Arc Patel

P253 Association of Defective Monocyte-Derived Macrophage Phagocytosis with Clinical Phenotypes in Stable COPD

Introduction Macrophages play an important role in clearing inhaled particles and bacteria from the lung, thus maintaining its sterility. Defective phagocytosis of bacteria has been demonstrated in both alveolar and monocyte-derived macrophages (MDMs) from COPD patients and may play a role in the aetiology of the frequent exacerbator phenotype. We hypothesised that defective phagocytosis may also be associated with lower airway bacterial colonisation (LABC) and other clinical parameters in stable COPD. Methods Whole blood and sputa were collected from stable patients in the London COPD cohort. Stable COPD was defined as no symptom-defined exacerbations recorded on prospectively completed diary cards in the preceding four weeks and subsequent two weeks. Monocytes were isolated from the whole blood and cultured with GM-CSF (2 ng/ml) for 12 days to generate MDMs. MDM phagocytosis of fluorescently-labelled polystyrene beads, Haemophilus influenzae (HI) and Streptococcus pneumoniae (SP) was measured by fluorimetry. LABC was defined as detection of HI, SP or Moraxella catarrhalis (MC) in sputum using quantitative PCR. Results MDMs were cultured from 26 COPD patients. 54% were male, mean age 70.0 years (SD 8.3), FEV1 predicted 55.3% (20.3), 46% were current smokers, median daily inhaled corticosteroid (ICS) dose was 1000 (640–2000) mcg (beclomethasone equivalent dose) and median exacerbation frequency per year was 1.8 (1.0–2.9) based on diary card events. Phagocytosis of HI was significantly less with increasing exacerbation frequency (p=0.002, r=–0.58, Figure 1), although no significant associations were demonstrated between exacerbation frequency and phagocytosis of inert beads or SP (p=0.27 and p=0.22 respectively). 13 patients (50%) with LABC did not demonstrate any significant difference in phagocytosis of either beads (p=0.29), HI (p=0.66) or SP (p=0.88) compared with non-colonised patients. There was no significant association between phagocytosis of beads, HI or SP with age, FEV1%predicted, smoking pack year history, ICS dose or BMI (all p>0.05). Abstract P253 Figure 1 Conclusion In stable COPD patients, decreasing phagocytosis of HI was associated with increasing exacerbation frequency. Phagocytosis was not related to LABC suggesting that macrophage activity alone may not determine bacteria colonisation. Further work is needed to elucidate the mechanisms of reduced phagocytosis in COPD and its relationship to exacerbation frequency.

P214 Gastro-Oesophageal Reflux Symptoms During COPD Exacerbations

Introduction Gastro-oesophageal reflux disease (GORD) has been associated with increased COPD exacerbation frequency (Terada et al, Thorax 2008) and was independently associated with the frequent exacerbator phenotype in the ECLIPSE study (Hurst et al, NEJM 2010). We aimed to quantify any changes in GORD symptoms during COPD exacerbations. Methods Outpatients from the London COPD cohort completed the Frequency Scale for the Symptoms of Gastro-oesophageal reflux (FSSG) and Hull Airway Reflux Questionnaire (HARQ) during stable-state clinic visits and at exacerbation, within a week of symptom-onset, and prior to systemic therapy. FSSG and HARQ scores range from 0–48 and 0–70 respectively, with significant reflux thought to be associated with scores of ≥8 and ≥13 respectively. Exacerbations were defined using our usual symptomatic criteria from daily diary cards (Seemungal et al, AJRCCM 1998). Results 42 COPD patients had a mean±SD age of 72.7±8.7 years, 64% male, 17% current smokers, median(IQR) 46 (19.71) pack years, mean±SD stable FEV1 1.22±0.64L and 50.7±21.5%predicted, BMI 27.7±7.4kg/m2. 13 (31%) patients had a diagnosis of GORD, of whom 12 (92%) were taking regular acid suppression therapy. There was a median (IQR) interval of 141(80,233) days between the stable and exacerbation visits. Although median (IQR) FSSG and HARQ scores were higher at exacerbation compared to the stable state, this was not statistically significant due to high variability (6.5 (4.0,13.0) vs 5.0 (1.5,11.5), p=0.247 and 15.5 (9.0,23) vs 18.5 (11.5,24.5), p=0.096 respectively). 16/42 (38%) patients had a high FSSG score (≥8) in the stable state compared with 20/42 (48%) at exacerbation (p=0.378). 10/42 (24%) changed from a low stable FSSG to a high score at exacerbation. 27/42 (64%) patients had a high HARQ score (≥13) in the stable state compared with 30/42 (71%) at exacerbation (p=0.483). 7/42 (17%) changed from a low stable HARQ to a high score at exacerbation. Conclusions GORD symptom scores are not significantly higher during acute COPD exacerbations. Due to high variability, approximately one fifth of COPD patients had a low GORD symptom score when stable and high scores at exacerbation implicating worsening reflux in some exacerbations. Further work is required to understand the potential mechanisms. Abstract P214 Figure 1

