Libor Fila

Gene Expression Profiling of Burkholderia cenocepacia at the Time of Cepacia Syndrome: Loss of Motility as a Marker of Poor Prognosis?

ABSTRACT Cepacia syndrome (CS) is a fatal septic condition that develops in approximately 20% of cystic fibrosis (CF) patients chronically infected with the Burkholderia cepacia complex (Bcc). The most common causative agent is Burkholderia cenocepacia, a clinically dominant Bcc species that contains the globally distributed epidemic strain sequence type 32 (ST32). Using microarrays, we compared the transcriptomes of ST32 isolates from the bloodstream at the time of CS with their sputum counterparts recovered 1 to 2 months prior to the development of CS. Global gene expression profiles of blood isolates revealed greater activities of the virulence genes involved in the type III secretion system, the bacterial exopolysaccharide cepacian, and quorum sensing, while reduced expression was demonstrated for flagellar genes. Furthermore, a nonmotile phenotype (as evaluated by a swimming motility assay) was identified in blood isolates from 6 out of 8 patients with CS; this phenotype was traceable to 24 months prior to the onset of CS. Loss of motility was not observed in any of the 89 ST32 isolates recovered over the course of chronic infection from 17 patients without CS. In conclusion, the gene expression of Bcc bacteria disseminated during CS has been elucidated for the first time. This study demonstrated marked differences at the transcriptome level between isogenic ST32 isolates that are attributable to the stage and site of infection. The finding of a nonmotile B. cenocepacia isolate may serve as a warning sign for the development of CS in the near future.

Characterization of Staphylococcus aureus strains isolated from Czech cystic fibrosis patients: high rate of ribosomal mutation conferring resistance to MLSB antibiotics as a result of long-term and low-dose azithromycin treatment

Staphylococcus aureus is one of the most frequent pathogens infecting the respiratory tract of patients with cystic fibrosis (CF). This study was the first to examine S. aureus isolates from CF patients in the Czech Republic. Among 100 S. aureus isolates from 92 of 107 observed patients, we found a high prevalence of resistance to macrolide-lincosamide-streptogramin B (MLS(B)) antibiotics (56%). More than half of the resistant strains (29 of 56) carried a mutation in the MLS(B) target site. The emergence of MLS(B) resistance and mutations conferring resistance to MLS(B) antibiotics was associated with azithromycin treatment (p=0.000000184 and p=0.000681, respectively). Methicillin resistance was only detected in 3% of isolates and the rate of resistance to other antibiotics did not exceed 12%. The prevalence of small-colony variant (SCV) strains was relatively low (9%) and eight of nine isolates with the SCV phenotype were thymidine dependent. The study population of S. aureus was heterogeneous in structure and both the most prevalent community-associated and hospital-acquired clonal lineages were represented. Of the virulence genes, enterotoxin genes seg (n=52), sei (n=49), and sec (n=16) were the most frequently detected among the isolates. The PVL genes (lukS-PV and lukF-PV) have not been revealed in any of the isolates.

Ivacaftor in cystic fibrosis adults: Czech experience with six years of follow-up

AIMS Ivacaftor is a revolutionary treatment option for cystic fibrosis (CF) patients with G551D and other gating mutations. The aim of this study was to evaluate the clinical status of patients on ivacaftor who were followed for up to 6 years together with an evaluation of ivacaftor therapy in one patient with an initial FEV1 less than 40% of predicted value. METHODS Data on development of clinical status and sinopulmonary-related therapies were obtained from patient health records during ivacaftor treatment lasting for up to six years and were compared with an equivalent period before ivacaftor administration. RESULTS Five CF adults with a median age 28.6 years (range 21.4-35.6 years) with median FEV1 45% pred. (range 16-85% pred.) were included in the study. Four subjects were also participants in the STRIVE and PERSIST studies. Altogether, twenty-four patient-years of ivacaftor treatment were analyzed. The median FEV1 decline per year decreased from -4.5 to -0.9% pred. (P = 0.043). Reduction in number of days on antibiotic treatment and hospital stays was 21% (P < 0.001) and 75% (P = 0.003), respectively. Improvement and stabilization of lung function was observed for up to six years of treatment. In a patient with severe airway obstruction, an increase in the FEV1 value (30.4% from baseline) was documented during the first twelve months of treatment. CONCLUSION Ivacaftor therapy resulted in improved and stabilized lung function in up to six years of treatment with a reduction in number of days on antibiotic treatment and hospital stays. Its efficiency was also displayed in a patient with severe airway obstruction.

