The Risk of Spread of Infection During Craniotomy/Craniostomy on Patients with Active Coronavirus Disease 2019 (COVID-19) Infection: Myth or Fact?

OBJECTIVES: Craniotomies/craniostomies have been categorized as aerosol-generating procedures and are presumed to spread coronavirus disease 2019 (COVID-19). However, the presence of severe acute respiratory distress syndrome coronavirus 2 virus in the generated bone dust has never been proved. Our objective is to evaluate the presence of virus in the bone dust (aerosol) generated during emergency neurosurgical procedures performed on patients with active COVID-19. This would determine the true risk of disease transmission during the surgery. METHODS: Ten patients with active COVID-19 infection admitted to our institute in 1 month required emergency craniotomy/craniostomy. The bone dust and mucosal scrapings form paranasal sinuses (if opened) collected during these procedures were tested for the virus using reverse transcription polymerase chain reaction. The entire surgical team was observed for any symptoms related to COVID-19 for 14 days following surgery. RESULTS: Nine patients had moderate viral load in their nasopharyngeal cavity, as detected on reverse transcription polymerase chain reaction. None of the samples of bone dust from these 10 patients tested positive. Mucosal scrapping obtained in 1 patient in which mastoid air cells were inadvertently opened tested negative as well. No health workers from the operating room developed COVID-19-related symptoms. CONCLUSIONS: The bone dust generated during craniotomy/stomy of active patients does not contain the virus. The procedure on an active patient is unlikely to spread the disease. However, a study with larger cohort would be confirmatory.

COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is associated with substantial morbidity and mortality. Although much has been learned in the first few months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation, and many patients with anosmia show no or only minor respiratory symptoms1. Studies in animals infected experimentally with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, provide opportunities to study aspects of the disease that are not easily investigated in human patients. Although the severity of COVID-19 ranges from asymptomatic to lethal2, most experimental infections provide insights into mild disease3. Here, using K18-hACE2 transgenic mice that were originally developed for SARS studies4, we show that infection with SARS-CoV-2 causes severe disease in the lung and, in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Furthermore, we show that infusion of convalescent plasma from a recovered patient with COVID-19 protected against lethal disease. Mice developed anosmia at early time points after infection. Notably, although pre-treatment with convalescent plasma prevented most signs of clinical disease, it did not prevent anosmia. Thus, K18-hACE2 mice provide a useful model for studying the pathological basis of both mild and lethal COVID-19 and for assessing therapeutic interventions.

Numerical evaluation of spray position for improved nasal drug delivery.

Topical intra-nasal sprays are amongst the most commonly prescribed therapeutic options for sinonasal diseases in humans. However, inconsistency and ambiguity in instructions show a lack of definitive knowledge on best spray use techniques. In this study, we have identified a new usage strategy for nasal sprays available over-the-counter, that registers an average 8-fold improvement in topical delivery of drugs at diseased sites, when compared to prevalent spray techniques. The protocol involves re-orienting the spray axis to harness inertial motion of particulates and has been developed using computational fluid dynamics simulations of respiratory airflow and droplet transport in medical imaging-based digital models. Simulated dose in representative models is validated through in vitro spray measurements in 3D-printed anatomic replicas using the gamma scintigraphy technique. This work breaks new ground in proposing an alternative user-friendly strategy that can significantly enhance topical delivery inside human nose. While these findings can eventually translate into personalized spray usage instructions and hence merit a change in nasal standard-of-care, this study also demonstrates how relatively simple engineering analysis tools can revolutionize everyday healthcare. Finally, with respiratory mucosa as the initial coronavirus infection site, our findings are relevant to intra-nasal vaccines that are in-development, to mitigate the COVID-19 pandemic.

HPV-Related Multiphenotypic Sinonasal Carcinoma: A Unique Case.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC), originally known as HPV-related carcinoma with adenoid cystic carcinoma-like features, is a recently described neoplasm that presents only in the sinonasal tract, displays features of both a surface-derived carcinoma and a salivary gland carcinoma, and is associated with high-risk HPV, specifically HPV type 33. Majority of the cases display high-grade histologic features, but HMSC paradoxically behaves in a relatively indolent fashion. Distinguishing HMSC from other histologic mimickers is essential as the management and prognosis are significantly different. In this article, we present a unique case of HMSC and review the literature.

