The pulmonary microbiome: recent advances in understanding its role in chronic lung allograft dysfunction.

PURPOSE OF REVIEW: Lung transplantation presents a rescue therapy for those with end-stage lung disease. Survival in lung transplant patients remains limited due to chronic lung allograft dysfunction (CLAD), a range of pathologic manifestations leading to graft loss. The mechanisms underlying CLAD remain poorly understood, and the lung microbiome has been suggested as a potential contributor to this condition. This review aims to explore how the pulmonary microbiome is impacted by lung transplantation, and how alterations in this microbiome may contribute to the pathogenesis of CLAD. RECENT FINDINGS: The pulmonary microbiome is made up of a range of microorganisms, and it varies considerably in lung transplant patients when compared with healthy controls. The lung microbiome changes over the early transplant period, and the composition of species appears to have an impact on inflammatory responses within the lungs. A number of studies have shown that an increase in bacterial biomass in the allograft, and enrichment with the genera Proteobacteria, or more specifically, Pseudomonas species, is associated with CLAD. SUMMARY: This area of research is still in its infancy; however, the suggestion that changes in the composition of the microbiome and enrichment with certain species may predispose to the pathologic changes that underlie CLAD indicate that modulation of the microbiome may be of use in potential future therapeutics.

An Alternative to Traditional Bedside Teaching During COVID-19: High-Fidelity Simulation-Based Study.

BACKGROUND: Bedside teaching is integral to medical education and has been highlighted to improve clinical and communication skills, as well as clinical reasoning. Despite the significant advantages of bedside teaching, its usage within medical education has been declining, and COVID-19 has added additional challenges. The pandemic has resulted in a significant reduction in opportunities to deliver bedside teaching due to risk of viral exposure, patients declining student interactions, and ward closures. Educators have therefore been required to be innovative in their teaching methods, leading to the use of online learning, social media platforms, and simulation. Simulation-based education allows for learning in a low-risk environment and affords the opportunity for deliberated repeated practice with case standardization. The results demonstrate that simulation-based training can increase students' confidence, increase the rates of correct clinical diagnoses, and improve retention of skills and knowledge when compared with traditional teaching methods. OBJECTIVE: To mitigate the impact of COVID-19 upon bedside teaching for third year students at Hull York Medical School amid closure of the cardiorespiratory wards, a high-fidelity simulation-based model of traditional bedside teaching was designed and implemented. The objectives of the teaching session were to enable students to perform history taking and a focused cardiorespiratory clinical examination in a COVID-19-safe environment using SimMan 3G. METHODS: Four clinical teaching fellows with experience of simulation-based medical education scripted histories for 2 common cardiorespiratory cases, which were asthma and aortic stenosis. The simulation sessions were designed for students to take a focused cardiorespiratory history and clinical examination using SimMan 3G. All cases involved dynamic vital signs, and the simulator allowed for auscultation of an ejection systolic murmur and wheezing in accordance with the cases chosen. Key aspects of the pathologies, including epidemiology, differential diagnoses, investigations, and management, were summarized using an interactive PowerPoint presentation, followed by a debriefing session. RESULTS: In total, 12 third year medical students undertook the sessions, and overall feedback was highly positive. Of the 10 students who completed the feedback questionnaires, 90% (n=9) felt more confident in their clinical examination skills following the teaching; 100% (n=10) of the students responded that they would recommend the session to a colleague; and implementation of regular simulation was frequently requested on feedback. These results are in keeping with the current literature. CONCLUSIONS: Bedside teaching continues to face ongoing challenges from the COVID-19 pandemic as well as declining patient recruitment and fluctuations in clinical findings. The support for simulation-based medical education is derived from high-quality studies; however, studies describing the use of this technology for bedside teaching in the undergraduate curriculum are limited. The authors describe a highly effective teaching session amid the pandemic, which allowed for maintenance of staff and student safety alongside continued education during a challenging time for educators globally.

High-resolution Metatranscriptomic Characterization of the Pulmonary RNA Virome After Lung Transplantation.

