Chiara Mondino

Diagnostic Performance of an Electronic Nose, Fractional Exhaled Nitric Oxide, and Lung Function Testing in Asthma

BACKGROUND Analysis of exhaled breath by biosensors discriminates between patients with asthma and healthy subjects. An electronic nose consists of a chemical sensor array for the detection of volatile organic compounds (VOCs) and an algorithm for pattern recognition. We compared the diagnostic performance of a prototype of an electronic nose with lung function tests and fractional exhaled nitric oxide (FENO) in patients with atopic asthma. METHODS A cross-sectional study was undertaken in 27 patients with intermittent and persistent mild asthma and in 24 healthy subjects. Two procedures for collecting exhaled breath were followed to study the differences between total and alveolar air. Seven patients with asthma and seven healthy subjects participated in a study with mass spectrometry (MS) fingerprinting as an independent technique for assessing between group discrimination. Classification was based on principal component analysis and a feed-forward neural network. RESULTS The best results were obtained when the electronic nose analysis was performed on alveolar air. Diagnostic performance for electronic nose, FENO, and lung function testing was 87.5%, 79.2%, and 70.8%, respectively. The combination of electronic nose and FENO had the highest diagnostic performance for asthma (95.8%). MS fingerprints of VOCs could discriminate between patients with asthma and healthy subjects. CONCLUSIONS The electronic nose has a high diagnostic performance that can be increased when combined with FENO. Large studies are now required to definitively establish the diagnostic performance of the electronic nose. Whether this integrated noninvasive approach will translate into an early diagnosis of asthma has to be clarified. TRIAL REGISTRATION EUDRACT https://eudralink.emea.europa.eu; Identifier: 2007-000890-51; and clinicaltrials.gov; Identifier: NCT00819676.

The Electronic Nose in Respiratory Medicine

Several volatile organic compounds have been identified in exhaled breath in healthy subjects and patients with respiratory diseases by gas chromatography/mass spectrometry. Identification of selective patterns of volatile organic compounds in exhaled breath could be used as a biomarker of inflammatory lung diseases. An electronic nose (e-nose) is an artificial sensor system that generally consists of an array of chemical sensors for detection of volatile organic compound profiles (breathprints) and an algorithm for pattern recognition. E-noses are handheld, portable devices that provide immediate results. E-noses discriminate between patients with respiratory disease, including asthma, COPD and lung cancer, and healthy control subjects, and also among patients with different respiratory diseases. E-nose breathprints are associated with airway inflammation activity. In combination with other ‘omics’ platforms, e-nose technology might contribute to the identification of new surrogate markers of pulmonary inflammation and subphenotypes of patients with respiratory diseases, provide a molecular basis to a personalized pharmacological treatment, and facilitate the development of new drugs.

Diagnosing Nonimmediate Reactions to Penicillins by in vivo Tests

Background: Maculopapular and urticarial rashes are nonimmediate manifestations common during penicillin treatment; the former often represent cell-mediated hypersensitivity. Our objectives were to assess the incidence of allergy in adults reporting nonimmediate manifestations during penicillin therapy and to evaluate the diagnostic potential of patch tests, delayed-reading skin tests and challenges in such cases. Methods: We used prick and intradermal tests as well as patch tests with penicillin determinants, ampicillin, amoxicillin and any other suspect penicillins. We also performed challenges with the suspect antibiotics. Results: Such antibiotics were aminopenicillins in 93.1% of 259 patients, most of whom had suffered from maculopapular rashes followed by piperacillin (4.2%). Three subjects displayed immediate skin test positivity. Ninety-four subjects showed patch test and delayed intradermal test positivity to the culprit penicillin (90 to aminopenicillins and 4 to piperacillin) and were considered as having had delayed hypersensitivity reactions. Five of the 8 subjects who displayed delayed intradermal test positivity and patch test negativity to the suspect penicillin underwent challenges, 2 reacted positively to the responsible aminopenicillin. Among the remaining 154 with negative results in allergologic tests, 125 agreed to undergo challenges; only 3 reacted. In all, 98 patients – 93 of whom had experienced maculopapular rashes – displayed delayed hypersensitivity (94 to aminopenicillins and 4 to piperacillin). Conclusions: Both patch and intradermal tests are useful in evaluating nonimmediate reactions to penicillins, particularly maculopapular rashes. Patch test and delayed intradermal positivity together indicate delayed hypersensitivity. Intradermal testing appears to be slightly more sensitive than patch testing.

