Impact of bacterial strain acquisition in the lung of patients with COPD: the AERIS study.

BACKGROUND: Bacterial infections are associated with acute exacerbations of chronic obstructive pulmonary disease (AECOPD), but the mechanism is incompletely understood. METHOD: In a COPD observational study (NCT01360398), sputum samples were collected monthly at the stable state and exacerbation. Post-hoc analyses of 1307 non-typeable Haemophilus influenzae (NTHi) isolates from 20 patients and 756 Moraxella catarrhalis isolates from 38 patients in one year of follow-up were conducted by multilocus sequence typing (MLST). All isolates came from cultured sputum samples that were analyzed for bacterial species presence, apparition (infection not detected at the preceding visit), or acquisition (first-time infection), with the first study visit as a baseline. Strain apparition or new strain acquisition was analyzed by MLST. The odds ratio (OR) of experiencing an exacerbation vs. stable state was estimated by conditional logistic regression modelling, stratified by patient. RESULTS: The culture results confirmed a significant association with exacerbation only for NTHi species presence (OR 2.28; 95% confidence interval [CI]: 1.12-4.64) and strain apparition (OR 2.38; 95% CI: 1.08-5.27). For M. catarrhalis, although confidence intervals overlapped, the association with exacerbation for first-time species acquisition (OR 5.99; 2.75-13.02) appeared stronger than species presence (OR 3.67; 2.10-6.40), new strain acquisition (OR 2.94; 1.43-6.04), species apparition (OR 4.18; 2.29-7.63), and strain apparition (OR 2.78; 1.42-5.42). This may suggest that previous M. catarrhalis colonization may modify the risk of exacerbation associated with M. catarrhalis infection. CONCLUSIONS: The results confirm that NTHi and M. catarrhalis infections are associated with AECOPD but suggest different dynamic mechanisms in triggering exacerbations.

Final Analysis of a Trial of M72/AS01E Vaccine to Prevent Tuberculosis.

BACKGROUND: Results of an earlier analysis of a trial of the M72/AS01E candidate vaccine against Mycobacterium tuberculosis showed that in infected adults, the vaccine provided 54.0% protection against active pulmonary tuberculosis disease, without evident safety concerns. We now report the results of the 3-year final analysis of efficacy, safety, and immunogenicity. METHODS: From August 2014 through November 2015, we enrolled adults 18 to 50 years of age with M. tuberculosis infection (defined by positive results on interferon-γ release assay) without evidence of active tuberculosis disease at centers in Kenya, South Africa, and Zambia. Participants were randomly assigned in a 1:1 ratio to receive two doses of either M72/AS01E or placebo, administered 1 month apart. The primary objective was to evaluate the efficacy of M72/AS01E to prevent active pulmonary tuberculosis disease according to the first case definition (bacteriologically confirmed pulmonary tuberculosis not associated with human immunodeficiency virus infection). Participants were followed for 3 years after the second dose. Participants with clinical suspicion of tuberculosis provided sputum samples for polymerase-chain-reaction assay, mycobacterial culture, or both. Humoral and cell-mediated immune responses were evaluated until month 36 in a subgroup of 300 participants. Safety was assessed in all participants who received at least one dose of M72/AS01E or placebo. RESULTS: A total of 3575 participants underwent randomization, of whom 3573 received at least one dose of M72/AS01E or placebo, and 3330 received both planned doses. Among the 3289 participants in the according-to-protocol efficacy cohort, 13 of the 1626 participants in the M72/AS01E group, as compared with 26 of the 1663 participants in the placebo group, had cases of tuberculosis that met the first case definition (incidence, 0.3 vs. 0.6 cases per 100 person-years). The vaccine efficacy at month 36 was 49.7% (90% confidence interval [CI], 12.1 to 71.2; 95% CI, 2.1 to 74.2). Among participants in the M72/AS01E group, the concentrations of M72-specific antibodies and the frequencies of M72-specific CD4+ T cells increased after the first dose and were sustained throughout the follow-up period. Serious adverse events, potential immune-mediated diseases, and deaths occurred with similar frequencies in the two groups. CONCLUSIONS: Among adults infected with M. tuberculosis, vaccination with M72/AS01E elicited an immune response and provided protection against progression to pulmonary tuberculosis disease for at least 3 years. (Funded by GlaxoSmithKline Biologicals and Aeras; ClinicalTrials.gov number, NCT01755598.).

Drivers of year-to-year variation in exacerbation frequency of COPD: analysis of the AERIS cohort.

