Pleural infection is usually defined using pleural fluid biochemical characteristics, given that only ~30% of cases are culture positive, but the relationship between these characteristics and pleural space bacterial concentration is unclear. We developed an assay to estimate bacterial ‘load’ using quantitative polymerase chain reaction (PCR) to determine 16S rRNA gene copy number in pleural fluid samples (this gene is present in all bacteria). This enabled us to explore the relationship between patient characteristics and pleural fluid bacterial ‘load’.
Methods
Pleural infection samples were obtained from the Second Multicentre Intrapleural Sepsis randomised controlled Trial (MIST2), REC no. 04/MRE5/53. DNA was extracted using the FastDNA SPIN Kit. Quantitative PCR (qPCR) of the 16S rRNA gene was undertaken using the ultra-pure Power SYBR Green PCR reagent and primers that amplified the 467 nt V3–4 region of the 16S rRNA gene. A 3-step thermal cycling profile was empirically determined to give optimal results. Ten-fold dilutions of Acidothermus cellulolyticus DNA were used to estimate sample 16S rRNA gene concentration. All PCRs were performed in duplicate. Melt-curve analyses and agarose gel electrophoresis of qPCR amplicons were used to ensure absence of non-specific PCR products.
Results
172 pleural fluid samples were analysed. Pleural fluid pH, culture status, appearance, LDH and glucose were all predictive of bacterial load (see Table). Patient C-reactive protein (CRP) and white cell count (WCC) were not significantly associated with bacterial load.
Conclusions
Bacterial ‘load’ was associated with acknowledged predictors for pleural infection. Such findings add further support to the utility of pH, glucose and LDH values as proxies for pleural infection, in the correct clinical context. Patient WCC and CRP were not significantly associated with bacterial ‘load’. This assay is limited in that it assesses total bacterial DNA (from viable and dead bacteria), rather than quantifying viable bacteria. Further, bacteria vary in their copy number of the 16S rRNA gene, dependent on species. Despite these limitations, our associations have reached a strong level of significance.
Haemophilus influenzae is emerging as the commonest pathogenic microorganism isolated from severe asthmatic airways; it is associated with sputum neutrophilia and may confer steroid resistance. Previous metagenomics studies in asthma were limited by lack of consistent clinical phenotyping and inadequate sequencing depth for species-level bacterial identification. We hypothesise that chronic bacterial infection constitutes a 'treatable trait' in non-eosinophilic severe asthma but its prevalence, clinical phenotype and reliable biomarkers need to be defined. Non-typeable Haemophilus influenzae strains (NTHi) can persist within the epithelium and cause episodic airways infections. Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes which protect against pulmonary bacterial infection, and respond to NTHi in the presence of professional antigen presenting cells (APCs) via MR1, a molecule expressed ubiquitously in many cell types including epithelial cells. It is not known whether NTHi-infected epithelial cells directly activate MAIT cells.
Aims
(1) Identify and characterise the sub-phenotype of severe asthmatics with chronic bacterial airways infection using modern molecular microbiological techniques (2) Determine whether MAIT cells directly respond to intra-epithelial infection with NTHi
Methods
Analysis of induced sputum samples from stable well-phenotyped severe asthmatics using culture, MALDI-TOF, RT-qPCR and metagenomic sequencing of total DNA extracts using the MiSeq platform. In vitro co-culture of MAIT cells with NTHi-infected bronchial epithelial cell lines (BEAS2B) or primary human airway epithelial cells. MAIT cell activation measured using intracellular cytokine staining.
Results
In a cohort of patients with severe asthma (n=23) H. influenzae was commonly cultured and subsequently identified as the dominant bacterial species by metagenomic sequencing (n=8, Median% Total bacterial reads=87.8%). Clinically significant infection was confirmed using a validated H. influenzae plasmid-based RT-qPCR assay. H. influenzae culture positive patients had sputum neutrophilia and lower FeNO. NTHi induces modest MAIT cell production of IFN-gamma which is partly MR1 dependent.
Conclusions
H. influenzae is a clinically-relevant pathogen in severe asthma that can be identified reliably using molecular microbiological methods. Ongoing analysis of a larger patient cohort will allow full characterisation of this clinical phenotype. MAIT cells are able to recognise airway epithelial cells infected with viable intracellular NTHi in the absence of APCs.
Laboratory culture of pleural infection samples is positive in only 30% of cases, probably related to antibiotic usage and fastidious or unculturable organisms such as some anaerobes. Previous studies using capillary sequencing of the 16S rRNA gene improves rates of organism identification, but is unable to resolve the polymicrobiality thought to be present in anaerobic infection. We used ultra-deep pyrosequencing to definitively characterise anaerobic pleural infection.
Methods
Pleural infection samples were obtained from the Second Multicentre Intrapleural Sepsis randomised controlled Trial (MIST2), REC no. 04/MRE5/53. DNA was extracted using the FastDNA SPIN Kit. Modified ‘fusion’ primers amplified the V4–6 regions of the 16S rRNA gene. Subsequent pyrosequencing was performed on the Roche 454 GS FLX instrument. Data analyses were performed using the open souce ‘Quantitative Insights Into Microbial Ecology’ platform. Strategies were used to control for contamination.
