infancy was associated with lasting epithelial metabolic reprogramming, which was characterized by (1) significant increase in glucose uptake and differential utilization of glucose by epithelium; (2) altered preferences for metabolism of several carbon and energy sources; and (3) significant sexual dimorphism in metabolic parameters, with RSV-induced metabolic changes most pronounced in male epithelium. In summary, our study supports the proposed phenomenon of metabolic reprogramming of epithelial cells associated with RSV infection in infancy and opens exciting new venues for pursuing mechanisms of RSV-induced epithelial barrier dysfunction in early life.

Variability in instructions for performance of nasopharyngeal swabs across Canada in the era of COVID-19 - what type of swab is actually being performed?

BACKGROUND: The primary method of surveillance for the presence of SARS-CoV-2 is with nasopharyngeal swabs. Given the significant demand for nasopharyngeal swabs, a large number of previously untrained and unfamiliar staff are now performing this test. It was noted that there was significant heterogeneity in instructions for performing nasopharyngeal swabs in Canada, in contrast to the guidance provided by the Centers for Disease Control and Prevention (CDC), and the Pan American Health Organization (PAHO). The objective of this study was to review the instructions provided across Canada and contrast them to those of the CDC and PAHO. METHODS: A standard series of steps for nasopharyngeal swab performance was outlined based on the CDC, PAHO, and New England Journal of Medicine instructions. A comprehensive search was performed in August 2020 to identify nasopharyngeal swab guidelines provided by public health in the provinces and territories of Canada. Regional health authority guidance was also collected. Instructions provided were contrasted against the standardized steps. RESULTS: Instructions were identified for all provinces and territories, and for 81 regional health authorities. From the provincial and territorial guidelines, 10/13 (77%) cleared the nasal passages before swab insertion, 11/13 (85%) tilted the patient's head back slightly, 12/13 (92%) inserted the swab parallel to the palate, but only 3/13 (23%) inserted the swab to at least a depth of two-thirds the distance between the patient's nose and ear. A clear majority (81%) of regional health authority guidelines followed their respective provincial guidelines. For depth of insertion, Quebec provided a pictogram but no distance or technique for estimation. Six provinces and territories - Northwest Territories, Nunavut, Ontario, Saskatchewan, Prince Edward Island and Alberta - recommended 4 cm or one-half the distance from nostrils to ear. British Columbia and Manitoba recommended a 7 cm depth of insertion. Nova Scotia recommended one-half to two-thirds the distance from nose to ear. Lastly, Newfoundland, New Brunswick and the Yukon recommended an insertion from nose to the external ear canal. CONCLUSION: There is significant heterogeneity in guidance for nasopharyngeal swab performance across Canada. The instructions provided by the majority of provinces and territories in Canada would not be effective in reaching the nasopharynx.

A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection.

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.

Coronavirus Disease-19 and Rhinology/Facial Plastics.

This review summarizes the challenges and adaptations that have taken place in rhinology and facial plastics in response to the ongoing coronavirus disease-19 pandemic. In particular, the prolonged exposure and manipulation of the nasal and oral cavities portend a high risk of viral transmission. We discuss evidence-based recommendations to mitigate the risk of viral transmission through novel techniques and device implementation as well as increasing conservative management of certain pathologies.

Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients.

