2-Alkyl-4-quinolone quorum sensing molecules are biomarkers for culture-independent Pseudomonas aeruginosa burden in adults with cystic fibrosis.

Introduction. Pseudomonas aeruginosa produces quorum sensing signalling molecules including 2-alkyl-4-quinolones (AQs), which regulate virulence factor production in the cystic fibrosis (CF) airways.Hypothesis/Gap statement. Culture can lead to condition-dependent artefacts which may limit the potential insights and applications of AQs as minimally-invasive biomarkers of bacterial load.Aim. We aimed to use culture-independent methods to explore the correlations between AQ levels and live P. aeruginosa load in adults with CF.Methodology. Seventy-five sputum samples at clinical stability and 48 paired sputum samples obtained at the beginning and end of IV antibiotics for a pulmonary exacerbation in adults with CF were processed using a viable cell separation technique followed by quantitative P. aeruginosa polymerase chain reaction (qPCR). Live P. aeruginosa qPCR load was compared with the concentrations of three AQs (HHQ, NHQ and HQNO) detected in sputum, plasma and urine.Results. At clinical stability and the beginning of IV antibiotics for pulmonary exacerbation, HHQ, NHQ and HQNO measured in sputum, plasma and urine were consistently positively correlated with live P. aeruginosa qPCR load in sputum, compared to culture. Following systemic antibiotics live P. aeruginosa qPCR load decreased significantly (P<0.001) and was correlated with a reduction in plasma NHQ (plasma: r=0.463, P=0.003).Conclusion. In adults with CF, AQ concentrations correlated more strongly with live P. aeruginosa bacterial load measured by qPCR compared to traditional culture. Prospective studies are required to assess the potential of systemic AQs as biomarkers of P. aeruginosa bacterial burden.

Weight gain during acute treatment of an initial pulmonary exacerbation is associated with a longer interval to the next exacerbation in adults with cystic fibrosis.

Weight gain during treatment for a cystic fibrosis exacerbation http://ow.ly/f1zl30dU9AO.

Diagnostic and prognostic significance of systemic alkyl quinolones for P. aeruginosa in cystic fibrosis: A longitudinal study.

BACKGROUND: Pulmonary P. aeruginosa infection is associated with poor outcomes in cystic fibrosis (CF) and early diagnosis is challenging, particularly in those who are unable to expectorate sputum. Specific P. aeruginosa 2-alkyl-4-quinolones are detectable in the sputum, plasma and urine of adults with CF, suggesting that they have potential as biomarkers for P. aeruginosa infection. AIM: To investigate systemic 2-alkyl-4-quinolones as potential biomarkers for pulmonary P. aeruginosa infection. METHODS: A multicentre observational study of 176 adults and 68 children with CF. Cross-sectionally, comparisons were made between current P. aeruginosa infection using six 2-alkyl-4-quinolones detected in sputum, plasma and urine against hospital microbiological culture results. All participants without P. aeruginosa infection at baseline were followed up for one year to determine if 2-alkyl-4-quinolones were early biomarkers of pulmonary P. aeruginosa infection. RESULTS: Cross-sectional analysis: the most promising biomarker with the greatest diagnostic accuracy was 2-heptyl-4-hydroxyquinoline (HHQ). In adults, areas under the ROC curves (95% confidence intervals) for HHQ analyses were 0.82 (0.75-0.89) in sputum, 0.76 (0.69-0.82) in plasma and 0.82 (0.77-0.88) in urine. In children, the corresponding values for HHQ analyses were 0.88 (0.77-0.99) in plasma and 0.83 (0.68-0.97) in urine. Longitudinal analysis: Ten adults and six children had a new positive respiratory culture for P. aeruginosa in follow-up. A positive plasma HHQ test at baseline was significantly associated with a new positive culture for P. aeruginosa in both adults and children in follow-up (odds ratio (OR)=6.67;-95% CI:-1.48-30.1;-p=0.01 and OR=70; 95% CI: 5-956;-p<0.001 respectively). CONCLUSIONS: AQs measured in sputum, plasma and urine may be used to diagnose current infection with P. aeruginosa in adults and children with CF. These preliminary data show that plasma HHQ may have potential as an early biomarker of pulmonary P. aeruginosa. Further studies are necessary to evaluate if HHQ could be used in clinical practice to aid early diagnosis of P. aeruginosa infection in the future.

Glutamine supplementation in cystic fibrosis: A randomized placebo-controlled trial.

