The microbiome of chronic rhinosinusitis: culture, molecular diagnostics and biofilm detection.

BACKGROUND: Bacteria and fungi are believed to influence mucosal inflammation in chronic rhinosinusitis (CRS). However their presence and relationship to disease is debated. This study used multiple detection methods to compare microbial diversity and microbial abundance in healthy and diseased sinonasal mucosa. The utility of contemporary detection methods is also examined. METHODS: Sinonasal mucosa was analyzed from 38 CRS and 6 controls. Bacterial and fungal analysis was performed using conventional culture, molecular diagnostics (polymerase chain reaction coupled with electrospray ionization time-of-flight mass spectrometry) and fluorescence in situ hybridization. RESULTS: Microbes were detected in all samples, including controls, and were often polymicrobial. 33 different bacterial species were detected in CRS, 5 in control patients, with frequent recovery of anaerobes. Staphylococcus aureus and Propionibacterium acnes were the most common organisms in CRS and controls, respectively. Using a model organism, FISH had a sensitivity of 78%, and a specificity of 93%. Many species were detected in both CRS and controls however, microbial abundance was associated with disease manifestation. CONCLUSIONS: This study highlights some cornerstones of microbial variations in healthy and diseased paranasal sinuses. Whilst the healthy sinus is clearly not sterile, it appears prevalence and abundance of organisms is critical in determining disease. Evidence from high-sensitivity techniques, limits the role of fungi in CRS to a small group of patients. Comparison with molecular analysis suggests that the detection threshold of FISH and culture is related to organism abundance and, furthermore, culture tends to select for rapidly growing organisms.

Comparative bibliometric analysis of publications by past Royal Australasian College of Surgeons research scholarship recipients.

BACKGROUND: The Royal Australasian College of Surgeons awards scholarships to surgeons, surgical trainees and recipients focused on developing their clinical knowledge and improving outcomes for patients. A bibliometric analysis of research scholarship recipients publications and h-index scores was conducted to understand the benefits of receiving these scholarships. METHODS: A bibliometric analysis of Royal Australasian College of Surgeons scholarship recipients in 2015 was performed using Open Researcher and Contributor ID (ORCID), Scopus, Google Scholar, ResearchGate, LinkedIn and PubMed to identify the number of publications, h-index scores, field-weighted citation impact and the relative citation ratio. RESULTS: Nineteen research scholarship recipients authored 842 publications, with 491 (58%) published after completion of their scholarship. Seven recipients published 50% or more of their articles in the 5 years since completion. Five recipients have each published more than 45 articles since 2015. H-index scores varied between Scopus and Google Scholar (overall range: 4-34). Scopus identified the most publications, followed by ResearchGate. Determining publication numbers for recipients was problematic due to self-reporting in some databases (i.e. Google Scholar, ResearchGate), variations in author names (i.e. maiden to married name), duplication of publications and the inclusion of supplementary material (i.e. extra tables) in self-reporting databases. Field-weighted citation impact and relative citation ratio values exceeded 1 on 12 occasions demonstrating recipients are more cited than the global average. CONCLUSION: Continuous tracking of publication rates and h-index scores of scholarship recipients demonstrates recipients' continuing interest in advancing and disseminating medical knowledge to improve patient outcomes. The 2015 scholarship recipients publication numbers continued to increase after their scholarship tenure.

Variation in Human Research Ethics Committee and governance processes throughout Australia: a need for a uniform approach.

BACKGROUND: In Australia, ethics committees across different states vary in application, requirement and process for the ethical review and approval for clinical research. This may lead to confusion and delays in the enablement of multicentre research projects. This study explores the effect of differing processes for Ethics and Governance in the establishment of the CovidSurg-Cancer study during the global COVID-19 pandemic. METHODS: An anonymous, structured web-based questionnaire was designed using the Research Electronic Data Capture application (REDCap) platform to capture consultant surgeons, fellows, and trainees experience in the ethics application process. 'CovidSurg-Cancer' was an international multicentre collaborative study to assess the impact of COVID-19 on the outcomes of patients undergoing cancer surgery. The ethics process to set up this observational study was used as to explore the differing processes applied across Australia. RESULTS: The CovidSurg-Cancer study was successfully set up in 14 hospitals. Four hospitals approved the study directly as an audit. Of the remaining sites, 10 ethics applications underwent Human Research Ethics Committee review following which two (14%) were subsequently approved as an audit activity and eight hospitals (57%) were given formal ethical approval with waiver of consent. Ethics application acceptance from another Australian Human Research Ethics Committee was provided with six applications; however, only three were reciprocated without the requirement for further agreements. A third of (30%) respondents suggested that the details of the application pathway, process and documentation were unclear. CONCLUSION: Ethics processes are varied across Australia with considerable repetition. A centralized, harmonized application process would enhance collaborative research.