S25 Arterial Stiffness and Airway Infection and Inflammation During COPD Exacerbations

Introduction Cardiovascular risk is elevated following COPD exacerbations requiring systemic therapy (Donaldson et al, Chest 2010:137; 1091–7). We have recently shown that a potential mechanism is increased arterial stiffness during COPD exacerbations compared to the stable state (Patel et al, AJRCCM 2012:185; A5853). We hypothesised that arterial stiffness may be mediated by airway infection and inflammation during COPD exacerbations. Methods We used aortic pulse wave velocity (aPWV) as a non-invasive validated measure of arterial stiffness using Vicorder apparatus (Skidmore Medical, UK) in outpatients from the London COPD cohort when they presented within seven days of exacerbation symptom onset and prior to any systemic therapy. These events were defined using our usual symptomatic criteria (Seemungal et al, AJRCCM 1998:157; 1418–22). We collected spontaneous sputum at the same clinic visit to identify typical bacterial (S pneumoniae, H influenzae and M catarrhalis) and rhinovirus infection using PCR. We also measured IL-6 and IL-8 in sputum supernatant using commercial ELISA kits (R&D Systems, USA). Results 32 exacerbating COPD patients produced a spontaneous sputum sample during the same clinic visit as an aPWV measurement. Their stable clinical characteristics were: 69% male; 25% current smokers; median (IQR) 39 (21.74) pack year smoking history; mean±SD age 72.4±8.1 years; FEV1 1.18±0.41L and 46.8±18.0%predicted; FEV1/FVC ratio 0.44±0.15 and BMI 25.4±3.8 kg/m2. Arterial stiffness was higher in patients with airway infection at exacerbation presentation (mean ±SD aPWV 12.3±2.3 (n=19) vs 10.8±1.8 (n=13) m/s, p=0.030) (Figure 1A). Arterial stiffness was strongly correlated with sputum IL-6 at exacerbation (n=32, rho=0.495, p=0.003) (Figure 1B) but not IL-8 (n=31, rho=0.100, p=0.591). Conclusions Arterial stiffness is related to airway infection and inflammation during COPD exacerbations. Interventions to prevent and reduce airway infection and inflammation may lower cardiovascular risk during COPD exacerbations. Abstract S25 Figure 1

P76 The effect of outside temperature on daily physical activity in COPD patients