RATIONALE: Pulmonary infection and malnutrition in cystic fibrosis are associated with decreased survival. Glutamine has a possible anti-microbial effect, with a specific impact against Pseudomonas aeruginosa. We aimed to test the hypothesis that oral glutamine supplementation (21 g/day) for 8 weeks in adults with cystic fibrosis would decrease pulmonary inflammation and improve clinical status. METHODS: The study design was a randomized double-blind placebo-controlled study design with an iso-nitrogenous placebo. The primary analysis was intention to treat, and the primary outcome was change in induced sputum neutrophils. RESULTS: Thirty-nine individuals were recruited and thirty-six completed the study. Glutamine supplementation had no impact on any of the outcome measures in the intention-to-treat analysis. In the per protocol analysis, glutamine supplementation was associated with an increase in induced sputum neutrophils (P = 0.046), total cells (P = 0.03), and in Pseudomonas isolation agar colony forming units (P = 0.04) compared to placebo. CONCLUSIONS: There was no effect of glutamine supplementation on markers of pulmonary inflammation in the intention-to-treat analysis.

Pseudomonas aeruginosa quorum sensing molecules correlate with clinical status in cystic fibrosis.

Pseudomonas aeruginosa produces quorum sensing signal molecules that are potential biomarkers for infection.A prospective study of 60 cystic fibrosis patients with chronic P. aeruginosa, who required intravenous antibiotics for pulmonary exacerbations, was undertaken. Clinical measurements and biological samples were obtained at the start and end of the treatment period. Additional data were available for 29 of these patients when they were clinically stable.Cross-sectionally, quorum sensing signal molecules were detectable in the sputum, plasma and urine of 86%, 75% and 83% patients, respectively. They were positively correlated between the three biofluids. Positive correlations were observed for most quorum sensing signal molecules in sputum, plasma and urine, with quantitative measures of pulmonary P. aeruginosa load at the start of a pulmonary exacerbation. Plasma concentrations of 2-nonyl-4-hydroxy-quinoline (NHQ) were significantly higher at the start of a pulmonary exacerbation compared to clinical stability (p<0.01). Following the administration of systemic antibiotics, plasma 2-heptyl-4-hydroxyquinoline (p=0.02) and NHQ concentrations (p<0.01) decreased significantly.In conclusion, quorum sensing signal molecules are detectable in cystic fibrosis patients with pulmonary P. aeruginosa infection and are positively correlated with quantitative measures of P. aeruginosa. NHQ correlates with clinical status and has potential as a novel biomarker for P. aeruginosa infection.

Quality of life and healthcare utilisation in cystic fibrosis: a multicentre study.

The aim of our study was to discover the health status and healthcare utilisation associated with pulmonary exacerbations in cystic fibrosis (CF) and chronic Pseudomonas aeruginosa infection. Patients with CF from five UK CF centres attended two visits, 8-12 weeks apart. They were classified at visit 1 as being in one of the three health states: no current pulmonary exacerbation; "mild" (no hospitalisation) pulmonary exacerbation; and "severe" (hospitalisation) pulmonary exacerbation. All patients completed the Cystic Fibrosis Questionnaire-Revised (CFQ-R) and EuroQol (EQ-5D) and a clinical form, and forced expiratory volume in 1 s (FEV1) was measured at visits 1 and 2. Annual healthcare utilisation data were collected. 94 patients of mean±sd age 28.5±8.2 yrs and FEV1 58.7±26.8% were recruited. 60 patients had no pulmonary exacerbation, 15 had a mild and 19 had a severe pulmonary exacerbation at visit 1. EQ-5D and CFQ-R data showed that the worse the exacerbation, the poorer the health-related quality of life (HRQoL). There were strong relationships between the CFQ-R and EQ-5D domain scores. The mean rate of pulmonary exacerbations per patient per year was 3.6 (1.5 in hospital and 2.2 at home). The mean length of stay per hospital pulmonary exacerbation was 9 days. As exacerbation status worsens, patients experience worse HRQoL. There is a significant healthcare burden associated with treatment of pulmonary exacerbation and long-term prophylaxis.

Microbial infections in eight genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis.

BACKGROUND: The authors have previously reported genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) based on expression of 88 human genes. AIM: To attempt to reproduce these findings, determine the specificity of this signature to CFS/ME, and test for associations between CFS/ME subtype and infection. METHODS: Expression levels of 88 human genes were determined in blood of 62 new patients with idiopathic CFS/ME (according to Fukuda criteria), six patients with Q-fever-associated CFS/ME from the Birmingham Q-fever outbreak (according to Fukuda criteria), 14 patients with endogenous depression (according to DSM-IV criteria) and 29 normal blood donors. RESULTS: In patients with CFS/ME, differential expression was confirmed for all 88 genes. Q-CFS/ME had similar patterns of gene expression to idiopathic CFS/ME. Gene expression in patients with endogenous depression was similar to that in the normal controls, except for upregulation of five genes (APP, CREBBP, GNAS, PDCD2 and PDCD6). Clustering of combined gene data in CFS/ME patients for this and the authors' previous study (117 CFS/ME patients) revealed genomic subtypes with distinct differences in SF36 scores, clinical phenotypes, severity and geographical distribution. Antibody testing for Epstein-Barr virus, enterovirus, Coxiella burnetii and parvovirus B19 revealed evidence of subtype-specific relationships for Epstein-Barr virus and enterovirus, the two most common infectious triggers of CFS/ME. CONCLUSIONS: This study confirms the involvement of these genes in CFS/ME.