Part 2: Blockchain technology in health care.

Blockchain technology is one of the many disruptive technologies of the Fourth Industrial Revolution that will irrevocably change the way we live and work. These technologies are well embedded in the areas of global finance, health care and defence, to name a few. This review focuses on the relevance of blockchain technology to health care. Blockchain technology will be the unifying platform for sharing patient data currently inaccessible due to the siloed architecture of legacy software systems, and as a result potentially be the basis for precision or individualized patient treatment. It will also strengthen digital security of sensitive patient data that is presently a lucrative target for cyber criminals. In the current COVID-19 environment, clinicians will rely more on telehealth to reduce person-to-person contact. This service can be delivered by the clinical team with confidence in the veracity of the patient data made accessible through the blockchain platform. Smart contracts written on the blockchain platform will reduce the possibility of international humanitarian aid to low- and middle-income countries being misspent. The pharmaceutical supply chain industry is adopting blockchain technology to ensure supply chain provenance. Similarly, the health insurance industry recognizes how the blockchain ecosystem can improve services to its members and expedite reimbursements to clinicians.

Part 1: Artificial intelligence technology in surgery.

Artificial intelligence (AI) is one of the disruptive technologies of the fourth Industrial Revolution that is changing our work practices. This technology is in use in highly diverse industries including health care, defence, insurance and e-commerce. This review focuses on the relevance of AI to surgery. AI will aid surgeons with diagnostic decision-making, patient selection for surgery as well as improve patient pre- and post-operative care and management. Ethical considerations of AI with respect to patient rights and data privacy are highlighted. A further challenge is how best to present to national regulators a pragmatic way to assess AI as 'software as a medical device'. This relates to the ramifications for the adoption of AI technology in clinical practice, and its subsequent public funding support and reimbursement. It is evident that AI technology has important applications in surgery in the 21st century. The establishment of a key work programme in this area will be important if surgeons are to fully utilize AI in surgery.

Safe surgery during the coronavirus disease 2019 crisis.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has created a global pandemic. Surgical care has been impacted, with concerns raised around surgical safety, especially in terms of laparoscopic versus open surgery. Due to potential aerosol transmission of SARS-CoV-2, precautions during aerosol-generating procedures and production of surgical plume are paramount for the safety of surgical teams. METHODS: A rapid review methodology was used with evidence sourced from PubMed, Departments of Health, surgical colleges and other health authorities. From this, a working group of expert surgeons developed recommendations for surgical safety in the current environment. RESULTS: Pre-operative testing of surgical patients with reverse transcription-polymerase chain reaction does not guarantee lack of infectivity due to a demonstrated false-negative rate of up to 30%. All bodily tissues and fluids should therefore be treated as a potential source of COVID-19 infection during operative management. Caution must be taken, especially when using an energy source that produces surgical plumes, and an appropriate capture device should also be used. Limiting the use of such devices or using lower energy devices is desirable. To reduce perceived risks association with desufflation of pneumoperitoneum during laparoscopic surgery, an appropriate suction irrigator system, attached to a high-efficiency particulate air filter, should be used. Additionally, appropriate use of personal protective equipment by the surgical team is necessary during high-risk aerosol-generating procedures. CONCLUSIONS: As a result of the rapid review, evidence-based guidance has been produced to support safe surgical practice.

Personal protective equipment and evidence-based advice for surgical departments during COVID-19.

