Koirobi Haldar

Acute exacerbations of chronic obstructive pulmonary disease: identification of biologic clusters and their biomarkers.

RATIONALE Exacerbations of chronic obstructive pulmonary disease (COPD) are heterogeneous with respect to inflammation and etiology. OBJECTIVES Investigate biomarker expression in COPD exacerbations to identify biologic clusters and determine biomarkers that recognize clinical COPD exacerbation phenotypes, namely those associated with bacteria, viruses, or eosinophilic airway inflammation. METHODS Patients with COPD were observed for 1 year at stable and exacerbation visits. Biomarkers were measured in sputum and serum. Viruses and selected bacteria were assessed in sputum by polymerase chain reaction and routine diagnostic bacterial culture. Biologic phenotypes were explored using unbiased cluster analysis and biomarkers that differentiated clinical exacerbation phenotypes were investigated. MEASUREMENTS AND MAIN RESULTS A total of 145 patients (101 men and 44 women) entered the study. A total of 182 exacerbations were captured from 86 patients. Four distinct biologic exacerbation clusters were identified. These were bacterial-, viral-, or eosinophilic-predominant, and a fourth associated with limited changes in the inflammatory profile termed “pauciinflammatory.” Of all exacerbations, 55%, 29%, and 28% were associated with bacteria, virus, or a sputum eosinophilia. The biomarkers that best identified these clinical phenotypes were sputum IL-1β, 0.89 (area under receiver operating characteristic curve) (95% confidence interval [CI], 0.83–0.95); serum CXCL10, 0.83 (95% CI, 0.70–0.96); and percentage peripheral eosinophils, 0.85 (95% CI, 0.78–0.93), respectively. CONCLUSIONS The heterogeneity of the biologic response of COPD exacerbations can be defined. Sputum IL-1β, serum CXCL10, and peripheral eosinophils are biomarkers of bacteria-, virus-, or eosinophil-associated exacerbations of COPD. Whether phenotype-specific biomarkers can be applied to direct therapy warrants further investigation.

Association Between Pathogens Detected Using Quantitative Polymerase Chain Reaction With Airway Inflammation in COPD at Stable State and Exacerbations

BACKGROUND: Relationships between airway inflammation and respiratory potentially pathogenic microorganisms (PPMs) quantified using quantitative polymerase chain reaction (qPCR) in subjects with COPD are unclear. Our aim was to evaluate mediators of airway inflammation and their association with PPMs in subjects with COPD at stable state and during exacerbations. METHODS: Sputum from 120 stable subjects with COPD was analyzed for bacteriology (colony-forming units; total 16S; and qPCR targeting Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae), differential cell counts, and inflammatory mediators using the Meso-Scale Discovery Platform. Subjects were classified as colonized if any PPM was identified above the threshold of detection by qPCR. Symptoms were quantified using the visual analog scale. RESULTS: At stable state, 60% of subjects were qPCR positive for H influenzae, 48% for M catarrhalis, and 28% for S pneumoniae. Elevated sputum concentrations of IL-1β, IL-10, and tumor necrosis factor (TNF)-α were detected in samples qPCR positive for either H influenzae or M catarrhalis. Bacterial loads of H influenzae positively correlated with IL-1β, IL-8, IL-10, TNF-α, and symptoms; and M catarrhalis correlated with IL-10 and TNF-α. H influenzae qPCR bacterial load was an independent predictor of sputum TNF-α and IL-1β. In 55 subjects with paired exacerbation data, qPCR bacterial load fold change at exacerbation in M catarrhalis but not H influenzae correlated to changes in sputum TNF-α and IL-1β concentrations. CONCLUSIONS: At stable state, H influenzae is associated with increased airway inflammation in COPD. The relationship between bacterial load changes of specific pathogens and airway inflammation at exacerbation and recovery warrants further investigation.