Cross-sectional and longitudinal multilocus sequence typing of pseudomonas aeruginosa in cystic fibrosis sputum samples.

Multilocus sequence typing (MLST) is a genetic typing tool designed to provide information about the relatedness of isolates at the core genome level. The utility of MLST in regard to cystic fibrosis (CF)-related infection with Pseudomonas aeruginosa is unknown. The molecular clock speed of the MLST genes was studied using 219 colonies isolated longitudinally from 49 patients with CF. A cross-sectional study examining 27 to 46 colonies per sputum sample for samples from 16 patients was also undertaken. The molecular clock speed was estimated to be 2.05 x 10(-5) (upper 95% confidence limit) or 4.75 x 10(-6) (50% confidence limit) point mutations per nucleotide per year. In the cross-sectional study, 50% of patients were infected with more than one sequence type. There was evidence of point mutations, recombination events, and coinfection with epidemic and unique strains. A clonal complex that was highly genetically distinct from the rest of the P. aeruginosa population was identified. The MLST scheme uses genes with an appropriate clock speed and provides useful information about the genetic variation of P. aeruginosa within and between patients with CF.

Association between hypermutator phenotype, clinical variables, mucoid phenotype, and antimicrobial resistance in Pseudomonas aeruginosa.

The presence of hypermutator Pseudomonas aeruginosa was associated with poorer lung function in patients at the Adult West Midlands CF Unit. Mucoid isolates were more likely to be hypermutators. The presence of resistant mutant subpopulations was associated with hypermutator phenotype but was not good enough to be used as a test for this phenotype.

Environmental Burkholderia cepacia complex isolates in human infections.

Members of the Burkholderia cepacia complex (Bcc), found in many environments, are associated with clinical infections. Examining diverse species and strains from different environments with multilocus sequence typing, we identified > 20% of 381 clinical isolates as indistinguishable from those in the environment. This finding links the natural environment with the emergence of many Bcc infections.

Cross-infection in cystic fibrosis: the knowledge and behaviour of adult patients.

INTRODUCTION: The knowledge and behaviour of adult patients with cystic fibrosis (CF) regarding cross-infection are ill understood. METHODS: A questionnaire was designed to investigate this at the West Midlands Adult CF Centre. RESULTS: 94 patients completed the questionnaire. 54%, 36% and 46% had "no idea" of the lifetime risk of contracting Burkholderia cepacia complex, epidemic strains of Pseudomonas aeruginosa, and MRSA, respectively. 25-33% did not know the consequences of infection with these bacteria. 35% mixed with other people with CF, 6.5% during physiotherapy or nebulizer use. Most respondents did not think quality of life was significantly linked with segregation from other patients with CF. CONCLUSIONS: Adults with CF, at least in the West Midlands, have poor knowledge of the risk and consequences of cross-infection. A significant proportion ignored advice not to mix with other patients, although segregation was not thought to impact upon quality of life. This suggests that more education about the risks of cross-infection would be beneficial.

Reliability of multilocus sequence typing of the Burkholderia cepacia complex in cystic fibrosis.

INTRODUCTION: Infection with the Burkholderia cepacia complex is an important cause of morbidity and mortality in cystic fibrosis (CF). We investigated the molecular clock speed of the seven genes used in the multilocus sequence typing (MLST) scheme for these bacteria. METHODS: At least two isolates, separated by months to years, from each of 20 patients were typed using MLST. In total 41 isolates, providing 128 isolate-years, were analyzed. Mutation and recombination rates were estimated assuming a Poisson distribution. RESULTS: Out of 20 patients, 15 had no change in sequence type over time (mean 7.07 years, range 1.09 to 14.24). One patient had strain replacement. Three patients had evidence of recombination involving one of the seven housekeeping genes, and one patient had evidence of recombination of two genes. The mutation rate was estimated as 2.36x10(-6) per nucleotide per year (50% confidence limit) and 1.02x10(-5) per nucleotide per year (upper 95% confidence limit). The rate of nucleotide changes due to recombination events was estimated as 0.676 to 0.839 per year (95% confidence limits). CONCLUSIONS: B. cepacia complex housekeeping genes have a slow molecular clock speed and MLST provides a robust and reliable typing technique for isolates from this complex. A low rate of point mutation was found, with a higher rate of recombination events, in keeping with previous cross-sectional epidemiological data. The study also demonstrated, for the first time, recombination in a longitudinal in vivo study.