BACKGROUND: Inconsistencies regarding the use of appropriate personal protective equipment (PPE) have raised concerns for the safety of surgical staff during the coronavirus disease 2019 (COVID-19) pandemic. This rapid review synthesizes the literature and includes input from clinical experts to provide evidence-based guidance for surgical services. METHODS: The rapid review comprised of targeted searches in PubMed and grey literature. Pertinent findings were discussed by a working group of clinical experts, and consensus recommendations, consistent with Australian and New Zealand Government guidelines, were formulated. RESULTS: There was a paucity of high-quality primary studies specifically investigating appropriate surgical PPE for healthcare workers treating patients possibly infected with COVID-19. SARS-CoV-2 is capable of aerosol, droplet and fomite transmission, making it essential to augment standard infection control measures with appropriate PPE, especially during surgical emergencies and aerosol-generating procedures. All biological material should be treated a potential source of SARS-COV-2. Staff must have formal training in the use of PPE and should be supervised by a colleague during donning and doffing. Patients with suspected or confirmed COVID-19 should wear a surgical mask during transfer to and from theatre. Potential solutions exist in the literature to extend the use of surgical P2/N95 respirators in situations of limited supply. CONCLUSION: PPE is advised for all high-risk procedures and when a patient's COVID-19 status is unknown. Surgical departments should facilitate staggered rostering, remote meeting attendance, and self-isolation of symptomatic staff. Vulnerable surgical staff should be identified and excluded from operations with a high risk of COVID-19 infection.

Screening and testing for COVID-19 before surgery.

BACKGROUND: Preoperative screening for coronavirus disease 2019 (COVID-19) aims to preserve surgical safety for both patients and surgical teams. This rapid review provides an evaluation of current evidence with input from clinical experts to produce guidance for screening for active COVID-19 in a low prevalence setting. METHODS: An initial search of PubMed (until 6 May 2020) was combined with targeted searches of both PubMed and Google Scholar until 1 July 2020. Findings were streamlined for clinical relevance through the advice of an expert working group that included seven senior surgeons and a senior medical virologist. RESULTS: Patient history should be examined for potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hyposmia and hypogeusia may present as early symptoms of COVID-19, and can potentially discriminate from other influenza-like illnesses. Reverse transcription-polymerase chain reaction is the gold standard diagnostic test to confirm SARS-CoV-2 infection, and although sensitivity can be improved with repeated testing, the decision to retest should incorporate clinical history and the local supply of diagnostic resources. At present, routine serological testing has little utility for diagnosing acute infection. To appropriately conduct preoperative testing, the temporal dynamics of SARS-CoV-2 must be considered. Relative to other thoracic imaging modalities, computed tomography has the greatest utility for characterizing pulmonary involvement in COVID-19 patients who have been diagnosed by reverse transcription-polymerase chain reaction. CONCLUSION: Through a rapid review of the literature and advice from a clinical expert working group, evidence-based recommendations have been produced for the preoperative screening of surgical patients with suspected COVID-19.

Effect of nerve growth factor and keratinocyte growth factor on wound healing of the sinus mucosa.

The healing of the sinuses after sinus surgery is often compromised by the development of adhesions. The aim of this study was to determine whether nerve growth factor (NGF) and keratinocyte growth factor (KGF) aid epithelial and fibroblast wound healing after surgery. Two in vitro models were used to compare their effect on wound closure rates and expression of cell adhesion (E-cadherin), tight junction formation (zona occludens-1), cell proliferation (proliferative cell nuclear antigen and Ki67), and ciliogenesis (Foxj1 and beta tubulin IVb) genes by real-time reverse transcription polymerase chain reaction. Epithelial cells from sinonasal tissue were seeded in collagen-coated Transwells, creating an air-liquid interface, and a submergent model was used for fibroblasts. In epithelial cells, NGF (20-50 ng/mL) significantly decreased wound areas to <40% compared with controls that were still 70% of their original sizes by 24 hours (p<0.05). E-cadherin and zona occludens-1 expression were up-regulated by 20- and 2.5-fold, respectively, relative to controls. KGF (5-100 ng/mL) slowed fibroblast proliferation by reducing (0.8-fold) and Ki67 (0.25-fold) expression. NGF (10 ng/mL) slowed Ki67 expression by 0.5-fold. NGF accelerated in vitro epithelial wound closure while NGF and KGF reduced fibroblast proliferation.

Response of peripheral blood lymphocytes to fungal extracts and staphylococcal superantigen B in chronic rhinosinusitis.