Airway bacteria measured by quantitative polymerase chain reaction and culture in patients with stable COPD: relationship with neutrophilic airway inflammation, exacerbation frequency, and lung function

Background Potentially pathogenic microorganisms can be detected by quantitative real-time polymerase chain reaction (qPCR) in sputum from patients with COPD, although how this technique relates to culture and clinical measures of disease is unclear. We used cross-sectional and longitudinal data to test the hypotheses that qPCR is a more sensitive measure of bacterial presence and is associated with neutrophilic airway inflammation and adverse clinical outcomes. Methods Sputum was collected from 174 stable COPD subjects longitudinally over 12 months. Microbial sampling using culture and qPCR was performed. Spirometry and sputum measures of airway inflammation were assessed. Findings Sputum was qPCR-positive (>106 copies/mL) in 77/152 samples (Haemophilus influenzae [n=52], Moraxella catarrhalis [n=24], Streptococcus pneumoniae [n=19], and Staphylococcus aureus [n=7]). Sputum was culture-positive in 50/174 samples, with 49 out of 50 culture-positive samples having pathogen-specific qPCR bacterial loads >106 copies/mL. Samples that had qPCR copy numbers >106/mL, whether culture-positive or not, had increased sputum neutrophil counts. H. influenzae qPCR copy numbers correlated with sputum neutrophil counts (r=0.37, P<0.001), were repeatable within subjects, and were >106/mL three or more times in 19 patients, eight of whom were repeatedly sputum culture-positive. Persistence, whether defined by culture, qPCR, or both, was associated with a higher sputum neutrophil count, lower forced expiratory volume in 1 second (FEV1), and worsened quality of life. Interpretation qPCR identifies a significant number of patients with potentially bacteria-associated neutrophilic airway inflammation and disease that are not identified by traditional culture-based methods.

Acute Exacerbations of Chronic Obstructive Pulmonary Disease

RATIONALE Exacerbations of chronic obstructive pulmonary disease (COPD) are heterogeneous with respect to inflammation and etiology. OBJECTIVES Investigate biomarker expression in COPD exacerbations to identify biologic clusters and determine biomarkers that recognize clinical COPD exacerbation phenotypes, namely those associated with bacteria, viruses, or eosinophilic airway inflammation. METHODS Patients with COPD were observed for 1 year at stable and exacerbation visits. Biomarkers were measured in sputum and serum. Viruses and selected bacteria were assessed in sputum by polymerase chain reaction and routine diagnostic bacterial culture. Biologic phenotypes were explored using unbiased cluster analysis and biomarkers that differentiated clinical exacerbation phenotypes were investigated. MEASUREMENTS AND MAIN RESULTS A total of 145 patients (101 men and 44 women) entered the study. A total of 182 exacerbations were captured from 86 patients. Four distinct biologic exacerbation clusters were identified. These were bacterial-, viral-, or eosinophilic-predominant, and a fourth associated with limited changes in the inflammatory profile termed “pauciinflammatory.” Of all exacerbations, 55%, 29%, and 28% were associated with bacteria, virus, or a sputum eosinophilia. The biomarkers that best identified these clinical phenotypes were sputum IL-1β, 0.89 (area under receiver operating characteristic curve) (95% confidence interval [CI], 0.83–0.95); serum CXCL10, 0.83 (95% CI, 0.70–0.96); and percentage peripheral eosinophils, 0.85 (95% CI, 0.78–0.93), respectively. CONCLUSIONS The heterogeneity of the biologic response of COPD exacerbations can be defined. Sputum IL-1β, serum CXCL10, and peripheral eosinophils are biomarkers of bacteria-, virus-, or eosinophil-associated exacerbations of COPD. Whether phenotype-specific biomarkers can be applied to direct therapy warrants further investigation.