The association between exacerbation aetiology and exacerbation frequency is poorly understood. We analysed 2-year follow-up data from a prospective observational study of patients with chronic obstructive pulmonary disease (COPD) (www.clinicaltrials.gov identifier number NCT01360398) to evaluate year-to-year variation in exacerbation frequency and related aetiology. A total of 127 patients underwent blood and sputum sampling monthly and at exacerbation to detect respiratory infections and eosinophilic inflammation; 103 continued into year 2 and 88 completed both years. The most common bacterial species at stable state and exacerbation was Haemophilus influenzae. Among infrequent exacerbators (one exacerbation per year), the incidence of viral infection at exacerbation was high (60.0% (95% CI 35.1-81.7%) in year 1 and 78.6% (53.4-94.2%) in year 2). Those with more frequent exacerbations tended to have higher relative incidence of bacterial than viral infection. Patients with at least two additional exacerbations in year 2 versus year 1 had a higher risk of H. influenzae colonisation at stable state than those with at least two fewer exacerbations, as detected by culture (OR 1.43 (95% CI 0.71-2.91) versus 0.63 (0.40-1.01), p=0.06) and PCR (1.76 (95% CI 0.88-3.51) versus 0.56 (0.37-0.86), p<0.01). This was not seen with other infection types or eosinophilic inflammation. Analysis of the same cohort over 2 years showed, for the first time, that changes in yearly COPD exacerbation rate may be associated with variations in H. influenzae colonisation.

Patients with Chronic Obstructive Pulmonary Disease harbour a variation of Haemophilus species.

H. haemolyticus is often misidentified as NTHi due to their close phylogenetic relationship. Differentiating between the two is important for correct identification and appropriate treatment of infective organism and to ensure any role of H. haemolyticus in disease is not being overlooked. Speciation however is not completely reliable by culture and PCR methods due to the loss of haemolysis by H. haemolyticus and the heterogeneity of NTHi. Haemophilus isolates from COPD as part of the AERIS study (ClinicalTrials - NCT01360398) were speciated by analysing sequence data for the presence of molecular markers. Further investigation into the genomic relationship was carried out using average nucleotide identity and phylogeny of allelic and genome alignments. Only 6.3% were identified as H. haemolyticus. Multiple in silico methods were able to distinguish H. haemolyticus from NTHi. However, no single gene target was found to be 100% accurate. A group of omp2 negative NTHi were observed to be phylogenetically divergent from H. haemolyticus and remaining NTHi. The presence of an atypical group from a geographically and disease limited set of isolates supports the theory that the heterogeneity of NTHi may provide a genetic continuum between NTHi and H. haemolyticus.

Ampicillin resistance in Haemophilus influenzae from COPD patients in the UK.

BACKGROUND: Haemophilus influenzae is commonly isolated from the airways of COPD patients. Antibiotic treatment may cause the emergence of resistant H. influenzae strains, particularly ampicillin-resistant strains, including ß-lactamase-negative ampicillin resistance (BLNAR) strains. Genetic identification using ftsI sequencing is the optimum method for identifying mutations within BLNAR strains. The prevalence of BLNAR in COPD patients during the stable state has not been reported. We investigated the antibiotic resistance patterns of H. influenzae present in the sputum of stable COPD patients, focusing on ampicillin resistance; the prevalence of enzyme and non-enzyme-mediated ampicillin resistance was determined. A subset of patients was followed up longitudinally to study H. influenzae strain switching and antibiotic sensitivity changes. PATIENTS AND METHODS: Sputum sampling was performed in 61 COPD patients, with 42 samples obtained at baseline; H. influenzae was detected by polymerase chain reaction in 28 samples. In all, 45 patients completed the follow-up for 2 years; 24 H. influenzae isolates were obtained. RESULTS: Disk diffusion showed the highest antibiotic resistance in the penicillin antibiotic group (eg, 67% for ampicillin) and macrolides (eg, 46% for erythromycin), whereas all isolates were susceptible to quinolones. Of the 16 isolates resistant to ampicillin, 9 (56%) were ß-lactamase positive. The ß-lactamase-negative isolates were further investigated; none of these fulfilled the phenotypic BLNAR classification criteria of ampicillin minimum inhibitory concentration >1 µg/mL, and only one demonstrated an ftsI mutation. Frequent H. influenzae strain switching was confirmed using multilocus sequence typing and was associated with changes in the antibiotic sensitivity pattern. CONCLUSION: We observed an overidentification of ampicillin resistance by disk diffusion. The majority of ampicillin resistance was due to enzyme production. H. influenzae strain changes during the stable state may be associated with a change in antibiotic sensitivity; this has implications for empirical antibiotic prescribing.

A prospective, observational cohort study of the seasonal dynamics of airway pathogens in the aetiology of exacerbations in COPD.