BACKGROUND: Previous studies have suggested that chronic rhinosinusitis may result from a hypersensitivity response of the nasal mucosa to the presence of fungal antigens or staphylococcal superantigens in the nasal mucus. Both of these groups of antigens are present so frequently in the nasal mucus of patients with chronic rhinosinusitis that their presence together is likely to be a common event. OBJECTIVE: The objective of this study was to determine whether the combined presence of fungal antigens and staphylococcal superantigens exert a synergistic proinflammatory effect on peripheral blood lymphocytes from patients with chronic rhinosinusitis. METHODS: Peripheral blood lymphocytes were extracted from patients with chronic rhinosinusitis with and without nasal polyposis (n = 7 for both groups) and normal controls (n = 7). These cells were cultured for 48 hours after the addition of fungal extracts (Aspergillus and Alternaria), staphylococcal superantigen type B (SEB), or a combination of these two antigens. Real-time polymerase chain reaction was used to determine the level transcription of interleukinL-5 and interferon-gamma genes. RESULTS: Fungal extracts alone resulted in minimal changes in the levels of cytokine expression in peripheral blood lymphocytes. SEB increased the expression of IFN-gamma, and this effect was magnified by the addition of SEB and fungal extracts together to the culture medium. There were no differences in the magnitude of responses seen in patients with and without polyps nor between patients with chronic rhinosinusitis and normal controls. CONCLUSION: SEB exerts a powerful proinflammatory effect on peripheral blood lymphocytes and fungal extracts may act synergistically to promote this action.

The use of chitosan-dextran gel shows anti-inflammatory, antibiofilm, and antiproliferative properties in fibroblast cell culture.

BACKGROUND: Chitosan-dextran gel has been used as an antihemostatic agent and antiadhesive agent after endoscopic sinus surgery. Because Staphylococcus aureus biofilms have been implicated in recalcitrant chronic rhinosinusitis, this study aimed to further investigate the (i) anti-inflammatory, (ii) bacterial biofilm inhibition, (iii) antiproliferative effects, and (iv) wound-healing properties of chitosan and chitosan-dextran gel. METHODS: Fibroblasts were isolated from human nasal tissue and were used to determine the effects of chitosan and chitosan-dextran gel on (i) cell proliferation, (ii) wound healing, (iii) inflammation in fibroblast cultures challenged with superantigens S. aureus enterotoxin B (SEB) and toxic shock syndrome toxin (TSST), and (iv) on S. aureus biofilms. RESULTS: Chitosan was highly effective at reducing IL-8 expression after TSST and SEB challenge. Chitosan was also effective at reducing IL-8 expression of nonchallenged fibroblasts showing its anti-inflammatory effects on fibroblasts in a diseased state. Chitosan-dextran gel showed strong antibiofilm properties at 50% (v/v) concentration in vitro. Dextran, on its own, showed antibiofilm properties at 1.25% (w/v) concentration. Chitosan, on its own, reduced proliferation of fibroblasts to 82% of control proliferation and chitosan-dextran gel reduced proliferation of the fibroblasts to 0.04% of control proliferation. Relative to the no treatment controls, chitosan-dextran gel significantly delayed the wound-healing rate over the first 48 hours of the experiment. CONCLUSION: Chitosan-dextran gel reduced fibroblast proliferation and wound-healing time, showing a possible mechanism of reducing adhesions in the postsurgical period. Chitosan reduced IL-8 levels, showing its anti-inflammatory properties. Chitosan-dextran gel and dextran treatment showed antibiofilm properties in our model.

Noninvasive Staphylococcus aureus biofilm determination in chronic rhinosinusitis by detecting the exopolysaccharide matrix component poly-N-acetylglucosamine.