Introduction COPD is characterised by breathlessness, leading to reduced physical activity. Breathlessness varies dependent upon the degree and intensity of physical activity (1). Common logic dictates that weather variation such as temperature and sunshine could influence physical activity in the general population, especially walking. The aims of this study were to evaluate the effect of weather variation on daily activity and the variation in activity during the days of the week in COPD patients. Methods Fifty-five stable COPD outpatients from the London COPD cohort wore a pedometer (Yamax Digi-Walker SW-200) daily for 30 days during waking hours. We excluded exacerbating patients and those using walking sticks or oxygen. All patients were asked to record their step count on daily diary cards. Weather data for London Heathrow were obtained with permission from the British Atmospheric Data Centre. Stable COPD was defined as having no symptom-defined exacerbations in the preceding six weeks and subsequent two weeks, according to prospectively-collected diary cards, with no change in long-term inhaled and oral medications in the preceding two weeks. Results The clinical characteristics of the 55 patients were mean (±SD) age 70.3 (8.7) years; FEV1% predicted 51.0% (±14.1); male gender 67%; current smoker 25%, BMI 27.2 kg/m2. The mean (±SD) daily step count was 4327 (±2944). COPD patients were more active as temperatures increased (Figure 1A) and on sunny as opposed to no sunshine days (Figure 1B, p<0.001). Figure 1C shows that mean weekday activity is significantly higher than mean weekend activity (p<0.001), mainly due to less activity on Sunday. Abstract 76 Figure 1 Conclusion Daily activity in stable COPD markedly affected by outdoor temperature and sunshine and patients had highest activity during weekdays. Further work is required to ascertain whether some patients are more active than others. Reference Pitta F et al., Quantifying Physical Activity in Daily Life with Questionnaires and Motion Sensors in COPD, ERJ 27, no. 5 (April 2006): 1040–1055.

P119 Changes in airway and systemic IL-18 levels at exacerbation in COPD patients

Introduction IL-18 is a proinflammatory cytokine implicated in COPD pathophysiology, causing pulmonary inflammation and emphysema in murine models. IL-18 receptor expression is increased on alveolar macrophages in COPD.1 IL-18 levels are elevated in serum and sputum of stable COPD patients. We hypothesised that airway and systemic IL-18 concentrations increase further at exacerbation. Methods Sputa and sera prospectively collected from the London COPD cohort were analysed using ELISA (eBioscience®, Vienna). Patients had an FEV1=80% predicted and FEV1/FVC ratio=0.7. Baseline was defined as at least 6 weeks after, and 2 weeks before, an exacerbation. An exacerbation was defined as an increase for two consecutive days in respiratory symptoms, with at least one major symptom (dyspnoea, sputum purulence or volume) plus another major or minor (wheeze, cold, sore throat or cough) symptom. Exacerbation frequency was calculated from daily diary cards collected over the previous 12 months. When unavailable, patients recall of exacerbations over the preceding year was used. Frequent exacerbators had ≥2 exacerbations in the preceding year, infrequent exacerbators <2. Results 94 COPD patients had serum analysed, of whom 48 also had sputum analysed. 60% were male, mean age was 71.6 years (SD 8.5), mean FEV1 predicted 50.6% (18.1). Sputum IL-18 levels increased significantly from baseline to exacerbation (median 1.46 log10 pg/ml (IQR 0.96–2.01) vs 1.95 (0.96–2.23), n=48, p=0.023, Abstract P119 figure 1). However, serum IL-18 concentrations were not significantly greater at exacerbation compared to paired baseline levels (median 2.17 log10 pg/ml (1.97–2.37) vs 2.08 (1.91–2.36), n=31, p=0.299).There was no correlation between baseline serum IL-18 concentrations and exacerbation frequency (n=94, ρ=0.096, p=0.357) or FEV1% predicted (n=93, ρ=0.081, p=0.443). No significant difference was found in baseline serum IL-18 concentrations between frequent and infrequent exacerbators (median 148 pg/ml (95–235; n=46) vs 120 (78–219; n=48), p=0.431). There was no correlation between baseline serum IL-18 concentrations and paired sputum levels (n=42, ρ=−0.149, p=0.348).Abstract P119 Figure 1 Paired sputum IL-18 levels at baseline and exacerbation. Conclusions Sputum but not serum IL-18 increases at COPD exacerbation. Treatment options for exacerbations are limited and there is a need for novel anti-inflammatories. The results of this study suggest IL-18 as a potential target for exacerbation therapy.

