Cystic fibrosis-related deaths in infancy and the effect of newborn screening.

Although newborn screening for cystic fibrosis (CF) is widely advocated, hard evidence in its favor is difficult to obtain, partly because of a dramatically improved life expectancy. Between 1985--1989 infants, born in Wales and the West Midlands were randomized to newborn CF screening by heel-prick immunoreactive trypsin (IRT) measurement or diagnosis by clinical presentation. Eligible children with CF who died in the first 5 years of life were identified from the local pediatricians and from the National UK CF Survey. In all, 230,076 infants were randomized to be screened, while 234,510 were unscreened. One hundred seventy-six CF children were identified, of whom 7 died in the first 5 years of life, 3 having presented with meconium ileus. Median age of diagnosis in the screened group was 8 weeks. On an intention to treat analysis, all 4 nonmeconium ileus-related deaths occurred in the unscreened group (Fisher's exact test, P < 0.05). However, the clinical presentation of 2 of these infants led to them being diagnosed prior to 8 weeks, i.e., earlier than would have been likely by screening. In conclusion, newborn screening has the potential to decrease infant CF deaths, but if it is to be successful, identification and treatment must occur as soon as possible after birth.

Lipopolysaccharide (LPS) from Burkholderia cepacia is more active than LPS from Pseudomonas aeruginosa and Stenotrophomonas maltophilia in stimulating tumor necrosis factor alpha from human monocytes.

Whole cells and lipopolysaccharides (LPSs) extracted from Burkholderia cepacia, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Escherichia coli were compared in their ability to stimulate tumor necrosis factor alpha (TNF-alpha) from the human monocyte cell line MonoMac-6. B. cepacia LPS, on a weight-for-weight basis, was found to have TNF-alpha-inducing activity similar to that of LPS from E. coli, which was approximately four- and eightfold greater than the activity of LPSs from P. aeruginosa and S. maltophilia, respectively. The LPS-stimulated TNF-alpha production from monocytes was found to be CD14 dependent. These results suggest that B. cepacia LPS might play a role in the pathogenesis of inflammatory lung disease in cystic fibrosis, and in some patients it might be responsible, at least in part, for the sepsis-like cepacia syndrome.

A melanin pigment purified from an epidemic strain of Burkholderia cepacia attenuates monocyte respiratory burst activity by scavenging superoxide anion.

The acquisition of Burkholderia cepacia in some cystic fibrosis patients is associated with symptoms of acute pulmonary inflammation that may be life threatening. The ability of lipopolysaccharide (LPS) from B. cepacia to prime a monocyte cell line for enhanced superoxide anion generation was investigated and compared with the priming activities of LPSs from Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Escherichia coli. The human monocyte cell line MonoMac-6 (MM6) was primed overnight with different LPSs (100 ng/ml), and the respiratory burst was triggered by exposure to opsonized zymosan (125 micrograms/ml). Superoxide generation was detected by enhanced chemiluminescence with Lucigenin. B. cepacia LPS was found to prime MM6 cells to produce more superoxide anion than P. aeruginosa or S. maltophilia LPS, and this priming response was CD14 dependent. In addition, the inhibition of respiratory burst responses in monocytes by a bacterial melanin-like pigment purified from an epidemic B. cepacia strain was investigated. The melanin-like pigment was isolated from tyrosine-enriched media on which B. cepacia had been grown and was purified by gel filtration, anion ion-exchange chromatography, and ethanol precipitation. The scavenging potential of the melanin-like pigment for superoxide anion radical (*O2-) generated during the respiratory burst was confirmed with superoxide produced from a cell-free system with xanthine-xanthine oxidase and detected by electron paramagnetic resonance spectroscopy with the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide. The addition of melanin during the LPS priming stage had no effect on the subsequent triggering of the respiratory burst, but melanin inhibited *O2- detection when added at the triggering stage of the respiratory burst. We conclude that melanin-producing B. cepacia may derive protection from the free-radical-scavenging properties of this pigment.

Maintenance of growth in cystic fibrosis despite reduction in pancreatic enzyme supplementation.

Twenty one children with cystic fibrosis were advised to decrease their pancreatic enzyme supplement (PES) dose to less than 10,000 units lipase/kg/day. Mean PES dosage was significantly decreased in 15 patients from 18,380 to 8647 units lipase/kg/day. There were no significant changes in energy or fat intake, but there were significant increases in weight SD score, height SD score, and weight/height ratio.

Transmission and prevalence of Burkholderia cepacia in Welsh cystic fibrosis patients.

