Davinder Garcha

Changes in prevalence and load of airway bacteria using quantitative PCR in stable and exacerbated COPD

Background Prevalence and load of airway bacteria in stable and exacerbated chronic obstructive pulmonary disease (COPD) has been previously studied using microbiological culture. Molecular techniques, such as quantitative PCR (qPCR), may be more informative. Methods In this study, 373 sputum samples from 134 COPD outpatients were assessed for prevalence and load of typical airway bacteria (Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis) by multiplex qPCR, with 176 samples analysed for atypical bacteria. Paired stable and exacerbation typical bacteria data were compared in 52 patients. We compared routine culture with qPCR in 177/373 samples. Results Typical bacteria were more prevalent in exacerbation than stable-state paired samples: 30/52 (57.7%) vs. 14/52 (26.9%); p=0.001. In patients who were bacteria-positive at both time points, mean (±1 SEM) load was significantly higher at exacerbation than stable state (108.5(±0.3) vs. 107.2(±0.5) cfu/ml), constituting a 20-fold increase (p=0.011). qPCR was more discriminatory at detecting typical bacteria than microbiological culture (prevalence 59.3% vs. 24.3%; p<0.001). At stable state, higher airway bacterial load correlated with more severe airflow limitation (FEV1%predicted) (r=−0.299; p=0.033) and higher inhaled corticosteroid dosage (r=0.382; p=0.008). Mean C-reactive protein was higher in bacterial-associated exacerbations (35.0 Vs 25.1 mg/L; p=0.032). Conclusions Airway bacterial prevalence and load increase at COPD exacerbations and are an aetiological factor. qPCR is more discriminatory than culture, identifying higher airway bacterial prevalence. Exacerbations associated with bacterial detection showed a higher mean C-reactive protein level. In the stable state, airway bacterial load is related to more severe airflow limitation and higher inhaled corticosteroid dosage used.

Cardiovascular Risk, Myocardial Injury, and Exacerbations of Chronic Obstructive Pulmonary Disease

RATIONALE Patients with chronic obstructive pulmonary disease (COPD) have elevated cardiovascular risk, and myocardial injury is common during severe exacerbations. Little is known about the prevalence, magnitude, and underlying mechanisms of cardiovascular risk in community-treated exacerbations. OBJECTIVES To investigate how COPD exacerbations and exacerbation frequency impact cardiovascular risk and myocardial injury, and whether this is related to airway infection and inflammation. METHODS We prospectively measured arterial stiffness (aortic pulse wave velocity [aPWV]) and cardiac biomarkers in 98 patients with stable COPD. Fifty-five patients had paired stable and exacerbation assessments, repeated at Days 3, 7, 14, and 35 during recovery. Airway infection was identified using polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS COPD exacerbation frequency was related to stable-state arterial stiffness (rho = 0.209; P = 0.040). Frequent exacerbators had greater aPWV than infrequent exacerbators (mean ± SD aPWV, 11.4 ± 2.1 vs. 10.3 ± 2.0 ms(-1); P = 0.025). Arterial stiffness rose by an average of 1.2 ms(-1) (11.1%) from stable state to exacerbation (n = 55) and fell slowly during recovery. In those with airway infection at exacerbation (n = 24) this rise was greater (1.4 ± 1.6 vs. 0.7 ± 1.3 ms(-1); P = 0.048); prolonged; and related to sputum IL-6 (rho = 0.753; P < 0.001). Increases in cardiac biomarkers at exacerbation were higher in those with ischemic heart disease (n = 12) than those without (n = 43) (mean ± SD increase in troponin T, 0.011 ± 0.009 vs. 0.003 ± 0.006 μg/L, P = 0.003; N-terminal pro-brain natriuretic peptide, 38.1 ± 37.7 vs. 5.9 ± 12.3 pg/ml, P < 0.001). CONCLUSIONS Frequent COPD exacerbators have greater arterial stiffness than infrequent exacerbators. Arterial stiffness rises acutely during COPD exacerbations, particularly with airway infection. Increases in arterial stiffness are related to inflammation, and are slow to recover. Myocardial injury is common and clinically significant during COPD exacerbations, particularly in those with underlying ischemic heart disease.

Human rhinovirus infection during naturally occurring COPD exacerbations

Human rhinovirus (HRV) infection is an important trigger of exacerbations of chronic obstructive pulmonary disease (COPD) but its role in determining exacerbation frequency phenotype or the time-course of HRV infection in naturally occurring exacerbations is unknown. Sputum samples from 77 patients were analysed by real-time quantitative PCR for both HRV (388 samples), and Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis (89 samples). Patients recorded worsening of respiratory symptoms on daily diary cards, from which exacerbations were identified. HRV prevalence and load at exacerbation presentation were significantly higher than in the stable state (prevalence 53.3% versus 17.2%, respectively; p<0.001) but 0% by day 35 post-exacerbation. HRV load was higher in patients with cold symptoms (p=0.046) or sore throats (p=0.006) than those without. 73% of bacterium-negative but HRV-positive exacerbations were bacterium-positive by day 14. Patients with HRV detected at exacerbation had a higher exacerbation frequency (interquartile range) of 3.01 (2.02–5.30) per year compared with patients without HRV (2.51 (2.00–3.51)) (p=0.038). HRV prevalence and load increased at COPD exacerbation, and resolved during recovery. Frequent exacerbators were more likely to experience HRV infection. Secondary bacterial infection is common after HRV infection, and provides a possible mechanism for exacerbation recurrence and a potential target for novel therapies. Increased HRV prevalence and load are aetiological factors in natural COPD exacerbations and frequent exacerbations http://ow.ly/tlscz

Inflammatory thresholds and the species-specific effects of colonising bacteria in stable chronic obstructive pulmonary disease

BackgroundThere has been increasing interest in the use of newer, culture-independent techniques to study the airway microbiome of COPD patients. We investigated the relationships between the three common potentially pathogenic microorganisms (PPMs) Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, as detected by quantitative PCR (qPCR), and inflammation and health status in stable patients in the London COPD cohort.MethodsWe prospectively collected sputum, serum and plasma samples for analysis of airway bacterial presence and load, and airway and systemic inflammation from 99 stable COPD patients between January 2011 and October 2012. Health status was measured with St George’s Respiratory Questionnaire and COPD Assessment Test.ResultsAirway inflammation and plasma fibrinogen, but not C-reactive protein, were greater in samples with PPM detection (p < 0.001, p = 0.049 and p = 0.261, respectively). Increasing total bacterial load was associated with increasing airway (p < 0.01) but not systemic inflammation (p > 0.05). Samples with high total bacterial loads had significantly higher airway inflammation than both samples without PPM detection and those with lower loads. Haemophilus influenzae presence was associated with significantly higher levels of airway but not systemic inflammation for all given pathogen loads (p < 0.05), and was significantly greater than with other PPMs. No association was observed between inflammation and health status (p > 0.05).ConclusionsAirway and systemic inflammation, as measured by fibrinogen, is greater in stable COPD patients with PPMs detected using the culture-independent qPCR technique. The airway, but not systemic inflammatory response, appears to have a total pathogen-load threshold and appears attributable to Haemophilus influenzae. However, discordance between inflammation and health status was observed.

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.

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

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.

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.