Phase II studies of nebulised Arikace in CF patients with Pseudomonas aeruginosa infection.

RATIONALE: Arikace is a liposomal amikacin preparation for aerosol delivery with potent Pseudomonas aeruginosa killing and prolonged lung deposition. OBJECTIVES: To examine the safety and efficacy of 28 days of once-daily Arikace in cystic fibrosis (CF) patients chronically infected with P aeruginosa. METHODS: 105 subjects were evaluated in double-blind, placebo-controlled studies. Subjects were randomised to once-daily Arikace (70, 140, 280 and 560 mg; n=7, 5, 21 and 36 subjects) or placebo (n=36) for 28 days. Primary outcomes included safety and tolerability. Secondary outcomes included lung function (forced expiratory volume at one second (FEV1)), P aeruginosa density in sputum, and the Cystic Fibrosis Quality of Life Questionnaire-Revised (CFQ-R). RESULTS: The adverse event profile was similar among Arikace and placebo subjects. The relative change in FEV1 was higher in the 560 mg dose group at day 28 (p=0.033) and at day 56 (28 days post-treatment, 0.093L±0.203 vs -0.032L±0.119; p=0.003) versus placebo. Sputum P aeruginosa density decreased >1 log in the 560 mg group versus placebo (days 14, 28 and 35; p=0.021). The Respiratory Domain of the CFQ-R increased by the Minimal Clinically Important Difference (MCID) in 67% of Arikace subjects (560 mg) versus 36% of placebo (p=0.006), and correlated with FEV1 improvements at days 14, 28 and 42 (p<0.05). An open-label extension (560 mg Arikace) for 28 days followed by 56 days off over six cycles confirmed durable improvements in lung function and sputum P aeruginosa density (n=49). CONCLUSIONS: Once-daily Arikace demonstrated acute tolerability, safety, biologic activity and efficacy in patients with CF with P aeruginosa infection.

Early Pseudomonas aeruginosa infection in individuals with cystic fibrosis: is susceptibility testing justified?

OBJECTIVES: To test the presumption that Pseudomonas aeruginosa isolates responsible for initial lung infection in individuals with cystic fibrosis (CF) are invariably susceptible to antipseudomonal agents. METHODS: Antibiotic susceptibility was determined (MIC and Etest) in two populations of P. aeruginosa associated with initial lung infection. Population 1: environmental isolates (n=78). Population 2: clinical isolates responsible for first infection in previously non-infected patients (85 isolates from 85 patients). Susceptibility or resistance was determined using current BSAC guidelines; ninth version (2009). RESULTS: The majority (≥ 90%) of isolates in both bacterial populations were susceptible to the front-line antipseudomonal agents; colistin, ciprofloxacin, tobramycin, ceftazidime, amikacin and meropenem. Up to 10% of isolates were resistant to one or more antibiotics. A single isolate from each population would be defined as resistant to tobramycin based on a breakpoint (>128 mg/L) that has been suggested for use in patients receiving inhaled therapy. CONCLUSIONS: The high prevalence of susceptibility found in P. aeruginosa isolates associated with initial infection contrasts with the high prevalence of resistance found in isolates from chronic CF lung infection. However, susceptibility in early isolates cannot be presumed. Until further data are obtained from clinically based studies, susceptibility tests should continue to be performed to assist the choice of antibiotics for treatment of early infection.

Assessment of fluorescent in situ hybridization and PCR-based methods for rapid identification of Burkholderia cepacia complex organisms directly from sputum samples.