From 1987 to 1994, 16 of 162 cystic fibrosis (CF) patients attending CF clinics at three different hospitals in South Wales, U.K. were found to have respiratory secretions colonized with Burkholderia cepacia (B. cepacia). Bacteriological typing by polymerase chain reaction (PCR) ribotyping demonstrated seven strains of B. cepacia among these 16 CF patients. This typing confirmed that cross-infection was the mechanism of colonization in six of the nine patients who were colonized at the paediatric CF clinic at the University Hospital of Wales in Cardiff, and in three of the six patients who were colonized at the adult CF clinic at Llandough Hospital in Cardiff (cross-infection rate nine of 16 patients or 56%). A search was made for a nosocomial source, with screening of wards and clinics. Swabs from fomites produced four positive cultures for B. cepacia. Two isolates had the same PCR ribotype as that of the previous CF room occupant. To establish prevalence of B. cepacia among CF children living throughout Wales, respiratory secretions were cultured from 151 of 186 CF children (age < 16 years). This failed to demonstrate B. cepacia colonization other than in the CF patients already identified.

A comparative study of Fusobacterium necrophorum strains from human and animal sources by phenotypic reactions, pyrolysis mass spectrometry and SDS-PAGE.

Fusobacterium necrophorum strains from human infection (21) were compared with strains from animals (17 biotype A, 2 biotype AB, 4 biotype B, 1 biotype unknown), and the type strain NCTC 10575 in conventional tests reaction patterns (CTRPs), SDS-PAGE and pyrolysis mass spectrometry (PMS). Classifications from the three approaches showed one major consensus group comprising all human strains, and another comprising animal biotype A strains. Animal biotype B strains and one animal strain, designated with some doubt to biotype A, were outliers of the consensus 'human strain' group. Again, animal biotype AB strains were outliers of the consensus 'animal biotype A group', as was the type strain, which was clearly atypical in conventional tests and PMS. Colonial and microscopic characters showed good discrimination between the major consensus groups. However, only haemagglutination and the API-ZYM leucine arylamidase of the biochemical tests discriminated well between these groups. The 'animal biotype A group' clearly corresponds to F. necrophorum subsp. necrophorum, but synonymy of F. necrophorum subsp. funduliforme with the group of human strains was less certain. The latter subspecies was described solely on the basis of animal strains, all of biotype B, but each of four animal biotype B strains in this study was an outlier of the 'human strain group' in one or more of the characterisation approaches. Strains of F. necrophorum causing human infection were clearly distinct from the biotype A strains commonly found in animal infection. This has implications for the validity of animal models of human necrobacillosis. In view of these differences, it would be useful to have a validated designation for strains causing human infection. However, it would be premature to assume that the definition of F. necrophorum subsp. funduliforme encompasses the human strains in the absence of confirmatory DNA-homology and 16S rRNA-sequencing studies.

Lack of evidence of nosocomial cross-infection by Burkholderia cepacia among Danish cystic fibrosis patients.

Burkholderia cepacia isolates from nine of the ten Danish cystic fibrosis (CF) patients known between 1975 and the present day to carry this organism were investigated. Eight distinct genotypes were found with polymerase chain reaction ribotyping and pulsed-field gel electrophoresis. The results indicate that there is little patient-to-patient cross-infection with Burkholderia cepacia within the Danish CF population, even though the majority of patients attend the same CF clinic on a regular basis.

Characterisation of Burkholderia cepacia from cystic fibrosis patients living in Wales by PCR ribotyping.

Polymerase chain reaction (PCR) ribotyping detects differences in the intergenic spacer between the 16S and 23SrRNA genes. This method was applied to Burkholderia cepacia isolates from 16 Welsh cystic fibrosis (CF) patients attending three different clinics. Amplification of the intergenic spacer followed by an additional digestion step with TaqI restriction endonuclease identified seven distinct electrophoretic patterns among the patient isolates. Each of the seven patterns was distinct from that of the so called "epidemic strain" commonly isolated from patients attending clinics elsewhere in the UK. Two environmental isolates from the hospital clinics and four NCTC reference strains gave different patterns. The simplicity of the method lends itself to use in a general microbiological laboratory.

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.

Screening for cystic fibrosis.

Neonatal screening for cystic fibrosis (CF) reduces short-term morbidity but its long term effects remain to be demonstrated. The best available method is the assay of immunoreactive trypsin in dried blood spots, and specificity can be improved by adding direct or indirect genetic analysis. Pregnancies known to be at risk of CF can also be screened by molecular methods, and affected pregnancies terminated. The application of genetic testing to whole communities, to detect unknown heterozygotes, raises many questions which require consideration by society and the health professions. The development of effective treatment of the basic abnormality of cell function in CF would enhance the need for neonatal screening, and possibly reduce the requirement for abortion.

Neonatal screening for cystic fibrosis in Wales and the West Midlands: clinical assessment after five years of screening.