BACKGROUND: The aetiology of acute exacerbations of COPD (AECOPD) is incompletely understood. Understanding the relationship between chronic bacterial airway infection and viral exposure may explain the incidence and seasonality of these events. METHODS: In this prospective, observational cohort study (NCT01360398), patients with COPD aged 40-85 years underwent sputum sampling monthly and at exacerbation for detection of bacteria and viruses. Results are presented for subjects in the full cohort, followed for 1 year. Interactions between exacerbation occurrence and pathogens were investigated by generalised estimating equation and stratified conditional logistic regression analyses. FINDINGS: The mean exacerbation rate per patient-year was 3.04 (95% CI 2.63 to 3.50). At AECOPD, the most common bacterial species were non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis, and the most common virus was rhinovirus. Logistic regression analyses (culture bacterial detection) showed significant OR for AECOPD occurrence when M. catarrhalis was detected regardless of season (5.09 (95% CI 2.76 to 9.41)). When NTHi was detected, the increased risk of exacerbation was greater in high season (October-March, OR 3.04 (1.80 to 5.13)) than low season (OR 1.22 (0.68 to 2.22)). Bacterial and viral coinfection was more frequent at exacerbation (24.9%) than stable state (8.6%). A significant interaction was detected between NTHi and rhinovirus presence and AECOPD risk (OR 5.18 (1.92 to 13.99); p=0.031). CONCLUSIONS: AECOPD aetiology varies with season. Rises in incidence in winter may be driven by increased pathogen presence as well as an interaction between NTHi airway infection and effects of viral infection. TRIAL REGISTRATION NUMBER: Results, NCT01360398.

Correction: Relationships between Mucosal Antibodies, Non-Typeable Haemophilus influenzae (NTHi) Infection and Airway Inflammation in COPD.

[This corrects the article DOI: 10.1371/journal.pone.0167250.].

Efficacy of 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine against acute otitis media and nasopharyngeal carriage in Panamanian children - A randomized controlled trial.

We previously reported 10-valent pneumococcal non-typeable Haemophilus influenzae (NTHi) protein D conjugate vaccine (PHiD-CV) efficacy in a double-blind randomized trial (ClinicalTrials.gov: NCT00466947) against various diseases, including acute otitis media (AOM). Here, we provide further analyses. In the Panamanian subset, 7,359 children were randomized (1:1) to receive PHiD-CV or control vaccine at age 2/4/6 and 15-18 months. Of these, 2,000 had nasopharyngeal swabs collected. AOM cases were captured when parents sought medical attention for children with AOM symptoms; surveillance was enhanced approximately 2 y into the study through regular telephone calls or home visits by study personnel, who advised parents to visit the clinic if their child had AOM symptoms. Mean follow-up was 31.4 months. Clinical AOM (C-AOM) cases were assessed by physicians and confirmed by otorhinolaryngologists. Middle ear fluid samples, taken from children with C-AOM after specific informed consent, and nasopharyngeal samples were cultured for pathogen identification. For 7,359 children, 2,574 suspected AOM cases were assessed by a primary healthcare physician; 649 cases were C-AOM cases as per protocol definition. From the 503 MEF samples collected, 158 resulted in a positive culture. In the intent-to-treat cohort (7,214 children), PHiD-CV showed VE against first C-AOM (24.0% [95% CI: 8.7, 36.7]) and bacterial (B-AOM) episodes (48.0% [20.3, 66.1]) in children <24 months, which declined thereafter with age. Pre-booster VE against C-AOM was 30.7% [12.9, 44.9]; post-booster, -6.7% [-36.4, 16.6]. PHiD-CV VE was 17.7% [-6.1, 36.2] against moderate and 32.7% [-20.5, 62.4] against severe C-AOM. VE against vaccine-serotype pneumococcal NPC was 31.2% [5.3, 50.3] 3 months post-booster, and 25.6% [12.7, 36.7] across all visits. NTHi colonization rates were low and no significant reduction was observed. PHiD-CV showed efficacy against C-AOM and B-AOM in children younger than 24 months, and reduced vaccine-serotype NPC.

Relationships between Mucosal Antibodies, Non-Typeable Haemophilus influenzae (NTHi) Infection and Airway Inflammation in COPD.