BACKGROUND: Lung transplantation provides a unique opportunity to investigate the constituents and temporal dynamics of the human pulmonary microbiome after lung transplantation. For methodological reasons, prior studies using metagenomics have detected DNA viruses but not demonstrated the presence of RNA viruses, including those that are common community acquired. In this proof-of-concept study, we aimed to further characterize the pulmonary microbiome after lung transplantation by using metagenomic next-generation sequencing (mNGS), with a particular focus on the RNA virome. METHODS: We performed a single-center longitudinal study of lower respiratory tract RNA viruses and bacteria using bronchoalveolar lavage at postoperative day 1 and week 6 analyzed with total RNA sequencing (metatranscriptomics). Five primary and 5 repeat transplant recipients were recruited. RESULTS: mNGS identified 5 RNA viruses (nil in the normal saline control), including 4 species of human rhinovirus not previously reported in Australia: A7 (HRV-A7), C22 (HRV-C22), B52 (HRV-B52), and B72 (HRV-B72). Overall, 12/20 specimens were virus positive in 7/10 cases. Human parainfluenza virus 3 was the most frequent virus in 7/20 specimens in 5/10 cases. In this small study, we did not detect a significant difference in abundance and diversity of RNA viruses and bacteria at postoperative day 1 and 6 wk, nor differences between retransplant recipients and primary lung transplant recipients. CONCLUSIONS: Our study demonstrates how mNGS can also identify RNA viruses within the human pulmonary virome, including novel RNA viruses, and paves the way for a greater understanding of the complex relationships among the constituents of the pulmonary infectome.

The Impact of Resistant Bacterial Pathogens including Pseudomonas aeruginosa and Burkholderia on Lung Transplant Outcomes.

Pseudomonas and Burkholderia are gram-negative organisms that achieve colonization within the lungs of patients with cystic fibrosis, and are associated with accelerated pulmonary function decline. Multidrug resistance is a hallmark of these organisms, which makes eradication efforts difficult. Furthermore, the literature has outlined increased morbidity and mortality for lung transplant (LTx) recipients infected with these bacterial genera. Indeed, many treatment centers have considered Burkholderia cepacia infection an absolute contraindication to LTx. Ongoing research has delineated different species within the B. cepacia complex (BCC), with significantly varied morbidity and survival profiles. This review considers the current evidence for LTx outcomes between the different subspecies encompassed within these genera as well as prophylactic and management options. The availability of meta-genomic tools will make differentiation between species within these groups easier in the future, and will allow more evidence-based decisions to be made regarding suitability of candidates colonized with these resistant bacteria for LTx. This review suggests that based on the current evidence, not all species of BCC should be considered contraindications to LTx, going forward.

Lung transplantation: a review of the optimal strategies for referral and patient selection.

One of the great challenges of lung transplantation is to bridge the dichotomy between supply and demand of donor organs so that the maximum number of potential recipients achieve a meaningful benefit in improvements in survival and quality of life. To achieve this laudable goal is predicated on choosing candidates who are sufficiently unwell, in fact possessing a terminal respiratory illness, but otherwise fit and able to undergo major surgery and a prolonged recuperation and rehabilitation stage combined with ongoing adherence to complex medical therapies. The choice of potential candidate and the timing of that referral is at times perhaps more art than science, but there are a number of solid guidelines for specific illnesses to assist the interested clinician. In this regard, the relationship between the referring clinician and the lung transplant unit is a critical one. It is an ongoing and dynamic process of education and two way communication, which is a marker of the professionalism of a highly performing unit. Lung transplantation is ultimately a team effort where the recipient is the key player. That principle has been enshrined in the three consensus position statements regarding selection criteria for lung and heart-lung transplantation promulgated by the International Society for Heart and Lung Transplantation over the last two decades. During this period, the number of indications for lung transplantation have broadened and the number of contraindications reduced. Risk management is paramount in the pre- and perioperative period to effect early successful outcomes. While it is not the province of this review to reiterate the detailed listing of those factors, an overview position will be developed that describes the rationale and evidence for selected criteria where that exists. Importantly, the authors will attempt to provide an historical and experiential basis for making these important and life-determining decisions. The reviews of this paper are available via the supplementary material section.

The lung microbiome and transplantation.

PURPOSE OF REVIEW: Lung transplantation survival is still limited compared with other solid organ transplant modalities, due to a range of factors that are continuing to be elucidated. However, new research is emerging which indicates that the microbiome of the lungs, and of other organs, may have important implications for immune response and mediating transplant outcomes. Pathogenic bacterial and viral species are known to have deleterious effects on the allograft, but taking a more global view, and considering the overall composition of the community of microbial species may undercover a greater understanding of the complex interplay involved in allograft dysfunction. RECENT FINDINGS: The microbiome appears to have an important modulatory role on immune response in both normal development, and after transplantation. A range of microbial species contributes to the resident microscopic community, with the gut and blood microbiomes having a significant impact on the function of the lungs and resistance to infection. Movement of species from within and outside the respiratory tract occurs in the early transplant period, leading to a modified donor microbiome in the recipient. There is evidence to suggest that chronic lung allograft dysfunction may be facilitated by the respiratory microbiome and interactions with immune cells within the allograft. SUMMARY: Further investigation of the respiratory microbiome, interactions with the microbiome of other organs and impact on immune and allograft function posttransplantation is needed. Promising insights are being gained regarding certain microbial profiles which may be associated with negative outcomes, and the mechanisms through which this occurs. As our understanding expands, the ability to modify the microbiome offers novel treatment strategies for combating allograft dysfunction.

