rRNA sequencing in molecular microbiological diagnosis of bacterial infections in the autopsy setting

Diagnosing the aetiology of infectious diseases at autopsy, such as pneumonia, meningitis, sepsis or SUDI, is complicated due to issues including post mortem contamination, difficulty culturing fastidious organisms and subjective interpretation of polymicrobial cultures. Death of organisms may also occur post mortem, especially if antibiotics were given to the patient, but residual DNA from non-viable organisms, amenable to molecular detection, may remain. The 16S rRNA gene is present in all bacteria with conserved and hyper-variable regions along its length, allowing amplification and sequencing of all bacterial 16S sequences present in a sample. 16S sequencing offers potential advantages over culture-based diagnostics and is increasingly used in clinical practice. It has been used to identify bacteria in formalin fixed paraffin embedded (FFPE) surgical pathology specimens but its use has not been reported in autopsy diagnosis. This talk will summarise a study aimed to assess the utility of 16S sequencing as an adjunctive microbiological test in the autopsy. Our preliminary work has used post mortem lung tissue samples from children dying with pneumonia as part of the Pneumonia Etiology Research for Child Health (PERCH) project. The technique has identified known pathogens in some cases and provided additional diagnostic information in others. The presentation will discuss the technical aspects of 16S sequencing from FFPE and autopsy material, and the issues surrounding its application to diagnosis in comparison with standard culture based diagnostics on surgical/autopsy material

Successful awake proning is associated with improved clinical outcomes in patients with COVID-19: single-centre high-dependency unit experience

The SARS-CoV-2 can lead to severe illness with COVID-19. Outcomes of patients requiring mechanical ventilation are poor. Awake proning in COVID-19 improves oxygenation, but on data clinical outcomes is limited. This single-centre retrospective study aimed to assess whether successful awake proning of patients with COVID-19, requiring respiratory support (continuous positive airways pressure (CPAP) or high-flow nasal oxygen (HFNO)) on a respiratory high-dependency unit (HDU), is associated with improved outcomes. HDU care included awake proning by respiratory physiotherapists. Of 565 patients admitted with COVID-19, 71 (12.6%) were managed on the respiratory HDU, with 48 of these (67.6%) requiring respiratory support. Patients managed with CPAP alone 22/48 (45.8%) were significantly less likely to die than patients who required transfer onto HFNO 26/48 (54.2%): CPAP mortality 36.4%; HFNO mortality 69.2%, (p=0.023); however, multivariate analysis demonstrated that increasing age and the inability to awake prone were the only independent predictors of COVID-19 mortality. The mortality of patients with COVID-19 requiring respiratory support is considerable. Data from our cohort managed on HDU show that CPAP and awake proning are possible in a selected population of COVID-19, and may be useful. Further prospective studies are required

Clinical characteristics of chylothorax: results from the International Collaborative Effusion database

BackgroundChylothorax is an uncommon medical condition for which limited data are available regarding the contemporary aetiology, management and outcomes. The goal of this study was to better define these poorly characterised features.MethodsThe medical records of adult patients diagnosed with chylothorax at 12 centres across Europe, America and South Africa from 2009-2021 were retrospectively reviewed. Descriptive and inferential statistics were performed.Results77 patients (median age 69 years, male to female ratio 1.5) were included. Subacute dyspnoea was the most typical presenting symptom (66%). The commonest cause of chylothorax was malignancy (68.8%), with lymphoma accounting for 62% of these cases. Other aetiologies were trauma (13%), inflammatory/miscellaneous conditions (11.7%) and idiopathic cases (6.5%). At the initial thoracentesis, the pleural fluid appeared milky in 73%, was exudative in 89% and exhibited triglyceride concentrations >100 mg·dL-1 in 88%. Lymphangiography/lymphoscintigraphy were rarely ordered (3%), and demonstration of chylomicrons in pleural fluid was never ascertained. 67% of patients required interventional pleural procedures. Dietary measures were infrequently followed (36%). No patient underwent thoracic duct ligation or embolisation. Morbidity included infections (18%), and thrombosis in malignant aetiologies (16%). The 1-year mortality was 47%. Pleural fluid protein >3.5 mg·dL-1 (sub-distribution hazard ratio (SHR) 4.346) or lactate dehydrogenase -1 (SHR 10.21) increased the likelihood of effusion resolution. Pleural fluid protein ≤3.5 mg·dL-1 (HR 4.047), bilateral effusions (HR 2.749) and a history of respiratory disease (HR 2.428) negatively influenced survival.ConclusionChylothoraces have a poor prognosis and most require pleural interventions. Despite the standard recommendations, lymphatic imaging is seldom used, nor are dietary restrictions followed