Non-typeable Haemophilus influenzae (NTHi) is a key pathogen in COPD, being associated with airway inflammation and risk of exacerbation. Why some patients are susceptible to colonisation is not understood. We hypothesised that this susceptibility may be due to a deficiency in mucosal humoral immunity. The aim of our study (NCT01701869) was to quantify the amount and specificity of antibodies against NTHi in the lungs and the associated risk of infection and inflammation in health and COPD. Phlebotomy, sputum induction and bronchoscopy were performed on 24 mild-to-moderate COPD patients and 8 age and smoking-matched controls. BAL (Bronchoalveolar lavage) total IgG1, IgG2, IgG3, IgM and IgA concentrations were significantly increased in COPD patients compared to controls. NTHi was detected in the lungs of 7 of the COPD patients (NTHi+ve-29%) and these patients had a higher median number of previous exacerbations than NTHi-ve patients as well as evidence of increased systemic inflammation. When comparing NTHi+ve versus NTHi-ve patients we observed a decrease in the amount of both total IgG1 (p = 0.0068) and NTHi-specific IgG1 (p = 0.0433) in the BAL of NTHi+ve patients, but no differences in total IgA or IgM. We observed no evidence of decreased IgG1 in the serum of NTHi+ve patients, suggesting this phenomenon is restricted to the airway. Furthermore, the NTHi+ve patients had significantly greater levels of IL-1ß (p = 0.0003), in BAL than NTHi-ve COPD patients.This study indicates that the presence of NTHi is associated with reduced levels and function of IgG1 in the airway of NTHi-colonised COPD patients. This decrease in total and NTHI-specific IgG1 was associated with greater systemic and airway inflammation and a history of more frequent exacerbations and may explain the susceptibility of some COPD patients to the impacts of NTHi.

Randomized controlled study of the safety and immunogenicity of pneumococcal vaccine formulations containing PhtD and detoxified pneumolysin with alum or adjuvant system AS02V in elderly adults.

Six vaccine formulations containing AS02V or alum (aluminum phosphate [AlPO4]) adjuvant with pneumococcal proteins, pneumococcal histidine triad D (PhtD), and/or detoxified pneumolysin (dPly), either as a polysaccharide carrier in an 8-valent pneumococcal conjugate vaccine (8PCV) or as free (unconjugated) proteins, were evaluated in adults -65 to 85 years of age. In this phase I observer-blind study, 167 healthy subjects were randomized to receive two doses (days 0 and 60) of 10 or 30 µg PhtD-dPly plus AS02V or alum, 8PCV plus AS02V or alum, or one dose (day 0) of 23-valent polysaccharide pneumococcal vaccine (23PPV) as a control (placebo on day 60). The safety, reactogenicity, and antibody-specific responses to these vaccines were evaluated. No vaccine-related serious adverse events were reported. The incidences of solicited local and specific general (fatigue and myalgia) symptoms tended to be higher in the AS02V groups than in other groups. Anti-PhtD and anti-Ply antibody responses were observed in all groups except the control group. One month post-dose 2, the anti-PhtD and anti-Ply antibody geometric mean concentrations tended to be higher with AS02V than with alum, higher with a dose of 30 µg than with 10 µg for PhtD-dPly and higher with 30-µg PhtD-dPly formulations than with conjugated PhtD and dPly (8PCV) formulations. Functional antibody responses, measured by an opsonophagocytic activity assay, tended to be higher with 8PCV than with 23PPV. In conclusion, vaccine formulations containing free or conjugated PhtD and dPly had acceptable reactogenicity and safety profiles in elderly adults. Immune responses were enhanced with an AS02V-adjuvanted formulation containing free 30-µg PhtD-dPly compared to those with alum adjuvant and conjugated proteins. (This study has been registered at ClinicalTrials.gov under registration no. NCT00756067.).

Effects of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D-conjugate vaccine on nasopharyngeal bacterial colonization in young children: a randomized controlled trial.

BACKGROUND: This study evaluated the effects of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D-conjugate vaccine (PHiD-CV) on nasopharyngeal bacterial colonization compared with the 7-valent pneumococcal conjugate vaccine (7vCRM) in young children. METHODS: A randomized controlled trial in the Netherlands, initiated 2 years after 7vCRM introduction, was conducted between 1 April 2008 and 1 December 2010. Infants (N = 780) received either PHiD-CV or 7vCRM (2:1) at 2, 3, 4, and 11-13 months of age. Nasopharyngeal samples taken at 5, 11, 14, 18, and 24 months of age were cultured to detect Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus. Polymerase chain reaction assays quantified H. influenzae and S. pneumoniae and confirmed H. influenzae as nontypeable (NTHi). Primary outcome measure was vaccine efficacy (VE) against NTHi colonization. RESULTS: In both groups, NTHi colonization increased with age from 33% in 5-month-olds to 65% in 24-month-olds. Three months postbooster, VE against colonization was 0.5% (95% confidence interval [CI], -21.8% to 18.4%) and VE against acquisition 10.9% (95% CI, -31.3% to 38.9%). At each sampling moment, no differences between groups in either NTHi prevalence or H. influenzae density were detected. Streptococcus pneumoniae (range, 39%-57%), M. catarrhalis (range, 63%--69%), and S. aureus (range, 9%-30%) colonization patterns were similar between groups. CONCLUSIONS: PHiD-CV had no differential effect on nasopharyngeal NTHi colonization or H. influenzae density in healthy Dutch children up to 2 years of age, implying that herd effects for NTHi are not to be expected. Other bacterial colonization patterns were also similar.