Chronic Pseudomonas aeruginosa infection definition: EuroCareCF Working Group report.

Chronic pulmonary infection with P. aeruginosa develops in most patients with cystic fibrosis (CF); by adulthood 80% of patients are infected and chronic P. aeruginosa infection is the primary cause of increased morbidity and mortality in CF. Chronic infection is preceded by an intermittent stage of infection. The initial stage is characteristically followed by the gradual emergence of mucoid variants of the colonizing strains and a rise in anti-Pseudomonas antibodies. In addition to optimizing existing therapeutic strategies, effective new agents need to be identified. Studies in patients with CF are particularly challenging: the progressive nature of the disease and the wide variation in severity influence considerably the outcome of drug testing. A validated, universally accepted, and clinically useful classification of patients infected with P. aeruginosa, particularly those chronically infected, is needed that can be used as both a criterion for patient selection for clinical trials and as a study endpoint.

Pulmonary exacerbation: towards a definition for use in clinical trials. Report from the EuroCareCF Working Group on outcome parameters in clinical trials.

Pulmonary exacerbations represent a key outcome variable in clinical trials of cystic fibrosis (CF). As there is variation in the trigger for use of intravenous antibiotics compared to the use of oral antibiotics or new nebulised therapy for treatment of exacerbations, the consensus view is that use of intravenous antibiotics cannot be regarded as the key defining character for an exacerbation on its own. The consensus view is that the clinical need for additional treatment as indicated by a recent change in clinical parameters provides the best definition of an exacerbation. Which parameters to include as well as the problems associated with the use of scoring systems and symptom clusters are being discussed.

Serum cystatin C level for better assessment of glomerular filtration rate in cystic fibrosis patients treated by amikacin.

BACKGROUND AND OBJECTIVE: Monitoring of renal function in cystic fibrosis (CF) patients is essential. The dosage regimen of amikacin is regularly modified according to the patient's glomerular filtration rate (GFR). The aim of the study was to evaluate the use of cystatin C (CyC) for monitoring amikacin therapy along with other markers of renal tubular and glomerular function, and damage [N-acetyl-beta-d glucosaminidase (NAG), creatinine level and creatinine clearance]. METHODS: We compared the GFR, estimated from the serum concentrations of creatinine (Cockcroft-Gault formula) and CyC (Grubb's formula). Seventy-one patients (mean age 12 years; range 4-28 years) with CF were treated by intermittent intravenous infusion of amikacin. Tubular nephrotoxicity was investigated by measurement of urine NAG/urine creatinine ratio (U-NAG/U-creatinine). Concentrations of all markers were measured before starting amikacin therapy and at days 3, 5, 7, 10 and 12. Fluorescence polarization analysis, turbidimetry, enzymatic phototometric creatinine deaminase method and fluorimetry were used for determination of serum amikacin, serum CyC, creatinine and urine NAG activity. Receiver operating characteristic (ROC) analysis was performed to assess the influence of GFR estimated from serum creatinine and serum CyC for the prediction of amikacin clearance during aminoglycoside therapy. RESULTS: Significant differences in the rate of U-NAG/U-creatinine were noted before and after treatment with amikacin (P < 0.001). Serum creatinine levels and creatinine clearance at the end of amikacin therapy (12th day) did not show any significant differences in comparison with the levels measured before the start of therapy (0th day). At days 5, 7, 10 and 12, serum CyC levels showed a significant elevation (P < 0.001), and CyC clearance showed a significant decrease (P < 0.001) in comparison with the levels measured at day 0. The ratio of amikacin clearance/creatinine clearance decreased with therapy whereas the amikacin clearance/CyC and amikacin clearance/CyC clearance increased. CONCLUSION: We showed that the rate of U-NAG/U-creatinine is a suitable marker for monitoring tubular nephrotoxicity in CF patients. Serum creatinine and estimated creatinine clearance are modest predictors of GFR in CF patients. CyC appears to be a better marker of GFR than serum creatinine concentration or creatinine clearance in our study. Serum CyC levels and CyC clearance showed greater ability to predict amikacin clearance during therapy than creatinine clearance.

Genetic variants of transferrin in cystic fibrosis.