Randomized controlled trial of urokinase versus placebo for nondraining malignant pleural effusion

Rationale: Patients with malignant pleural effusion experience breathlessness, which is treated by drainage and pleurodesis. Incomplete drainage results in residual dyspnea and pleurodesis failure. Intrapleural fibrinolytics lyse septations within pleural fluid, improving drainage. Objectives: To assess the effects of intrapleural urokinase on dyspnea and pleurodesis success in patients with nondraining malignant effusion. Methods: We conducted a prospective, double-blind, randomized trial. Patients with nondraining effusion were randomly allocated in a 1:1 ratio to intrapleural urokinase (100,000 IU, three doses, 12-hourly) or matched placebo. Measurements and Main Results: Co–primary outcome measures were dyspnea (average daily 100-mm visual analog scale scores over 28 d) and time to pleurodesis failure to 12 months. Secondary outcomes were survival, hospital length of stay, and radiographic change. A total of 71 subjects were randomized (36 received urokinase, 35 placebo) from 12 U.K. centers. The baseline characteristics were similar between the groups. There was no difference in mean dyspnea between groups (mean difference, 3.8 mm; 95% confidence interval [CI], −12 to 4.4 mm; P = 0.36). Pleurodesis failure rates were similar (urokinase, 13 of 35 [37%]; placebo, 11 of 34 [32%]; adjusted hazard ratio, 1.2; P = 0.65). Urokinase was associated with decreased effusion size visualized by chest radiography (adjusted relative improvement, −19%; 95% CI, −28 to −11%; P < 0.001), reduced hospital stay (1.6 d; 95% CI, 1.0 to 2.6; P = 0.049), and improved survival (69 vs. 48 d; P = 0.026). Conclusions: Use of intrapleural urokinase does not reduce dyspnea or improve pleurodesis success compared with placebo and cannot be recommended as an adjunct to pleurodesis. Other palliative treatments should be used. Improvements in hospital stay, radiographic appearance, and survival associated with urokinase require further evaluation. Clinical trial registered with ISRCTN (12852177) and EudraCT (2008-000586-26)

Multiple dimensions of health locus of control in a representative population sample: ordinal factor analysis and cross-validation of an existing three and a new four factor model

<p>Abstract</p> <p>Background</p> <p>Based on the general approach of locus of control, health locus of control (HLOC) concerns control-beliefs due to illness, sickness and health. HLOC research results provide an improved understanding of health related behaviour and patients' compliance in medical care. HLOC research distinguishes between beliefs due to Internality, Externality powerful Others (POs) and Externality Chance. However, evidences for differentiating the POs dimension were found. Previous factor analyses used selected and predominantly clinical samples, while non-clinical studies are rare. The present study is the first analysis of the HLOC structure based on a large representative general population sample providing important information for non-clinical research and public health care.</p> <p>Methods</p> <p>The standardised German questionnaire which assesses HLOC was used in a representative adult general population sample for a region in Northern Germany (N = 4,075). Data analyses used ordinal factor analyses in LISREL and Mplus. Alternative theory-driven models with one to four latent variables were compared using confirmatory factor analysis. Fit indices, chi-square difference tests, residuals and factor loadings were considered for model comparison. Exploratory factor analysis was used for further model development. Results were cross-validated splitting the total sample randomly and using the cross-validation index.</p> <p>Results</p> <p>A model with four latent variables (Internality, Formal Help, Informal Help and Chance) best represented the HLOC construct (three-dimensional model: normed chi-square = 9.55; RMSEA = 0.066; CFI = 0.931; SRMR = 0.075; four-dimensional model: normed chi-square = 8.65; RMSEA = 0.062; CFI = 0.940; SRMR = 0.071; chi-square difference test: p < 0.001). After excluding one item, the superiority of the four- over the three-dimensional HLOC construct became very obvious (three-dimensional model: normed chi-square = 7.74; RMSEA = 0.059; CFI = 0.950; SRMR = 0.079; four-dimensional model: normed chi-square = 5.75; RMSEA = 0.049; CFI = 0.965; SRMR = 0.065; chi-square difference test: p < 0.001). Results were confirmed by cross-validation. Results based on our large community sample indicated that western general populations separate health-related control-beliefs concerning formal and informal assistance.</p> <p>Conclusions</p> <p>Future non-clinical HLOC studies in western cultures should consider four dimensions of HLOC: Internality, Formal Help, Informal Help and Chance. However, the standardised German instrument needs modification. Therefore, confirmation of our results may be useful. Future research should compare HLOC structure between clinical and non-clinical samples as well as cross-culturally.</p

