Sarah Thurston

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.

Blood and sputum eosinophils in COPD; relationship with bacterial load

BackgroundSputum and blood eosinophil counts predict corticosteroid effects in COPD patients. Bacterial infection causes increased airway neutrophilic inflammation. The relationship of eosinophil counts with airway bacterial load in COPD patients is uncertain. We tested the hypothesis that bacterial load and eosinophil counts are inversely related.MethodsCOPD patients were seen at stable state and exacerbation onset. Sputum was processed for quantitative polymerase chain reaction detection of the potentially pathogenic microorganisms (PPM) H. influenzae, M. catarrhalis and S. pneumoniae. PPM positive was defined as total load ≥1 × 104copies/ml. Sputum and whole blood were analysed for differential cell counts.ResultsAt baseline, bacterial counts were not related to blood eosinophils, but sputum eosinophil % was significantly lower in patients with PPM positive compared to PPM negative samples (medians: 0.5% vs. 1.25% respectively, p = 0.01). Patients with PPM positive samples during an exacerbation had significantly lower blood eosinophil counts at exacerbation compared to baseline (medians: 0.17 × 109/L vs. 0.23 × 109/L respectively, p = 0.008), while no blood eosinophil change was observed with PPM negative samples.ConclusionsThese findings indicate an inverse relationship between bacterial infection and eosinophil counts. Bacterial infection may influence corticosteroid responsiveness by altering the profile of neutrophilic and eosinophilic inflammation.

Streptococcus pseudopneumoniae Identification by Pherotype: a Method To Assist Understanding of a Potentially Emerging or Overlooked Pathogen

ABSTRACT The recent identification of Streptococcus pseudopneumoniae (pseudopneumococcus) has complicated classification schemes within members of the “mitis” streptococcal group. Accurate differentiation of this species is necessary for understanding its disease potential and identification in clinical settings. This work described the use of the competence-stimulatory peptide ComC sequence for identification of S. pseudopneumoniae. ComC sequences from clinical sources were determined for 17 strains of S. pseudopneumoniae, Streptococcus pneumoniae, and Streptococcus oralis. An additional 58 ComC sequences from a range of sources were included to understand the diversity and suitability of this protein as a diagnostic marker for species identification. We identified three pherotypes for this species, delineated CSP6.1 (10/14, 79%), CSP6.3 (3/14, 21%), and SK674 (1/14, 7%). Pseudopneumococcal ComC sequences formed a discrete cluster within those of other oral streptococci. This suggests that the comC sequence could be used to identify S. pseudopneumoniae, thus simplifying the study of the pathogenic potential of this organism. To avoid confusion between pneumococcal and pseudopneumococcal pherotypes, we have renamed the competence pherotype CSP6.1, formerly reported as an “atypical” pneumococcus, CSPps1 to reflect its occurrence in S. pseudopneumoniae.

Airway bacteria and respiratory symptoms are common in ambulatory HIV-positive UK adults

The widespread use of antiretroviral therapy (ART) and Pneumocystis pneumonia prophylaxis has led to significant declines in opportunistic infection rates in people with chronic HIV infection. Despite this, pulmonary disease is more common in these patients than HIV-negative individuals [1]. Observational studies suggest that while bacterial pneumonia is reduced by ART, it still occurs relatively frequently even after CD4 T-cell reconstitution [2, 3]. Non-recurrent HIV-associated bacterial pneumonia is 10 times more likely than in uninfected individuals [3, 4], and is now the most common infection and the leading cause of death after sepsis [5]. Airway bacterial colonisation and respiratory symptoms are common in ambulatory HIV-positive UK adults http://ow.ly/Nug5A

S24 Profile of the Airway Microbiome in COPD Using MALDI-TOF

Introduction Bacterial infections are well known to have an association with chronic obstructive pulmonary disease (COPD), with Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis often seen at exacerbation. Many microbiome studies have relied on molecular methods which can be prolonged and expensive. The use of matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) offers an efficient and economical approach to the evaluation of the airway microbiome, necessary to increase knowledge of the role of the microbial community in COPD. Aims To identify the range and diversity of bacteria which form the airway microbiome in COPD To consider whether the MALDI-TOF is an appropriate method for this study. Methods 44 patients from the London COPD cohort produced 65 sputum samples at baseline and exacerbation mean (±SD) age 73.7 years (±9.4); predicted FEV1 50.7% (±18.12); male gender 77.5%; exacerbation samples 47.7%. Sputum samples are initially sent to the NHS Royal Free Trust microbiology lab where traditional culture methods are performed. Once processed the agar plates were collected and different colonies are spotted onto target plates and loaded onto the MALDI-TOF MALDI-TOF profile spectrum is then generated allowing for data interpretation. Isolates that were defined as pathogenic were removed from the data with those classified as typical bacteria were then included separately. Results: Streptococcus, Staphylococcus, Rothia and Neisseria are the most dominant genera seen in baseline and exacerbation NRTF samples at the genus level contributing to 85, 28, 20 and 18 percent respectively. MALDI-TOF identified 25 NRTF groups at genus level in the COPD samples. In the typical pathogenic genus groups Haemophilus is the most dominant group identified by this method. Conclusion The MALDI-TOF identifies a diverse bacterial range in COPD patients including pathogenic organisms and those defined as NRTF The MALDI-TOF is quick and inexpensive, as well as efficient which is an advantage over molecular assays. Abstract S24 Figure 1

S17 Impact of COPD severity and sputum production on antibiotic resistance

Introduction Bacterial infections are a well-known trigger for exacerbations of COPD. A variety of antibiotics are regularly prescribed for this group of patients but the risk and frequency of antibiotic resistance in the COPD population is less understood. Routine culture data can be evaluated to establish resistance prevalence and patterns. Methods Culture data were collected from the sputum samples of 293 patients in the London COPD cohort over a period of 5 years (01/01/2006–31/12/2010) mean (±SD) months in study 28.4(±19.9); age 69.9 years (±8.9); predicted FEV1 47.8% (±16.5); male gender 58%; exacerbation samples 48.9%; sputum producers 77.5%. Identification of bacterial presence was established and where clinically indicated drug sensitivity tests (DSTs) were performed. A resistant sample was reported as any bacterial isolate resistant to at least one antimicrobial agent. Results 92/293 (31.4%) patients had at least one bacteria positive sample over the study period. 87/92 (94.6%) patients had samples where DSTs were performed on bacteria positive samples. Resistance was observed in 69/87 (79.3%) patients. 30/293 (10.2%) patients were resistant to all samples where DSTs were performed. 227/293 (77.5%) of patients were sputum producers. There was no significant relationship between predicted FEV1 and antibiotic resistance frequency in this cohort (X2-test; p=0.577). Patients who were classified as regular sputum producers were more likely to exhibit resistance in culture positive bacteria (p=0.048). Conclusion Results from this analysis conclude that an estimated 23.5% of COPD patients will develop resistance to an antimicrobial agent within 28.4 months of follow-up with sputum producers being at a higher risk. This study highlights the importance of investigating sputum samples with determination of resistance patterns. Information on resistance patterns and transmission of resistance in COPD can allow more appropriate and targeted antibiotic therapy for COPD exacerbations with improved outcomes.

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.