Validation of leukotriene B4 measurements in exhaled breath condensate.

Abstract:Objective: To qualitatively validate an enzyme immunoassay to measure leukotriene B4 in exhaled breath condensate. Exhaled breath condensate is a new non-invasive method to monitor airway inflammation.¶Subjects: Twenty-two subjects with different lung diseases attended the outpatient clinic on one occasion for exhaled breath condensate collection.¶Methods: Samples were pooled together and purified by reverse-phase high-performance liquid chromatography. The fractions eluted were assayed for leukotriene B4 by enzyme immunoassay.¶Results: A single peak of leukotriene B4-like immunoreactivity co-eluting with leukotriene B4 standard (retention time: 24 min) was identified by enzyme immunoassay. Reverse phase-high performance liquid chromatography peak of leukotriene B4 was clearly separated from those of 6-trans- leukotriene B4 (retention time: 14 min) and leukotriene B5 (retention time: 18 min) for which the antiserum used in the enzyme immunoassay had the highest cross-reactivity. Leukotriene B4 recovery was 64%.¶Conclusions: This study provides evidence for the presence of leukotriene B4 in the exhaled breath condensate and the specificity of the enzyme immunoassay used.

Liquid chromatography/mass spectrometry analysis of exhaled leukotriene B4 in asthmatic children

BackgroundThe role of leukotriene (LT) B4, a potent inflammatory mediator, in atopic asthmatic and atopic nonasthmatic children is largely unknown. The lack of a gold standard technique for measuring LTB4 in exhaled breath condensate (EBC) has hampered its quantitative assessment in this biological fluid. We sought to measure LTB4 in EBC in atopic asthmatic children and atopic nonasthmatic children. Exhaled nitric oxide (NO) was measured as an independent marker of airway inflammation.MethodsFifteen healthy children, 20 atopic nonasthmatic children, 25 steroid-naïve atopic asthmatic children, and 22 atopic asthmatic children receiving inhaled corticosteroids were studied. The study design was of cross-sectional type. Exhaled LTB4 concentrations were measured using liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS) with a triple quadrupole mass spectrometer. Exhaled NO was measured by chemiluminescence with a single breath on-line method. LTB4 values were expressed as the total amount (in pg) of eicosanoid expired in the 15-minute breath test. Kruskal-Wallis test was used to compare groups.ResultsCompared with healthy children [87.5 (82.5–102.5) pg, median and interquartile range], exhaled LTB4 was increased in steroid-naïve atopic asthmatic [255.1 (175.0–314.7) pg, p < 0.001], but not in atopic nonasthmatic children [96.5 (87.3–102.5) pg, p = 0.59)]. Asthmatic children who were receiving inhaled corticosteroids had lower concentrations of exhaled LTB4 than steroid-naïve asthmatics [125.0 (25.0–245.0) pg vs 255.1 (175.0–314.7) pg, p < 0.01, respectively]. Exhaled NO was higher in atopic nonasthmatic children [16.2 (13.5–22.4) ppb, p < 0.05] and, to a greater extent, in atopic steroid-naïve asthmatic children [37.0 (31.7–57.6) ppb, p < 0.001] than in healthy children [8.3 (6.1–9.9) ppb]. Compared with steroid-naïve asthmatic children, exhaled NO levels were reduced in asthmatic children who were receiving inhaled corticosteroids [15.9 (11.5–31.7) ppb, p < 0.01].ConclusionIn contrast to exhaled NO concentrations, exhaled LTB4 values are selectively elevated in steroid-naïve atopic asthmatic children, but not in atopic nonasthmatic children. Although placebo control studies are warranted, inhaled corticosteroids seem to reduce exhaled LTB4 in asthmatic children. LC/MS/MS analysis of exhaled LTB4 might provide a non-invasive, sensitive, and quantitative method for airway inflammation assessment in asthmatic children.

