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Background In response to rising TB notification rates in England, universal strain typing was introduced in 2010. We evaluated the acceptability, effectiveness and cost-effectiveness of the TB strain typing service (TB-STS). Methods We conducted a mixed-methods evaluation using routine laboratory, clinic and public health data. We estimated the effect of the TB-STS on detection of false positive Mycobacterium tuberculosis diagnoses (2010–2012); contact tracing yield (number of infections or active disease per pulmonary TB case); and diagnostic delay. We developed a deterministic age-structured compartmental model to explore the effectiveness of the TB-STS, which informed a cost-effectiveness analysis. Results Semi-structured interviews explored user experience. Strain typing identified 17 additional false positive diagnoses. The TB-STS had no significant effect on contact tracing yield or diagnostic delay. Mathematical modelling suggested increasing the proportion of infections detected would have little value in reducing TB incidence in the white UK-born population. However, in the non-white UK-born and non-UK-born populations, over 20 years, if detection of latent infection increases from 3% to 13% per year, then TB incidence would decrease by 11%; reducing diagnostic delay by one week could lead to 25% reduction in incidence. The current TB-STS was not predicted to be cost-effective over 20 years (£95 628/quality-adjusted life-years). Interviews found people had mixed experiences, but identified broader benefits, of the TB-STS. Conclusions To reduce costs, improve efficiency and increase effectiveness, we recommend changes to the TB-STS, including discontinuing routine cluster investigations and focusing on reducing diagnostic delay across the TB programme. This evaluation of a complex intervention informs the future of strain typing in the era of rapidly advancing technologies.

Background Long-term antibiotic therapy is used to prevent exacerbations of COPD but there is uncertainty over whether this reduces airway bacteria. The optimum antibiotic choice remains unknown. We conducted an exploratory trial in stable patients with COPD comparing three antibiotic regimens against placebo. Methods This was a single-centre, single-blind, randomised placebo-controlled trial. Patients aged ≥45 years with COPD, FEV1

Introduction and objectives Drug-induced hepatotoxicity is a common complication of tuberculosis treatment.1 Guidelines based on expert opinion are available, but the natural history of liver enzyme measurements over the course of treatment and the most effective approach to monitoring on treatment remains unclear. We investigated the pattern of liver enzyme levels in the REMoxTB trial to describe the magnitude and timing of elevations, along with factors that could influence enzyme patterns, and related this to liver function monitoring. Methods Patients received either standard tuberculosis treatment (2EHRZ/4HR), or a four-month regimen with moxifloxacin substituted for ethambutol (isoniazid arm, 2MHRZ/2MHR) or isoniazid (ethambutol arm, 2EMRZ/2MR). Liver function tests were performed at weeks 0, 2, 4, 8, 12, 17, and during adverse events. The Chi Square or Fisher’s exact test was used for testing proportions among groups, log rank test for comparison of time, and Students t test for comparison of means. Results 639 patients were allocated to receive standard therapy as controls, 654 to the isoniazid arm, and 635 to the ethambutol arm (see Table 1). 60 patients (9.4%) taking standard therapy developed a peak ALT/AST ≥ 3 × ULN at median time 28 days (IQR 14–56). The mean difference in time to reach peak ALT was 7 days between isoniazid-containing regimens and the ethambutol arm, and a higher proportion of Asian patients elevated ALT/AST ≥ 3 × ULN in isoniazid-containing arms (51.0% vs 26.3%, (p Discussion Monitoring liver function routinely for the first two months of HRZE therapy would have detected approximately 75% of patients with a peak enzyme elevation of ≥3 × ULN, and we would recommend this as a standard of care based on these results. However, there is reassurance that over 90% of patients completed therapy without an ALT/AST result ≥3 xULN. HIV positive and Asian patients were at higher risk of liver enzyme elevation and there was a shorter time to peak ALT/AST for those receiving isoniazid. Reference Thompson N, Caplin M, Hamilton M, et al. Anti-tuberculosis medication and the liver: dangers and recommendations in management. Eur Respir J 1995;8:1384–1388.