BACKGROUND: Genetic polymorphism of serum transferrin (Tf) was studied in order to differentiate between protein genetic variants and congenital disorders of glycosylation (CDG), further focusing on unusual findings. METHODS: Screening of Tf hypoglycosylation was carried out by isoelectric focusing with direct immunofixation and Coomassie blue staining in 100 healthy controls and a group of 1247 patients with various symptoms and diagnoses. RESULTS: Of the seven different genotypes detected, a significantly higher (p = 0.004) frequency of Tf C1C2 was found among 92 patients with cystic fibrosis; only the most severe DF508 mutation (in either homozygous or heterozygous form) was regularly present in the carriers of this Tf genotype, in contrast to those with the Tf C1C1 variant. CONCLUSIONS: Association of Tf C2 allele with various malfunctions has been noticed before, but is so far unresolved. This is the a report on increased frequency of Tf C1C2 genotype found in cystic fibrosis. Analysis of larger samples and independent confirmation of our results are needed.

Association of tumour necrosis factor alpha variants with the CF pulmonary phenotype.

BACKGROUND: The pulmonary phenotype in patients with cystic fibrosis (CF), even in those with the same CF transmembrane conductance regulator (CFTR) genotype, is variable and must therefore be influenced by secondary genetic factors as well as environmental factors. Possible candidate genes that modulate the CF lung phenotype may include proinflammatory cytokines. One such protein is tumour necrosis factor alpha (TNFalpha), a member of the immune system. METHODS: Three polymorphic loci in the promoter (-851c/t, -308g/a, -238g/a) and one polymorphic locus in intron 1 (+691g ins/del) of the TNFalpha gene were typed by a single nucleotide primer extension assay in CF patients and healthy controls. Spirometric data and first age of infection with Pseudomonas aeruginosa were collected retrospectively from patients' medical records. RESULTS: An association was found between the TNFalpha +691g ins/del polymorphic locus and severity of CF lung disease. Patients heterozygous for +691g ins and +691g del were more likely to have better pulmonary function (mean (SD) forced expiratory volume in 1 second (FEV1) 79.7 (12.8)% predicted) than patients homozygous for +691g ins (mean (SD) FEV1 67.5 (23.0)% predicted; p = 0.008, mean difference 12.2%, 95% CI 3.5 to 21.0). Also, patients heterozygous for +691g ins and +691g del were more likely to have an older first age of infection with P aeruginosa (mean (SD) 11.4 (6.0) years) than patients homozygous for +691g ins (mean (SD) 8.3 (4.6) years; p = 0.018, mean difference 3.1 years, 95% CI 0.5 to 5.6). An association was also found with the -851c/t polymorphic locus. In the group of patients with more severe FEV1% predicted, a higher proportion of patients were homozygous for the -851c allele than in the other group of patients (p = 0.04, likelihood ratio chi2, odds ratio = 2.4). CONCLUSION: TNFalpha polymorphisms are associated with the severity of CF lung disease in Czech and Belgian patients with CF.

[Evaluation of three dosage regimens of amikacin using pharmacokinetic models in patients with cystic fibrosis]. / Vyhodnocení trí dávkových rezimu amikacinu farmakokinetickým modelováním u pacientu s cystickou fibrózou.

BACKGROUND: Once-daily administration of aminoglykosides is routinely used, but comparative efficacy data for patients with cystic fibrosis are not available. METHODS AND RESULTS: The aim of the this study was to compare the predicted pharmacodynamic (PD) activity of amikacin at 28 mg/kg/den administered every 24 hod.(q24h), q12h, and q8h. Pharmacokinetic (PK) data were derived from analysis of the amikacin serum concentration from 42 CF children patients. Individual pharmacokinetics values were used to construct serum concentration--versus time curves and to determine various indices (c peak/MIC ratio and time during the concentration was less than the MIC--T < MIC) for all three dose regimens described above. MIC (minimal inhibitory concentration) for Pseudomonas aeruginosa was 4 mg/l. Significantly lower c peak/MIC but shorter T < MIC were noted when regimens of q8h versus q12h (p < 0.001), q8h vs. q24h (p < 0.001) and q12h vs. q24h (p < 0.001) were compared. This analysis suggests that the potential advantage of achieving a greater c peak/MIC with once-daily aminoglycoside administration may be neutralized by the significantly greater T < MIC in CF patients compared with that achieved with multiple-daily-dosing regimens. CONCLUSIONS: Routine use of once daily amikacin administration could not be recommended until the clinical data confirming efficiency of this dose modality are available.

