Analysis of volatile organic compounds (VOCs) in breath holds great promise for noninvasive diagnostic applications. However, concentrations of VOCs in breath may change quickly, and actual and previous uptakes of exogenous substances, especially in the clinical environment, represent crucial issues. We therefore adapted proton-transfer-reaction-time-of-flight-mass spectrometry for real time breath analysis in the clinical environment. For reasons of medical safety, a 6 m long heated silcosteel transfer line connected to a sterile mouth piece was used for breath sampling from spontaneously breathing volunteers and mechanically ventilated patients. A time resolution of 200 ms was applied. Breath from mechanically ventilated patients was analyzed immediately after cardiac surgery. Breath from 32 members of staff was analyzed in the post anesthetic care unit (PACU). In parallel, room air was measured continuously over 7 days. Detection limits for breath-resolved real time measurements were in the high pptV/low ppbV range. Assignment of signals to alveolar or inspiratory phases was done automatically by a matlab-based algorithm. Quickly and abruptly occurring changes of patients' clinical status could be monitored in terms of breath-to-breath variations of VOC (e.g. isoprene) concentrations. In the PACU, room air concentrations mirrored occupancy. Exhaled concentrations of sevoflurane strongly depended on background concentrations in all participants. In combination with an optimized inlet system, the high time and mass resolution of PTR-ToF-MS provides optimal conditions to trace quick changes of breath VOC profiles and to assess effects from the clinical environment.
Breath volatile organic compound (VOC) analysis can open a non-invasive window onto pathological and metabolic processes in the body. Decades of clinical breath-gas analysis have revealed that changes in exhaled VOC concentrations are important rather than disease specific biomarkers. As physiological parameters, such as respiratory rate or cardiac output, have profound effects on exhaled VOCs, here we investigated VOC exhalation under respiratory manoeuvres. Breath VOCs were monitored by means of real-time mass-spectrometry during conventional FEV manoeuvres in 50 healthy humans. Simultaneously, we measured respiratory and hemodynamic parameters noninvasively. Tidal volume and minute ventilation increased by 292 and 171% during the manoeuvre. FEV manoeuvre induced substance specific changes in VOC concentrations. pET-CO2 and alveolar isoprene increased by 6 and 21% during maximum exhalation. Then they decreased by 18 and 37% at forced expiration mirroring cardiac output. Acetone concentrations rose by 4.5% despite increasing minute ventilation. Blood-borne furan and dimethyl-sulphide mimicked isoprene profile. Exogenous acetonitrile, sulphides, and most aliphatic and aromatic VOCs changed minimally. Reliable breath tests must avoid forced breathing. As isoprene exhalations mirrored FEV performances, endogenous VOCs might assure quality of lung function tests. Analysis of exhaled VOC concentrations can provide additional information on physiology of respiration and gas exchange.
There is a need for standardisation in sampling and analysis of breath volatile organic compounds (VOCs) in order to minimise ubiquitous confounding effects. Physiological factors may mask concentration changes induced by pathophysiological effects. In humans, unconscious switching of oral and nasal breathing can occur during breath sampling, which may affect VOC patterns. Here, we investigated exhaled VOC concentrations in real-time while switching breathing routes. Breath from 15 healthy volunteers was analysed continuously by proton transfer reaction time-of-flight mass spectrometry during paced breathing (12 breaths min). Every two minutes breathing routes were switched (Setup-1: Oral → Nasal → Oral → Nasal; Setup-2: OralNasal → NasalOral → OralNasal → NasalOral). VOCs in inspiratory and alveolar air and respiratory and hemodynamic parameters were monitored quantitatively in parallel. Changing of the breathing routes and patterns immediately affected exhaled VOC concentrations. These changes were reproducible in both setups. In setup-1 cardiac output and acetone concentrations remained constant, while partial pressure of end-tidal CO (pET-CO), isoprene and furan concentrations inversely mirrored tidal-volume and minute-ventilation. HS (hydrogen-sulphide), CHS (allyl-methyl-sulphide), CHO (isopropanol) and CHO increased during oral exhalation. CHS increased during nasal exhalations. CHO steadily decreased during the whole measurement. In setup-2 pET-CO, CHS (dimethyl-sulphide), isopropanol, limonene and benzene concentrations decreased whereas, minute-ventilation, HS and acetonitrile increased. Isoprene and furan remained unchanged. Breathing route and patterns induced VOC concentration changes depended on respiratory parameters, oral and nasal cavity exposure and physico-chemical characters of the compounds. Before using breath VOC concentrations as biomarkers it is essential that the breathing modality is defined and strictly monitored during sampling.
Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants. Bacterial growth is still the diagnostic 'gold standard', but is very time consuming. MAP-specific volatile organic compounds (VOCs) above media could accelerate cultural diagnosis. The aim of this project was to assess the kinetics of a VOC profile linked to the growth of MAP in vitro. The following sources of variability were taken into account: five different culture media, three different MAP strains, inoculation with different bacterial counts, and different periods of incubation. Needle-trap microextraction was employed for pre-concentration of VOCs, and gas chromatography-mass spectrometry for subsequent analysis. All volatiles were identified and calibrated by analysing pure references at different concentration levels. More than 100 VOCs were measured in headspaces above MAP-inoculated and control slants. Results confirmed different VOC profiles above different culture media. Emissions could be assigned to either egg-containing media or synthetic ingredients. 43 VOCs were identified as potential biomarkers of MAP growth on Herrold's Egg Yolk Medium without significant differences between the tree MAP strains. Substances belonged to the classes of alcohols, aldehydes, esters, ketones, aliphatic and aromatic hydrocarbons. With increasing bacterial density the VOC concentrations above MAP expressed different patterns: the majority of substances increased (although a few decreased after reaching a peak), but nine VOCs clearly decreased. Data support the hypotheses that (i) bacteria emit different metabolites on different culture media; (ii) different MAP strains show uniform VOC patterns; and (iii) cultural diagnosis can be accelerated by taking specific VOC profiles into account.
BackgroundSpecies of Mycobacteriaceae cause serious zoonotic diseases in mammals, for example tuberculosis in humans, dogs, parrots, and elephants (caused by Mycobacterium tuberculosis) and in ruminants and humans (caused by M. bovis and M. caprae). Pulmonary diseases, lymphadenitis, skin diseases, and disseminated diseases can be caused by non-tuberculous mycobacteria (NTM). Diagnosis and differentiation among Mycobacterium species are currently done by culture isolation. The established diagnostic protocols comprise several steps that allow species identification. Detecting volatile organic compounds (VOCs) above bacterial cultures is a promising approach towards accelerating species identification via culture isolation. The aims of this project were to analyse VOCs in the headspace above 13 different species of mycobacteria, to define VOC profiles that are unique for each species, and to compile a set of substances that indicate the presence of growing mycobacteria in general.Materials & methodsVOCs were measured in the headspace above 17 different mycobacterial strains, all cultivated on Herrold’s Egg Yolk Medium and above pure media slants that served as controls. For pre-concentration of VOCs, needle-trap micro-extraction was employed. Samples were subsequently analysed using gas chromatography-mass spectrometry. All volatiles were identified and calibrated by analysing pure reference substances.ResultsMore than 130 VOCs were detected in headspace above mycobacteria-inoculated and control slants. Results confirmed significant VOC emissions above all mycobacterial species that had grown well. Concentration changes were measurable in vials with visually assessed bacterial growth and vials without apparent growth. VOCs above mycobacterial cultures could be grouped into substances that were either higher or equally concentrated, lower or equally concentrated, or both as those above control slants. Hence, we were able to identify 17 substances as potential biomarkers of the presence of growing mycobacteria in general.ConclusionsThis study revealed species-specific VOC profiles for eleven species of mycobacteria that showed visually apparent bacterial growth at the time point of analysis.
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