Human Papillomavirus-Related Carcinoma With Adenoid Cystic-like Features of the Sinonasal Tract (Also Known as Human Papillomavirus-Related Multiphenotypic Sinonasal Carcinoma).

Human papillomavirus (HPV)-related carcinoma with adenoid cystic-like features is a rare, recently recognized entity restricted to the sinonasal tract. By definition, it is associated with high-risk HPV infection, particularly with HPV type 33. In most cases, tumors are composed of dual cell populations, including predominant basaloid myoepithelial cells and usually inconspicuous ductal cells. Solid components with focal cribriform or tubular patterns, abrupt keratinization within tumor nests, and squamous dysplasia of the surface epithelium are characteristics of HPV-related carcinoma with adenoid cystic-like features. The immunohistochemistry of p16 followed by high-risk HPV testing may help in the differential diagnosis. Recent studies have demonstrated that the morphologic features of this entity are more diverse than initially believed. Surgical resection is the prime alternative for treatment. According to the limited data, the prognosis of this disease may be better than that of other sinonasal carcinomas.

Human papillomavirus-related multiphenotypic sinonasal carcinoma: An emerging tumor type with a unique microscopic appearance and a paradoxical clinical behaviour.

Human papillomavirus (HPV) is well established as a causative factor in most squamous cell carcinomas of the oropharynx (OPSCC). Indeed, a growing awareness over the past two decades that HPV-OPSCC is a distinct form of head and neck cancer has had a profound impact on diagnostic and clinical practices. The sinonasal tract is a second anatomic "hot spot" for HPV-related head and neck carcinomas, but certain pathologic features and the clinical behavior of HPV-related carcinomas at this site remain unclear. The enigmatic nature of HPV-positive sinonasal carcinomas is especially true for an emerging form recently designated as HPV-related multiphenotypic sinonasal carcinoma (HMSC). HMSC has come to the attention of the pathology community largely owing to its highly unusual microscopic appearance: it exhibits mixed salivary gland (e.g. adenoid cystic carcinoma) and squamous differentiation. At the same time, HMSC is largely unknown by the clinical community despite an unexpected clinical behavior that could affect therapy. HMSC is characterized by high grade histologic features, locally destructive growth, advanced T stage, and a propensity for local recurrence; and yet it appears to have little potential for metastatic spread or lethal behavior. This review will describe the unique pathologic features of HMSC, discuss its distinction from adenoid cystic carcinoma and squamous cell carcinoma, and draw attention to a behavior that departs from the expected clinical course of most high grade carcinomas of the head and neck.

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.

Respiratory viral detection in the paranasal sinuses of patients with cystic fibrosis.

BACKGROUND: Pulmonary colonization with antibiotic-resistant organisms in patients with cystic fibrosis (CF) is often preceded by upper-airway infections. Although there is a well-described relationship between pulmonary respiratory viral infections and overall disease progression of CF, the pathogenicity of respiratory viral infections in the paranasal sinuses of patients with CF remains unknown. With recent advances in respiratory virus detection techniques, this study sought to detect the presence of respiratory viruses in the paranasal sinuses of patients with CF in comparison with healthy controls and to correlate the viral presence with clinical measures of sinonasal disease. METHODS: This prospective individual cohort study compared 24 patients with CF with 14 healthy controls. Basic demographics, clinical measures of disease and respiratory viral screens (commercial multiplex) obtained directly from the paranasal sinuses were compared between the two groups. RESULTS: Respiratory viruses were detected in 33% of patients with CF (8/24) compared with 0% of the healthy controls (0/14) (p = 0.017). Respiratory viruses were only detected during the winter months, and the most commonly identified were influenza A and human rhinovirus strains. There was no statistical difference in the 22-Item Sino-Nasal Outcome Test (SNOT-22) scores (p = 0.93) or modified Lund-Kennedy scores (p = 0.74) between patients with CF with a positive viral test and those without a positive result. CONCLUSIONS: Respiratory viral detection is more commonly detected in the paranasal sinuses of patients with CF compared with healthy controls. Although respiratory viral presence did not correlate with a worse clinical severity of sinonasal disease, these findings may provide insight into the pathophysiology of CF and open new avenues for potential targeted therapy.