Background: Immunological ex vivo assays to diagnose tuberculosis (TB) have great potential but have largely been blood-based and poorly evaluated in active TB. Lung sampling enables combined microbiological and immunological testing and uses higher frequency antigen-specific responses than in blood. Methods: A prospective evaluation was undertaken of a flow cytometric assay measuring the percentage of interferon-γ synthetic CD4+ lymphocytes following stimulation with purified protein derivative of Mycobacterium tuberculosis (PPD) in bronchoalveolar lavage fluid from 250 sputum smear-negative individuals with possible TB. A positive assay was defined as >1.5%. Results: Of those who underwent lavage and were diagnosed with active TB, 95% (106/111) had a positive immunoassay (95% CI 89% to 98%). In 139 individuals deemed not to have active TB, 105 (76%) were immunoassay negative (95% CI 68% to 82%). Of the remaining 24% (34 cases) with a positive immunoassay, a substantial proportion had evidence of untreated TB; in two of these active TB was subsequently diagnosed. Assay performance was unaffected by HIV status, disease site or BCG vaccination. In culture-positive pulmonary cases, response to PPD was more sensitive than nucleic acid amplification testing (94% vs 73%). The use of early secretory antigen target-6 (ESAT-6) responses in 71 subjects was no better than PPD, and 19% of those with culture-confirmed TB and a positive PPD immunoassay had no detectable response to ESAT-6. Conclusions: These findings suggest that lung-orientated immunological investigation is a potentially powerful tool in diagnosing individuals with sputum smear-negative active TB, regardless of HIV serostatus.

Background: Lower respiratory tract infections (LRTI) are a common reason for consulting general practitioners (GPs). In most cases the aetiology is unknown, yet most result in an antibiotic prescription. The aetiology of LRTI was investigated in a prospective controlled study. Methods: Eighty adults presenting to GPs with acute LRTI were recruited together with 49 controls over 12 months. Throat swabs, nasal aspirates (patients and controls), and sputum (patients) were obtained and polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR) assays were used to detect Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, influenza viruses (AH1, AH3 and B), parainfluenza viruses 1–3, coronaviruses, respiratory syncytial virus, adenoviruses, rhinoviruses, and enteroviruses. Standard sputum bacteriology was also performed. Outcome was recorded at a follow up visit. Results: Potential pathogens were identified in 55 patients with LRTI (69%) and seven controls (14%; p

Background: Tuberculosis notification rates for London have risen dramatically in recent years. Molecular typing of Mycobacterium tuberculosis has contributed to our understanding of the epidemiology of tuberculosis throughout the world. This study aimed to assess the degree of recent transmission of M tuberculosis in London and subpopulations of the community with high rates of recent transmission. Methods: M tuberculosis isolates from all persons from Greater London diagnosed with culture positive tuberculosis between 1 July 1995 and 31 December 1997 were genetically fingerprinted using IS 6110 restriction fragment length polymorphism (RFLP) typing. A structured proforma was used during record review of cases of culture confirmed tuberculosis. Cluster analysis was performed and risk factors for clustering were examined in a univariate analysis followed by a logistic regression analysis with membership of a cluster as the outcome variable. Results: RFLP patterns were obtained for 2042 isolates with more than four copies of IS 6110 ; 463 (22.7%) belonged to 169 molecular clusters, which ranged in size from two (65% of clusters) to 12 persons. The estimated rate of recent transmission was 14.4%. Young age (0–19 years) (odds ratio (OR) 2.65, 95% confidence interval (CI) 1.59 to 4.44), birth in the UK (OR 1.55, 95% CI 1.04 to 2.03), black Caribbean ethnic group (OR 2.19, 95% CI 1.15 to 4.16), alcohol dependence (OR 2.33, 95% CI 1.46 to 3.72), and streptomycin resistance (OR 1.82, 95% CI 1.15 to 2.88) were independently associated with an increased risk of clustering. Conclusions: Tuberculosis in London is largely caused by reactivation or importation of infection by recent immigrants. Newly acquired infection is also common among people with recognised risk factors. Preventative interventions and early diagnosis of immigrants from areas with a high incidence of tuberculosis, together with thorough contact tracing and monitoring of treatment outcome among all cases of tuberculosis (especially in groups at higher risk of recent infection), remains most important.