In routine clinical practice, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is determined by reverse-transcription PCR (RT-PCR). In the current pandemic, a more rapid and high-throughput method is in growing demand. Here, we validated the performance of a new antigen test (LUMIPULSE) based on chemiluminescence enzyme immunoassay. A total of 313 nasopharyngeal swabs (82 serial samples from 7 infected patients and 231 individual samples from 4 infected patients and 215 uninfected individuals) were analyzed for SARS-CoV-2 with quantitative RT-PCR (RT-qPCR) and then subjected to LUMIPULSE. We determined the cutoff value for antigen detection using receiver operating characteristic curve analysis and compared the performance of the antigen test with that of RT-qPCR. We also compared the viral loads and antigen levels in serial samples from seven infected patients. Using RT-qPCR as the reference, the antigen test exhibited 55.2% sensitivity and 99.6% specificity, with a 91.4% overall agreement rate (286/313). In specimens with > 100 viral copies and between 10 and 100 copies, the antigen test showed 100% and 85% concordance with RT-qPCR, respectively. This concordance declined with lower viral loads. In the serially followed patients, the antigen levels showed a steady decline, along with viral clearance. This gradual decline was in contrast with the abrupt positive-to-negative and negative-to-positive status changes observed with RT-qPCR, particularly in the late phase of infection. In summary, the LUMIPULSE antigen test can rapidly identify SARS-CoV-2-infected individuals with moderate to high viral loads and may be helpful for monitoring viral clearance in hospitalized patients.

Traumatic Nonmissile Penetrating Transnasal Anterior Skull Base Fracture and Brain Injury with Cerebrospinal Fluid Leak: Intraoperative Leak Detection and an Effective Reconstruction Procedure for a Localized Skull Base Defect Especially After Coronavirus Disease 2019 Outbreak.

BACKGROUND: Cerebrospinal fluid (CSF) leakage after penetrating skull base injury is relatively rare compared with close head injuries involving skull base fractures. CASE DESCRIPTION: We report the case of a 65-year-old man who had presented with epistaxis and serous rhinorrhea. When he had fallen to the ground near his bee boxes, a garden pole had poked into his right nostril. He had instantly removed the pole from his nostril himself. However, immediately after removal of the pole, he had developed nasal bleeding and serous rhinorrhea. He then drove to our emergency room. Computed tomography showed pneumocephalus with a minor cerebral contusion in the left frontal lobe and a penetrating injury in the left anterior skull base. His CSF leakage had not resolve spontaneously within 1 week after the injury with strict bed rest. We repaired the CSF leakage using a fat (adipose tissue)-on-fascia autograft plug and caulked the defect in the anterior skull base with the fat-on-fascia graft (FFG) plug through the left nostril with endoscopic guidance. The CSF rhinorrhea was successfully controlled. Intranasal local application of fluorescein aided in the detection of the direction of flow of the CSF leakage. CONCLUSIONS: Endonasal endoscopic caulking of a skull base defect using an FFG plug can be useful to treat CSF leakage due to the localized skull base defect, especially in the coronavirus disease 2019 pandemic. It is simple, inexpensive, and timesaving. It requires no special skills nor sophisticated instruments that can cause aerosolization, reducing the risk of infection during the surgery.

Virus isolation and genotype identification of human respiratory syncytial virus in Guizhou Province, China

ABSTRACT To investigate the genetic variation and molecular epidemiology characteristics of Human Respiratory Syncytial Virus (HRSV) in Guizhou Province, nasopharyngeal aspirates were collected from patients with acute respiratory infection (ARI) in Guizhou Provincial People's Hospital, from December 2017 to March 2018, and inoculated to Hep-2 cells to isolate HRSV. Cells that showed cytopathic effect (CPE) were then confirmed by indirect immunofluorescence assay and reverse transcription. The sequence of the PCR products was determined for HRSV isolates, and the genetic variation was analyzed. Out of 196 nasopharyngeal aspirate samples, HRSV were isolated in 39. The second hypervariable region at the 3' terminal of glycoprotein gene (HVR2) sequence analysis showed that subgroup A was dominant. Seventy-nine percent of the isolates belonged to subgroup A, ON1 genotype, and 21 % belonged to subgroup B, BA9 genotype, which indicates that the dominant HRSV circulating in Guizhou Province was subgroup A, genotype ON1, co-circulating with a less prevalent subgroup B, genotype BA9.

[Detection of bovine herpesvirus 4 DNA in cattle by realtime PCR.]