Systemic and pulmonary inflammation is independent of skeletal muscle changes in patients with chronic obstructive pulmonary disease

Background Nutritional depletion is an important manifestation of chronic obstructive pulmonary disease (COPD), which has been related to systemic inflammation. It remains unclear to what degree airway inflammation contributes to the presence or progression of nutritional depletion. Objectives To determine whether airway inflammation and lung bacterial colonization are related to nutritional status or predict progressive weight loss and muscle atrophy in patients with COPD. Methods Body composition using dual energy X-ray absorptiometry, indices of airway inflammation, and bacterial colonization were measured in 234 COPD patients. Systemic inflammation was assessed from serum C reactive protein (CRP) and circulating total and differential leukocyte counts. Nutritional depletion was defined as a body mass index (BMI) less than 21 kg/m2 and/or fat-free mass index (FFMI) less than 15 or 17 kg/m2 in women and men, respectively. FFMI was calculated as the fat-free mass (FFM) corrected for body surface area. Measurements were repeated in 94 patients after a median 16-month follow-up. Regression analysis was used to assess the relationships of weight change and FFM change with indices of bacterial colonization and airway and systemic inflammation. Results Nutritional depletion occurred in 37% of patients. Lung function was worsened in patients with nutritional depletion compared to those without (forced expiratory volume in 1 second 1.17 L versus 1.41 L, mean difference 0.24, 95% confidence interval 0.10 to 0.38, P<0.01). There were no differences in airway inflammation and bacterial colonization in patients with and without nutritional depletion. At baseline, BMI correlated positively with serum CRP (rs=0.14, P=0.04). Change in weight and change in FFM over time could not be predicted from baseline patient characteristics. Conclusion Nutritional depletion and progressive muscle atrophy are not related to airway inflammation or bacterial colonization. Overspill of pulmonary inflammation is not a key driver of muscle atrophy in COPD.

Microbiome balance in sputum determined by PCR stratifies COPD exacerbations and shows potential for selective use of antibiotics

Background While a subgroup of patients with exacerbations of chronic obstructive pulmonary disease (COPD) clearly benefit from antibiotics, their identification remains challenging. We hypothesised that selective assessment of the balance between the two dominant bacterial groups (Gammaproteobacteria (G) and Firmicutes (F)) in COPD sputum samples might reveal a subgroup with a bacterial community structure change at exacerbation that was restored to baseline on recovery and potentially reflects effective antibiotic treatment. Methods Phylogenetically specific 16S rRNA genes were determined by quantitative real time PCR to derive a G:F ratio in serial sputum samples from 66 extensively-phenotyped COPD exacerbation episodes. Results Cluster analysis based on Euclidean distance measures, generated across the 4 visit times (stable and exacerbation day: 0,14 and 42) for the 66 exacerbation episodes, revealed three subgroups designated HG, HF, and GF reflecting predominance or equivalence of the two target bacterial groups. While the other subgroups showed no change at exacerbation, the HG cluster (n = 20) was characterized by G:F ratios that increased significantly at exacerbation and returned to baseline on recovery (p<0.00001); ratios in the HG group also correlated positively with inflammatory markers and negatively with FEV1. At exacerbation G:F showed a significant receiver-operator-characteristic curve to identify the HG subgroup (AUC 0.90, p<0.0001). Conclusions The G:F ratio at exacerbation can be determined on a timescale compatible with decisions regarding clinical management. We propose that the G:F ratio has potential for use as a biomarker enabling selective use of antibiotics in COPD exacerbations and hence warrants further clinical evaluation.