BACKGROUND: The role that bacterial biofilms might play in recalcitrant forms of chronic rhinosinusitis (CRS) is increasingly being recognized. However, the detection of bacteria existing in this form, using standard culture, is limited by their unique metabolically inactive properties. All current biofilm diagnostic modalities require invasive mucosal biopsies, which limit their use to the operating theatre. METHODS: Twenty CRS patients and 5 controls were enrolled in a prospective study to assess the feasibility of noninvasively diagnosing S. aureus biofilms by detecting the biofilm matrix polysaccharide poly-N-acetylglucosamine (PNAG). An immunofluorescence protocol was developed for PNAG detection and compared with both standard microbiological cultures and fluorescence in situ hybridization (FISH). RESULTS: Thirteen of 20 CRS patients had evidence of S. aureus biofilm formation using FISH. Of these, 12 had detectable PNAG. Interestingly none of the S. aureus FISH-negative patients were PNAG-positive despite the presence of coagulase-negative Staphylococci biofilms, some of which may exhibit PNAG in their pathogenic forms. The development of a noninvasive S. aureus biofilm diagnostic test provides a reliable means to identify a high-risk group of CRS patients who harbor S. aureus biofilms. The ability to be used outside of the perioperative period to assess surgical efficacy, guide management, and evaluate new treatment modalities provides a significant advance in this field of research and clinical practice. CONCLUSION: This study has confirmed the feasibility of noninvasive detection of S. aureus biofilms with a simple test that produces results comparable to the more invasive methods that are currently relied upon.

Bacterial-induced epithelial damage promotes fungal biofilm formation in a sheep model of sinusitis.

BACKGROUND: Fungal biofilms have been discovered in chronic rhinosinusitis (CRS) patients, but factors contributing to their establishment are obscure. A recent animal study showed bacterial co-inoculation was required. We examine the role of 4 bacterial species and a cilia toxin on fungal biofilm formation in a sheep sinusitis model. The importance of epithelial integrity on fungal biofilm formation is also examined. METHODS: Forty-eight frontal sinuses were inoculated with Aspergillus fumigatus alone, with 1 of 4 bacteria, or a cilia toxin. Bacterial and fungal biofilm was determined using confocal scanning laser microscopy. Inflammation and cilia integrity were assessed using light microscopy and transmission electron microscopy, respectively. RESULTS: No fungal biofilm formed when inoculated alone. Florid fungal biofilm developed in more than 75% of sinuses associated with bacterial biofilm of all species, except Haemophilus influenzae, which failed to establish bacterial biofilm. Fungal biofilm also established in association with cilia toxin. Significant cilial damage was incited by all bacterial biofilms and cilia toxin, and was associated with fungal proliferation. Fungal biofilm formation did not significantly increase mucosal inflammation or epithelial damage over that caused by the bacteria or cilia toxin alone. CONCLUSION: Bacterial biofilms cause sinonasal mucosal inflammation and epithelial injury, which provides conditions appropriate for fungal biofilm proliferation. The role of cilia in sinonasal mucosal defense against fungal organisms has been demonstrated. Without such an insult, fungal biofilms fail to proliferate in occluded sinuses. Improving cilial recovery postoperatively and treating bacterial biofilms may be key factors in reducing recalcitrance in allergic fungal rhinosinusitis patients.

Fungicidal activity of lysozyme is inhibited in vitro by commercial sinus irrigation solutions.

BACKGROUND: Lysozyme is an innate immune peptide with bactericidal and fungicidal activity (FA). Despite increased expression of lysozyme protein in chronic rhinosinusitis (CRS) sinus mucosa, CRS patients experience repeated bacterial and/or fungal infections. Commercial sinus irrigation solutions are often used to provide symptomatic relief. However, one of the mechanisms of action of lysozyme involves ionic interactions with the microbial cell wall, which may be inhibited by ionic solutions such as commercial sinus irrigation solutions. OBJECTIVE: Determine if the FA of lysozyme is reduced in the presence of solutions with increasing ionic strength and inhibited in the presence of commercial sinus irrigation solutions. METHODS: Using an in vitro colony-forming unit (CFU) assay, the FA of lysozyme (5 µM) was tested against a fungi commonly isolated from CRS patients, Aspergillus fumigatus, in solutions of increasing ionic strength or commercial sinus irrigation solutions. FA was presented as percent of control. RESULTS: FA of lysozyme against A. fumigatus was 95% in a 21-mM ionic strength solution. However, with increasing ionic strength, FA decreased and was abolished in a 46-mM ionic strength solution. Commercial sinus irrigation solutions abolished the FA of lysozyme against A. fumigatus. CONCLUSION: The in vitro FA of lysozyme is dependent on the ionic strength of the solution. The use of sinus irrigation solutions should be further evaluated with regard to maintaining functional activity of cationic antimicrobial peptides involved in sinonasal innate immunity.