Serum alpha-glutathione S-transferase as a sensitive marker of hepatocellular damage in patients with cystic fibrosis.

The aim of the study was to evaluate serum a-glutathione S-transferase (s-GSTA) levels in patients with cystic fibrosis (CF) and to compare s-GSTA with other liver function tests and with a hepatic ultrasound scan (US). The cytosolic enzyme, alpha-glutathione S-transferase is predominantly found in the liver and is distributed uniformly in the liver tissue. In our study s-GSTA levels were measured in 37 CF patients aged 1 to 28 years (mean age 10.4 years, 24 males). The control group consisted of 27 patients aged 2 to 17 years (mean age 8.5 years, 18 males). The presence of hepatobiliary abnormalities was assessed by clinical examination, ultrasound scan, s-GSTA, and conventional liver enzymes: alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST) and gama-glutamyl transferase (GMT). The calculated 5-95 % range of s-GSTA for the control group was 0.098-2.54 microg/l, for the CF group 0.43-9.76 microg/l. Mean s-GSTA level in the control group was 1.55 microg/l (S.D.=1.57), and 2.05 micro/l (S.D.=2.60) in the CF group. In the group of CF patients, the serum levels were significantly higher than in the control group (P<0.01). No significant correlation existed in the CF group between s-GSTA and conventional liver tests (ALT, AST, ALP and GMT). Four patients in the CF group had hepatobiliary abnormalities detectable by conventional liver tests, s-GSTA and US. Four patients had abnormal s-GSTA, while conventional liver tests and US were normal. One other patient had abnormal hepatic US, but normal standard liver tests and s-GSTA. The study has suggested that a raised s-GSTA level might be a marker of possible pathological changes of the hepatobiliar system in CF patients. Serum GSTA seems to be a more sensitive marker than transaminases for the monitoring of hepatocellular integrity and as an early predictor of hepatic damage.

Lung transplantation for cystic fibrosis: immune system and autoimmunity.

BACKGROUND: In the current study we focused on changes in the immune parameters of patients with CF after lung transplantation (Tx), with particular emphasis on the interaction of the immune system, infection, the autoimmune phenomenon observed in some CF patients, and immunosuppression. MATERIAL AND METHODS: Seven transplant patients with CF were investigated, 3 men and 4 women; the average age at Tx was 24.2 years (20.2-32.3). The parameters of both humoral immunity (immunoglobulins, complement, CRP, antinuclear and antineutrophil cytoplasmic antibodies) and cellular immunity (T and B lymphocytes, NK cells) were traced. RESULTS: We observed marked initial hyperimmunoglobulinemia, with a sharp drop in immunoglobulin levels within 1 month after Tx. Positivity for antineutrophil cytoplasmic antibodies (ANCA) was found in 3 patients before Tx. A strong ANCA positivity persisted 2 months after Tx despite deep introductory immunosuppression. In one patient ANCA positivity, after a transient negative result at months 2 and 12 after Tx, reappeared one year after Tx. The Burkholderia cepacia infections found in 2 patients proved to be lethal. CONCLUSIONS: In our series of CF lung transplant recipients, we found Burkholderia cepacia infection to be a risk factor. The robust appearance of autoantibodies and their persistent positivity for many months despite deep immunosuppression is a remarkable feature observed in some CF patients.

Insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in cystic fibrosis: a positive effect of antibiotic therapy and hyperalimentation.

UNLABELLED: Patients with cystic fibrosis (CF) are underweight and growth retarded. This study tested the link between serum insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein-3 (IGFBP-3) levels and body height, nutritional status, pulmonary function tests and activity of inflammation in 92 subjects with CF (age 2.1-18.8 y). It also analysed the effect of short-term antibiotic treatment and hyperalimentation on IGF-I and IGFBP-3 levels in 33 subjects (age 3.6-33.7y) on 41 occasions. Both IGF-I (-1.19 +/- 0.17 SD) and IGFBP-3 levels (-0.66 +/- 0.12 SD; both p < 0.0001 vs 0) were decreased in cross-sectional measurements. Their standardized values were inversely proportional to age (IGF-I: r = -0.23, p = 0.03; IGFBP-3: r = -0.29, p = 0.005) and positively correlated with SDS of height (IGF-I: r = 0.40, p < 0.0001; IGFBP-3: r = 0.36, p = 0.0005) and of mid-arm circumference (IGF-I: r = 0.39, p = 0.0001; IGFBP-3: r = 0.38, p = 0.0002), and with pulmonary function tests. After a short-term course of intensive antibiotic therapy and hyperalimentation, IGF-I normalized (from -0.66 +/- 0.20 to 0.00 +/- 0.25 SD; p < 0.0001) and IGFBP-3 increased (from -0.78 +/- 0.15 to -0.53 +/- 0.16 SD; p = 0.002). IGFBP-3 correlated inversely with erythrocyte sedimentation rate (r = -0.40, p = 0.01). CONCLUSION: The levels of IGF-I and IGFBP-3 are markedly decreased in patients with CF and tend to normalise after a short course of antibiotic treatment and hyperalimentation.