INTRODUCTION: BoHV-4 is poorly understood. Data on the circulation of the virus among animals and its role in infectious diseases insufficient. Aimes and goals. Development of real-time PCR for detecting the BoHV-4 and studying the frequency of its presence in samples from sick animals. MATERIAL AND METHODS: The nucleotide sequences of the glycoprotein L gene served as a target for amplification. The sequences of reference strains published in GenBank were used to analyze and design the primers. Studies were conducted in 3 regions of Western Siberia on 5 large dairy farms. RESULTS: 27.7% of samples contained the virus. The virus was present as a monoagent in nasal cavity of calves (80.0%), lungs (46.2%) and bronchial lymph nodes (38.5%) in pneumonia. In the cases of diarrhea the virus was detected in 20%, and in cows with gynecological pathology in 10.0%. In respiratory diseases of calves the virus was detected in association with BoHV-1 (21.6%) and BoCV (20.3%), and in gynecological pathology of cows with BVDV1 (6%). DISCUSSION: According to the phylogenetic analysis of 5 identified virus isolates, four belonged to the American branch and one to the European branch. The circulation of American strains occurred in the territory of the Republic of Kazakhstan (1), Tyumen (1) and Novosibirsk (2) regions, and the European - in the Novosibirsk region. CONCLUSION: The search for viruses involved to the infectious pathology, as well as studying the genetic diversity of viruses circulating on a particular farm including imported from other countries, is relevant.

The Use of Procalcitonin for Prediction of Pulmonary Bacterial Coinfection in Children With Respiratory Failure Associated With Viral Bronchiolitis.

Objectives. Viral bronchiolitis is a frequent cause of pediatric hospitalization and respiratory failure. Procalcitonin (PCT) is a biomarker used to identify serious bacterial infection and can distinguish bacterial and viral infections. Concomitant bacterial pneumonia is not rare in viral bronchiolitis and can lead to a worse clinical course. This study examined the use of PCT in pediatric patients with respiratory failure attributed to viral bronchiolitis to predict concomitant bacterial pneumonia. Methods. This prospective descriptive study evaluated children less than 4 years of age who underwent endotracheal intubation for respiratory failure due to viral bronchiolitis. PCT levels and endotracheal aspirate cultures were obtained at admission. Bacterial pneumonia was defined as at least moderate growth of a single pathogenic organism from endotracheal culture. PCT levels were evaluated in groups with and without concomitant bacterial pneumonia. Results. Thirty-five patients were enrolled between February 2013 and May 2015. All subjects tested positive for at least 1 viral pathogen by nasal wash polymerase chain reaction or enzyme immunoassay. The top viruses obtained were respiratory syncytial virus (n = 15, 42.8%) and rhinovirus (n = 8, 22.9%). The incidence of bacterial pneumonia was 60% (21/35). The PCT median was 0.93 ng/mL (interquartile range = 0.25-6.64) in the bacterial pneumonia group and 1.85 ng/mL (interquartile range = 0.28-7.94) in the nonbacterial pneumonia group. No correlation was found between PCT and bronchiolitis with bacterial coinfection (P = .74). Conclusion. Incidence of bacterial coinfection in patients with respiratory failure and viral bronchiolitis was high. PCT did not predict concomitant bacterial pneumonia in children with viral bronchiolitis.

Comparative Viral Sampling in the Sinonasal Passages; Different Viruses at Different Sites.