Aspergillus fumigatus biofilm on primary human sinonasal epithelial culture.

BACKGROUND: Bacterial biofilms have been implicated in chronic rhinosinusitis (CRS). However, direct evidence in support of fungal biofilms in sinus disease is lacking in the literature. This study was designed to develop and characterize an in vitro Aspergillus fumigatus biofilm model on primary human sinonasal epithelial cell culture. METHODS: Sinonasal biopsy specimens harvested during endoscopic sinus surgery of six CRS patients and three pituitary tumor (control) patients were cultured in Dulbecco's modified Eagle media (DMEM; Invitrogen)/Hams F12 airway media to encourage epithelial cell proliferation. Epithelial cells separated by immunomagnetic beads were seeded in tissue culture-treated Y-shaped microslides. At confluence the primary cultures were inoculated with A. fumigatus spores. Fungus was allowed to germinate and form biofilms under two in vitro conditions: (1) static (no flow through of media) and (2) continuous flow coculture (continuous flow movement of media). At regular intervals cocultures were stained with FUN-1, concanavalin A-alexa fluor 488, and examined by confocal scanning laser microscopy. Comstat software was used to assess biomass and thickness. RESULTS: A. fumigatus formed three-dimensional biofilm structures with parallel-packed, cross-linked hyphae and channels/passages. Metabolically active hyphae showed orange-red fluorescing intravacuolar structures. Extracellular matrix (ECM) between/around the hyphae fluoresced intense green. A. fumigatus biofilms development occurred in five stages: (1) conidial attachment to epithelial cells, (2) hyphal proliferation, (3) ECM production, (4) hyphal parallel packing and cross-linking, and (5) channel/pores formation. Mature biofilms showed basal conidial, middle hyphal, and superficial ECM layers. Biofilms formed under flow conditions displayed more robust and faster growth kinetics when compared with that under static conditions, with a thick, stocky, wrinkly/undulating hyphal growth and extensive ECM production. The differences in biomass and average thickness of the cocultures under static and flow conditions were statistically significant after similar periods of incubation (p = 0.0002; p < 0.0001, respectively). CONCLUSION: To our knowledge, this is the first article of an in vitro model characterizing A. fumigatus biofilm formation using primary human sinonasal epithelium under different growth conditions.

Methylglyoxal-infused honey mimics the anti-Staphylococcus aureus biofilm activity of manuka honey: potential implication in chronic rhinosinusitis.

OBJECTIVES/HYPOTHESIS: Low pH, hydrogen peroxide generation, and the hyperosmolarity mechanisms of antimicrobial action are ubiquitous for all honeys. In addition, manuka honey has been shown to contain high concentrations of methylglyoxal (MGO), contributing the relatively superior antimicrobial activity of manuka honey compared to non-MGO honeys. In high concentrations, manuka honey is effective in killing Staphylococcus aureus biofilms in vitro. Lower concentrations of honey, however, are desirable for clinical use as a topical rinse in chronic rhinosinusitis in order to maximize the tolerability and practicality of the delivery technique. This study, therefore, was designed to evaluate the contribution of MGO to the biofilm-cidal activity of manuka honey, and furthermore determine whether the antibiofilm activity of low-dose honey can be augmented by the addition of exogenous MGO. STUDY DESIGN: In vitro microbiology experiment. METHODS: Five S. aureus strains (four clinical isolates and one reference strain) were incubated to form biofilms using a previously established in vitro dynamic peg model. First, the biofilm-cidal activities of 1) manuka honey (790 mg/kg MGO), 2) non-MGO honey supplemented with 790 mg/kg MGO, and 3) MGO-only solutions were assessed. Second, the experiment was repeated using honey solutions supplemented with sufficient MGO to achieve concentrations exceeding those seen in commercially available manuka honey preparations. RESULTS: All honey solutions containing a MGO concentration of 0.53 mg/mL or greater demonstrated biofilm-cidal activity; equivalent activity was achieved with ≥1.05 mg/mL MGO solution. CONCLUSIONS: MGO is only partially responsible for the antibiofilm activity of manuka honey. Infusion of MGO-negative honey with MGO, however, achieves similar cidality to the equivalent MGO-rich manuka honey.