Breath tests cover the fraction of nitric oxide in expired gas (), volatile organic compounds (VOCs), variables in exhaled breath condensate (EBC) and other measurements. For EBC and for , official recommendations for standardised procedures are more than 10 years old and there is none for exhaled VOCs and particles. The aim of this document is to provide technical standards and recommendations for sample collection and analytic approaches and to highlight future research priorities in the field. For EBC and, new developments and advances in technology have been evaluated in the current document. This report is not intended to provide clinical guidance on disease diagnosis and management.Clinicians and researchers with expertise in exhaled biomarkers were invited to participate. Published studies regarding methodology of breath tests were selected, discussed and evaluated in a consensus-based manner by the Task Force members.Recommendations for standardisation of sampling, analysing and reporting of data and suggestions for research to cover gaps in the evidence have been created and summarised.Application of breath biomarker measurement in a standardised manner will provide comparable results, thereby facilitating the potential use of these biomarkers in clinical practice.

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Aim To determine whether the low C-peptide levels (<50 pmol/l) produced by the pancreas for decades after onset of Type 1 diabetes have clinical significance. Methods We evaluated fasting C-peptide levels, duration of disease and age of onset in a large cross-sectional series (n=1272) of people with Type 1 diabetes. We then expanded the scope of the study to include the relationship between C-peptide and HbA1c control (n=1273), as well as diabetic complications (n=324) and presence of hypoglycaemia (n=323). The full range of C-peptide levels was also compared with 1,5-Anhydroglucitol, a glucose responsive marker. Results C-peptide levels declined for decades after diagnosis, and the rate of decline was significantly related to age of onset (P<0.0001), after adjusting for disease duration. C-peptide levels > 10 pmol/l were associated with protection from complications (e.g. nephropathy, neuropathy, foot ulcers and retinopathy; P=0.03). Low C-peptide levels were associated with poor metabolic control measured by HbA1c (P<0.0001). Severe hypoglycaemia was associated with the lowest C-peptide levels compared with mild (P=0.049) or moderate (P=0.04) hypoglycaemia. All levels of measurable C-peptide were responsive to acute fluctuations in blood glucose levels as assessed by 1,5-Anhydroglucitol (P<0.0001). Conclusions Low C-peptide levels have clinical significance and appear helpful in characterizing groups at-risk for faster C-peptide decline, complications, poorer metabolic control and severe hypoglycaemia. Low C-peptide levels may be a biomarker for characterizing at-risk patients with Type 1 diabetes.

This study aimed to establish and evaluate a bovine respiratory model of experimentally induced acute C. psittaci infection. Calves are natural hosts and pathogenesis may resemble the situation in humans. Intrabronchial inoculation of C. psittaci strain DC15 was performed in calves aged 2–3 months via bronchoscope at four different challenge doses from 106 to 109 inclusion-forming units (ifu) per animal. Control groups received either UV-inactivated C. psittaci or cell culture medium. While 106 ifu/calf resulted in a mild respiratory infection only, the doses of 107 and 108 induced fever, tachypnea, dry cough, and tachycardia that became apparent 2–3 days post inoculation (dpi) and lasted for about one week. In calves exposed to 109 ifu C. psittaci, the respiratory disease was accompanied by severe systemic illness (apathy, tremor, markedly reduced appetite). At the time point of most pronounced clinical signs (3 dpi) the extent of lung lesions was below 10% of pulmonary tissue in calves inoculated with 106 and 107 ifu, about 15% in calves inoculated with 108 and more than 30% in calves inoculated with 109 ifu C. psittaci. Beside clinical signs and pathologic lesions, the bacterial load of lung tissue and markers of pulmonary inflammation (i.e., cell counts, concentration of proteins and eicosanoids in broncho-alveolar lavage fluid) were positively associated with ifu of viable C. psittaci. While any effect of endotoxin has been ruled out, all effects could be attributed to infection by the replicating bacteria. In conclusion, the calf represents a suitable model of respiratory chlamydial infection. Dose titration revealed that both clinically latent and clinically manifest infection can be reproduced experimentally by either 106 or 108 ifu/calf of C. psittaci DC15 while doses above 108 ifu C. psittaci cannot be recommended for further studies for ethical reasons. This defined model of different clinical expressions of chlamydial infection allows studying host-pathogen interactions.

Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP) emits volatile organic compounds (VOCs). Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold’s egg yolk medium in dilutions of 10-0, 10-2, 10-4 and 10-6. Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME), thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to diagnose MAP infections in animals and to identify different bacterial strains and origins.

Experimental models are critical for the understanding of lung health and disease and are indispensable for drug development. However, the pathogenetic and clinical relevance of the models is often unclear. Further, the use of animals in biomedical research is controversial from an ethical perspective.The objective of this task force was to issue a statement with research recommendations about lung disease models by facilitating in-depth discussions between respiratory scientists, and to provide an overview of the literature on the available models. Focus was put on their specific benefits and limitations. This will result in more efficient use of resources and greater reduction in the numbers of animals employed, thereby enhancing the ethical standards and translational capacity of experimental research.The task force statement addresses general issues of experimental research (ethics, species, sex, age, and models, gene editing). The statement also includes research recommendations on modelling asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, lung infections, acute lung injury and pulmonary hypertension.The task force stressed the importance of using multiple models to strengthen validity of results, the need to increase the availability of human tissues and the importance of standard operating procedures and data quality.

Differential ion mobility spectrometry (DMS) is a method to detect volatile organic compounds (VOC) in the ppt range. This study assessed whether VOC analysis using DMS could discriminate subjects with an experimentally induced chronic intestinal infection caused by Mycobacteria from non-infected controls. The animal model consisted of two groups of goats orally infected with two different doses of Mycobacterium avium subspecies paratuberculosis (MAP) and one group of non-infected healthy controls (each group: n = 6). Using DMS, exhaled breath and headspace of feces were analyzed on-line on an individual basis 9 months after inoculation of MAP. Data analysis included peak detection, cluster analysis, selection of discriminating VOC features (Mann-Whitney U test), and classification using a support-vector-machine. Taking the background of ambient air conditions into account, VOC analysis of exhaled breath as well as of feces revealed significant differences between chronically infected animals and non-infected controls. In both specimens, increasing as well as decreasing VOC features could be attributed to infection. Discrimination between infected and non-infected animals was sharper analyzing exhaled breath compared to headspace of feces. In exhaled breath, at least two VOC features were found to increase in a dose-dependent manner with increasing doses of MAP inoculated. Results of this study provide strong evidence that DMS analysis of exhaled breath has the potential to become a valuable tool for non-invasive assessment of VOC specifically related to certain diseases or infections.