S13 Differentiated human rhinovirus loads in stable COPD and at exacerbation

Introduction Human rhinoviruses (HRV) are the main aetiological agents of virus-associated COPD exacerbations (Seemungal et al, 2001). However the importance of the level of viral load as a trigger for naturally occurring exacerbations is not fully understood. We aimed to assess and quantify HRV prevalence and load in stable and exacerbating patients from the London COPD cohort. Methods A real-time qPCR protocol was utilised to detect HRV presence and quantify load in sputum samples taken at baseline (n=58) and at COPD exacerbation onset (n=57). COPD patients were defined as stable if exacerbation-free for at least 42 days since the previous exacerbation and more than 14 days before the next. Exacerbations were defined using our usual symptom criteria; an increase in respiratory symptoms for two consecutive days, with at least one symptom being a major symptom; dyspnoea, sputum purulence or volume and the other a major or minor symptom; wheeze, cold sore throat, and cough (Anthonisen criteria). A χ2 test was used to compare HRV prevalence of the two disease phases, and an independent-samples t test was used to compare the differences in viral load. Results Sixty-four patients provided 115 sputum samples: mean age 70.5 years (SD±8.1); FEV1 45.8% predicted (±20.0%); current smoker 31.3%. There is a significantly higher prevalence of HRV at exacerbation onset, 31.6% (18/57) compared to baseline 15.5% (9/58) (p=0.042). Similarly, the mean viral load was significantly greater at exacerbation onset 1.70 (±1.67) log10 pfu/ml than baseline 0.30 (±0.69) log10 pfu/ml (p=0.025), exhibiting a 25-fold increase in viral load from baseline to exacerbation. 6.9% (4/58) of patients were positive for HRV at baseline only, 26.3% (15/57) at exacerbation only and 8.8% (4/45) at both time points (p=0.006). Conclusions HRV load is significantly greater at COPD exacerbation than when detected in the stable state. This emphasises the importance of HRV as a key trigger of COPD exacerbations. HRV can be detected in the stable state; however the loads are very low suggesting asymptomatic carriage rather than chronic infection.

P46 Gender differences in the prevalence of comorbidities in COPD patients

Introduction Effective recognition and appropriate management of comorbidities is an important aspect of modern COPD care. In particular, cardiovascular diseases are a leading cause of morbidity and mortality. There is increasing interest in the differential impact of gender in COPD. This study aims to characterise gender differences in the profile of comorbidities in COPD. Methods We analysed the recruitment records of 386 well-characterised patients enrolled into the London COPD Cohort. Comorbidities, medication, age, gender, height, weight, spirometry, St Georges Respiratory Questionnaire (SGRQ) and MRC dyspnoea scores were recorded. Results There were no significant differences between females (n=164) and males (n=222) in terms of mean ± SD age; 67.5±8.3 vs 69.0±9.0 years, p=0.102, median (IQR) smoking pack year history 47 (27–62) vs 44 (27–65), p=0.769 or body mass index 25.3 (22.1–29.4) vs 25.5 (22.8–29.0), p=0.311. Females had milder airflow limitation at recruitment with a higher mean ± SD FEV1 % predicted; 53.5±19.9 vs 46.4±18.9, p<0.001. Following adjustment for FEV1 % predicted, there were no gender differences in SGRQ (51.2±18.6 vs 49.8±19.5, p=0.127) or MRC dyspnoea scores (3.0 (2.0, 4.0) vs 3.0 (2.0, 4.0), p=0.104). Cardiovascular conditions were more common in male COPD patients (Abstract P46 figure 1), whereas of the clinically significant comorbidities, only osteoporosis was more common in females (10.4% vs 2.7%, p=0.001).Abstract P46 Figure 1 Gender differences in the prevalence of cardiovascular diseases in COPD. MI, myocardial infarction, CVA, cerebrovascular accident, TIA, transient ischaemic attack; PVD, peripheral vascular disease; IHD, ischaemic heart disease, CV, cardiovascular. Conclusions The excess cardiovascular disease in COPD patients is predominantly found in men despite lower gender differences in cardiovascular risk factors such as smoking history, diabetes, hypercholesterolaemia and hypertension. This may partly represent under-diagnosis of cardiovascular disease in COPD patients. Clinical vigilance must be maintained to identify and optimally manage important comorbidities in all COPD patients, although clinicians should be aware of the increased prevalence of cardiovascular disease in men and osteoporosis in women.