A novel RNA-based in situ hybridization to detect Seneca Valley virus in neonatal piglets and sows affected with vesicular disease.

Seneca Valley virus (SVV) is the causative agent of an emerging vesicular disease in swine, which is clinically indistinguishable from other vesicular diseases such as foot-and-mouth disease. In addition, SVV has been associated with neonatal mortality in piglets. While a commercial SVV qRT-PCR is available, commercial antibodies are lacking to diagnose SVV infections by immunohistochemistry (IHC). Thus, a novel in situ hybridization technique-RNAscope (ISH) was developed to detect SVVRNA in infected tissues. From a total of 78 samples evaluated, 30 were positive by qRT-PCR and ISH-RNA, including vesicular lesions of affected sows, ulcerative lesions in the tongue of piglets and various other tissues with no evidence of histological lesions. Nineteen samples were negative for SVV by qRT-PCR and ISH-RNA. The Ct values of the qRT-PCR from ISH-RNA positive tissues varied from 12.0 to 32.6 (5.12 x 106 to 5.31 RNA copies/g, respectively). The ISH-RNA technique is an important tool in diagnosing and investigating the pathogenesis of SVV and other emerging pathogens.

Enumerating Virus-Like Particles and Bacterial Populations in the Sinuses of Chronic Rhinosinusitis Patients Using Flow Cytometry.

There is increasing evidence to suggest that the sinus microbiome plays a role in the pathogenesis of chronic rhinosinusitis (CRS). However, the concentration of these microorganisms within the sinuses is still unknown. We show that flow cytometry can be used to enumerate bacteria and virus-like particles (VLPs) in sinus flush samples of CRS patients. This was achieved through trialling 5 sample preparation techniques for flow cytometry. We found high concentrations of bacteria and VLPs in these samples. Untreated samples produced the highest average bacterial and VLP counts with 3.3 ± 0.74 x 10(7) bacteria ml(-1) and 2.4 ± 1.23 x 10(9) VLP ml(-1) of sinus flush (n = 9). These counts were significantly higher than most of the treated samples (p < 0.05). Results showed 10(3) and 10(4) times inter-patient variation for bacteria and VLP concentrations. This wide variation suggests that diagnosis and treatment need to be personalised and that utilising flow cytometry is useful and efficient for this. This study is the first to enumerate bacterial and VLP populations in the maxillary sinus of CRS patients. The relevance of enumeration is that with increasing antimicrobial resistance, antibiotics are becoming less effective at treating bacterial infections of the sinuses, so alternative therapies are needed. Phage therapy has been proposed as one such alternative, but for dosing, the abundance of bacteria is required. Knowledge of whether phages are normally present in the sinuses will assist in gauging the safety of applying phage therapy to sinuses. Our finding, that large numbers of VLP are frequently present in sinuses, indicates that phage therapy may represent a minimally disruptive intervention towards the nasal microbiome. We propose that flow cytometry can be used as a tool to assess microbial biomass dynamics in sinuses and other anatomical locations where infection can cause disease.

Biological characteristics and propagation of human rhinovirus-C in differentiated sinus epithelial cells.

Information about the basic biological properties of human rhinovirus-C (HRV-C) viruses is lacking due to difficulties with culturing these viruses. Our objective was to develop a cell culture system to grow HRV-C. Epithelial cells from human sinuses (HSEC) were differentiated at air-liquid interface (ALI). Differentiated cultures supported 1-2 logs growth of HRV-C15 as detected by quantitative RT-PCR. Two distinguishing features of HRVs are acid lability and optimal growth at 33-34 °C. We used this system to show that HRV-C15 is neutralized by low pH (4.5). In contrast to most HRV types, replication of HRV-C15 and HRV-C41 was similar at 34 and 37°C. The HSEC ALI provides a useful tool for quantitative studies of HRV-C replication. The ability of HRV-C to grow equally well at 34°C and 37°C may contribute to the propensity for HRV-C to cause lower airway illnesses in infants and children with asthma.

Apoptotic and proliferative status in HPV (+) and HPV (-) inverted papilloma patients. Correlation with local recurrence and clinicopathological variables.

