Francis J. Gilchrist

The effect of long-term untreated growth hormone deficiency (GHD) and 9 years of GH replacement on the quality of life (QoL) of GH-deficient adults

background Quality of life (QoL) is reduced in GH‐deficient adults compared with the normal population. Further support for the role of GH in the maintenance of QoL is derived from short‐term studies of GH replacement in severely GH‐deficient adults; these have predominantly reported beneficial effects, although the degree of improvement has often been modest. To date, however, there are few data to demonstrate whether this beneficial effect on QoL is maintained in the long term.

Variation in hydrogen cyanide production between different strains of Pseudomonas aeruginosa

There is increasing interest in using the cyanogenic properties of Pseudomonas aeruginosa to develop a nonmicrobiological method for its detection. Prior to this, the variation in cyanide production between different P. aeruginosa strains needs to be investigated. Hydrogen cyanide (HCN) released into the gas phase by 96 genotyped P. aeruginosa samples was measured using selected ion flow tube-mass spectrometry after 24, 48, 72 and 96 h of incubation. The HCN produced by a range of non-P. aeruginosa cultures and incubated blank plates was also measured. All P. aeruginosa strains produced more HCN than the control samples, which generated extremely low levels. Analysis across all time-points demonstrated that nonmucoid samples produced more HCN than the mucoid samples (p=0.003), but this relationship varied according to strain. There were clear differences in the headspace HCN concentration for different strains. Multivariate analysis of headspace HCN for the commonest strains (Liverpool, Midlands_1 and Stoke-on-Trent, UK) revealed a significant effect of strain (p<0.001) and a borderline interaction of strain and phenotype (p=0.051). This evidence confirms that all P. aeruginosa strains produce HCN but to varying degrees and generates interest in the possible future clinical applications of the cyanogenic properties of P. aeruginosa.

Hydrogen cyanide, a volatile biomarker of Pseudomonas aeruginosa infection

Since we first recognized the regular presence of gaseous hydrogen cyanide, HCN, in the headspace of plate cultures of the bacterium Pseudomonas aeruginosa, PA, derived from sputum of cystic fibrosis, CF, patients, and following crucial ion chemistry research that allowed accurate quantification of gaseous HCN by selected ion flow tube mass spectrometry, we have carried out many further in vitro and in vivo studies. We have measured HCN in the headspace of various PA culture types, planktonic and biofilm, significant numbers of genetically identified PA strains together with studies of HCN in the mouth-exhaled and nose-exhaled breath of healthy children and adults and those with CF. The major findings are: (i) virtually all strains of PA release HCN when cultured in vitro, as shown by the investigation of more than 150 genetically differentiated strains, both mucoid and non-mucoid. (ii) HCN is present in the mouth-exhaled breath of adults and children, but is at lower concentrations in children. Its concentration is below the detection limit in nose-exhaled breath of healthy people. (iii) HCN is present in both mouth-exhaled and nose-exhaled breath of patients with CF, suggesting the presence of PA in the lower airways as indicated by clinical microbiological cultures. With confirmation of these findings by further research and clinical trials, nose-exhaled breath HCN measurements could be an additional diagnostic tool to detect the early presence of PA in the lower airways and a non-invasive monitor to enhance the likelihood of its eradication.

Hydrogen cyanide concentrations in the breath of adult cystic fibrosis patients with and without Pseudomonas aeruginosa infection

Elevated concentrations of hydrogen cyanide (HCN) have been detected in the headspace of Pseudomonas aeruginosa (PA) cultures and in the breath of children with cystic fibrosis (CF) and PA infection. The use of mouth-exhaled breath HCN as a marker of PA infection in adults is more difficult to assess as some without PA infection generate HCN in their mouths. The analysis of breath exhaled via the nose, thereby avoiding volatile compounds produced in the mouth, will demonstrate elevated concentrations of HCN in adult CF patients chronically infected with PA. Using selected ion flow mass spectrometry (SIFT-MS), the mouth and the nose-exhaled breaths of 20 adult CF patients; 10 with chronic PA infection and 10 free from PA infection, were analysed for HCN. Acetone and ethanol were also measured as controls. SIFT-MS allows direct sampling and analysis of single breath exhalations, obviating the need to collect samples into bags or onto traps, which can compromise samples. HCN was detected in the mouth-exhaled breath of patients in both groups and in the nose-exhaled breath of patients with chronic PA infection. The difference in median (IQR) nose-exhaled HCN between the groups is statistically significant (11 (0.8-18) ppbv versus 0 (0-3.2) ppbv, p = 0.03). The concentrations of acetone and ethanol in nose-exhaled and mouth-exhaled breath are in keeping with previous studies. HCN in nose-exhaled breath is a biomarker of chronic airway infection with PA in adults with CF. Its application as a non-invasive diagnostic test for early PA infection warrants further investigation.