Several species within the Burkholderia cepacia complex (BCC) have emerged as significant opportunistic pathogens of patients with cystic fibrosis (CF). BCC infection is typically associated with a poor clinical prognosis and decreased survival. These factors, combined with the existence of highly transmissible epidemic strains, have resulted in strict segregation of BCC- and non-BCC-infected patients to minimize cross infection. Accurate and rapid diagnosis of infections is essential to enable appropriate patient management. However, the rapidly evolving taxonomy of BCC poses a considerable challenge to diagnostics. In the present study, we assessed a commercially available fluorescent in situ hybridization (FISH) assay (seaFAST Cystic Fibrosis I kit) and a novel rRNA gene-based PCR assay for the rapid identification of BCC-positive sputa, irrespective of the BCC species. We report that, while the FISH assay fails to identify all BCC species, it does identify the majority of species, including the two most clinically relevant species, B. multivorans and B. cenocepacia. The sensitivity of the assay applied to sputum was limited by nonspecific background fluorescence. While sputum processing was optimized to minimize background, the resulting sensitivity for BCC detection was 8 x 10(5) CFU/ml. In contrast, the novel PCR assay reported herein exhibits 100% sensitivity and specificity for all BCC species and can detect 10(4) CFU/ml when applied to sputum. This novel rRNA gene-based assay is currently the most sensitive BCC-specific PCR assay for the detection of BCC direct from clinical samples and as such is a valuable addition to the field of BCC diagnostics.

Transmission of Burkholderia cepacia complex: evidence for new epidemic clones infecting cystic fibrosis patients in Italy.

To analyze national prevalence, genomovar distribution, and epidemiology of the Burkholderia cepacia complex in Italy, 225 putative B. cepacia complex isolates were obtained from 225 cystic fibrosis (CF) patients attending 18 CF centers. The genomovar status of these isolates was determined by a polyphasic approach, which included whole-cell protein electrophoresis and recA restriction fragment length polymorphism (RFLP) analysis. Two approaches were used to genotype B. cepacia complex isolates: BOX-PCR fingerprinting and pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments. A total of 208 (92%) of 225 isolates belonged to the B. cepacia complex, with Burkholderia cenocepacia as the most prevalent species (61.1%). Clones delineated by PFGE were predominantly linked to a single center; in contrast, BOX-PCR clones were composed of isolates collected either from the same center or from different CF centers and comprised multiple PFGE clusters. Three BOX-PCR clones appeared of special interest. One clone was composed of 17 B. cenocepacia isolates belonging to recA RFLP type H. These isolates were collected from six centers and represented three PFGE clusters. The presence of insertion sequence IS 1363 in all isolates and the comparison with PHDC reference isolates identified this clone as PHDC, an epidemic clone prominent in North American CF patients. The second clone included 22 isolates from eight centers and belonged to recA RFLP type AT. The genomovar status of strains with the latter RFLP type is not known. Most of these isolates belonged to four different PFGE clusters. Finally, a third clone comprised nine B. pyrrocinia isolates belonging to recA RFLP type Se 13. They represented three PFGE clusters and were collected in three CF centers.

Antimicrobial activity of murine lung cells against Staphylococcus aureus is increased in vitro and in vivo after elafin gene transfer.

Staphylococcus aureus is a pathogen often found in pneumonia and sepsis. In the context of the resistance of this organism to conventional antibiotics, an understanding of the regulation of natural endogenous antimicrobial molecules is of paramount importance. Previous studies have shown that both human and mouse airways express a variety of these molecules, including defensins, cathelicidins, and the four-disulfide core protein secretory leukocyte protease inhibitor. We demonstrate here by culturing mouse tracheal epithelial cells at an air-liquid interface that, despite the production of Defb1, Defb14, and Defr1 in this system, these cells are unable to clear S. aureus when exposed to this respiratory pathogen. Using an adenovirus (Ad)-mediated gene transfer strategy, we show that overexpression of elafin, an anti-elastase/antimicrobial molecule (also a member of the four-disulfide core protein family), dramatically improves the clearance of S. aureus. In addition, we also demonstrate that this overexpression is efficient in vivo and that intratracheal instillation of Ad-elafin significantly reduced the lung bacterial load and demonstrates concomitant anti-inflammatory activity by reducing neutrophil numbers and markers of lung inflammation, such as bronchoalveolar lavage levels of tumor necrosis factor and myeloperoxidase. These findings show that an increased antimicrobial activity phenotype is provided by the elafin molecule and have implications for its use in S. aureus-associated local and systemic infections.

Burkholderia cenocepacia and Burkholderia multivorans: influence on survival in cystic fibrosis.