Inverted papilloma (IP) is a rare sinonasal benign lesion characterized by aggressive biological behavior. Our aim was to evaluate the expression of various proliferation and apoptotic markers and the presence of HPV genotypes in paraffin sections gathered from surgically treated IP patients. Immunohistochemistry for PCNA, bax, cytochrome c and caspase-8 and flow cytometry for the detection of apoptosis, necrosis and ki67 expression were performed. The identification of various HPV subtypes was achieved by nested PCR amplification. Nasal polyps (NP) and specimens from normal nasal epithelium (NE) were used as controls. PCNA was more frequently expressed in IP compared to NE (p=0.04) and caspase-8 and bax staining were less frequently observed in IP compared to NP (p=0.004 and p=0.01 respectively) and NE (p=0.003 and p=0.01, respectively). IP and NP presented significantly higher Ki67 flow cytometry values compared to NE (p<0.001 and p=0.02 respectively). Cytochrome c was more frequently expressed in IP specimens with more prominent inflammation (p=0.02). A low HPV DNA detection rate was observed. Neither HPV status nor any of the apoptotic or proliferative markers studied was associated with the patients' clinicopathological characteristics. Increased Ki67 appeared to correlate with disease recurrence (p=0.01). Increased PCNA and Ki67 and decreased bax and caspase-8 expression indicate that cell proliferation is increased while apoptosis is inhibited in IP, explaining its biological behavior.

Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C.

A recently recognized human rhinovirus species C (HRV-C) is associated with up to half of HRV infections in young children. Here we propagated two HRV-C isolates ex vivo in organ culture of nasal epithelial cells, sequenced a new C15 isolate and developed the first, to our knowledge, reverse genetics system for HRV-C. Using contact points for the known HRV receptors, intercellular adhesion molecule-1 (ICAM-1) and low-density lipoprotein receptor (LDLR), inter- and intraspecies footprint analyses predicted a unique cell attachment site for HRV-Cs. Antibodies directed to binding sites for HRV-A and -B failed to inhibit HRV-C attachment, consistent with the alternative receptor footprint. HRV-A and HRV-B infected HeLa and WisL cells but HRV-C did not. However, HRV-C RNA synthesized in vitro and transfected into both cell types resulted in cytopathic effect and recovery of functional virus, indicating that the viral attachment mechanism is a primary distinguishing feature of HRV-C.

Is chronic rhinosinusitis caused by persistent respiratory virus infection?

BACKGROUND: Many chronic rhinosinusitis (CRS) patients recall an upper respiratory tract infection as the inciting event of their chronic illness. Viral infections have been shown to cause obstruction of the osteomeatal complex, which is likely to be a critical step in the development of CRS. There is clear overlap between the pathogenesis of CRS and asthma. Infections with respiratory viruses in childhood increase the risk of subsequently developing asthma. Viral infections in established asthmatics are associated with acute exacerbations. We sought to determine whether respiratory viruses could be detected within the sinonasal mucosa of CRS patients using polymerase chain reaction (PCR) techniques. METHODS: Sinus mucosa was sampled from 13 patients with CRS and 2 patients with normal sinuses. PCR was used to look for common respiratory viruses (parainfluenza 1, 2, and 3; respiratory syncytial virus [RSV]; human metapneumovirus [hMPV]; adenovirus [ADV]; rhinovirus; coronavirus; bocavirus [BoV]; cytomegalovirus [CMV]; and influenza A and B). RESULTS: No respiratory viruses were detected in any of the samples. CONCLUSION: Persistence of respiratory viruses within the sinonasal mucosa is unlikely to be a cause of ongoing inflammation in CRS. The possibility remains that a transient viral infection provides the initial inflammatory stimulus.

Identification of the novel KI Polyomavirus in paranasal and lung tissues.