Bronchoalveolar lavage in children with cystic fibrosis: how many lobes should be sampled?

Background The European Respiratory Society guidance on bronchoalveolar lavage (BAL) in children was published in 2000. It recommended taking one BAL specimen from the most affected lobe or from the right middle lobe in diffuse disease. In 2007, the European Respiratory Society modified the recommendations for children with cystic fibrosis (CF), suggesting two BAL specimens (right middle lobe and the lingula or the most affected lobe). Objective To determine if BAL samples from one or two lobes give the full picture of lower airway infection in children with CF. Design, setting and patients A retrospective review of all paediatric patients with CF who underwent flexible bronchoscopy between May 2007 and May 2009 was undertaken. Main outcome measures As BAL specimens from all six lobes were collected, the BAL results were reviewed to identify if positive cultures would have been missed if only one lobe (right middle or most affected) or two lobes (right middle plus the lingula or most affected) had been sampled. Results The results of 39 bronchoscopic procedures in 31 children were reviewed. The BAL samples were taken from 6 lobes in all 39 procedures. Had only one lobe been used, 26 positive cultures (14 organisms) would have been missed in 11 patients. Had two lobes been used, 12 positive cultures (8 organisms) would have been missed in 7 patients. Conclusion A single-lobe BAL is insufficient in assessing patients with CF for lower airway infection. Even when BAL specimens are taken from two lobes, a number of infections may be missed.

Quantification by SIFT-MS of volatile compounds emitted by in vitro cultures of S. aureus, S. pneumoniae and H. influenzae isolated from patients with respiratory diseases

In the light of the exciting recent developments in the detection of Pseudomonas aeruginosa in the breath of cystic fibrosis patients by measuring exhaled HCN, an in vitro study has been conducted to identify and quantify the volatile compounds emitted into the gas phase by other respiratory pathogens. Selected ion flow tube mass spectrometry (SIFT-MS) was used to investigate clinical isolates of Staphylococcus aureus (SA), Streptococcus pneumoniae (SP), and Haemophilus influenzae (HI). Six volatile compounds, mainly alcohols, ketones and aldehydes, were found to be elevated in the headspace of SA cultures and eight were elevated in the SP cultures. It is clear from the mass spectra that a number of other compounds were present at low levels that, as yet, cannot be identified by SIFT-MS alone. Only indole and ethanol were somewhat elevated in the headspace of some of the HI cultures. Principal component analyses (PCA) indicated that the SA cultures clearly separated into two distinct groups in terms of their volatile compound emissions. This may relate to genetic or phenotypic differences. It is postulated that the very efficient production of ethanol and acetaldehyde by SA and SP may provide indicators of airways infection by these bacteria if the levels of these compounds are elevated in exhaled breath above those levels expected due to their normal endogenously produced levels. Plans are in place to check this postulate by breath analysis studies involving patients with pulmonary infections with these organisms.

Quantification of hydrogen cyanide and 2-aminoacetophenone in the headspace of Pseudomonas aeruginosa cultured under biofilm and planktonic conditions

Hydrogen cyanide (HCN) and 2-aminoacetophenone (2-AA; H2NC6H4COCH3) are possible biomarkers of pulmonary Pseudomonas aeruginosa (PA) infection that could be used in an exhaled breath test. All factors affecting their production need to be investigated, including the culture conditions: planktonic (free-floating) or biofilm (non-motile communities attached to a solid surface). In vivo, the change from planktonic to biofilm growth is signalled when a certain population density is reached. Using selected ion flow tube mass spectrometry, SIFT-MS, we have analysed HCN and 2-AA produced by 12 genotyped PA samples, cultured under both planktonic and biofilm conditions after 24, 48, 72 and 96 hours of incubation. The 12 samples included 3 different strains (genotypes), 50% of which had a mucoid phenotype and 50% had a non-mucoid phenotype. All samples produced significant concentrations of HCN; median (25th to 75th percentiles, IQR) concentration: 144 (61–512) parts-per-billion by volume (ppbv). Multivariate analysis showed HCN production varied dependent on genotype (p = 0.0014), culture duration (p = 0.005) and phenotype (p < 0.001) but not culture conditions (planktonic/biofilm). Much smaller concentrations of 2-AA were detected, median (IQR) concentration 1.8 (1.3–3) ppbv, despite which, multivariate analysis showed production was affected by genotype (p < 0.001) and culture duration (p = 0.007) but not phenotype or culture conditions. These data show that biofilm formation does not affect HCN production by PA and supports its use as a biomarker of PA infection. The concentrations of 2-AA are much lower than previous studies have shown. The reason for this is unclear but it raises questions about its suitability as a biomarker of PA infection.