Results
172 pleural fluid samples were available, 98 of which were successfully sequenced. 32/98 samples contained anaerobes (defined when ≥10% of sequences in a sample represented anaerobes). Fusobacteriales, particularly Fusobacterium nucleatum, and Bacteroidales, particularly Prevotella spp. were commonly found although other anaerobes were seen (see Figure). Anaerobic pleural infection was usually polymicrobial, with an estimated 4–5 operational taxonomic units (“species”) per sample. Particular patterns of co-infection were Fusobacteriumnucelatum and Streptococcus ‘milleri’ group although Prevotella spp. ± Fusobacterium spp. ± Porphyromonas spp. ± Treponema spp. also co-infected several samples. Many species were found that have not been previously documented, including Atopobium rimae, Cryptobacterium curtum, Lactobacillus spp., Stomatobaculum spp., Oribacterium spp., Prevotella baroniae, Prevotella dentalis/Hallella seregens, Prevotella scopos, Fretibacterium spp., Tanerella forsythia, Treponema denticola, lecithinolyticum, maltophilum, medium and socranskii. Intriguingly, the original isolation and description of almost all these anaerobes were from the oropharynx and some have never been detected at other body sites.
Conclusions
Anaerobic infection occurs in ~33% of cases of pleural infection and is typically polymicrobial. Sequencing revealed many anaerobic bacteria never previously isolated in the pleural space. These bacteria have a strong association with the oropharynx, particularly the gingival crevices. Such findings add to our understanding of the mechanism of development of pleural infection.
This table contains a full account of people who contributed in different ways to the development of the Clinical Practice Guideline. This includes people who were actively involved in the Delphi study. The Table summarises their roles in the process, their affiliations and expertise, and possible conflicts of interest.
Abstract Background Colorectal cancer is associated with secondary sarcopenia (muscle loss) and myosteatosis (fatty infiltration of muscle) and patients who exhibit these host characteristics have poorer outcomes following surgery. Furthermore, patients who undergo curative advanced rectal cancer surgery such as pelvic exenteration, are at risk of skeletal muscle loss due to immobility, malnutrition and a post-surgical catabolic state. Neuromuscular electrical stimulation (NMES) may be a feasible adjunctive treatment to help ameliorate these adverse side-effects. Hence, the purpose of this study is to investigate NMES as an adjunctive pre- and post-operative treatment for rectal cancer patients in the radical pelvic surgery setting and to provide early indicative evidence of efficacy in relation to key health outcomes. Method In a phase II, Double-blind, randomised controlled study, 58 patients will be recruited and randomised (1:1) to either a treatment (NMES plus standard care) or placebo (sham-NMES plus standard care) group. The intervention will begin two weeks pre-operatively and continue for eight weeks after exenterative surgery. The primary outcome will be change in mean skeletal muscle attenuation, a surrogate marker of myosteatosis. Sarcopenia, quality of life, inflammatory status and cancer specific outcomes will also be assessed. Discussion This pilot study will provide study important preliminary evidence of the potential for this adjunctive treatment. It will provide guidance on subsequent development of phase 3 studies on the clinical benefit of NMES for rectal cancer patients in the radical pelvic surgery setting. Trial Registration ClinicalTrials.gov Identifier: NCT04065984; Registered August 22, 2019; Recruiting.
This file contains the survey content used for the first round of a Delphi Study relating to: Delphi Consensus Study and Clinical Practice Guideline Development for Functional Electrical Stimulation to support mobility in people with an upper motor neuron lesion
This file contains a summary of the analysis of the first survey in the Delphi Consensus Study. It focuses on all items that were failed to reach consensus of over 75% of Expert Panellists indicating that they ‘Agreed’ or ‘Strongly Agreed’ to the statement. It also includes examples of free text data written by people who did not ‘Agree’ or ‘Strongly Agree’ to the statement. The free text data were used to refine the statements that did not reach consensus, for use in the second round of the Delphi study.
Abstract Background Over the past 20 years Functional Electrical Stimulation (FES) has grown in clinical use to support walking in people with lower limb weakness or paralysis due to upper motor neuron lesions. Despite growing consensus regarding its benefits, provision across the UK and internationally is variable. This study aimed to explore stakeholder views relating to the value of a clinical guideline focusing on service provision of FES to support walking, how people might use it and what should be included. Methods A mixed methods exploration sought the views of key stakeholders. A pragmatic online survey ( n = 223) focusing on the study aim was developed and distributed to the email distribution list of the UK Association for Chartered Physiotherapists Interested in Neurology (ACPIN). In parallel, a qualitative service evaluation and patient public involvement consultation was conducted. Two group, and seven individual interviews were conducted with: FES-users ( n = 6), their family and carers ( n = 3), physiotherapists ( n = 4), service providers/developers ( n = 2), researchers ( n = 1) and distributors of FES ( n = 1). Descriptive analysis of quantitative data and framework analysis of qualitative data were conducted. Results Support for clinical guideline development was clear in the qualitative interviews and the survey results. Survey respondents most strongly endorsed possible uses of the clinical guideline as ensuring best practice and supporting people seeking access to a FES service. Data analysis and synthesis provided clear areas for inclusion in the clinical guidelines, including current research evidence and consensus relating to who is most likely to benefit and optimal service provision as well as pathways to access this. Specific areas for further investigation were summarised for inclusion in the first stage of a Delphi consensus study. Conclusions Key stakeholders believe in the value of a clinical guideline that focuses on the different stages of service provision for FES to support walking. A Delphi consensus study is being planned based on the findings.
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