Background: With the emergence of the microbiome as an important factor in health and disease in the respiratory tract standardised, validated techniques are required for its accurate characterisation. No standardised technique has been reported specifically for viral sampling in the sinonasal passages. Aim: To optimise viral sampling techniques from the sinonasal cavity. Methods: Sterile cytology brushes were used under endoscopic guidance to sample the sinonasal mucosa at time of endoscopic sinus surgery at both the middle and inferior meatuses (MM and IM). DNA and RNA were extracted from the samples and underwent PCR or RT-PCR testing, respectively, for a panel of 15 common upper respiratory tract viruses. Results: Twenty-four adult patients were recruited for this study. 18/24 (75%) patients were positive for virus in at least one site, while 8/24 (33%) were positive for virus at both sites. The mean number of viruses identified at the two sites were similar (0.875 ± 0.899 at the MM vs. 0.750 ± 1.032 at the IM). 6/24 (25%) of patients showed no virus at either site, while 3/24 (12.5%) demonstrated the same viral species at both sites. Conclusion: Although the number of viruses present at different sites with the nasal cavity are similar, discord exists in the viral species between sites. It is therefore recommended that both sites are sampled in the clinical and research setting better to characterise the viral species within the nasal cavity.

First detection of influenza A virus genes from wild raccoons in Japan.

Serological surveys have shown that wild raccoons are exposed to influenza A viruses (IAVs); however, no genetic evidence for this IAV infection has been found. In the present study, we first detected IAV genes in wild raccoons captured during periods other than the wintering season of migratory waterfowl and epidemic season of influenza in Japan. Viral matrix (M) and nucleoprotein (NP) genes were detected by a conventional reverse transcription-polymerase chain reaction assay from three suckling siblings and one juvenile without any noticeable clinical signs, although the NP gene could not be detected from one sibling. The sequences of M gene fragments detected from the rectal swabs of three suckling siblings were comparable with each other but different from those detected from the nasal swab of the juvenile raccoon caught from a different site. The sequences of NP gene fragments detected from two suckling siblings were also comparable. These genetic evidences suggest that IAV is maintained among raccoon populations in the northern part of Japan. Further genetic and virological investigation of IAV infection in wild raccoons is needed to better understand the IAV ecology in the field.

Nation-wide surveillance of human acute respiratory virus infections between 2013 and 2015 in Korea.

The prevalence of eight respiratory viruses detected in patients with acute respiratory infections (ARIs) in Korea was investigated through analysis of data recorded by the Korea Influenza and Respiratory Viruses Surveillance System (KINRESS) from 2013 to 2015. Nasal aspirate and throat swabs specimens were collected from 36 915 patients with ARIs, and viral nucleic acids were detected by real-time (reverse-transcription) polymerase chain reaction for eight respiratory viruses, including human respiratory syncytial viruses (HRSVs), influenza viruses (IFVs), human parainfluenza viruses (HPIVs), human coronaviruses (HCoVs), human rhinovirus (HRV), human adenovirus (HAdV), human bocavirus (HBoV), and human metapneumovirus (HMPV). The overall positive rate of patient specimens was 49.4% (18 236/36 915), 5% of which carried two or more viruses simultaneously. HRV (15.6%) was the most predominantly detected virus, followed by IFVs (14.6%), HAdV (7.5%), HPIVs (5.8%), HCoVs (4.2%), HRSVs (3.6%), HBoV (1.9%), and HMPV (1.6%). Most of the ARIs were significantly correlated with clinical symptoms of fever, cough, and runny nose. Although HRV and HAdV were frequently detected throughout the year in patients, other respiratory viruses showed apparent seasonality. HRSVs and IFVs were the major causative agents of acute respiratory diseases in infants and young children. Overall, this study demonstrates a meaningful relationship between viral infection and typical manifestations of known clinical features as well as seasonality, age distribution, and co-infection among respiratory viruses. Therefore, these data could provide useful information for public health management and to enhance patient care for primary clinicians.

A novel polyomavirus from the nasal cavity of a giant panda (Ailuropoda melanoleuca).