Validation of 8-isoprostane and prostaglandin E2 measurements in exhaled breath condensate

AbstractObjective:To qualitatively validate radioimmunoassays for 8-isoprostane and prostaglandin (PG) E2 in exhaled breath condensate. Subjects:Twenty-two subjects with different lung diseases attended the outpatient clinic on one occasion for exhaled breath condensate collection. Methods:Samples were pooled together and purified by reverse phase high performance liquid chromatography (RP-HPLC). The eluted fractions were assayed for 8-isoprostane-like immunoreactivity and PGE2-like immunoreactivity by radioimmunoassays. In addition, simultaneous measurements of exhaled breath condensate unextracted samples with two anti-8-isoprostane and anti-PGE2 sera with different cross-reactivity were performed. Results:A single peak of 8-isoprostane-like immunoreactivity and PGE2-like immunoreactivity co-eluting with 8-isoprostane (retention time: 13 min) and PGE2 (retention time: 21 min) standards, respectively, was identified by radioimmunoassays. Testing with two different antisera showed similar results for both 8-isoprostane-like immunoreactivity (limits of agreement = 4.5 pg/ml and – 4.1 pg/ml, n = 12) and PGE2-like immunoreactivity (limits of agreement = 6.1 pg/ ml and – 6.1 pg/ml, n = 12). Conclusion: This study provides evidence for the specificity of the radioimmunoassays for 8-isoprostane and PGE2 in exhaled breath condensate. This is critical for proposing these markers as a non-invasive way for monitoring airway inflammation.

Liquid chromatography-mass spectrometry measurement of leukotrienes in asthma and other respiratory diseases

Leukotrienes (LTs), including cysteinyl-LTs (LTC4, LTD4 and LTE4) and LTB4, are potent inflammatory lipid mediators which have been involved in the pathophysiology of respiratory diseases. LC-MS/MS techniques for measuring LT concentrations in sputum supernatants, serum, urine and exhaled breath condensate (EBC) have been developed. In asthmatic adults, reported LTB4 and LTE4 concentrations in sputum range from 79 to 7,220 pg/ml and from 11.9 to 891 pg/ml, respectively. Data on sputum LT concentrations in healthy subjects are not available. In EBC, reported LTE4 concentrations range from 38 to 126 pg/ml (95% CI) in adult asthma patients and from 34 to 48 pg/ml in healthy subjects. LTB4 concentrations in EBC range from 175 to 315 pg/ml (interquartile range) in asthmatic children, and from 25 to 245 pg/ml in healthy children. Enabling an accurate quantitative assessment of LTs in biological fluids, LC-MS/MS techniques provide a valuable tool for exploring the pathophysiological role of LTs in respiratory disease and might be useful for assessing the effects of therapeutic intervention. This review presents the analytical aspects of the LC-MS/MS techniques for measuring LT concentrations in biological fluids and discusses their potential utility for the assessment of airway inflammation and monitoring of pharmacological treatment in patients with asthma phenotypes and other respiratory diseases.

Electronic Nose and Exhaled Breath NMR-based Metabolomics Applications in Airways Disease