Infectious Disease Transmission in Solid Organ Transplantation: Donor Evaluation, Recipient Risk, and Outcomes of Transmission.

In 2016, the Transplantation Society of Australia and New Zealand, with the support of the Australian Government Organ and Tissue authority, commissioned a literature review on the topic of infectious disease transmission from deceased donors to recipients of solid organ transplants. The purpose of this review was to synthesize evidence on transmission risks, diagnostic test characteristics, and recipient management to inform best-practice clinical guidelines. The final review, presented as a special supplement in Transplantation Direct, collates case reports of transmission events and other peer-reviewed literature, and summarizes current (as of June 2017) international guidelines on donor screening and recipient management. Of particular interest at the time of writing was how to maximize utilization of donors at increased risk for transmission of human immunodeficiency virus, hepatitis C virus, and hepatitis B virus, given the recent developments, including the availability of direct-acting antivirals for hepatitis C virus and improvements in donor screening technologies. The review also covers emerging risks associated with recent epidemics (eg, Zika virus) and the risk of transmission of nonendemic pathogens related to donor travel history or country of origin. Lastly, the implications for recipient consent of expanded utilization of donors at increased risk of blood-borne viral disease transmission are considered.

Transplanting the pulmonary virome: Dynamics of transient populations.

BACKGROUND: Lung transplantation provides a unique opportunity to investigate the dynamics of the human pulmonary virome that is transplanted within the donor lungs. The pulmonary virome comprises both "resident" and "transient" viruses. In this study we aimed to analyze the dynamics of the "transient" members. METHODS: We conducted a single-center, prospective, longitudinal investigation of community-acquired respiratory viruses detected in nasopharyngeal swabs, swabs of explanted and donor lungs, and serial bronchoalveolar lavages post-transplant. RESULTS: Fifty-two consecutive lung transplant recipients were recruited (bilateral:heart‒lung:bilateral lung-liver = 48:2:2) (age [mean ± SD] 48 ± 15 years, range 20 to 63 years; 27 males and 25 females). Follow-up was 344 ± 120 (range 186 to 534) days. Seventeen of 45 explanted lungs were positive for influenza A and/or B (A = 14, B = 2, A+B = 1), despite recipient vaccination and negative nasal swabs, and 4 of 45 had human rhinovirus and 2 of 45 parainfluenza. Donor swabs showed influenza (A = 1, B = 1) and rhinovirus (n = 3). Day 1 lavage showed influenza A (n = 28), rhinovirus (n = 9), and parainfluenza (n = 1). Forty-seven of 52 recipients had a positive lavage for virus (38 of 47 on multiple lavages). Influenza persisted for 59 ± 38 (range 4 to 147) days in 27 of 52, and 14 had a single isolate. Rhinovirus persisted for 95 ± 84 (range 22 to 174) days in 13 of 52, and 13 had a single isolate. Analysis of 118 paired transbronchial biopsies and lavage demonstrated no association between viruses and acute cellular rejection (Fisher's exact test, 2 tailed, p = 1.00). CONCLUSIONS: Using a sensitive uniplex polymerase chain reaction we found that the transplanted pulmonary virome often includes community-acquired respiratory viruses, including influenza, which are variably persistent but not associated with acute rejection.

Introduction to Techniques and Methodologies for Characterizing the Human Respiratory Virome.

There have been great advances in the methodologies available for the detection of respiratory viruses. Accompanying this, our knowledge surrounding the impact of these viruses has also made a great leap forward. We have come a long way from the once commonly accepted belief that the lower respiratory tract was sterile and that the detection of any microbial species must represent a breach in host defence and likely be associated with symptomatic infection. With the advent of molecular detection techniques and improvements in sequencing-based methodologies to make these tools more accessible and cost effective, we now know that there is an abundant and diverse ecosystem within the lower-respiratory tract. This chapter will outline the clinical impact of the human respiratory virome, techniques for sampling the lower respiratory tract, the evolution of the diagnostic tools available, and the current limitations in our instruments and knowledge in this area. The human respiratory virome is an exciting new area of research that will continue to grow with the aid of the methodologies outlined in the following chapters and the advent of even more efficient tools in the future.

Viruses in bronchiectasis: a pilot study to explore the presence of community acquired respiratory viruses in stable patients and during acute exacerbations.