73 Influence of treatment with sildenafil on lung function and exercise tolerance in cystic fibrosis adults

Objectives: Phosphodiesterase 5 inhibitors (IPDE5) are able to restore chloride transport in F508del cystic fibrosis (CF) airway epithelium. CF patients with severe lung disease are at risk of pulmonary hypertension and impaired exercise tolerance. Treatment with IPDE5 is a promising strategy in CF. Methods: We conducted a study in 12 stable F508del homozygous CF adults (5 males and 7 females) with median (IQR) age of 25.9 (22.8–27.6) years without Burkholderia cepacia complex airway colonization. We administered sildenafil for 30 days and evaluated pulmonary function, respiratory symptoms and exercise tolerance before and after treatment using spirometry (forced expiratory volume in 1 sec. [FEV1]), 6-minute walking test (6-minute walking distance [6-MWD]) and CFQ-R quality of life questionaire (physical and respiratory domain [PHYS and RESP, respectively]). 8 patients completed the study (2 dropped due to pulmonary exacerbation and further 2 due to headaches). Exercise tolerance changed significantly (median and IQR; Wilcoxon test): 6-MWD 618 (612–692) vs. 577 (562–633) m, p = 0.012, and PHYS 93.8 (68.8–100.0) vs. 89.6 (60.4–97.9), p = 0.043, whereas pulmonary function and respiratory symptoms did not. Transient headaches were common adverse events during the first 1−2 weeks of sildenafil treatment in 7 of the 12 patients. Conclusion: We conclude that short-term sildenafil treatment in CF adults led to an improvement of exercise tolerance without changing pulmonary function and respiratory symptoms. Supported by the project (Ministry of Health, Czech Republic) for conceptual development of research organization 00064203 (University Hospital Motol, Prague, Czech Republic).

What matters in chronic Burkholderia cenocepacia infection in cystic fibrosis: Insights from comparative genomics

Burkholderia cenocepacia causes severe pulmonary infections in cystic fibrosis (CF) patients. Since the bacterium is virtually untreatable by antibiotics, chronic infections persist for years and might develop into fatal septic pneumonia (cepacia syndrome, CS). To devise new strategies to combat chronic B. cenocepacia infections, it is essential to obtain comprehensive knowledge about their pathogenesis. We conducted a comparative genomic analysis of 32 Czech isolates of epidemic clone B. cenocepacia ST32 isolated from various stages of chronic infection in 8 CF patients. High numbers of large-scale deletions were found to occur during chronic infection, affecting preferentially genomic islands and nonessential replicons. Recombination between insertion sequences (IS) was inferred as the mechanism behind deletion formation; the most numerous IS group was specific for the ST32 clone and has undergone transposition burst since its divergence. Genes functionally related to transition metal metabolism were identified as hotspots for deletions and IS insertions. This functional category was also represented among genes where nonsynonymous point mutations and indels occurred parallelly among patients. Another category exhibiting parallel mutations was oxidative stress protection; mutations in catalase KatG resulted in impaired detoxification of hydrogen peroxide. Deep sequencing revealed substantial polymorphism in genes of both categories within the sputum B. cenocepacia ST32 populations, indicating extensive adaptive evolution. Neither oxidative stress response nor transition metal metabolism genes were previously reported to undergo parallel evolution during chronic CF infection. Mutations in katG and copper metabolism genes were overrepresented in patients where chronic infection developed into CS. Among professional phagocytes, macrophages use both hydrogen peroxide and copper for their bactericidal activity; our results thus tentatively point to macrophages as suspects in pathogenesis towards the fatal CS.

Immunonutrition in patients with cyctic fibrosis leads to drop of serum amyloid A and increase of oxidative stress

The aim of the present study is to evaluate of the impact of immunonutrition on parameters of oxidative stress and inflammation in patients with cystic fibrosis and malnutrition. In the 30 patients with cystic fibrosis and long-term enteral nutrition support for malnutrition the effect of standard and immunonutrion sipping on oxidative stress and inflammatory activity parameters was compared. Malonyldialdehyde (MDA) as parameter of oxidative stress and serum amyloid A (SAA), interleukin 1 and 6, hsCRP, IgM, IgA, IgG as parameters of inflammatory activity were examined. Immunonutrition decreased SAA to 17.6 mg/L comparing to 25.6 mg/L when standard nutrition was given (p = 0.014). MDA was 0.66 µM on standard and 0.96 µM on immunonutrition support (p<0.01). The significant negative correlation was recorded between MDA and SAA, hs-CRP, interleukin 6, IgA and IgG. In conclusion, the application of immunonutrition in patients with cystic fibrosis and malnutrition is associated with drop of SAA but with the rise of MDA.

