Inverse relation between vitamin D and serum total immunoglobulin G in the Scandinavian Cystic Fibrosis Nutritional Study.

BACKGROUND/OBJECTIVES: The hallmark of cystic fibrosis (CF) is chronic lung inflammation. The severity of lung disease is closely correlated with immunoglobulin G (IgG) levels. Beyond its contribution to the bone health, the importance of vitamin D has not been fully recognized owing to the lack of human studies providing evidence of its benefit. In the context of the recently described immunomodulatory functions of vitamin D, we aimed to assess the relationship between vitamin D and IgG levels. SUBJECTS/METHODS: Eight hundred and ninety-six CF patients were included (0.53-65.9 years) from seven centers in Denmark, Norway and Sweden. Serum 25-hydroxyvitamin D (25OHD) and total IgG were measured, spirometry was carried out and vitamin D intake data were gathered using a 7-day dietary food record. Multiple linear regression analyses were performed for IgG and forced expiratory volume in 1λs (FEV1) as dependent variables, and serum 25OHD, daily food and supplemented vitamin D sources of intake as independent variables. The model was controlled for age, gender, genotype, CF-related diabetes, season, infection/colonization status, long-term oral corticosteroid treatment, long-term treatment with macrolide antibiotics, pancreatic insufficient phenotype and body mass index z-score. RESULTS: Serum total IgG levels were negatively associated with serum 25OHD (adjusted R (2) = 0.376; beta = -0.02; P<0.001), supplemented vitamin D intake per kg bodyweight (adjusted R (2) = 0.375; beta = -0.82; P < 0.001) and total vitamin D intake per kg bodyweight (adjusted R (2) = 0.398; beta = -0.60; P = 0.002). Serum 25OHD was positively associated with FEV1 (adjusted R (2) = 0.308; beta = 0.0007; P = 0.025). CONCLUSIONS: Increasing vitamin D intake may positively modulate inflammation in CF. This study supports the proposed role of vitamin D in the immune system during infection and substantiates prospective studies.

Co-morbidity of cystic fibrosis and celiac disease in Scandinavian cystic fibrosis patients.

BACKGROUND: The co-morbidity of cystic fibrosis (CF) and celiac disease (CD) has been reported sporadically since the 1960s. To our knowledge, this is the first time a systematic screening is performed in a large cohort of CF patients. METHODS: Transglutaminase-IgA (TGA), endomysium-IgA (EMA) and total IgA in serum were measured in 790 CF patients (48% females, 86% with pancreatic insufficiency). Six patients were diagnosed with CD prior to the study, all receiving a gluten-free diet. Patients with elevated TGA (>50 Units/mL) and a positive EMA test were offered a gastroscopy obtaining mucosal biopsies from the duodenum. RESULTS: Four new cases of CD were diagnosed. Two additional patients had positive serological tests, but normal biopsies. In total, 10 cases of CD (1.2%, 1:83) indicate a prevalence rate about three times higher than the general prevalence of CD in Norway and Sweden. No CD patients were detected in the Danish CF cohort. Patients diagnosed with untreated CD reported symptoms typical of both CF and CD (poor weight gain, loose and/or fatty stools, fatigue, irritability, abdominal pain). They improved after introduction of a gluten-free diet. CONCLUSIONS: Systematic screening for CD in a Scandinavian cohort of CF patients revealed a higher prevalence of CD than in the general population. Clinical signs of CD are difficult to differentiate from CF with malabsorption, and patients may go undiagnosed for a long time. In a population where CD is common we recommend screening for CD in patients with CF.

Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients.

In total, 40 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients were included in this study. Twenty of these were collected in 1994 and 1997, from six CF patients, and the rest were collected from different CF patients in 2000 and 2001. The relative expression of mRNA for the efflux pump protein MexY was determined by real-time PCR and correlated with susceptibilities to amikacin and tobramycin. The chromosomal genes mexZ, rplY, galU, PA5471 and nuoG, which were found to have a role in the gradual increase in MICs of aminoglycoside antibiotics in laboratory mutants of P. aeruginosa, were analysed. MexY mRNA overproduction was found in 17/20 isolates collected in 1994 and 1997, and was correlated with decreased susceptibility to aminoglycosides. Alteration of the MexXY-OprM efflux system has been the main mechanism of resistance to aminoglycoside antibiotics in CF P. aeruginosa isolates over the 3-year period. In several isolates, expression of the PA5471 gene product might have some effect on elevated MICs of aminoglycosides. Inactivation of rplY, galU and/or nuoG may explain the gradual increase in MICs of aminoglycosides in laboratory mutants but probably not in the CF environment, as rplY and galU were unaltered in all isolates, and nuoG was not expressed in only one isolate. No 16S rRNA A-site mutations were found in any of the four copies of the gene in 13 investigated isolates.

Arbitrarily primed PCR and sequencing of 16S rDNA for epidemiological typing and species identification of Burkholderia cepacia isolates from Swedish patients with cystic fibrosis reveal genetic heterogeneity.