Introduction and objectives The current standard treatment for pulmonary tuberculosis (TB) has been in use for several decades and the major risks associated with each of the four drugs (HRZE) are well recognised. However, large prospective trials with regular review and documentation of adverse events while taking HRZE are lacking. We used the incidence of grade 3 and 4 adverse events (AEs) and serious adverse events (SAEs) in patients taking HRZE in the REMoxTB trial to investigate the overall tolerability of the regimen. Methods Grade 3 or 4 AEs and SAEs (of any grade) for patients taking standard TB therapy were analysed. Events were labelled as occurring in the intensive phase, continuation phase or in follow-up (up to 18 months after enrolment). ANOVA and chi-square testing was used to test for significant differences in the incidence of events across the treatment phases. Logistic regression was used to investigate associations between baseline characteristics and on-treatment SAEs and withdrawal from treatment, death or relapse/treatment failure. Results 201 (31.5%) of 639 patients taking standard therapy experienced grade 3/4 AEs or SAEs during treatment. AEs, SAEs, and withdrawals from treatment occurred most frequently in the intensive phase (see Table). Of 116 SAEs reported 84 (72.4%) improved or resolved and were most commonly respiratory (16.4%), gastrointestinal (6.9%), and infection (5.2%) related. There were 10 deaths in follow-up due to suicide, trauma, TB relapse, and acute illness. Logistic regression detected a significant association between on-treatment SAEs and withdrawal (p Discussion AEs and SAEs were predominantly reported in the intensive phase, probably due to a combination of TB and effects of medication. However most deaths occurred in follow-up and were unrelated, emphasising the impact that social circumstances have on TB patients. The lack of significant association between SAEs during treatment and relapse/treatment failure provides reassurance that a complicated treatment period can end with successful treatment of TB. The association between SAEs on treatment and lower weights at baseline and HIV infection reinforces the need to monitor these patients closely.

Background The pan London “Find andTreat” tuberculosis (TB) service includes a mobile digital X-ray unit (MXU) screening service which has been shown to be cost effective among hard-to-reach groups (homeless people, substance users and prisoners). This study compared the impact of current practice of hostel staff encouraging MXU screening for TB among homeless people with the addition of peer educators with direct experience of TB and/or homelessness on screening uptake. Design/methods Between February 2012 and October 2013 London homeless hostels accessing MXU TB screening were randomised to intervention or control arms by minimisation, balancing on hostel size (≤43 beds), and previous screening uptake level (≤50%). Sites with a previous MXU uptake of >80% were excluded. At intervention sites, peers worked with hostel staff to encourage screening (through contacting and speaking with residents). The primary outcome was the proportion of eligible residents screened for TB. Blinding of participants and observers was not possible due to the nature of the intervention. Analysis was performed by intention to treat (ITT). Results Of 59 hostels considered for eligibility, 46 were randomised (24 allocated to the control and 22 to the intervention arm – Figure 1). Across all sites, median uptake was 44% (IQR: 26,59). Control hostels had a total of 1192 residents [median uptake 45%, (IQR: 33,55)]. Intervention sites had 1150 residents [median uptake 40%, (IQR: 25,61)]. Using Poisson regression to account for the clustered study design, size of hostel, and previous screening uptake, there was no evidence for peer educators increasing uptake of screening – adjusted risk ratio 0.98% (95% CIs: 0.80,1.20). Conclusion This study found no evidence for peer educators increasing the uptake of MXU TB screening. The wide confidence intervals associated with this result mean that we may be missing a 20% effect, which could be explained by a pragmatic study design where sites were not naive to the intervention as peers had previously been involved in screening sessions at many hostels. This study was supported by NIHR Programme Grant for Applied Research (RP-PG-0407–10340). Trial registration number: ISRCTN17270334.

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

In patients receiving treatment for pulmonary tuberculosis (TB), change in sputum culture from positive to negative is the principal outcome measure of a therapeutic response in both clinical practice1 2 and drug trials.3 Patients will often stop producing sputum early in the course of treatment.4 We have tested the assumption that “no sputum” means that the patient is “culture negative”, as this has never been confirmed experimentally. We prospectively followed 16 patients with newly diagnosed pulmonary TB. Sputum samples were collected at diagnosis, during weeks 1 and 2, at months 1, 2 and 4, and on completing treatment. Those patients who were not producing sputum spontaneously had specimens collected by induction. This was performed in a purpose-built negative pressure isolation chamber (Elwyn E Roberts Isolators, Shropshire, UK) where …