Vanishing Lung Syndrome in a Cystic Fibrosis Patient

Fig. 1. Chest X-ray and chest CT. (a) Posteroanterior chest X-ray shows hyperlucent upper parts of both lung fields, approx. upper half on the right side and upper third on the left side. Residual lung parenchyma has increased bronchovascular markings; (b) Chest CT (sagittal reconstruction) at the level of left hilum shows vanishing lung in the upper third of left lung; (c) Chest CT (coronal reconstruction) at the level of main bronchi shows bronchiectasis in the left upper lobe ending in severe emphysematous changes of lung parenchyma, vanishing lung in both upper zones, large bulla in the right lower lobe and consolidation in basolateral part of the right upper lobe.

273 Influenza vaccination in CF adults: Coverage level and adverse reactions

Objectives Influenza increases morbidity in CF individuals. Annual vaccination against influenza is a recommended measure in people with chronic respiratory diseases including CF. However, there are worries about adverse reactions of influenza vaccine in some patients and coverage level is generally low in the Czech Republic (approximately 5% of population). Methods We conducted a study in CF adults in Prague CF Centre regarding to coverage level and occurrence of adverse reactions after influenza vaccination using interviews during visits of CF clinic or by phone or e-mail in January 2015. Results 112 CF adults (59 males) with mean±SD age 27.3±6.2 years and mean±SD FEV 1 68±26 pred. were interviewed. Thirty-two (28.6%) patients received influenza vaccine. Among them, three patients experienced adverse reaction of mild intensity. In those who did not received a vaccination, eleven subjects reported adverse reactions in the past (two of them described it as severe), four patients experienced prolonged pulmonary exacerbations and others (58.0% of study group) had no interest in vaccination. Together, current or previous adverse reactions were reported in 14 (12.5%) subjects. Level of vaccination was not associated with sex, age or lung function. Conclusion Coverage level of influenza vaccination in Czech CF adults is low. The main reason is no interest for vaccination. Adverse reactions were quite frequent but usually with only mild intensity.

Lung cancer, pulmonary emphysema and pleural effusion: An autopsy study

OBJECTIVES To determine the exact incidence of lung cancer, pulmonary emphysema and pleural effusion we decided to carry out an autopsy study. METHODS In this autopsy study carried out over two years, we compared the results of autopsy findings with the clinical data in accompanying records of the deceased. RESULTS Among the 708 deceased subjects, there were 398 males and 310 females with a median age of 71 years. At autopsy, 55 cases of lung carcinoma (BCA) were found, of which 24 have not been identified during life (44%). Among the deceased with BCA, emphysema was also observed at autopsy in 40% of the cases. Pulmonary emphysema was described macroscopically in 28% of the full set of 708 deceased, whereas the accompanying records of the deceased described this condition in only 12% of the cases. Microscopic changes compatible with emphysema were identified in 54% of the examined lungs. Pleural effusions were described in the accompanying records of 13% of the deceased, while the autopsies showed this condition in 33% of the deceased. BCA was accompanied by effusion in 25% of the cases. CONCLUSIONS The obtained results show that the studied conditions are present in more cases than are reported by clinicians. The study confirms the commonly accepted association between lung cancer and emphysema.

WS8.2 Pulmonary arterial hypertension in adult CF lung transplantation candidates

Objectives: Lung transplantation is the most effective means of improving survival and quality of life in patients with end-stage CF. Our aim was to determine outcomes of referrals, waiting times and factors associated with a poor outcome. Methods: Data were retrieved retrospectively from our electronic patient record system for all adult patient referred to UK Transplant centres over a 6 year period. Results: 48 patients [27 female, median (range) age at referral 29.7 yrs (16.5–49.5), FEV1 25% predicted (13−41) and BMI 20.2 kg/m 2 (16.6–29.7) were referred for lung transplantation. Median time from initial discussion to referral was 80 days (13–1090). 3 patients died prior to first appointment and 5 are waiting to be seen. 40 patients were assessed and 26 accepted (4 died after first appointment, 2 not accepted and 8 currently under follow up). Of the 26 accepted, 16 have received a transplant [waiting time on active list 177 days (28–865)], 3 died on the active list, 3 were removed (significant clinical deterioration) and 4 are currently waiting. Total referral process from initial assessment to transplant was 1.6 yrs (0.7−3.2). Fifteen patients died during the referral process; there were no significant differences in clinical severity when compared to patients who were transplanted. However patients who died during the process had a significantly longer time from initial discussion to local assessment [112 (13–1090) vs. 56 (29–440) days, p = 0.0481]. Conclusion: Early discussion is important and current waiting times need to be taken into consideration. Increased psychological input is essential as an initial delay in the process may be associated with poor outcome.