Characterization of a novel 21-kb deletion, CFTRdele2,3(21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central and East Europe.

We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, viz., a deletion that is frequently observed in Central and Eastern Europe. The mutation, termed CFTRdele2,3(21 kb), deletes 21,080 bp spanning introns 1-3 of the CFTR gene. Transcript analyses have revealed that this deletion results in the loss of exons 2 and 3 in epithelial CFTR mRNA, thereby producing a premature termination signal within exon 4. In order to develop a simple polymerase chain reaction assay for this allele, we defined the end-points of the deletion at the DNA sequence level. We next screened for this mutation in a representative set of European and European-derived populations. Some 197 CF patients, including seven homozygotes, bearing this mutation have been identified during the course of our study. Clinical evaluation of CFTRdele2,3(21 kb) homozygotes and a comparison of compound heterozygotes for deltaF508/CFTRdele2,3(21 kb) with pairwise-matched deltaF508 homozygotes indicate that this deletion represents a severe mutation associated with pancreatic insufficiency and early age at diagnosis. Current data show that the mutation is particularly common in Czech (6.4% of all CF chromosomes), Russian (5.2%), Belorussian (3.3%), Austrian (2.6%), German (1.5%), Polish (1.5%), Slovenian (1.5%), Ukrainian (1.2%), and Slovak patients (1.1%). It has also been found in Lithuania, Latvia, Macedonia and Greece and has sporadically been observed in Canada, USA, France, Spain, Turkey, and UK, but not in CF patients from Bulgaria, Croatia, Romania or Serbia. Haplotype analysis has identified the same extragenic CF-haplotype XV-2c/KM. 19 "A" and the same infrequent intragenic microsatellite haplotype 16-33-13 (IVS8CA-IVS 17bTA-IVS 17bCA) in all examined CFTRdele2,3(21 kb) chromosomes, suggesting a common origin for this deletion. We conclude that the 21-kb deletion is a frequent and severe CF mutation in populations of Eastern- and Western-Slavic descent.

[Cystic fibrosis--a disease of adolescents and adults?]. / Cystická fibróza--nemoc dospívajících a dospelých?

BACKGROUND: Cystic fibrosis (CF) is no longer a childhood disease. Since the identification of the gene in 1989 research has made advances and changed views on the pathogenesis, diagnosis and treatment. The objective of the present work is to make doctors treating adult patients familiar with modern therapeutic methods and their value. METHODS AND RESULTS: In the CF Centre of the Faculty Hospital in Prague Motol 349 patients are followed up on a long-term basis, incl. 95 who died since 1985. Hundred and twenty six (36.1%) patients survived to the age of 18 years, of those 41 died and 85 patients live. Comparison of semilongitudinal data of a group of 83 patients born before 1975 whose treatment during childhood and puberty was inadequate and 196 patients born in 1976-90 treated by modern methods proved the great effect of treatment on the course and prognosis of the disease. The median age at death increased during from 12.2 years in 1985-90 to 18.8 years in 1991-1998 (p = 0.004). The nutritional status of adult patients is satisfactory in 40.4%, poor in 33.3% and marginal in 26.3%. A normal pulmonary function was recorded in 17.5%, 22.8% are severely affected, the majority of patients (59.7%) has values within 40 to 80% of normal levels. CONCLUSIONS: Modern intensive treatment improved the prognosis and quality of live in patients with CF. Critical deterioration of the clinical condition shifted to the threshold of adult age. It is therefore essential that doctors treating such patients should be familiar with this issue.

Antineutrophil cytoplasmic autoantibodies (ANCA) in children with cystic fibrosis.

