Clinical and microbiological features of Inquilinus sp. isolates from five patients with cystic fibrosis

Patients with cystic fibrosis (CF) may be colonized with unusual gram-negative bacilli whose identification is difficult and clinical impact unclear. We describe the clinical and microbiological features of five colonizations with organisms belonging to the recently described genus Inquilinus in CF patients. Isolates were identified from Burkholderia cepacia selective medium by means of 16S rRNA analysis. All of them were resistant to colistin, penicillins, cephalosporins, and monobactams but exhibited a remarkable susceptibility to imipenem. One of the five patients was transiently colonized with a nonmucoid isolate, whereas the four other patients were persistently colonized over the period of follow-up (8 to 21 months) with mucoid isolates. Pulsed-field gel electrophoresis of SpeI-digested genomic DNA was powerful for strain genotyping and demonstrated the clonality of Inquilinus sp. colonization for the two patients tested. Clinical evolution after the onset of Inquilinus was heterogeneous, but for at least one patient the lung function worsened and eradication of Inquilinus sp. was unsuccessful despite several imipenem courses. Finally, Inquilinus spp. may represent a new threat for CF patients due to their mucoid characteristic, their multiresistant pattern to antibiotics, and their ability to persist in the respiratory tract.     

Severity of chest disease in cystic fibrosis patients in relation to their genotypes.

A detailed comparison of the severity of chest disease with mutational status was carried out by cross sectional study of 127 cystic fibrosis patients, aged 1 to 31 years, living in Wales. Lung disease was classified according to severity, depending on pulmonary function tests (carried out on 76 patients) and chest radiograph status; information was obtained also on age at diagnosis in relation to severity of chest disease and colonisation with Pseudomonas species. Genotypes were determined by analysis for the mutations delta F508, delta I507, G551D, R553X, G542X, R117H, R560T, 1717--IG > A, and 621 + 1G > T. CF patients homozygous positive and heterozygous for the delta F508 deletion showed a significant decline of lung function with age. Unlike other studies, we did not find patients homozygous positive for the delta F508 deletion to have poorer lung function compared with heterozygous patients. Patients with the genotype 621 + IG > T/delta F508 tended to have more severe chest disease than the delta F508 homozygous patients in the same age group. There was some evidence that four patients heterozygous for R117H have mild chest disease.     

Histopathologic features of Burkholderia cepacia pneumonia in patients without cystic fibrosis.

We present the histopathologic features of fatal Burkholderia cepacia pneumonia in three adults (one man [age 44 years] and two women [aged 40 and 43 years]). In all patients, the pulmonary infiltrates initially were localized (right middle lobe, left upper lobe, and right middle lobe) but rapidly progressed. Two open-lung biopsies and one pneumonectomy specimen showed necrotizing granulomatous inflammation merging with areas of more conventional necrotizing bronchopneumonia In one patient, a mediastinal lymph node also showed stellate necrotizing granulomas. Vasculitis was absent. B. cepacia was cultured from the open-lung biopsies and bronchial wash specimens in two patients and from postmortem cultures of lung, subcarinal lymph nodes, and blood in the third. The histopathology in these patients resembles that of melioidosis, which is caused by a related organism, Burkholderia pseudomallei. B. cepacia needs to be considered in the differential diagnosis of necrotizing granulomatous inflammation. In addition, given the rarity with which B. cepacia is identified as a cause of pneumonia in the immunocompetent host, isolation of B. cepacia should trigger a workup for underlying immunodeficiency or lead to an investigation to exclude the possibility of a nosocomial infection.     

Dynamic colonisation by different Pneumocystis jirovecii genotypes in cystic fibrosis patients

Although asymptomatic carriers of Pneumocystis jirovecii with cystic fibrosis (CF) have been described previously, the molecular epidemiology of P. jirovecii in CF patients has not yet been clarified. This study identified the distribution and dynamic evolution of P. jirovecii genotypes based on the mitochondrial large-subunit (mt LSU) rRNA gene. The mt LSU rRNA genotypes of P. jirovecii isolates in 33 respiratory samples from CF patients were investigated using nested PCR and direct sequencing. Three different genotypes were detected: 36.3% genotype 1 (85C/248C); 15.1% genotype 2 (85A/248C); 42.4% genotype 3 (85T/248C); and 6% mixed genotypes. Patients studied during a 1-year follow-up period showed a continuous colonisation/clearance cycle involving P. jirovecii and an accumulative tendency to be colonised with genotype 3.     