Background There are high rates of active tuberculosis (TB) in London’s hard to reach groups (homeless people, substance misusers and prisoners). However no systematic data are available regarding the prevalence of latent TB infection (LTBI) and blood borne viruses (BBV) - HIV, Hepatitis B and C (HBV, HCV). Hard to reach groups often present late for healthcare. We undertook a TB/BBV screening programme to assess the prevalence of LTBI and co-infection with BBVs within homeless hostels, drug services and a prison in London. We also investigated management outcomes in those referred on to healthcare services. Design/method Recruitment took place from May 2011–June 2013. Service users screened for TB on a mobile chest x-ray unit and in prison using the static digital x-ray machine were approached and, with consent, blood was drawn for IGRA (Quantiferon In-Tube) and HIV, HCV and HBV. Results were provided to participants with onward referral to healthcare services in line with current guidance. Treatment outcomes were collected via telephone follow up one year post referral for the positive cases. Results Prison (n = 511) (Table 1) LTBI: 65(13%) participants were IGRA positive [3(5%) co-infected with HCV]. Of these, 37(57%) were referred for preventive treatment, 16(43%) did not attend (DNA) appointments and were discharged or were lost to follow up (LFU). Of the 15 who commenced treatment, 9(60%) completed treatment. HCV: 22 participants were positive and referred, of which 11(50%) DNA/LFU, 11(50%) were under review and none commenced treatment. HBV: 10 participants were positive, of which 6(60%) DNA/LFU, 4(40%) were under review and none commenced treatment. There were no HIV positives. Community (N=696) (Table 1) LTBI: 116(17%) participants were IGRA positive [22(19%) co-infected with HCV]. Of the 14(12­%) referred to services, 8(57%) DNA/LFU and 3(21%) commenced treatment (2 completed). HCV: 84 were positive and referred (8 new diagnosis), 42(50%) DNA/LFU/declined referral, and 37(44%) under review. Five (6%) commenced treatment, one completed. HBV: 9 participants had current infection, none started treatment; 6 were under review and 3 DNA/LFU. HIV: 9 participants were positive and known to healthcare services. Conclusion The prevalence of LTBI, BBVs and co-infection within the hard to reach group is high compared to the general population with fewer patients starting TB prophylaxis/HCV treatment. The high overall rates of DNA/LFU (47%) seen indicate that current approaches to onward referral and retention in care for this group appear to be poor and effective measures to improve engagement with clinical services are essential. This study was supported by NIHR Programme Grant for Applied Research (RP-PG-0407–10340).

Background Chest radiographs are commonly used for the diagnosis of tuberculosis and to assess the extent of disease. A relationship between the extent of disease as determined by smear grade and cavitation has been shown to predict 2-month smear results but little has been done to determine whether radiological severity reflects the bacterial burden at diagnosis. Design/methods Pre-treatment chest X-rays from 1837 subjects with smear positive pulmonary tuberculosis enrolled into the REMoxTB trial were reviewed retrospectively. Two clinicians blinded to clinical details using the Ralph et al scoring system 1 (comprised of the percentage of affected lung field and presence of cavitation) performed separate readings. An independent reader (a radiologist) reviewed discrepant results for quality assessment and cavity presence. The final cavity assessment was combined with the average percentage affected value to give a severity score (maximum score 100% + 40 if any cavitation). The Ralph score was compared to the time to positivity (TTP) of liquid cultures as measured by MGIT 960. The association between CXR severity score and time to positivity (log scale) was evaluated using Spearman’s rank correlation coefficient. A Welch t-test was used to compare TTP of those with high and low disease extent and those with and without cavitation. Results Matching sets of data were available for 1422 subjects. The median severity score was 53.75/140 (IQR 32.03–66.25) and median time to culture positivity 117 h (4.88 days). CXR severity score was weakly correlated with time to positivity (Spearman’s correlation -0.20, p Conclusions The radiological severity of pulmonary tuberculosis at diagnosis is weakly correlated with bacterial load as measured by TTP. This suggests that, in addition to bacterial burden, other factors such as immune response influence radiological appearances. References Ralph AP, Ardian M, Wiguna A, et al . A simple, valid, numerical score for grading chest X-ray severity in adult smear-positive pulmonary tuberculosis. Thorax . 2010;65:863–9

Since the mid 1980s the number of notified cases of TB in the UK has continued to rise with the largest increases noted in London and inner city areas.1 King George Hospital in Goodmayes, Essex provides clinical services to a population of approximately 230 000; 17% are non-white subjects including immigrants from countries with high rates of M tuberculosis infection and drug resistance. From September 1996 to July 1997 47 adult cases of culture proven TB were identified including seven with drug resistant isolates. None was identified by contact tracing. A previous TB audit of African born patients revealed …