KI is a novel polyomavirus identified in the respiratory secretions of children with acute respiratory symptoms. Whether this reflects a causal role of the virus in the human respiratory disease remains to be established. To investigate the presence of KIV in the respiratory tissue, we examined 20 fresh lung cancer specimens and surrounding normal tissue along with one paranasal and one lung biopsy from two transplanted children. KIV-VP1 gene was detected in 9/20 lung cancer patients and 2/2 transplanted patients. However, amplification of the sequence coding for the C-terminal part of the early region of KIV performed on the 11 positive cases was successful only in two malignant lung tissues, one surrounding normal tissue, and 1/2 biopsies tested. Phylogenetic analysis performed on the early region of KIV (including the four Italian isolates), BKV and JCV revealed the presence of three distinct clades. Within the KIV clade two sub-clades were observed. A sub-clade A containing the four Italian strains, and a sub-clade B comprising the Swedish and Australian isolates. Interestingly, the two Italian strains identified in normal tissue clustered together, whereas those detected in malignant tissue fell outside this cluster. In vitro studies are needed to investigate the transforming potential of KIV strains.

Histopathology and growth kinetics of influenza viruses (H1N1 and H3N2) in the upper and lower airways of guinea pigs.

Recent investigations have shown that guinea pigs are important for the study of influenza A virus (IAV) transmission. However, very little is known about IAV replication and histopathology in the guinea pig respiratory tract. Here, we describe viral growth kinetics, target cells and histopathology in the nasosinus, trachea and lungs of IAV-infected guinea pigs. We found that guinea pigs infected with either A/Puerto Rico/8/34 (H1N1) or A/Hong Kong/8/68 (H3N2) developed a predominantly upper airway infection with high nasal viral titres. IAV grew to moderate titres in the lungs but induced marked inflammatory responses, resulting in severe bronchopneumonia and alveolitis. Although non-lethal at the high dose of 2x10(6) p.f.u., infections with these IAV strains were associated with reduced weight gain. IAV infection in guinea pigs is characterized by extensive viral replication in the ciliated nasal epithelial cells followed by heavy nasal mucus secretion.

Detection of human papillomavirus DNA in benign and malignant sinonasal neoplasms.

Infections with human papillomaviruses are divided basically into three different infection types: those producing specific clinically visible lesions, those remaining subclinical, and those being latent. The assumed infection type thought to be present in tissue specimens has influence on the conclusions that can be made from an analysis, i.e. whether or not the HPV infection has a causal relationship with other epidemiological or molecular investigation observations. To determine whether HPV DNA detection in different entities of the upper aerodigestive tract represents a coincidental, persistent/latent or specific infection, 20 clinically intact mucosa specimens of the upper aerodigestive tract, 20 sinonasal polyps, 26 inverted papillomas, and 20 squamous cell carcinomas of the paranasal sinuses were investigated. HPV DNA was not detectable in specimens derived from clinically intact mucosa or in nasal polyps. Yet, three out of 26 inverted papillomas were HPV-positive, each showing double infection with HPV6 and 11. Four out of 20 squamous cell carcinomas were HPV16 positive. To our knowledge, we are presenting the first study contemporaneously analyzing benign as well as malignant non-proliferative and proliferative mucosal entities whilst applying identical methodical standards. The data corroborate the hypothesis that HPV DNA demonstration in tissue specimens represents a specific infection of the mucosa of the upper aerodigestive tract. It can thus be assumed that there is a causative involvement of HPV infections in the alteration of cell proliferation and in the case of infection with high risk HPV types even on progression to malignant transformation.

Pathogenicity, transmissibility, and tissue distribution of avian pneumovirus in turkey poults.

The pathogenicity, transmissibility, tissue distribution, and persistence of avian pneumovirus (APV) in turkey poults were investigated in three experiments. In the first experiment, we inoculated 2-wk-old commercial turkey poults oculonasally with APV alone or in combination with Bordetella avium. In the dually infected group, clinical signs were more severe, the virus persisted longer, the bacteria invaded more respiratory tissues, and the birds had higher antibody titer than the group exposed to APV or B. avium alone. In the second experiment, we studied the distribution of APV in different tissues in experimentally inoculated 2-wk-old commercial turkey poults. Only samples from sinuses, tracheas, and lungs were positive for APV by both reverse transcriptase-polymerase chain reaction and virus isolation. In the third experiment, we studied the ability of APV to spread among birds in 1-wk-old commercial turkey poults inoculated oculonasally. The virus was isolated and the viral RNA was detected in the inoculated and direct contact birds. The virus was not isolated, viral RNA was not detected, and no antibodies were detected in the indirect contact birds. These birds were placed in different cages in the same room where the airflow was directed from the infected toward the uninfected indirect contact group.