To investigate whether arbitrarily primed (AP)-PCR and/or 16S rDNA sequencing could be used as rapid methods for epidemiological typing and species identification of clinical Burkholderia isolates from patients with cystic fibrosis (CF), a total of 39 clinical B. cepacia isolates, including 33 isolates from 14 CF patients, were fingerprinted. ERIC-2 primer was used for AP-PCR. The AP-PCR clustering analysis resulted in 14 different clusters at a 70% similarity level. The AP-PRC patterns were individual despite considerable similarities. To sequence rDNA, a broad-range PCR was applied. The PCR product included four variable loops (V8, V3, V4 and V9) of the 16S ribosomal small subunit RNA gene. The multiple sequence alignment produced 12 different patterns, 5 of them including more than one isolate. Heterogeneity of the bases in the V3 region, indicating the simultaneous presence of at least two different types of 16S rRNA genes in the same cell, was revealed in 10 isolates. Most of the CF patients were adults who had advanced disease at follow-up. Both the sequencing and the AP-PCR patterns revealed genetic heterogeneity of isolates between patients. According to the results obtained, AP-PCR could advantageously be used for epidemiological typing of Burkholderia, whereas partial species identification could effectively be obtained by sequencing of the V3 region of the 16S RNA gene.

TNF-alpha and IL-8 in consecutive sputum samples from cystic fibrosis patients during antibiotic treatment.

Proinflammatory cytokines in sputum are useful markers of the activity of lung disease in cystic fibrosis (CF). Tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) concentrations in sputum of 10 CF patients were determined during exacerbation and IL-8 in sputum of 48 patients at a yearly follow-up when patients were in optimal clinical condition. In 9 patients of the former group, TNF-alpha levels were increased during exacerbation. In 4 patients, the peak occurred within 2 d (median value > 1500 ng/l), whereas the remaining 5 had peak values on days 3-6 (median value 720 ng/l). IL-8 levels were > 800 microg/l in all 10 patients, and in 9 cases there was a positive correlation between IL-8 and TNF-alpha. Baseline IL-8 levels of 48 patients showed considerable variation (median 207 microg/l, range 1.5-392). There was a significant correlation between IL-8 concentrations and current colonization with either Pseudomonas aeruginosa or Staphylococcus aureus in the lower airways (p = 0.002), immunoglobulin G levels (p = 0.02) and the severity of the pathological findings shown by chest X-ray (p = 0.008). High IL-8 and TNF-alpha values correlated with symptoms of deterioration. IL-8 levels seemed to be markers of both current bacterial colonization and the degree of lung damage.

Polymerase chain reaction for the detection of Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia in sputum of patients with cystic fibrosis.

Occurrence of Pseudomonas aeruginosa, Stenotrophomonas (Xanthomonas) maltophilia and Burkholderia (Pseudomonas) cepacia in sputum of cystic fibrosis (CF) patients was demonstrated with a simple and rapid polymerase chain reaction (PCR) technique. The PCR was performed with a set of three primer pairs based on 16S rRNA sequences after sputum preparation with dithiothreitol and NaOH lysis. All three pathogens could be individually detected by the use of this technique. To prevent carry-over contamination, dUTP and uracil-N-glycosylase were included in the reaction. The amplicons were visualized by agarose gel electrophoresis. Sputum culture was performed on all samples. Ninety specimens from CF patients were analysed. The sensitivity for the detection of P. aeruginosa was 37/40 (93%) compared to culture. Bacterial growth of P. aeruginosa was found in three cases, where PCR amplicons were not detected, while PCR was positive in five cases, where culture did not reveal the presence of this bacterium. For this reason, the specificity was 45/50 (90%). For S. maltophilia, the PCR was less sensitive than culture (positive in three of six cases). In our series, B. cepacia was detected by culture in one case and this was also detected by PCR. There were no false-positive PCR results regarding S. maltophilia or B. cepacia. Thus, combined PCR-based detection of these three clinically relevant bacteria in sputum samples from CF patients can be performed by a reliable technique in a relatively simple manner. The present data indicate a high sensitivity and specificity for P. aeruginosa. The lower sensitivity observed for the detection of S. maltophilia in sputum and B. cepacia, as estimated from laboratory strains, may depend on PCR conditions and genetic heterogeneity, respectively. The greatest gains with this method can be made when it is used for the early detection of P. aeruginosa in sputum-producing CF patients.

Bacterial colonisation with Xanthomonas maltophilia--a retrospective study in a cystic fibrosis patient population.

Xanthomonas maltophilia was isolated from 25 of 150 patients with cystic fibrosis during a period of 10 years (1983-1992). Twelve patients harboured X. maltophilia chronically, i.e. repeatedly for more than 6 months. No predisposing factors for the colonisation could be identified by studying the clinical and laboratory data of the patients, including preceding and concurrent bacterial colonisation with other bacteria, antibacterial treatments, pulmonary function and biochemical markers. Up to 2 years after the chronic colonisation was established no clinical deterioration could be verified, but the patients with X. maltophilia generally had a worse lung function at the latest follow-up (2-7 years after colonisation) than controls colonised with Pseudomonas aeruginosa (p < 0.05). Our data imply that X. maltophilia is a pathogen and the colonisation appears to follow the same pattern as the colonisation by P. aeruginosa. The development of resistance to different antibiotics, as revealed by analysis of the inhibition zones, was related to antibacterial treatment courses. X. maltophilia showed reduced sensitivity to the most commonly used antibiotics, ceftazidime and tobramycin.