P47 The impact of comorbid ischaemic heart disease on exercise capacity in COPD patients

Introduction Comorbid ischaemic heart disease (IHD) is associated with an adverse impact on health status, symptoms (ARJCCM 2011;183:A2614) and exacerbation recovery in COPD patients (ERJ 2010;954s:E5209). Any impact on exercise capacity is poorly understood. We aimed to assess and quantify differences in exercise capacity in stable COPD patients with and without IHD. Methods We assessed 6-min walking distance (6MWD) in accordance with ATS guidance (AJRCCM 2002;166:111–117) in patients from the London COPD cohort. All assessments were performed in the stable state with no symptom-defined exacerbations recorded on daily diary cards for 6 weeks prior and 2 weeks following the visit. Dyspnoea and fatigue were measured before and after the test using the Borg scale, as were saturations from a pulse oximeter. Data were analysed using unpaired t-tests, Mann–Whitney U, χ2 tests and multiple regression techniques. Results 115 patients had a 6MWD assessment, 19 (17%) had IHD (Abstract P47 table 1). COPD patients with IHD had a lower mean ± SD 6MWD than those without (310±138 vs 354±107 m) although this was not statistically significant (p=0.119). Following adjustment for age, gender, FEV1 % predicted, BMI and smoking pack year history, IHD was found to be independently related with a 66 m reduction in 6MWD (95% CI 5 to 127 m), p=0.035. Median (IQR) dyspnoea on the Borg scale before the test was not higher in those with IHD (1(1,2) vs 1(0,3), p=0.135), this increased more in those with IHD compared to those without during the test (2(1,3) vs 1(0,3), p=0.043). Fatigue measured on the Borg scale was higher at the start of the test in those with IHD (1.5(0,3) vs 0(0,2), p=0.038), however, the increase after the test was not different between the groups (0(0,2) vs 0(0,2), p=0.831). The mean ± SD pre-test oxygen saturations and post-test change were similar in those with and without IHD (93.8±2.6% vs 94.1±2.4%, p=0.684; −0.9±4.4% vs −1.4±3.2%, p=0.595).Abstract P47 Table 1 Clinical characteristics of COPD patients with and without comorbid ischaemic heart disease (IHD) All COPD patients (n=115) COPD without IHD† (n=96) COPD with IHD‡ (n=19) p Value† vs ‡ Age (years) 69.7±8.7 68.9±8.9 74.0±5.9 0.019 Male gender 67% 65% 79% 0.224 FEV1 (% predicted) 51.9±18.6 52.3±19.2 49.5±15.5 0.550 FEV1 (L) 1.36±0.61 1.37±0.64 1.29±0.46 0.583 BMI (kg/m2) 26.8±5.8 27.1±6.0 25.8±4.8 0.390 Current smoker 28% 28% 26% 0.872 Smoking (pack years) 46 (30,72) 44 (30,72) 57 (40,79) 0.110 Data are presented as percentage, mean ± SD or median (IQR) as appropriate. Conclusions Comorbid IHD is independently associated with a clinically significant lower exercise capacity in COPD patients. Such patients may have a higher level of fatigue before exercise and develop more dyspnoea during exercise. Such patients may be an appropriate target for further intervention such as tailored pulmonary rehabilitation.

S18 A comparison of prevalence and load of airway bacteria in COPD patients with paired stable and exacerbation state samples