Direct sputum analysis of Pseudomonas aeruginosa macrorestriction fragment genotypes in patients with cystic fibrosis

The distribution of bacterial populations in the airways of 13 patients with cystic fibrosis who were colonized for 6 – 23 years with Pseudomonas aeruginosa was investigated by genotyping of bacterial chromosomes directly isolated from 21 sputa. After removal of host material from sputum by hypotonic cell lysis and repetitive washing and centrifugation steps, agarose-embedded bacterial cells were lysed, residual eukaryotic DNA separated by field inversion gel electrophoresis, and the purified bacterial chromosomes subjected to macrorestriction fragment pattern and Southern analyses. Bacterial populations consisted of a single P. aeruginosa clone in 17 sputa, of which more than one clonal variant was apparent in two SpeI fragment fingerprints. Two clones of P. aeruginosa and another species co-existed in four samples. Genomically homogeneous populations of P. aeruginosa are characteristic for chronically colonized lungs in most cases of cystic fibrosis. Received: 25 April 1997     

Heterogeneity, persistence, and distribution of Pseudomonas aeruginosa genotypes in cystic fibrosis patients.

A collection of 222 isolates of Pseudomonas aeruginosa was obtained from the respiratory tract of 16 patients with cystic fibrosis over a 4- to 9-month period. Fourteen of these patients were unrelated, while the remaining two were siblings. Isolates were typed by conventional pyocin typing and also by the use of a DNA probe containing 741 bp immediately upstream of the exotoxin A structural gene and the initial 732 bp of the exotoxin A structural gene. By pyocin typing, 69% (11 of 16) of the patients were shown to harbor a single type that persisted in the lung throughout the study. By genotyping (DNA probe typing), all but three patients (13 of 16, 81%) harbored a single persistent genotype in their lungs. Six patients other than the sibling pair (6 of 14, 43%) shared a common genotype in their lungs as judged by DNA probing, and the pyocin type of these isolates was also identical. In four of these six patients, the shared genotype was also the persistent genotype. The sibling pair studied also carried a common genotype in their lungs as indicated by DNA probing, even though the pyocin type of these isolates varied. Results presented suggest that the majority of patients harbor a persistent strain in their lungs and that cross-colonization may occur.     

Prediction of mortality in patients with cystic fibrosis.

BACKGROUND. The majority of patients with cystic fibrosis die in early adulthood of lung disease. Lung transplantation is a treatment option for patients with advanced pulmonary disease, although the waiting period for organs may be as long as two years. Our purpose was to determine whether the risk of death due to respiratory failure could be predicted one or two years in advance on the basis of pulmonary function, blood gas levels, and nutritional status. METHODS. The study cohort consisted of 673 patients followed between 1977 and 1989. In each patient, pulmonary function, blood gas levels, nutritional status, and vital status were assessed between 1977 and 1987. Cox proportional-hazards regression analysis was used to compute the relative risk of death within one or two years after particular measurements. The effects of age and sex on mortality were also included in the analysis. RESULTS. One hundred ninety patients (28 percent) died during the study period. Overall, patients with a forced expiratory volume in one second (FEV1) less than 30 percent of the predicted value, a partial pressure of arterial oxygen below 55 mm Hg, or a partial pressure of arterial carbon dioxide above 50 mm Hg had two-year mortality rates above 50 percent. Among the laboratory measurements, the FEV1 was the most significant predictor of mortality, but age and sex were also significant in predicting risk. After adjustment for age and sex, the relative risk of death within two years was 2.0 (95 percent confidence interval, 1.9 to 2.2) for each decrement in the FEV1 of 10 percent below the predicted value. Among patients with the same FEV1, the relative risk of death was 2.0 (95 percent confidence interval, 1.5 to 2.6) in patients 10 years younger than other patients, and 2.2 (1.6 to 3.1) in female patients as compared with male patients. CONCLUSIONS. Patients with cystic fibrosis should be considered candidates for lung transplantation when the FEV1 falls below 30 percent of the predicted value. Female patients and younger patients may need to be considered for transplantation at an earlier stage.     