Investigating the role of pentraxin 3 as a biomarker for bacterial infection in subjects with COPD

Background Pentraxin 3 (PTX3) is an acute phase protein, involved in antibacterial resistance. Recent studies have shown PTX3 levels to be elevated in the presence of a bacterial infection and in a murine sepsis model. Objective We aim to investigate if sputum PTX3 can be used as a biomarker for bacterial infection in subjects with COPD. Materials and methods Sputum samples from 142 COPD patients (102 men) with a mean (range) age of 69 years (45–85) and mean (SD) post-bronchodilator percentage predicted forced expiratory volume in 1 second (FEV1) of 50% (19) were analyzed for PTX3, using a commercial assay at stable state and during an exacerbation. Association with bacteria, from culture, quantitative real-time polymerase chain reaction (qPCR) and colony-forming units (CFU) was investigated. Results The geometric mean (95% CI) PTX3 level at stable state was 50.5 ng/mL (41.4–61.7). PTX3 levels correlated with absolute neutrophil count in sputum (r=0.37; P<0.01), but not FEV1 or health status. There was a weak correlation between PTX3 and bacterial load (CFU: r=0.29, P<0.01; 16S qPCR: r=0.18, P=0.05). PTX3 was a poor predictor of bacterial colonization (defined as >105 CFU/mL at stable state) with a receiver-operating characteristic (ROC) area under the curve (AUC) of 0.59 and 95% confidence interval (CI) 0.43–0.76 (P=0.21). During an exacerbation, there was a modest increase in PTX3 (fold difference 0.15, 95% of difference 0.02–0.29; P=0.02), and PTX3 fared better at identifying a bacteria-associated exacerbation (ROC AUC 0.65, 95% CI 0.52–0.78, P=0.03). Conclusion PTX3 is associated with bacterial infection in patients with COPD, but its utility as a biomarker for identifying a bacteria-associated exacerbation warrants further studies.

S16 Molecular profiling of the airway microbiome in COPD

Introduction It is hypothesised that bacteria are important in the pathogenesis of COPD exacerbations and clinical expression of disease. To date, most bacteriological research in COPD has been performed using culture based methods. However, novel molecular approaches offer more detailed evaluation of the airway microbiome that may better inform the role of bacteria in COPD. Aims To characterise the microbial community in COPD and examine whether detectable changes occur with serial longitudinal assessment at stable, exacerbation, follow-up and recovery visits. Methods 115 COPD patients that were part of a clinical trial had sputum samples collected at the four time points. Patients received antibiotics and / or oral corticosteroids after clinical assessment to treat exacerbations. Follow-up and recovery samples were collected 2 and 6 weeks after the exacerbation sample. Real-time quantitative PCR (qPCR) was performed on sputum DNA using universal 16S gene primers and specific gene targets to quantify total bacterial load and the specific pathogens Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis and Staphylococcus aureus. In a subgroup of 30 patients, 454 high-throughput pyrosequencing was performed at each of the 4 visits to examine changes to the global microbiome. Results Quantitative PCR identified one or more pathogens in 94% of stable samples and 97% of exacerbation samples. There was no significant difference in the total bacterial load or any specific pathogen between longitudinal stable and exacerbation samples. 454 pyrosequencing identified Proteobacteria and Firmicutes to be the dominant groups contributing >80% of the sequence reads at phylum level. Haemophilus, Moraxella and Streptococcus were the dominant groups at genus level. Cluster analysis characterised three groups on the basis of the ratio of Proteobacteria to Firmicutes. No significant differences in patient characteristics were observed between microbiome clusters. There was no significant change across visits in the microbial community at either phylum or genus level. No treatment specific effects on the microbiome were observed. Conclusions Molecular profiling identifies heterogeneity in the airway microbiome of COPD patients, with dominance of pathogens routinely identified at culture. However, a precise role for bacteria in COPD remains unclear.