Introduction Airway bacterial infections are associated with COPD exacerbations. The most frequently identified bacteria in COPD are Haemophilus influenzae (HI), Moraxella catarrhalis (MC) and Streptococcus pneumoniae (SP) (Wilkinson et al, 2006), though studies have used culture techniques, with little data available on PCR methodology in airway infection. Using the London COPD cohort, we aimed to assess and quantify bacterial prevalence and load via quantitative PCR, in paired baseline and exacerbation sputum samples. Methods Quantitative PCR was utilised, measuring prevalence and load on paired baseline and exacerbation samples, with baseline samples obtained within 1 year prior to its paired exacerbation. SP, HI and MC gene targets were Spn9082; Haemophilus influenzae P4 lipoprotein gene; copB outer-membrane-protein gene, respectively. The baseline state was defined as being at least 6 weeks after, and 2 weeks before, an exacerbation. Exacerbation was defined as two consecutive days of at least two increased symptoms (Anthonisen criteria), at least one of which is a major symptom (dyspnoea; sputum purulence; sputum volume). Results Sixty-nine paired baseline and exacerbation sputum samples were obtained from 56 patients: mean (±SD) age 71.0 years (±8.4); predicted FEV1 46.4% (±17.0); male gender 60.4%; current smoker 30.2%. Bacteria were detected at significantly higher rate at exacerbation, being seen in 36/69 (52.2%) exacerbations, and 19/69 (27.5%) baseline samples (χ2-test; p=0.003). Mean bacterial load was significantly higher at exacerbation, with mean load of 8.3 (±1.1) log10 cfu/ml, compared with mean of 7.3 (±1.8) log10 cfu/ml at baseline (paired-samples t test; p<0.001), indicating a 10-fold overall-load increase at exacerbation. MC frequency increased significantly from 4.3% (3/69) at baseline to 17.4% (12/69) at exacerbation (p=0.014). Prevalence of HI (17.4% vs 26.1%) and SP (8.7% vs 20.3%) showed non-significant increases. Mean loads of SP and MC increased significantly from baseline to exacerbation (p=0.048; p=0.008, respectively). Conclusion Prevalence and load of airway bacteria in COPD increases from baseline to exacerbation. This confirms that bacteria play an important role in exacerbation aetiology, implicating increasing bacterial load as a key underlying mechanism, and emphasises the importance of prompt antibiotic therapy at COPD exacerbation.Abstract S18 Figure 1 Bacterial load at baseline and exacerbation of COPD as determined by quantitative PCR.

P110 Quantitative PCR-based detection and quantification of atypical bacteria at baseline and exacerbation of COPD

Introduction Airway bacterial infections are associated with exacerbations of COPD. The potential role of atypical bacteria as a trigger for exacerbations is not well understood. Atypical bacteria such as Chlamydophila pneumoniae (CP), Legionella pneumophila (LP) and Mycoplasma pneumoniae (MP) are difficult to culture as they are intracellular pathogens. LP can be detected by urinary antigen, and serology can be performed for MP, but these techniques give no indication as to the atypical bacterial load. Quantitative PCR (qPCR) offers an alternative approach to identification and quantification of bacteria in sputum. Methods Multiplex qPCR was used to detect and quantify CP, LP and MP in 238 samples prospectively collected from 87 patients in the London COPD Cohort: mean (±SD) age 71.4 (±8.1); predicted FEV1 43.4% (±17.5%); male gender 47.9%; current smoker 49.2%. Baseline (n=104), exacerbation (n=95), and follow-up (n=39) samples were tested: Baseline was defined as at least 6-weeks without exacerbation, and exacerbation was defined as 2 consecutive days of two symptoms (Anthonisen criteria), at least one of which is a major symptom (dyspnoea; sputum purulence; sputum volume). Follow-up involved taking samples 2 or 5 weeks post-exacerbation onset. Using a qPCR developed by our clinical diagnostic service, the CP, MP and LP gene targets were RNA-polymerase β-chain; P1 adhesin protein; and MIP respectively. Routine microbiological analysis was also performed on these samples. Results No samples were positive for the atypical organisms using culture. With qPCR analysis 6/238 samples (six separate patients) were positive for LP (2.5%), four at baseline and two at exacerbation/follow-up. One baseline sample was positive for MP (0.42%), and no samples were positive for CP. Atypical bacteria were present at 0.83% of exacerbations. Median (IQR) bacterial load was 4.3x104 cfu ml−1 (2.0x104–8.55x104) for LP PCR-positive samples; the MP-positive sample load was 2.64x107 cfu ml−1. Conclusion Quantitative PCR was more sensitive and informative than standard microbiological culture for the detection of atypical bacteria. Atypical bacteria in sputum were detected at very few exacerbations of COPD; moreover, when they were detected by qPCR, the load was low, indicating little or no significance in the aetiology of these events.