Recovery of mycobacteria from patients with cystic fibrosis.

Despite decontamination, overgrowth by pseudomonads renders cultural isolation of mycobacteria from respiratory specimens of patients with cystic fibrosis (CF) difficult or impossible. We performed a prospective study by comparing levels of reduction of overgrowth and recovery of mycobacteria using either pretreatment with N-acetyl-L-cysteine (NALC)-NaOH alone or pretreatment with NALC-NaOH and then with oxalic acid. From 406 specimens of 148 CF patients, 11 specimens were positive for mycobacteria, 5 of which grew mycobacteria after decontamination by either procedure. Three specimens grew mycobacteria only after decontamination with NALC-NaOH, whereas three specimens grew mycobacteria only after treatment with NALC-NaOH followed by oxalic acid but were overgrown after decontamination with NALC-NaOH. Thus, inactivation of mycobacteria by the more aggressive oxalic acid treatment offsets its beneficial effect of reducing the proportion of cultures overgrown with microorganisms other than mycobacteria.     

Circulating immune complexes in patients with cystic fibrosis in relation to clinical features

Immune complexes have been previously reported in the serum of patients with cystic fibrosis. This study was undertaken to relate the finding of complexes with the clinical features of the disease. Immune complexes detected by the ^I25I-Clq binding assay were found in the sera of 17/60 (28%) of patients with cystic fibrosis (CF). There was no association between the finding of raised levels of immune complexes and duration of chest symptoms, duration of daily sputum production, age, sex, weight, atopy, the presence or absence of malabsorption, pneumothorax, diabetes, Aspergillus precipitins or specific bacterial pathogens in the sputum. There was however a correlation between the finding of increasing circulating immune complexes and decrease in the respiratory function; forced expiratory volume in 1 sec (P< 0.001) and forced vital capacity (P< 0.005); also with weight (P<0.02). It is possible that the finding of immune complexes, at low levels, in cystic fibrosis are the result of tissue damage rather than its cause.     

Pancreatic ductal adenocarcinomas with cystic features: neither rare nor uniform.

Cystic tumors of the pancreas are uncommon but important because of their diverse pathology and biology. Their wide spectrum also includes cystic variants of otherwise solid tumors, such as cystic endocrine tumors, cystic acinar cell carcinomas and ductal adenocarcinomas with cystic changes. In this study, we screened pancreatic ductal adenocarcinomas and their variants for macrocystic changes and determined the nature of the cysts (neoplastic vs non-neoplastic). Of 483 tumors 38 (8%) had cystic features. The largest group consisted of 24 pancreatic ductal adenocarcinomas showing a large-gland pattern with small cysts whose diameter varied between 0.5 and 1.8 cm. The epithelial lining of these cysts was generally positive for CEA (83%) and/or MUC1 (71%) and MUC5AC (74%). p53 was positive in 57% of the cases. The second group of cystic tumors (8/483) showed degenerative cystic cavities with diameters ranging between 1 and 6 cm. This group consisted of poorly differentiated pancreatic ductal adenocarcinomas, undifferentiated carcinomas with or without osteoclast-like giant cells and one adenosquamous carcinoma. In the third group of cystic tumors there were four pancreatic ductal adenocarcinomas containing tumor-related retention cysts. Their epithelial cells were positive for MUC5AC, but negative for CEA, MUC1 and p53. The fourth group consisted of two pancreatic ductal adenocarcinomas showing closely attached pseudocysts caused by tumor-associated pancreatitis. The results indicate that a considerable number of pancreatic ductal adenocarcinomas and their variants display cystic features and must therefore be considered in the differential diagnosis of cystic neoplasms of the pancreas. Moreover, not all of the cystic structures we observed were neoplastic in nature. They may also represent non-neoplastic changes, such as retention cysts and inflammatory pseudocysts.     

Increased PIVKA-II concentrations in patients with cystic fibrosis.