Anti-neutrophil cytoplasmic antibodies (ANCA) represent a useful diagnostic tool in patients with small vessel vasculitis. Circulating ANCA specific for bactericidal/permeability increasing protein (BPI) have been recently reported in adult patients with cystic fibrosis (CF), an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene with consequent impaired function of a transmembrane chloride channel. To contribute to the better understanding of the significance of ANCA in this disease, we investigated ANCA presence and antigenic specificity in children with CF. Results were correlated with clinical status, immunological data, age and genotype. The indirect immunofluorescence pattern of a total of 71 children with CF indicated that 31 were c-ANCA positive, while seven were p-ANCA positive. In further ELISA studies of ANCA antigenic specificity, 51 out of 66 investigated samples were positive for BPI, and 14 out of 28 were positive for proteinase 3 (PR3). We found an association between levels of antibodies against PR3 with age and Pseudomonas infection. We did not, however, find any correlation between CFTR genotypes, Pseudomonas infection or paediatric parameters and the level of anti-BPI antibodies. High positivity of anti-BPI antibodies were seen even among the youngest CF patients, before the development of clinical signs of CF, indicating that formation of ANCA might be a very early event in the disease. Both anti-BPI and anti-PR3 antibodies may play a significant, although variable role, in the pathogenesis of CF.

Frequency of the delta F508 mutation and flanking marker haplotypes at the CF locus from 167 Czech families.

This study analyses distribution patterns of the delta F508 mutation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) gene and the cystic fibrosis (CF)-linked marker loci MET, D7S23, D7S399, and D7S8 in a sample of 167 (116 complete) CF families from Bohemia and Moravia (Czechoslovakia). DNA typing was performed by polymerase chain reaction amplification, restriction analysis, and agarose or polyacrylamide gel electrophoresis. The frequency of the delta F508 mutation in this sample is 67% and the frequency of the B haplotype is 77.6% on CF chromosomes. Linkage disequilibrium was found between delta F508 and all markers tested.

[Strategies in prenatal diagnosis of cystic fibrosis after the introduction of DNA analysis. Initial experience]. / Strategie prenatální diagnostiky cystické fibrózy po zavedení analýzy DNA. Prvé zkusenosti.

The authors describe their experience with the prenatal genetic diagnosis of cystic fibrosis (CF), using DNA analysis in the first trimester of pregnancy in three families with a 25% risk of CF. The authors examined polymorphisms of probes J3.11, met D, met H, KM-19 and XV-2c. All families were fully informative when one or two probes were used. In two families the development of unaffected children--carriers of the gene for CF was proved. In one of these foetuses in the 17th and 21st week false pathological values of microvilillous enzymes were assessed. With regard to this possibility the authors do not recommend to supplement the DNA analysis in the first trimester by biochemical examination of amniotic fluid. The results were confirmed by delivery of unaffected children. In one family DNA analysis revealed the development of an unaffected homozygote, the pregnancy was, however, terminated by a miscarriage. In women with an increased risk of abortion the authors recommend therefore to make the molecular genetic examination during the second trimester from amniotic fluid cells.

[Experience with rapid molecular genetic diagnosis using the polymerase chain reaction]. / Zkusenosti s metodou rychlé molekulárne genetické diagnostiky polymerázovou retezovou reakcí.

The authors give an account of their experience with 1000 amplifications of DNA by the polymerase chain reaction for the prenatal diagnosis of cystic fibrosis. The method is demonstrated on examples of examinations of the informativity value and prenatal diagnosis in the first trimester of pregnancy in families with a 25% risk of cystic fibrosis, using J 3.11 (Msp I), met H (Msp I), KM 19 (Pst I), CS 7 (Hha I), Mp6d9 (Msp I), XV 2c (Taq I) probes. The authors summarize methodical check-up and safety measures to ensure the reliability of diagnoses made by the PCR method.

Cystic fibrosis marker testing in Bohemia with polymerase chain reaction.

Conditions for assessing KM-19 probe detected by Pst-1 restriction fragment length polymorphism (RFLP) by means of polymerase chain reaction were provided. Computer controlled mechanical arm with waterbaths and cloned heat-stable DNA polymerase was used. Results of KM-19 allelic frequencies on 90 cystic fibrosis chromosomes are presented. Allele two frequency was -0.833.