Non-coding RNA molecules, particularly miRNA, regulate translation of mRNA and have been found to target expression of genes important in immune response, such as IFN-γ. Differences in blood transcriptome signatures and differential miRNA expression have been reported to discriminate between uninfected, active and latent tuberculosis. We analysed the ability of small RNA molecules (0–150 nucleotides) in blood plasma to act as biomarkers of tuberculosis treatment response. Total blood plasma small RNA (0–150 nucleotides) concentration was significantly higher (p -1 plasma, range 93–1603pg μL -1 ) than at the end of therapy at week 24 (median 86pg μL -1 plasma, range 16–1098pg μL -1 ). Expression analysis of small RNA genes revealed that, in 5 tuberculosis-infected HIV-1 negative individuals, 36 of 90 genes (> 2-fold, p 2 fold in active tuberculosis. Co-infection with HIV-1 was not found to change the expression of these six tested biomarkers.

Paradoxical reaction (PR) in tuberculosis (TB) is defined as transient worsening of symptoms and signs or the appearance of new lesions after beginning appropriate anti-tuberculosis chemotherapy. Recent studies suggest that PR occurs in 10–35% of patients. It is more common and more severe in HIV co-infected individuals with disseminated disease.1 PR is thought to be an immune mediated phenomenon but the reasons for its occurrence are unknown.1 Infection by Mycobacterium tuberculosis (MTB) results in highly variable outcomes between individuals. The characterisation of MTB strains by molecular typing techniques suggests this may be a reflection of the infecting organism, as well as host response and environmental factors. MTB strains with distinct genotypes have been shown to evoke different immunopathological events in mouse models2 and variable clinical manifestations …

Background Urban homeless people have high levels of disease and often present late for healthcare. Despite high rates of active TB in London’s large homeless population, limited data are available regarding the prevalence of latent TB infection (LTBI) and blood borne viruses (BBV) - HIV, Hepatitis B & C. We have undertaken a TB/BBV screening programme to assess the prevalence of LTBI, infection with BBV and co-infection within hard to reach groups (homeless people and substance misusers) in homeless hostels and residential drug services in London. Method Residents screened for TB on a mobile chest x-ray unit were approached and with consent, blood was drawn for TB IGRA (Quantiferon In-Tube) and BBV. Results were fed back to participants with onward referral as necessary. Results Of 413 eligible participants, 390 (94%) reported a history of homelessness. Of these 390 participants, 89% were male, 68% were 16–49 years of age and 66% UK born. 17% were IGRA positive, 1% HIV positive (all previously known), and 10% had current and 4% past Hepatitis C. 1% of those screened had current Hepatitis B infection, 10% past infection, 18% had vaccine induced protective levels of immunity and 71% had insufficient or no Hepatitis B immunity. 29% of subjects with Hepatitis C were LTBI co-infected. Multivariate analysis identified increasing age e.g. 30–49 age group (odd ratio [OR], 2.15; 95% confidence interval [CI95], 0.84–5.49) compared to the under 30, foreign birth (OR, 6.59; CI95, 3.50–12.39), smoking hard drugs (OR, 2.19; CI95, 1.02–4.64), and injecting hard drug (OR, 2.36; CI95, 1.08–5.16) such as heroin, crack or cocaine (although 95% of injectors also smoked hard drugs) as risk factors for LTBI. Injecting drug use was the only factor associated with increased risk for Hepatitis C infection (OR, 19.62; CI95, 8.23–46). Conclusion Extremely high rates of LTBI, Hepatitis C and co-infection are present in our urban study population. Despite targeted Hepatitis B vaccination programmes, a high proportion of participants appear unvaccinated. These levels of unmet need have major implications for public and personal healthcare planning and should be recognised through appropriate targeted health and social policy.

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.

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 FEV 1 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 (χ 2 -test; p=0.003). Mean bacterial load was significantly higher at exacerbation, with mean load of 8.3 (±1.1) log 10 cfu/ml, compared with mean of 7.3 (±1.8) log 10 cfu/ml at baseline (paired-samples t test; p 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.

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 FEV 1 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 4.3x10 4 cfu ml −1 (2.0x10 4 –8.55x10 4 ) for LP PCR-positive samples; the MP-positive sample load was 2.64x10 7 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.