Screening of the newborn for cystic fibrosis by measurement of immunoreactive trypsin has been undertaken on alternate weeks in Wales and the West Midlands for five years since 1985 to evaluate the possible clinical benefits of early diagnosis. Patients detected by screening and those diagnosed by clinical symptoms alone were assessed annually for differences in clinical, anthropometric, and biochemical variables. Fifty eight infants not considered to be at risk of cystic fibrosis (they did not present with meconium ileus and do not have a sibling with cystic fibrosis) have been detected by screening and they have been compared with 44 children who were diagnosed clinically. This latter group includes nine children whose screening was negative but who were recognised subsequently to have cystic fibrosis. The mean age at diagnosis of the screened group was significantly lower than that of the group diagnosed clinically. Excluding admissions for diagnostic tests for cystic fibrosis, the screened group spent a significantly shorter time in hospital during the first year of life. The results of all other comparisons made between the screened group and those diagnosed clinically were similar up to the age of 4 years.

Attitudes of parents of cystic fibrosis children towards neonatal screening and antenatal diagnosis.

Information on parents' attitudes towards neonatal screening for cystic fibrosis (CF) and antenatal diagnosis by chorion villus biopsy (CVS) has been derived from a detailed questionnaire administered to parents of CF babies diagnosed early following newborn screening (18 babies), and later on account of clinical criteria (11 babies). Screening was by measurement of immunoreactive trypsin (IRT) on Guthrie card blood spots, which was the basis of the Wales/West Midlands IRT Screening Survey, 1985-1989. Families questioned were from Wales. Most parents supported screening: parents of 15/18 (83%) screened babies and 10/11 (91%) unscreened babies. Following antenatal diagnosis, 15/29 (52%) of families would abort a CF foetus. Neither standard of education nor social class correlated with attitudes to screening or antenatal diagnosis, although these factors were related to the parents' knowledge of CF in general. Several families emphasised the importance of minimal delay between the initial mention of the possibility of CF on IRT testing and confirmation (or otherwise) of the diagnosis. Four mothers acknowledge temporary rejection of their babies during the period of uncertainty or following the procedures of diagnosis, emphasising that neonatal screening for CF can have a psychological impact on the parent-child bonding. Although most families supported neonatal screening for CF, this study underlines some of the difficulties which may be encountered during the procedure of screening for CF by IRT.

Human red blood cells inhibit endothelium-derived relaxing factor (EDRF) activity.

We have used a bioassay-cascade system to investigate the inhibitory effects of human red blood cells on EDRF activity. The vascular smooth muscle relaxant effect of EDRF released from an endothelium-intact pig coronary artery by the calcium ionophore A23187 was inhibited by washed red cells. This inhibition of EDRF activity by red cells was similar to that expected from their haemoglobin content. This study provides further evidence that in vivo EDRF acts as a local autocoid.

Neonatal screening for cystic fibrosis in Wales and the West Midlands: 1. Evaluation of immunoreactive trypsin test.

A study programme was set up in Wales and the West Midlands to evaluate serum immunoreactive trypsin screening for cystic fibrosis in neonates using blood spots collected for metabolic screening. By screening half the blood spots from each area, it was hoped to generate two comparable groups of fibrocystic children; those detected by screening and those not screened who would be diagnosed clinically. Over almost three years, more than 120,000 specimens were screened and 37 infants detected with cystic fibrosis. Four additional fibrocystic patients were missed on screening: two had negative immunoreactive trypsin values, of which one had meconium ileus, and two, although giving initial positive tests, were negative on follow up. Excluding infants known to be at risk, comparison of the numbers of children detected in the screened and unscreened groups showed more than a two-fold difference in favour of the screened group. There may be a large number of undiagnosed fibrocystic patients in the general population.

Enzyme immunoassay of immunoreactive trypsin in serum and blood spots.

An enzyme immunoassay method for the assay of serum immunoreactive trypsin (IRT) is described. The method is a two site binding assay carried out on microtitre plates as the solid phase. Wells were coated with affinity purified anti-human trypsin and biotinylated anti-trypsin and avidin-beta-galactosidase were used as the second antibody and detection system respectively. The assay was sensitive enough to determine IRT concentrations in either serum or dried blood spots. A good correlation was obtained when the method was compared with the Hoechst radioimmunoassay method.

Haptoglobin-haemoglobin complex in human plasma inhibits endothelium dependent relaxation: evidence that endothelium derived relaxing factor acts as a local autocoid.

Endothelium dependent relaxation of isometrically mounted rabbit aortic strip preparations was rapidly inhibited by human plasma at dilutions down to 1:1000. Gel filtration and ion exchange chromatography were used to demonstrate that this inhibitory activity was present in fractions containing haptoglobin. Purified haptoglobin itself possessed no inhibitory action against endothelium dependent relaxation, but the haptoglobin-haemoglobin complex did, consistent with the documented ability of haemoglobin to inhibit this phenomenon. The concentration of haemoglobin normally bound to haptoglobin is sufficient to account for the inhibitory properties of human plasma. This suggests that endothelium derived relaxing factor exerts no downstream intravascular effect in vivo and thus that its physiological dilator role is that of a local autocoid acting on subjacent smooth muscle.