BACKGROUND: Bronchiectasis is a chronic respiratory condition. Persistent bacterial colonisation in the stable state with increased and sometimes altered bacterial burden during exacerbations are accepted as key features in the pathophysiology. The extent to which respiratory viruses are present during stable periods and in exacerbations is less well understood. METHODS: This study aimed to determine the incidence of respiratory viruses within a cohort of bronchiectasis patients with acute exacerbations at a teaching hospital and, separately, in a group of patients with stable bronchiectasis. In the group of stable patients, a panel of respiratory viruses were assayed for using real time quantitative PCR in respiratory secretions and exhaled breath. The Impact of virus detection on exacerbation rates and development of symptomatic infection was evaluated. RESULTS: Routine hospital-based viral PCR testing was only requested in 28% of admissions for an exacerbation. In our cohort of stable bronchiectasis patients, viruses were detected in 92% of patients during the winter season, and 33% of patients during the summer season. In the 2-month follow up period, 2 of 27 patients presented with an exacerbation. CONCLUSIONS: This pilot study demonstrated that respiratory viruses are commonly detected in patients with stable bronchiectasis. They are frequently detected during asymptomatic viral periods, and multiple viruses are often present concurrently.

A novel sampling method to detect airborne influenza and other respiratory viruses in mechanically ventilated patients: a feasibility study.

BACKGROUND: Respiratory viruses circulate constantly in the ambient air. The risk of opportunistic infection from these viruses can be increased in mechanically ventilated patients. The present study evaluates the feasibility of detecting airborne respiratory viruses in mechanically ventilated patients using a novel sample collection method involving ventilator filters. METHODS: We collected inspiratory and expiratory filters from the ventilator circuits of mechanically ventilated patients in an intensive care unit over a 14-month period. To evaluate whether we could detect respiratory viruses collected in these filters, we performed a reverse transcription polymerase chain reaction on the extracted filter membrane with primers specific for rhinovirus, respiratory syncytial virus, influenza virus A and B, parainfluenza virus (type 1, 2 and 3) and human metapneumovirus. For each patient, we also performed a full virology screen (virus particles, antibody titres and virus-induced biomarkers) on respiratory samples (nasopharyngeal swab, tracheal aspirate or bronchoalveolar fluid) and blood samples. RESULTS: Respiratory viruses were detected in the ventilator filters of nearly half the patients in the study cohort (n = 33/70). The most common virus detected was influenza A virus (n = 29). There were more viruses detected in the inspiratory filters (n = 18) than in the expiratory filters (n = 15). A third of the patients with a positive virus detection in the ventilator filters had a hospital laboratory confirmed viral infection. In the remaining cases, the detected viruses were different from viruses already identified in the same patient, suggesting that these additional viruses come from the ambient air or from cross-contamination (staff or visitors). In patients in whom new viruses were detected in the ventilator filters, there was no evidence of clinical signs of an active viral infection. Additionally, the levels of virus-induced biomarker in these patients were not statistically different from those of non-infected patients (p = 0.33). CONCLUSIONS: Respiratory viruses were present within the ventilator circuits of patients receiving mechanical ventilation. Although no adverse clinical effect was evident in these patients, further studies are warranted, given the small sample size of the study and the recognition that ventilated patients are potentially susceptible to opportunistic infection from airborne respiratory viruses.

The Human Respiratory Microbiome: Implications and Impact.

Once considered a sterile site below the larynx, the tracheobronchial tree and parenchyma of the lungs are now known to harbor a rich diversity of microbial species including bacteria, viruses, fungi, and archaea. Many of these organisms, particularly the viruses which comprise the human respiratory virome, have not been identified, so their true role is unknown. It seems logical to conclude that a "healthy" respiratory microbiome exists which may be modified in disease states and perhaps by therapies such as antibiotics, antifungals, and antiviral treatments. It is likely that there is a critical relationship or equilibrium between components of the microbiome until such time as perturbations occur which lead to a state of dysbiosis or an "unhealthy" microbiome. The act of lung transplantation provides an extreme change to an individual's respiratory microbiome as, in effect, the donor respiratory microbiome is transplanted into the recipient. The mandatory ex-vivo period of the donor lungs appears to be associated with blooms of resident viral species in particular. Subsequently, allograft injury, rejection, and immune suppressive therapy all combine to create periods of dysbiosis which when combined with transient infections such as community acquired respiratory viruses may facilitate the development of chronic allograft dysfunction in predisposed individuals. As our understanding of the respiratory microbiome is rapidly expanding, based on the use of new-generation sequencing tools in particular, it is to be hoped that insights gained into the subtle relationship between the microbiome and the lung allograft will facilitate improved outcomes by directing novel therapeutic endeavors.