INTRODUCTION: Burkholderia cepacia infection has been associated with a poor prognosis for patients with cystic fibrosis (CF). It is now recognised that organisms classified as B cepacia comprise a number of distinct genomic species each known as a genomovar of the B cepacia complex (BCC). The outcome of infection for CF patients with individual genomovars is unknown. The clinical outcome of infection with the two most commonly isolated genomovars (B cenocepacia and B multivorans) was studied at a specialist CF centre between 1982 and 2003. METHODS: The numbers of patients who progressed from initial to chronic infection were assessed. Control groups were created by matching patients with chronic BCC infection by percentage forced expiratory volume in 1 second with patients with Pseudomonas aeruginosa infection. Outcome measures were survival time, deaths from "cepacia syndrome", rate of decline in spirometry and body mass index (BMI), and treatment requirements. RESULTS: Forty nine patients had an initial infection with either B multivorans (n = 16) or B cenocepacia (n = 33); 8/16 and 31/33, respectively, developed chronic infection (p<0.001). Deaths from "cepacia syndrome" occurred in both BCC groups. Patients with B cenocepacia infection had a shorter survival than patients with P aeruginosa infection (p = 0.01). There was no difference in survival between CF patients infected with B multivorans and P aeruginosa. There were no observed differences in changes in spirometry and BMI or treatment requirements between the BCC groups and respective controls. CONCLUSION: In CF, the genomovar status of BCC may influence both the likelihood of progression from initial to chronic infection and the overall survival of the patients.

Genotyping of Pseudomonas aeruginosa in cystic fibrosis suggests need for segregation.

BACKGROUND: Emerging resistance of Pseudomonas aeruginosa within cystic fibrosis (CF) populations is attributed to antibiotic pressure and spread of transmissible strains. We describe increasing resistance of P. aeruginosa isolates, resulting in the identification of two multiresistant strains and their impact on morbidity. METHODS: Susceptibility reports of all P. aeruginosa isolates since 1998 in our unit were reviewed. Isolates were submitted for genomic finger-printing by pulsed-field gel electrophoresis. Clinical measures and the consumption of treatment resources were compared between those harbouring resistant organisms and those with sensitive strains. RESULTS: Analysis of 407 reports from 43 patients revealed isolation of multiresistant (MR) organisms increased during 1999. Those harbouring MR strains consumed more resources than non-MR. Strain typing showed a new 'Sheffield' strain in seven patients (100% MR), and the 'Liverpool' strain in 10 patients (40% MR). Individuals in these groups consumed significantly more resources than 23 patients with unique, susceptible strains (4% MR). DISCUSSION: Increasing resistance in isolates of P. aeruginosa may herald the arrival of a transmissible strain in CF Units which though sometimes sensitive, may become multiply resistant and require more intensive treatment. We now segregate those with transmissible strains from each other and from those with unique strains.

Prevalence and clonality of Burkholderia cepacia complex genomovars in UK patients with cystic fibrosis referred for lung transplantation.

BACKGROUND: It has previously been reported that patients infected with Burkholderia cenocepacia (genomovar III) before lung transplantation have a poorer outcome than those with other B. cepacia complex infections. METHODS: An extensive study was conducted to determine the prevalence and clonality of B. cepacia complex genomovars isolated from patients referred for transplant assessment between 1989 to the present and, where appropriate, whether strain type was related to transplant outcome. RESULTS: Isolates from 29 patients were identified as B. cepacia complex organisms by molecular analysis. Thirteen patients (45%) were infected with the highly transmissible ET-12 strain of B. cenocepacia recA lineage III-A, while all remaining patients were infected with genetically unique B. cenocepacia, B. multivorans, and B. vietnamiensis strains. All previously reported deaths following transplantation were associated with ET-12 infection. CONCLUSIONS: The ET-12 strain is the predominant cause of B. cenocepacia infections in patients with cystic fibrosis referred to our pulmonary transplant centre and is associated with poor transplant outcomes using standard treatment regimens.

Epidemiology of Burkholderia cepacia complex colonisation in cystic fibrosis patients.

