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.
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 (p50.003), 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 (p50.051).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.
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.
During this 9-year study, small but significant declines in health were observed in GH-deficient adults who remained untreated. By contrast, the patients who received GH continuously experienced improvements in energy levels while all other areas of QoL were maintained. The beneficial effects of GH on QoL are therefore maintained with long-term GH replacement and obviate the reduction in QoL seen over time in untreated GH-deficient adults.
The SPACE study will assess exhaled breath hydrogen cyanide (HCN) concentrations as a marker of Pseudomonas aeruginosa (PA) infection in 240 children with cystic fibrosis (CF). It will use off-line selected ion flow tube mass spectrometry (SIFT-MS) analysis and so we needed to investigate which breath sampling bag material to use, the maximum storage time before analysis and the benefit of warming the bag samples. We studied 15 children with CF, 8 had chronic PA infection and 7 did not. Each exhaled directly into the instrument (on-line) and also into two 25 µm thick Nalophan (25N), two 70 µm Nalophan (70N) and two Tedlar® bags. Bags were stored at 20 or 37 °C. HCN concentrations were analysed at 1, 6, 24 and 48 h (off-line). Acetone and water vapour concentrations were also measured in parallel. Correlation between on-line and off-line concentrations measured by SIFT-MS was better for all compounds and bag types at 37 °C. The median (IQR) on-line HCN concentration was 8.9(4.4-13.7) parts per billion by volume, ppbv. Both on-line and off-line HCN concentrations were significantly higher in patients with PA infection than those without. At 37 °C the correlation between on-line and off-line HCN concentrations was good up to 6 h in the 25N bag (R(2) = 0.79) and up to 24 h for the 70N and Tedlar bags (R(2) = 0.82 and 0.86). The correlation between on- and off-line acetone concentrations at 37 °C was good up to 24 h in 25N, 70N and Tedlar bags (R(2) = 0.89, 0.93 and 0.97). In all three types of bag the water vapour concentration fell quickly and by 24 h was equivalent to that of lab air. Samples stored in Tedlar or 70N bags, warmed to 37 °C and analysed within 24 h, give HCN and acetone concentrations which correlate well with on-line measurements.
ObjectivesFlexible bronchoscopy with bronchoalveolar lavage (FB-BAL) is increasingly used for the microbiological confirmation of protracted bacterial bronchitis (PBB) in children with a chronic wet cough. At our centre, when performing FB-BAL for microbiological diagnosis we sample 6 lobes (including lingula) as this is known to increase the rate of culture positive procedures in children with cystic fibrosis. We investigated if this is also the case in children with PBB.MethodsWe undertook a retrospective case note review of 50 children investigated for suspected PBB between May 2011 and November 2013.ResultsThe median (IQR) age at bronchoscopy was 2.9 (1.7–4.4) years and the median (IQR) duration of cough was 11 (8.0–14) months. Positive cultures were obtained from 41/50 (82%) and 16 (39%) of these patients isolated ≥2 organisms. The commonest organisms isolated were Haemophilus influenzae (25 patients), Moraxella catarrhalis (14 patients), Staphylococcus aureus (11 patients) and Streptococcus pneumoniae (8 patients). If only one lobe had been sampled (as per the European Respiratory Society guidance) 17 different organisms would have been missed in 15 patients, 8 of whom would have had no organism cultured at all. The FB-BAL culture results led to an antibiotic other than co-amoxiclav being prescribed in 17/41 (41%) patients.ConclusionsBacterial distribution in the lungs of children with PBB is heterogeneous and organisms may therefore be missed if only one lobe is sampled at FB-BAL. Positive FB-BAL results are useful in children with PBB and can influence treatment.
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.
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