Serum vitamin K concentrations and prothrombin induced by absence of vitamin K (PIVK-II) concentrations were assayed in 43 patients with cystic fibrosis. Twenty nine showed a normal PIVKA-II and vitamin K concentrations; 14 showed an increased PIVKA-II concentration, in one of whom serum vitamin K was decreased. Although their vitamin K concentrations were normal, some patients with cystic fibrosis still had an increased PIVKA-II. There was a significant correlation between PIVKA-II concentrations and the administration of antibiotics, a factor which has not previously been considered responsible for an increase in PIVKA-II.     

Microbiology of airway disease in patients with cystic fibrosis.

Individuals with cystic fibrosis have abbreviated life spans primarily due to chronic airway infection. A limited number of types of organisms are responsible for these infections, with Staphylococcus aureus and Pseudomonas aeruginosa being of primary importance. In the pre-antibiotic era, greater than 90% of deaths due to infection were caused by S. aureus and death usually occurred in the first 2 years of life. With the advent of effective antistaphylococcal therapy, life spans increased and P. aeruginosa became the pathogen of primary importance. P. aeruginosa isolates recovered from patients with cystic fibrosis have a unique phenotypic characteristic referred to as "mucoid." The mucoid phenotype is due to the production of a mucoid exopolysaccharide. A mucoid exopolysaccharide is believed to play a central role in the establishment of chronic pseudomonal lung infection in these patients. A third organism, Pseudomonas cepacia, has recently been detected in the airways of older patients with cystic fibrosis and is associated with increased mortality. The virulence of P. cepacia is not understood, but the organism is extremely refractory to antimicrobial therapy. Other bacteria, including Haemophilus influenzae and members of the family Enterobacteriaceae, appear to play a secondary role in airway infection. Aspergillus fumigatus is the most important fungal agent causing allergic bronchopulmonary disease. The role of viruses has only recently been examined. At least in some patients with cystic fibrosis, respiratory syncytial virus may be important in predisposing to subsequent bacterial infections.     

Clinical and serological survey of pulmonary aspergillosis in patients with cystic fibrosis

A 5.5-year survey revealed 9 patients with allergic bronchopulmonary aspergillosis (ABPA) and 1 patient with a highly probable diagnosis among 200 Danish patients with cystic fibrosis (CF). The incidence of ABPA was 0.9 (0.4-1.7, 95% confidence limits) per 100 patients per year. All the patients with definite or probable ABPA had serum antibodies to the Aspergillus fumigatus catalase antigen, and in 5 patients the appearance of catalase antibodies during the incipient phase of ABPA was documented. 29 patients without ABPA also had catalase antibodies, but in lower levels than ABPA patients (p = 0.006). The 6-month prevalence rate of A. fumigatus in sputum was 80 and 72% in the 2 groups, respectively. The finding of catalase antibodies in some CF patients without ABPA may indicate the occurrence of a symptom-poor form of pulmonary aspergillosis.     

Pseudomonas aeruginosa flagellar antibodies in patients with cystic fibrosis.

An enzyme-linked immunosorbent assay specific for flagellum type (a or b) of Pseudomonas aeruginosa was used to detect serum immunoglobulin antibodies in 98 random outpatients and 14 colonized cystic fibrosis patients. Antibodies were detected to both types of flagella in addition to M-2 lipopolysaccharide. Titers to both flagellar antigens (FlAg) were 10 to 100 times higher in cystic fibrosis patients than in random outpatients of a comparable age group. Mean antibody titers against b-type FlAg were 454 for outpatients (ages newborn to 21 years), whereas the mean titer for cystic fibrosis patients (ages 6 to 21 years) was 51,520. Titers against a-type FlAg were generally lower, with mean outpatient titers of 68 and mean cystic fibrosis patient titers of 34,323. Differences were also seen in antibody titer against M-2 lipopolysaccharide, but these differences did not correspond to M-2 FlAg titers. In 98 random outpatients (ages newborn to 86 years), FlAg titers generally increased with age. To demonstrate further specificity of the enzyme-linked immunosorbent assay for flagellum antibody, Western blots were performed with selected high-titer cystic fibrosis patient sera. Sera that had a high titer (greater than 25,600) for b- or a-type FlAg showed a corresponding reactive band. These results demonstrate that flagellum antibodies are produced in humans in response to P. aeruginosa infection.