In Belgian cystic fibrosis (CF) clinics, sputum samples are evaluated on selective MAST medium routinely every 3 months. In this study, in 1993 and 1999, isolates were further examined by recA restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis of genomic DNA restricted with SpeI. In 1993, 12 patients were colonised with Burkholderia cepacia complex (Bcc): B. cenocepacia (n=6), B. multivorans (n=3), B. stabilis (n=3). Four patients were colonised with the same B. cenocepacia strain; two with the same B. stabilis strain. After 5 yrs, three B. cenocepacia- and one B. multivorans-colonised patients had died. In 1999, Bcc was isolated in 12 patients: B. multivorans (n=9), B. stabilis (n=1) and B. cenocepacia (n=2). Three patients were colonised by the same B. multivorans strain. Compared to matched controls, the 5 yr outcome was poor; four B. cepacia patients died and none of the control patients died. Lung-function evolution was poor. In conclusion, the rate of colonisation in Belgian cystic fibrosis patients is stable and low. Burkholderia cenocepacia was most prevalent in 1993; Burkholderia multivorans in 1999. The cross-infection rate is low. Three patients had transient colonisation. The impact of Burkholderia cepacia complex on morbidity in the Belgian cystic fibrosis population is high and not limited to Burkholderia cenocepacia.

Polyclonal outbreak of Burkholderia cepacia complex bacteraemia in haemodialysis patients.

We report a polyclonal outbreak of bacteraemia involving 24 patients at a haemodialysis facility in Recife (Brazil). During the outbreak period (4 June to 11 July, 2001), three Burkholderia cepacia complex strains were isolated from human blood and from various water samples collected at different sites in the haemodialysis unit and from dialysate fluids. Out of 14 patients with positive blood cultures, six were infected by Burkholderia cepacia complex bacteria: three with Burkholderia cepacia genomovar III, two with a first strain of Burkholderia vietnamiensis, and one with the Burkholderia cepacia genomovar III strain and a second B. vietnamiensis strain.

Identification of airborne dissemination of epidemic multiresistant strains of Pseudomonas aeruginosa at a CF centre during a cross infection outbreak.

BACKGROUND: Chronic Pseudomonas aeruginosa infection is a major cause of morbidity and mortality for individuals with cystic fibrosis (CF). P aeruginosa cross infection outbreaks have recently been reported at CF holiday camps and specialist centres. The mechanism of cross infection is unknown. A study was performed to look for the presence of epidemic strains of P aeruginosa in the environment of a CF centre during a cross infection outbreak and to examine their potential modes of spread between patients. METHODS: Microbiological sampling of the environment of the CF facility was performed, including room air sampling. Individual P aeruginosa strains were identified by bacterial fingerprinting. The typing patterns were compared with those of epidemic strains responsible for cross infection among the patients. RESULTS: Epidemic P aeruginosa strains were isolated from room air when patients performed spirometric tests, nebulisation, and airway clearance, but were not present in other areas of the inanimate environment of the CF centre. CONCLUSIONS: Aerosol dissemination may be the most important factor in patient-to-patient spread of epidemic strains of P aeruginosa during recent cross infection outbreaks at adult CF centres.

Increased treatment requirements of patients with cystic fibrosis who harbour a highly transmissible strain of Pseudomonas aeruginosa.

BACKGROUND: A group of patients who harbour the same highly transmissible strain of Pseudomonas aeruginosa were identified at a cystic fibrosis (CF) centre. Isolates of this strain display a number of unusual phenotypic features including resistance to most typical antipseudomonal antibiotics. A study was undertaken to see if there was a difference in treatment requirements between CF patients with chronic infection with their own unique P aeruginosa strains (group 1) and those who harbour a highly transmissible strain (group 2). METHODS: Data on treatment requirements for the year 2000 were collected from the case records of CF patients with chronic P aeruginosa infection who had received inpatient treatment. Patients co-infected with Burkholderia cepacia or other highly transmissible strains of P aeruginosa were excluded. RESULTS: There were 2/56 and 3/22 deaths in groups 1 and 2, respectively; these patients were excluded from the analysis. No difference was found between the two groups for mean age, % predicted forced expiratory volume in 1 second (FEV(1)), % predicted forced vital capacity (FVC), and body mass index. Patients in group 2 had a greater median (range) number of intravenous antibiotic days (60 (17-216) v 33 (4-237) days; p=0.01), inpatient days (39 (7-183) v 16 (1-172) days; p<0.01), and inpatient episodes (3 (1-9) v 2 (1-6); p<0.01), and more respiratory exacerbations (mean (SD) 8.2 (3.4) v 6.1 (3.2); p=0.01). CONCLUSIONS: Patients who harbour the highly transmissible P aeruginosa strain have a greater treatment burden than patients with CF who harbour their own unique strains. These findings support the need for microbiological surveillance for highly transmissible P aeruginosa and the implementation of infection control measures to prevent cross infection.