Pathogen identification in lower respiratory tract infection

Treatment of lower respiratory tract infection (pneumonia and pleural infection) relies on the use of empirical broad spectrum antibiotics, primarily because reliable pathogen identification occurs infrequently. Another consequence of poor rates of pathogen identification is that our understanding of the microbiology of these infections is incomplete. This thesis addresses some of these issues by combining the acquisition of high quality lower respiratory tract samples, free from nasooropharyngeal contamination, with novel molecular microbiological techniques in an attempt to increase rates of pathogen identification. Four main areas are examined; The role of so-called ‘atypical pneumonia’ bacteria in causing pleural infection. These pathogens have been previously identified in the pleural space infrequently and routine culture usually fails to isolate such bacteria. High sensitivity nested polymerase chain reaction (PCR) is a culture-independent technique which is used to undertake a systematic evaluation for these pathogens in pleural infection samples. The role of Pneumocystis jirovecii in pleural infection, either as a co-infecting pathogen or in monomicrobial infection. This fungus causes severe pneumonia, particularly in the immunosuppressed, but is increasingly recognised as a co-pathogen in community-acquired pneumonia, and is frequently isolated in the upper and lower respiratory tract in health. A high sensitivity real-time PCR assay is used to examine for this fungus. Ultra-deep sequencing of the 16S rRNA gene is used to perform a comprehensive microbial survey in samples taken from the multi-centre MIST2 study of pleural infection. The techniques employed allow analysis of polymicrobial samples and give very high taxonomic resolution, whilst incorporating methods to control for potential contamination. Further, these techniques provide confirmation of the results from the ‘atypical’ bacteria nested PCR study. Bedside ultrasound-guided percutaneous transthoracic needle aspiration (TNA) of consolidated lung is undertaken in patients with pneumonia, as part of the PIPAP study. An evaluation is undertaken of the efficacy and acceptability of TNA. Aspirate samples acquired are also processed using ultra-deep sequencing of the 16S rRNA gene. Other samples obtained as part of the PIPAP study, such as ‘control’ lung aspirates and ‘control’ pleural fluid samples, are similarly processed to enable calculation of sensitivity and specificity of the sequencing methodology.</p

Pathogen identification in lower respiratory tract infection

Treatment of lower respiratory tract infection (pneumonia and pleural infection) relies on the use of empirical broad spectrum antibiotics, primarily because reliable pathogen identification occurs infrequently. Another consequence of poor rates of pathogen identification is that our understanding of the microbiology of these infections is incomplete. This thesis addresses some of these issues by combining the acquisition of high quality lower respiratory tract samples, free from nasooropharyngeal contamination, with novel molecular microbiological techniques in an attempt to increase rates of pathogen identification. Four main areas are examined; The role of so-called ‘atypical pneumonia’ bacteria in causing pleural infection. These pathogens have been previously identified in the pleural space infrequently and routine culture usually fails to isolate such bacteria. High sensitivity nested polymerase chain reaction (PCR) is a culture-independent technique which is used to undertake a systematic evaluation for these pathogens in pleural infection samples. The role of Pneumocystis jirovecii in pleural infection, either as a co-infecting pathogen or in monomicrobial infection. This fungus causes severe pneumonia, particularly in the immunosuppressed, but is increasingly recognised as a co-pathogen in community-acquired pneumonia, and is frequently isolated in the upper and lower respiratory tract in health. A high sensitivity real-time PCR assay is used to examine for this fungus. Ultra-deep sequencing of the 16S rRNA gene is used to perform a comprehensive microbial survey in samples taken from the multi-centre MIST2 study of pleural infection. The techniques employed allow analysis of polymicrobial samples and give very high taxonomic resolution, whilst incorporating methods to control for potential contamination. Further, these techniques provide confirmation of the results from the ‘atypical’ bacteria nested PCR study. Bedside ultrasound-guided percutaneous transthoracic needle aspiration (TNA) of consolidated lung is undertaken in patients with pneumonia, as part of the PIPAP study. An evaluation is undertaken of the efficacy and acceptability of TNA. Aspirate samples acquired are also processed using ultra-deep sequencing of the 16S rRNA gene. Other samples obtained as part of the PIPAP study, such as ‘control’ lung aspirates and ‘control’ pleural fluid samples, are similarly processed to enable calculation of sensitivity and specificity of the sequencing methodology.This thesis is not currently available in ORA