Pseudomonas aeruginosa and cyanide production

Infection and subsequent chronic infection of the cystic fibrosis (CF) lung with organisms such as Pseudomonas aeruginosa probably occurs at an early stage in the life of a child suffering from CF. Clinicians worldwide are eager to isolate the organism as early as possible in an attempt to treat the patient before any permanent damage occurs and hopefully to eliminate the organism completely from the respiratory tract for many years. Young children are usually unable to expectorate sputum for microbiological analysis and cough swabs are relatively insensitive. Induced sputum techniques are generally not well tolerated in children and bronchoscopic manoeuvres are very invasive, requiring a general anaesthetic. There has, therefore, been interest in discovering whether there are any surrogate markers which could be helpful as a specific indication of the presence of P. aeruginosa . It has been postulated that cyanide could be one such compound. P. aeruginosa is one of a limited number of organisms which can synthesise cyanide 1. Synthesis occurs by the oxidative decarboxylation of glycine by a hydrogen cyanide synthase enzyme. This process also produces four electrons and four hydrogen ions per glycine molecule. Cyanide synthesis by P. aeruginosa occurs under microaerobic (O2<5%) conditions but is inactivated under both atmospheric oxygen and strictly anaerobic conditions 1, 2. Cyanide production by P. aeruginosa is maximised at temperatures between 34°C and 37°C 3. Studies which have investigated cyanogenesis in relation to P. aeruginosa using liquid or solid samples have assayed cyanide concentrations …

Quantification by SIFT-MS of volatile compounds emitted by Aspergillus fumigatus cultures and in co-culture with Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae

Following our recent in vitro study of the volatile compounds emitted into the gas phase by the respiratory pathogens Pseudomonas aeruginosa (PA), and most recently Staphylococcus aureus (SA), Streptococcus pneumoniae (SP) and Haemophilus influenzae (HI), we have extended this work to the investigation of the volatile compounds emitted by in vitro cultures of the common respiratory fungus Aspergillus fumigatus (AF). The measurements were achieved using selected ion flow tube mass spectrometry (SIFT-MS) by which real time analyses of trace volatile compounds can be achieved without disturbing the cultures. It is seen that copious amounts of ammonia and the organosulphur compounds methanethiol, dimethyl sulphide and dimethyl disulphide are produced by AF cultures. These may be sufficient to allow for non-invasive detection of the AF in the airways of infected patients by breath analysis. AF also efficiently absorbs and metabolises the aldehydes acetaldehyde, butanal and pentanal from the supportive medium (brain-heart infusion broth). Preliminary studies of the volatile compounds emitted by co-cultures of AF with PA, SA and SP revealed that the biomarker HCN (for PA) is not compromised by the presence of AF, and the organosulphur compounds (for AF) are not compromised by the presence of SA or SP.

Itraconazole and inhaled fluticasone causing hypothalamic-pituitary-adrenal axis suppression in adults with cystic fibrosis.

BACKGROUND Although there have been case reports of hypothalamic-pituitary-adrenal (HPA) axis suppression in patients with cystic fibrosis (CF) caused by the combination of oral itraconazole and inhaled fluticasone, to date no study has assessed the incidence of this potentially serious side effect. METHODS Synacthen tests were conducted on all patients with CF receiving itraconazole and inhaled fluticasone and an equal number of patients with CF receiving inhaled fluticasone but not itraconazole. Itraconazole levels were measured in patients receiving the therapy. RESULTS Twelve patients receiving itraconazole and fluticasone underwent synacthen tests. All 12 had abnormal synacthen test results and 10/12 (83%) had HPA axis suppression. Two patients had severe HPA axis suppression with a peak cortisol <75 nmol/L and further 3 patients had moderately severe suppression with a peak cortisol <250 nmol/L. In contrast, only 2/12 on fluticasone alone had HPA axis suppression (both mild). The median (range) basal cortisol levels were significantly lower in those patients receiving itraconazole and inhaled fluticasone compared to those on fluticasone alone (219(22-508)nmol/L v 348(41-738)nnmol/L, p=0.02), similar results were seen for peak cortisol levels (404(59-706)nmol/L v 672(432-1178)nmol/L, p<0.001) and cortisol rise (179(37-240)nmol/L v 368(210-539)nmol/L, p<0.001). The median (range) itraconazole level was 5.5(1.7-14.7)mg/L. Neither itraconazole levels nor fluticasone dose correlated with the degree of adrenal suppression. CONCLUSIONS In this study, all patients receiving itraconazole and inhaled fluticasone had abnormal synacthen test results. The incidence of HPA axis suppression with this treatment combination appears to be higher than that previously reported with itraconazole and inhaled budesonide.