A randomised clinical trial of nebulised tobramycin or colistin in cystic fibrosis.

Chronic infection with Pseudomonas aeruginosa is associated with progressive deterioration in lung function in cystic fibrosis (CF) patients. The purpose of this trial was to assess the efficacy and safety of tobramycin nebuliser solution (TNS) and nebulised colistin in CF patients chronically infected with P. aeruginosa. One-hundred and fifteen patients, aged > or = 6 yrs, were randomised to receive either TNS or colistin, twice daily for 4 weeks. The primary end point was an evaluation of the relative change in lung function from baseline, as measured by forced expiratory volume in one second % predicted. Secondary end points included changes in sputum P. aeruginosa density, tobramycin/colistin minimum inhibitory concentrations and safety assessments. TNS produced a mean 6.7% improvement in lung function (p=0.006), whilst there was no significant improvement in the colistin-treated patients (mean change 0.37%). Both nebulised antibiotic regimens produced a significant decrease in the sputum P. aeruginosa density, and there was no development of highly resistant strains over the course of the study. The safety profile for both nebulised antibiotics was good. Tobramycin nebuliser solution significantly improved lung function of patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa, but colistin did not, in this study of 1-month's duration. Both treatments reduced the bacterial load.

Insights into cystic fibrosis microbiology from the European tobramycin trial in cystic fibrosis.

The infection of the airways of cystic fibrosis patients by Pseudomonas aeruginosa is a complex, multistaged process that is associated with a deterioration of lung function. The complexity of the formation of biofilms and their interaction with the immune system means that treatment with antibiotics has been an uncertain science. Tobramycin nebuliser solution (TNS) is a novel formulation of the antibiotic tobramycin developed specifically for inhalation. A recent large trial comparing TNS with inhaled colistin provided an opportunity to define further the effect of antibiotic treatment on microbial infection. In the TNS group, the percentage of patients with a tobramycin minimal inhibitory concentration (MIC) > or = 4 mg l(-1) increased from 38 to 49%, and the percentage of patients with a colistin MIC > or = 4 mg l(-1) remained at 55%. In the colistin group, the percentage of patients with a colistin MIC > or = 4 mg l(-1) remained at 34%, whereas the percentage of patients with a tobramycin MIC > or = 4 mg l(-1) decreased from 27 to 16%. Furthermore, clinical and bacterial response to TNS and colistin was independent of the MIC at baseline. Neither antimicrobial therapy was associated with infection by Burkholderia cepacia or other inherently resistant pathogens. We conclude that conventional measures of antimicrobial resistance may underestimate the effectiveness of tobramycin and colistin when delivered at the high concentrations achieved with the TNS formulation.

Spread of a multiresistant strain of Pseudomonas aeruginosa in an adult cystic fibrosis clinic.

We initiated a prospective surveillance study to investigate possible Pseudomonas aeruginosa cross-infection in our cystic fibrosis centre. We characterised isolates by pyocin typing and pulsed-field gel electrophoresis. 22 (14%) of 154 patients with chronic P aeruginosa had isolates with similar and new pyocin and pulsed-field gel electrophoresis types. The shared isolates showed unusual phenotypic features: they were non-pigmented, non-motile, and resistant to a number of antipseudomonal antibiotics. Cross-infection by a multiresistant P aeruginosa strain has therefore occurred in patients attending our cystic fibrosis centre. We recommend microbiological surveillance in other cystic fibrosis centres.