BACKGROUND: Polyomaviruses infect a wide variety of mammalian and avian hosts with a broad spectrum of outcomes including asymptomatic infection, acute systemic disease, and tumor induction. METHODS: Viral metagenomics and general PCR methods were used to detected viral nucleic acid in the samples from a diseased and healthy giant pandas. RESULTS: A novel polyomavirus, the giant panda polyomavirus 1 (GPPyV1) from the nasal cavity of a dead giant panda (Ailuropoda melanoleuca) was characterized. The GPPyV1 genome is 5144 bp in size and reveals five putative open-reading frames coding for the classic small and large T antigens in the early region, and the VP1, VP2 and VP3 capsid proteins in the late region. Phylogenetic analyses of the large T antigen of the GPPyV1 indicated GPPyV1 belonged to a putative new species within genus Deltapolyomavirus, clustering with four human polyomavirus species. The GPPyV1 VP1 and VP2 clustered with genus Alphapolyomavirus. Our epidemiologic study indicated that this novel polyomavirus was also detected in nasal swabs and fecal samples collected from captive healthy giant pandas. CONCLUSION: A novel polyomavirus was detected in giant pandas and its complete genome was characterized, which may cause latency infection in giant pandas.

Respiratory syncytial virus subtype ON1/NA1/BA9 predominates in hospitalized children with lower respiratory tract infections.

Respiratory syncytial virus (RSV) infection is the leading cause of acute respiratory tract disease in children less than 5 years old. The aim of this study was to further elucidate the molecular properties and clinical characteristics of RSV infection. The study sample included 238 patients <5 years old who were hospitalized with clinical symptoms of upper or lower respiratory tract infection (URTI or LRTI) in the Pediatric Department at the First People's Hospital of Chenzhou, South China in 2014. We subjected nasopharyngeal aspirate (NPA) or nasal swab (NS) samples from the patients to indirect fluorescence assay screens. RSV G genes were amplified by reverse transcription-PCR (RT-PCR) and sequenced. Of the 238 patients screened, 64 (26.8%) were confirmed to have RSV infections. Of those 64 confirmed RSV infection cases, 39 (60.9%) had subtype BA9, 13 (20.3%) had the recently identified subtype ON1, 11 (17.2%) had subtype NA1, and 1 (1.6%) had subtype GB2. The predominant presentation was LRTI with coughing, sputum production, fever, and wheezing. RSV subtype NA1 and BA9 infections were found mostly in infants, whereas the age distribution of subtype ON1 infections was more uniform across the age bands. Phylogenetic analysis indicated that, compared with the prototype strain A2, all ON1 and most NA1 isolates had lost one potential N-glycosylation site at amino acid 251 and 249 due to T251K and N249Y substitution, respectively. These findings suggest that NA1, BA9, and ON1 are the dominant RSV subtypes causing respiratory tract infections in young children presenting to the hospital in South China. J. Med. Virol. 89:213-221, 2017. © 2016 Wiley Periodicals, Inc.

WU and KI polyomavirus infections in Filipino children with lower respiratory tract disease.

BACKGROUND: WU and KI are human polyomaviruses initially detected in the respiratory tract, whose clinical significance remains uncertain. OBJECTIVES: To determine the epidemiology, viral load and clinical characteristics of WU and KI polyomaviruses. STUDY DESIGN: We tested respiratory specimens collected during a randomized, placebo-controlled pneumococcal conjugate vaccine trial and related epidemiological study in the Philippines. We analyzed 1077 nasal washes from patients aged 6 weeks to 5 years who developed lower respiratory tract illness using quantitative real-time PCR for WU and KI. We collected data regarding presenting symptoms, signs, radiographic findings, laboratory data and coinfection. RESULTS: The prevalence and co-infection rates for WU were 5.3% and 74% respectively and 4.2% and 84% respectively for KI. Higher KI viral loads were observed in patients with severe or very severe pneumonia, those presenting with chest indrawing, hypoxia without wheeze, convulsions, and with KI monoinfection compared with co-infection. There was no significant association between viral load and clinical presentation for WU. CONCLUSIONS: These findings suggest a potential pathogenic role for KI, and that there is an association between KI viral load and illness severity.