Breathomics, the multidimensional molecular analysis of exhaled breath, includes analysis of exhaled breath with gas-chromatography/mass spectrometry (GC/MS) and electronic noses (e-noses), and metabolomics of exhaled breath condensate (EBC), a non-invasive technique which provides information on the composition of airway lining fluid, generally by high-resolution nuclear magnetic resonance (NMR) spectroscopy or MS methods. Metabolomics is the identification and quantification of small molecular weight metabolites in a biofluid. Specific profiles of volatile compounds in exhaled breath and metabolites in EBC (breathprints) are potentially useful surrogate markers of inflammatory respiratory diseases. Electronic noses (e-noses) are artificial sensor systems, usually consisting of chemical cross-reactive sensor arrays for characterization of patterns of breath volatile compounds, and algorithms for breathprints classification. E-noses are handheld, portable, and provide real-time data. E-nose breathprints can reflect respiratory inflammation. E-noses and NMR-based metabolomics of EBC can distinguish patients with respiratory diseases such as asthma, COPD, and lung cancer, or diseases with a clinically relevant respiratory component including cystic fibrosis and primary ciliary dyskinesia, and healthy individuals. Breathomics has also been reported to identify patients affected by different types of respiratory diseases. Patterns of breath volatile compounds detected by e-nose and EBC metabolic profiles have been associated with asthma phenotypes. In combination with other -omics platforms, breathomics might provide a molecular approach to respiratory disease phenotyping and a molecular basis to tailored pharmacotherapeutic strategies. Breathomics might also contribute to identify new surrogate markers of respiratory inflammation, thus, facilitating drug discovery. Validation in newly recruited, prospective independent cohorts is essential for development of e-nose and EBC NMRbased metabolomics techniques.

Exhaled and non-exhaled non-invasive markers for assessment of respiratory inflammation in patients with stable COPD and healthy smokers

We aimed at comparing exhaled and non-exhaled non-invasive markers of respiratory inflammation in patients with chronic obstructive pulmonary disease (COPD) and healthy subjects and define their relationships with smoking habit. Forty-eight patients with stable COPD who were ex-smokers, 17 patients with stable COPD who were current smokers, 12 healthy current smokers and 12 healthy ex-smokers were included in a cross-sectional, observational study. Inflammatory outcomes, including prostaglandin (PG) E2 and 15-F2t-isoprostane (15-F2t-IsoP) concentrations in exhaled breath condensate (EBC) and sputum supernatants, fraction of exhaled nitric oxide (FENO) and sputum cell counts, and functional (spirometry) outcomes were measured. Sputum PGE2 was elevated in both groups of smokers compared with ex-smoker counterpart (COPD: P  <  0.02; healthy subjects: P  <  0.03), whereas EBC PGE2 was elevated in current (P  =  0.0065) and ex-smokers with COPD (P  =  0.0029) versus healthy ex-smokers. EBC 15-F2t-IsoP, a marker of oxidative stress, was increased in current and ex-smokers with COPD (P  <  0.0001 for both) compared with healthy ex-smokers, whereas urinary 15-F2t-IsoP was elevated in both smoker groups (COPD: P  <  0.01; healthy subjects: P  <  0.02) versus healthy ex-smokers. FENO was elevated in ex-smokers with COPD versus smoker groups (P  =  0.0001 for both). These data suggest that the biological meaning of these inflammatory markers depends on type of marker and biological matrix in which is measured. An approach combining different types of outcomes can be used for assessing respiratory inflammation in patients with COPD. Large studies are required to establish the clinical utility of this strategy.

Investigational prostaglandin D2 receptor antagonists for airway inflammation

ABSTRACT Introduction: By activating DP1 and DP2 receptors on immune and non-immune cells, prostaglandin D2 (PGD2), a major metabolic product of cyclo-oxygenase pathway released after IgE-mediated mast cell activation, has pro-inflammatory effects, which are relevant to the pathophysiology of allergic airway disease. At least 15 selective, orally active, DP2 receptor antagonists and one DP1 receptor antagonist (asapiprant) are under development for asthma and/or allergic rhinitis. Areas covered: In this review, the authors cover the pharmacology of PGD2 and PGD2 receptor antagonists and look at the preclinical, phase I and phase II studies with selective DP1 and DP2 receptor antagonists. Expert opinion: Future research should aim to develop once daily compounds and increase the drug clinical potency which, apart from OC000459 and ADC-3680, seems to be relatively low. Further research and development of DP2 receptor antagonists is warranted, particularly in patients with severe uncontrolled asthma, whose management is a top priority. Pediatric studies, which are not available, are required for assessing the efficacy and safety of this novel drug class in children with asthma and allergic rhinitis. Studies on the efficacy of DP2 receptor antagonists in various asthma phenotypes including: smokers, obese subjects, early vs late asthma onset, fixed vs reversible airflow limitation, are required for establishing their pharmacotherapeutic role.