Multilocus sequence typing scheme that provides both species and strain differentiation for the Burkholderia cepacia complex.

A single multilocus sequence typing (MLST) scheme was developed for precise characterization of the opportunistic pathogens of Burkholderia cepacia complex (BCC), a group composed of at least nine closely related species. Seven conserved housekeeping genes were selected after a comparison of five Burkholderia species, and a collection of strains was subjected to nucleotide sequence analysis using a nested PCR amplification approach for each gene. MLST differentiated all nine current BCC species and identified 114 sequence types within a collection of 119 strains. No differentiation was found between strains recovered from environmental or clinical sources. The improved resolution in strain identification offered by MLST was able to identify previously characterized epidemic strain lineages and also demonstrated the presence of four novel potential species groups within the complex. There was also evidence for recombination having an important role in the recent evolution of individual BCC species. This highly transferable, validated, MLST scheme provides a new means to assist in species identification as well as unambiguous strain discrimination of the BCC by a single approach. It is also the first MLST scheme designed at the outset to incorporate multiple species and should facilitate global epidemiological investigations of the BCC.

The effect of oral clarithromycin on bronchial airway inflammation in moderate-to-severe stable COPD: a randomized controlled trial.

INTRODUCTION: COPD is characterized by bronchial neutrophilic inflammation. Clarithromycin is a macrolide antibiotic that has antibacterial and anti-inflammatory properties. Macrolide antibiotics have been shown to improve airway inflammation in diffuse pan-bronchiolitis but their role in COPD is undetermined. The aim of the study was to determine if 3 months of therapy with modified-release oral clarithromycin (Klaricid XL) 500 mg/day reduced bronchial airway inflammation in patients with moderate-to-severe stable COPD compared with placebo. METHODS: A prospective, double-blind controlled trial randomized patients with moderate-to-severe stable COPD to 3 months' therapy with oral modified-release clarithromycin 500 mg/day or placebo. Patients underwent saline sputum induction before and after treatment with clarithromycin. The effects of clarithromycin on sputum total cell and neutrophil counts, supernatant interleukin-8 (IL-8), leukotriene B(4) (LTB(4)), tumor necrosis factor (TNF)-alpha, neutrophil elastase (NE), and neutrophil chemotaxis were assessed in comparison with placebo. RESULTS: Of a total of 67 patients included in the trial, 31 were treated with clarithromycin and 36 with placebo. The groups were similar in age, body mass index, history of smoking, and spirometry. Of 60 evaluable patients, 26 and 34 completed 3 months' therapy with clarithromycin and placebo, respectively. Clarithromycin had no significant effect on sputum total cell count, neutrophil count, IL-8, LTB(4), TNFalpha levels or neutrophil elastase. However, clarithromycin did cause a small reduction in the neutrophil differential (p = 0.04 relative to placebo) and neutrophil chemotaxis (p = 0.058 relative to placebo). CONCLUSIONS: Oral clarithromycin 500 mg/day administered for 3 months had no significant effect on sputum neutrophil numbers or cytokine levels in patients with moderate-to-severe stable COPD. However, clarithromycin did cause a small reduction in the neutrophil differential and neutrophil chemotaxis. Further studies may be warranted to determine the clinical significance of these findings.

Community-acquired pneumonia mortality: a potential link to antibiotic prescribing trends in general practice.

BACKGROUND: Community prescribing of antibiotics has decreased substantially in the UK in recent years. We examine the association between pneumonia mortality and recent changes in community-based antibiotic prescribing for lower respiratory tract infections (LRTI). METHODS: Retrospective analysis of aggregated data for pneumonia mortality, influenza incidence, and antibiotic prescribing for LRTI in England and Wales during 12-week winter periods between 1993/94 and 1999/2000. RESULTS: Winter antibiotic prescribing for LRTI showed a 30.0% decline since 1995/96. Over the same period, there was a 50.6% increase in winter excess pneumonia mortality adjusted for influenza incidence. Negative binomial regression analysis showed that the incidence of influenza alone had a significant association with winter pneumonia mortality (P<0.001). The analysis also showed the reduction in antibiotic prescribing had a small but significant association with mortality (P<0.001), when simultaneously modelling for influenza incidence. CONCLUSIONS: Our findings suggest an association between recent reductions in antibiotic prescribing for LRTI in general practice and an increase in pneumonia mortality in England and Wales. This retrospective study of aggregate data represents the first attempt to assess the effect of limiting antibiotic prescribing on patient outcomes, and highlights the need to identify which patients benefit from antibiotic treatment for LRTI.