Face mask sampling reveals antimicrobial resistance genes in exhaled aerosols from patients with chronic obstructive pulmonary disease and healthy volunteers

Introduction The degree to which bacteria in the human respiratory tract are aerosolised by individuals is not established. Building on our experience sampling bacteria exhaled by individuals with pulmonary tuberculosis using face masks, we hypothesised that patients with conditions frequently treated with antimicrobials, such as chronic obstructive pulmonary disease (COPD), might exhale significant numbers of bacteria carrying antimicrobial resistance (AMR) genes and that this may constitute a previously undefined risk for the transmission of AMR. Methods Fifteen-minute mask samples were taken from 13 patients with COPD (five paired with contemporaneous sputum samples) and 10 healthy controls. DNA was extracted from cell pellets derived from gelatine filters mounted within the mask. Quantitative PCR analyses directed to the AMR encoding genes: blaTEM (β-lactamase), ErmB (target methylation), mefA (macrolide efflux pump) and tetM (tetracycline ribosomal protection protein) and six additional targets were investigated. Positive signals above control samples were obtained for all the listed genes; however, background signals from the gelatine precluded analysis of the additional targets. Results 9 patients with COPD (69%), aerosolised cells containing, in order of prevalence, mefA, tetM, ErmB and blaTEM, while three healthy controls (30%) gave weak positive signals including all targets except blaTEM. Maximum estimated copy numbers of AMR genes aerosolised per minute were mefA: 3010, tetM: 486, ErmB: 92 and blaTEM: 24. The profile of positive signals found in sputum was not concordant with that in aerosol in multiple instances. Discussion We identified aerosolised AMR genes in patients repeatedly exposed to antimicrobials and in healthy volunteers at lower frequencies and levels. The discrepancies between paired samples add weight to the view that sputum content does not define aerosol content. Mask sampling is a simple approach yielding samples from all subjects and information distinct from sputum analysis. Our results raise the possibility that patient-generated aerosols may be a significant means of AMR dissemination that should be assessed further and that consideration be given to related control measures.

S116 Cell-dissociated haemophilus influenzae and bacteria-associated inflammatory mediators in the airways of patients with chronic obstructive pulmonary disease

Background Patients with COPD have a susceptibility to respiratory tract infections associated with increased pulmonary inflammation. Bacteria can reside within the host as cell-associated (attached to host cells via adhesins, pili or biofilm formation) or cell-dissociated bacteria. It is unclear how bacteria-to-cell interactions affect pulmonary inflammation and whether these levels differ over an exacerbation time course. We sought to investigate the effects of Haemophilius influenzae cell-interaction upon airway inflammation and whether the levels of H. influenzae bacteria and cell-dissociated bacteria differ over an exacerbation time course. Methods Cell differential counts were carried out on sputum samples as per standard protocol. Bacterial DNA was extracted and H.influenzae was quantified using qPCR from the sputum plug (contains cell-associated and dissociated bacteria) and the sputum cell-free supernatant (cell-dissociated bacteria only). Inflammatory mediators (IL-1α, TNF-α, IL-8 and neutrophil elastase (NE)) were measured in the sputum supernatant using commercial assays. Results 63 patients (77% male; average age of 69 (45–88); FEV1 percentage predicted of 53%; mean percentage neutrophil count in sputum of 65%) at stable state were analysed. Levels of H. influenzae in the supernatant only correlated with the sputum total cell count (r=0.38; p=0.03). Levels of H. influenzae in the plug correlated with inflammatory mediators (sputum neutrophil percentage r=0.42, p=0.01; sputum macrophage percentage r=−0.35, p=0.04; IL-1α r=0.36, p=0.03; IL-8 r=0.49, p<0.01; NE r=0.40, p=0.02). The exacerbation time course in 10 paired COPD subjects was examined. There was no significant difference in H. influenzae levels in the plug (p=0.89) (figure 1A). However, there was a significant increase in levels in the supernatant over the exacerbation time course (p=0.05) (figure 1B). Conclusion H. influenzae levels in the sputum plug appear to have much more of an effect on airway inflammation than levels of cell-dissociated H. influenzae suggesting that cell-associated bacteria may be a driver of airway inflammation in COPD. Further investigation into this highly complicated relationship needs to be conducted. Abstract S116 Figure 1 Levels of H. influenzae in the sputum plug (A) and in the sputum supernatant (B) during an exacerbation time course in 10 paired subjects with COPD.